trends and perspectives in pharmaceutical sciences

Annual Meeting of the German Pharmaceutical Society – DPhG

Trends and Perspectives in Pharmaceutical SciencesConference Book

Annual Meeting of the German Pharmaceutical Society – DPhG

Frankfurt/Main September 24 – 26, 2014 at Goethe University

www.2014.dphg.de

Frankfurt/Main, September 24 – 26, 2014 at Goethe University

www.2014.dphg.deISBN 978-3-9816225-1-5

DPhG

Annual Meeting 2014 – Conference Book

Annual Meeting of the German Pharmaceutical Society DPhG

Trends and Perspectives in Pharmaceutical Sciences Conference Book Frankfurt/Main, September 24 26, 2014 at Goethe University

www.2014.dphg.de

Sponsors of the DPhG Annual Meeting 2014

MEDIEN FÜR DIE APOTHEKE

Institutional Sponsors

Förderer der DPhG-Jahrestagung 2014

DPhG Annual Meeting 2014 3

CONFERENCE COMMITTEES

Scientific Committee Prof. Dr. Thomas Efferth Prof. Dr. Christoph Friedrich Prof. Dr. Peter Gmeiner Prof. Dr. Ulrike Holzgrabe Prof. Dr. Ulrich Jaehde Prof. Dr. Jochen Klein Prof. Dr. Heyo Kroemer Prof. Dr. Peter Langguth Prof. Dr. Stefan Laufer Prof. Dr. Kristina Friedland Prof. Dr. Andreas Link Prof. Dr. Irmgard Merfort Prof. Dr. Klaus Mohr Dr. Olaf Queckenberg Prof. Dr. Peter Ruth Prof. Dr. Andrea Sinz Prof. Dr. Angelika Vollmar Prof. Dr. Hermann Wätzig Prof. Dr. Werner Weitschies Prof. Dr. Gerhard Winter

Organisation Committee Seniorprof. Dr. Theodor Dingermann Prof. Dr. Jennifer Dressman

PD. Dr. Gunter Eckert Prof. Dr. Robert Fürst Dr. Ann-Kathrin Häfner Dr. Bettina Hofmann Prof. Dr. Michael Karas PD. Dr. Thorsten Maier Prof. Dr. Rolf Marschalek Jun.-Prof. Dr. Eugen Proschak Dr. Bernd Sorg Dr. Michael Stein Dr. Mario Wurglics

4

ADDRESS OF WELCOME

Prof. Dr. Dieter Steinhilber DPhG President

Dear colleagues,

as President of the German Pharmaceutical Society (DPhG) and congress chair-

man it is a pleasure for me to welcome you at Frankfurt to attend our Annual

place for such a meeting which is part of the GU100 celebrations on the occa-

sion of the 100th

birthday of Goethe University. Furthermore, the meeting is

cosponsored by one of our most important partners, the Pharmaceutical Society

of Japan (PSJ).

We can present you an interesting scientific programme which focuses on

recent developments in all pharmaceutical disciplines. The meeting program

covers topics such as antiinflammatory drugs, anticancer drugs and epigenetics,

biotechnology, clinical pharmacy, drug design/medicinal chemistry/analytics,

natural compounds, biopharmaceutics as well as many aspects of pharmaceuti-

cal technology and drug delivery.

I thank the scientific and the organisation committee as well as the chair

persons of the sessions for creating an exciting programme. I would like to

thank all participants of the meeting for sharing their recent results and for

their contributions. The abstract book provides you with an overview about the

scientific posters and presentations and we hope, it will contribute to a success-

ful meeting and to scientific exchange and discussions.


TABLE OF CONTENTS

General Information ................................................................................................................................................................................. 6

Conference Program Overview ............................................................................................................................................................. 7

Plenary lectures ...................................................................................................................................................................................... 15

Scientific Sessions .................................................................................................................................................................................. 23

Antiinflammatory Drugs ................................................................................................................................................................................. 24 Neurodegeneration ........................................................................................................................................................................................... 29 Pharmaceutical Technology and Drug Delivery....................................................................................................................................... 33 Medicinal Chemistry (PSJ) .............................................................................................................................................................................. 38 Biomarker and Modeling ................................................................................................................................................................................ 42 Ligand Binding Assays ..................................................................................................................................................................................... 45 Computational Chemistry and Molecular Design ................................................................................................................................... 49 Natural Compounds ......................................................................................................................................................................................... 54 Analytics ............................................................................................................................................................................................................... 59 Case studies from Pharmaceutical Research and Development ........................................................................................................ 64 GPCR Medicinal Chemistry............................................................................................................................................................................. 69 Industrial Pharmacy ......................................................................................................................................................................................... 73 Biopharmaceutics and Pharmaceutical Technology .............................................................................................................................. 77 Anticancer and Epigenetic Drugs ................................................................................................................................................................. 82 Evidence-based Medication Management ................................................................................................................................................ 86 Optimizing Oral Drug Performance ............................................................................................................................................................. 90 Multitarget Drugs ............................................................................................................................................................................................. 94 Non-canonical GPCR-Signaling ................................................................................................................................................................... 99 Biopharmaceuticals/Biotechnology ........................................................................................................................................................... 103

Short lectures ........................................................................................................................................................................................ 108

Posters ..................................................................................................................................................................................................... 121

Antiinflammatory Drugs (AD01-AD19) .................................................................................................................................................... 122 Anticancer Drugs and Epigenetics (ACE01-ACE33) .............................................................................................................................. 128 Biotechnology (BT01-BT16) ......................................................................................................................................................................... 140 Clinical Pharmacy (CP01-CP15) .................................................................................................................................................................. 147 Drug Design/Medicinal Chemistry/Analytics (MC01-MC62) ............................................................................................................. 153 GPCR (G01-G14) .............................................................................................................................................................................................. 176 Natural Compounds (NC01-NC21) ............................................................................................................................................................ 182 Neuroactive Drugs (ND01-ND13) .............................................................................................................................................................. 189 Biopharmaceutics (BP01-BP09) .................................................................................................................................................................. 194 Pharmaceutical Technology and Drug Delivery (PT01-PT34) ............................................................................................................ 198

Authors Index ........................................................................................................................................................................................ 212

6

GENERAL INFORMATION INSTRUCTIONS FOR USING CONFERENCE WLAN If you are a member of an institution (e.g. a university), which is a member of the "eduroam" community, you should use the wireless network "eduroam". In most cases you can use the wireless network eduroam in the same way you are used to connect to the wireless network at your home institution. Please use your account and the domain of your institution (e.g. [email protected]). If your institution (e.g. a university) is not a member of the "eduroam" community, you have to obtain a guest-account

s-word. After authenticating successfully, you will have access to the Internet.

ABSTRACT AND POSTER NUMBERS

Each abstract has a unique identifier, a letter-number combination. Letters refer to the conference topic a contribution was assigned to. For example, a poster presentation in the area of Antiinflammatory Drugs might have the number AD.01. Short lectures SL Please note that in the case of poster presentations the abstract number is identical with the poster number.

Please refer to the authors index on page 212 for direct access to specific abstracts.

CONFERENCE OFFICE The Conference office is located at the Conference building (Otto-Stern-Zentrum (OSZ), S1 / S2). Opening hours: Wednesday, September 24th, 2014: 10.00 am 6.00 pm; Thursday, September 25th, 2014: 8.00 am 6.00 pm; Friday, September 26th, 2014: 8.00 am 12.00 am.

POSTER SESSIONS There will be two poster sessions:

Topics

Antiinflammatory Drugs, Anticancer Drugs /

Epigenetics and Drug Design / Medicinal Chemis-

try / Analytics

Biotechnology, Clinical Pharmacy, GPCR, Natural

Compounds, Neuroactive Drugs, Biopharmaceu-

tics and Pharmaceutical Technology/Drug Deliv-

ery

Session Wednesday, Sept. 24th, 6 pm until 10 pm

Thursday, Sept. 25th, 12 o' clock noon until 1.30

pm

Set-up Wednesday, Sept. 24th, before 6 pm Thursday, Sept. 25th, before 10 am

Dismantling Wednesday, Sept. 24th, after 10 pm Thursday, Sept. 25th, after 6 pm

CONFERENCE DINNER Separate registration necessary (special fee). Please refer to the Conference Office for registration and details. The Conference dinner will take place at the Casino on Campus Westend (no public parking available, please use subway U8 from station Uni Campus Riedberg to station Holzhausenstraße). For detailed maps of Campus Riedberg and Campus Westend see page 224.


CONFERENCE PROGRAM OVERVIEW

Pre-symposia and public talk

Tuesday, 23.9.2014

Bürgersymposium: Frankfurter Pharmaziegeschichte - Von Goethe bis Hoechst Ort: Campus Westend (Foyer PA Gebäude)

Advanced Course in Pharmacology (DGPT) 'Durchflusszytometrie: Anwendungen in pharmakologi-scher und pharmazeutischer Forschung Separate Anmeldung bei der DGPT erforderlich

Ort: OSZ (HS 3) 15.00-18.30 Uhr

Workshop Drittmittelförderung Ort: OSZ (HS 4)

15.00-15.15: Begrüßung, Christoph Friedrich, Marburg

15.00-15.10: Einführung, Prof. Dr. Detlef Neumann

14.00-16.00: Dos und Don'ts beim Antragschreiben, T. Hotopp, DFG Ort: OSZ (HS 4)

15.15-16.00: Zur Entwicklung der Pharmazie an der Johann-Wolfgang-Goethe-Universität Frankfurt, Prof. Dr. Axel Helmstädter

15.10-15.55: Einführung in die Durchflusszytometrie, Dr. Stefan Schnell

Fluoreszenz-aktiviertes Zellsortieren mithilfe der Mikrochip-Technologie, Dr. Martin Büscher

16.00-17.00: Horizon 2020, M. Ackermann, Nationale Kontakt-stelle Lebenswissenschaften Ort: OSZ (HS 4)

16.00-16.45: Zur Geschichte des Frankfurter Apothekenwesens, Dr. Caroline Seyfang

15.55-16.40: Durchflusszytometrie für die pharmakologische Charak-terisierung von GPCR Liganden, Prof. Dr. Erich Schneider

15.00-16.30: Beiratssitzung des VdPPhI Ort: OSZ (HS 5)

16.45-17.15: Kaffeepause 16.40-17.00: Kaffeepause

17.15-18.00: Die Entwicklung der Firma Hoechst unter besonderer Berücksichtigung ihrer Geschichte im Dritten Reich, Prof. Dr. Stephan H. Lindner

17.00-17.45: Chipzytometrie für die - o-marker-Analyse: Technologie und Anwendung, Dr. Christian Hennig

17.00-19.30: Mitgliederversamm-lung des VdPPhI Ort: OSZ (HS 5)

18.00-18.45: Eine Tradition der besonderen Art: Goethe und sein Verhältnis zur Pharmazie und zu Pharmazeuten, Prof. Dr. Christoph Friedrich

17.45-18.30: Standardisierung und Automatisierung durch-flusszytometrischer Assays in der pharmakologischen Forschung, Dr. Peter Engel

anschl. Empfang, Campus Wes-tend, Foyer PA Gebäude

8


Wednesday, 24.9.2014

Fachgruppen-Meetings

OSZ H2 OSZ H3 OSZ H4 OSZ H5 OSZ H6

9.00-10.30 Pharmazie 2020, D. Steinhilber, S. Laufer

10.30-12.00 Fachgruppe Pharm./Med. Chemie, P. Gmeiner

Fachgruppe Pharmakologie, J. Klein

Fachgruppe Klin. Pharmazie, K. Friedland

Fachgruppe Pharm. Biologie, A. Vollmar

Fachgruppe Pharm. Techno-logie, P. Langguth

Wednesday, 24.9.2014

Main Symposium (Congress language English)

13.00-13.30

Opening of the Annual DPhG Meeting 2014, Trends and Perspectives in Pharmaceutical Sciences

OSZ H1+H2

13.30-14.15

PL 1, Peter Ruth, New disease relevant functions of Ca2+-activated potassium channels OSZ H1+H2

14.15-15.00

PL 2, Shinji Yamashita, Streamlining the development of oral drug product: Role of researchers in academia

OSZ H1+H2

15.00-15.30

Coffee break OSZ

Short talks, parallel sessions I

15.30-17.00 OSZ H3 OSZ H4 OSZ H5

Antiinflammatory Drugs Chair: S. Laufer, D. Steinhilber

Neurodegeneration Chair: C. Culmsee, J. Klein

Pharmaceutical Technology and Drug Delivery Chair: L. Meinel, W. Weitschies

15.30 Jan Schwab, Resolvins, protectins and maresins as candidates to propagate resolution of in-flammation in lesions of the central nervous system (CNS)

15.30 Carsten Culmsee, New insights into Bid-mediated mitochondrial demise in neuronal cell death

15.30 Markus Thommes, Formulation strategies for poorly water soluble drugs

15.50 Andreas Köberle, Functional lipidomics reveals phosphatidylcholine-bound arachidonic acid as regulator of protein kinase B

15.55 Jochen Klein, Experimental stroke research: problems and opportunities

15.50 Stephan Reichl, Valid cell culture models of the human cornea for drug transport investigations - where are we?

16.10 Thorsten Maier, Nitro lipids as novel regulators of leukotriene biosynthesis

16.20 Carina Hohmann, Emerging options for pharma-ceutical care in stroke patients

16.10 Tessa Charlotte Lüh-mann, Protein engineering of fibro-blast growth factor 2 (FGF-2) for bioresponsive protein delivery


OSZ H3 OSZ H4 OSZ H5

16.30 Olivia Merkel, Ex vivo and in vivo siRNA delivery to activated T cells as novel anti-inflammatory asthma therapy

16.40 Amalia Dolga, SK channel modulation atten-uates mitochondrial dysfunc-tion, neuroinflammation, and neuronal cell death

16.30 Anne Seidlitz, In vitro estimation of drug transfer from paclitaxel-coated balloon catheters

16.45 Christoph Schmidt, Rational protein-engineering yields a minimised innate immune inhibitor with unique targeting properties

16.45 Miriam Pein, Self-developed sensor mem-branes for etongue sensors

17.00-18.00

Short lectures (5 min) OSZ H1+H2

Hayato Fukuda, Design, synthesis and biological evaluation of a stabilized resolvin E2 analogue Yudai Matsuda, Biosynthetic studies on fungal meroterpenoids and their fascinating chemistry Masahito Yoshida, Destruxin E, a potent negative regulator of osteoclast morphology: Solid-phase library synthesis and biological evaluation Tsuyoshi Saitoh, Design and synthesis of NF-κB inhibitors carring epoxyquinol moiety Marlene Barho, Structure-activity relationship studies on small molecule Bid-inhibitors Anna Junker, Synthesis and structure affinity relationships of dual chemokine receptor 2 and chemokine receptor 5 antagonists and development of a selective, fluorinated CCR2 ligand for PET studies Ann-Kathrin Schoenfeld, Testing of potential inhibitors of human heparanase in a fluorescence activity assay Rico Schwarz, Monitoring conformational changes in PPARβ/δ by cross-linking and mass spec-trometry Wenjin Li, A dynamic pH junction method for monitoring the catalytic activity of cerebroside sulfotransferase Dominique Lunter, Confocal Raman microscopic (CRM) methodology for the analysis of the penetration of pharmaceutical actives into the skin Julian Schichtel, Determination of the dissolution behaviour of celecoxib-Eudragit E 100-nanoparticles using cross-flow filtration Verena Gotta, Sensitivity of concentration-effect versus dose-effect analysis to detect small magnitudes of QTc prolongation in preclinical cardiovascular safety setting

18.00-22.00 Poster session I and welcome reception, OSZ

18.00-18.30 Außerordentliche Hauptver-sammlung der DPhG, OSZ H3

10


Thursday, 25.9.2014

8.30-9.15

PL 3, Rolf Hartmann, Interference with bacterial quorum sensing: a new antivirulence strategy OSZ H1+H2

9.15-10.00

PL 4, Nagayoshi Nagai Lecture, Masakatsu Shibasaki (PSJ), Recent progress in cooperative asymmetric catalysis

OSZ H1+H2

10.00-10.30

Coffee break

OSZ

Short talks, parallel sessions II

10.30-12.00 OSZ H3 OSZ H4 OSZ H5

Medicinal Chemistry (PSJ) Chair: N. Miyata, K. Tomioka

Biomarker and Modeling Chair: C. Kloft, T. Lehr

Ligand Binding Assays Chair: C. Müller, H. Wätzig

10.30 Kiyoshi Tomioka, Paradigm re-shift of medicinal chemistry in Japan

10.30 Markus Joerger, Implementation of dosing algorithms of anticancer drugs based on pharmacological biomarkers

10.30 Frank M. Boeckler, Biophysical techniques in fragment hit identification and lead optimization - A change of perspective?

10.55 Naoki Miyata, Design, synthesis and biologi-cal activity of lysine-specific demethylase (KDM) inhibitors

11.00 Thorsten Lehr, Mathematical modeling of amyloid beta for the diagnosis and treatment of Adisease

10.50 Christian Kramer, The impact of experimental uncertainty on decision mak-ing in drug design

11.20 Hiroshi Nagase, Synthesis of a novel opioid receptor agonist, SYK-146 with 1,3,5-trioxazatriquinane skeleton and its pharmacolo-gies

11.30 Rolf Burghaus, Understanding coagulation biomarkers and deriving clinically relevant surrogates by use of an in-silico coagula-tion model

11.20 Dominique Bonnet, Fluorescent probes to track GPCR binding and dimeriza-tion

11.45 Hiroaki Ohno, Gold-catalyzed annulations and their medicinal applica-tions

11.40 Yosuke Taniguchi, Development of triplex-forming oligonucleotide having artificial nucleoside analogues to inhibit the gene expression as an antigene strategy

12.00-13.30 Poster Session II and lunch break

OSZ

Short talks, parallel sessions III

13.30-15.00 OSZ H3 OSZ H4 OSZ H5 OSZ H6

Computational Chemistry and Mo-lecular Design. Chair: F. Böckler, O. Koch

Natural Compounds Chair: R. Fürst, A. Vollmar

Analytics Chair: M. Karas, A. Sinz

Case studies from Pharmaceutical Research and Devel-opment Chair: B. Cezanne, M. Wei-gandt

mailto:[email protected]



OSZ H3 OSZ H4 OSZ H5 OSZ H6

13.30 Andreas Bend-er, Integrating chemical and biological data for drug design and mode-of-action analysis

13.30 Verena Dirsch, Neolignans: from PPARγ RXRα

13.30 Dietrich Volmer, Analysis of vitamin D metabolic markers by mass spectrometry: Advantages and limitations of the gold standard method

13.30 Andrea Hane-feld, Dendritic cell-targeting cancer vaccine formulations for pulmonary and peroral delivery

13.45 Steve Maginn, A knowledge-based approach to assessing propensity for poly-morphism in the pharmaceutical crystalline solid form

13.50 Andreas Bechthold, Waking up biosyn-thetic gene clusters in a row

13.50 Andreas Römpp, High resolution MALDI imaging: Reliable molecular identifica-tion at cellular resolu-tion

13.50 Christoph Saal, Selection of solid state forms for new chemical entities: Challenges, opportu-nities, adventures and lessons learned

14.00 Thomas Exner, Direct integration of ligand-based NMR data into protein-ligand docking

14.10 Jennifer Her-mann and Florian Förster, Chondramides: setting the stage for actin binding compounds in cancer therapy

14.10 Kai Scheffler, Mass spectrometric characterization of biopharmaceuticals - possibilities, challeng-es and limitations

14.10 Matthias Winzer, Fast track formulation development for biotherapeutics

14.15 Alexander Dömling, Screening reaction pathway-driven very large chemical space: Discovery of potent mdm2-p53 antagonist

14.30 Johanna Liebl, Cdk5 inhibition potentiates imatinib responsiveness of Philadelphia chromo-some positive chronic myeloid leukemia cells

14.30 Ganna Kalayda, Fluorescent oxaliplatin analogue as a model for the anticancer drug oxaliplatin for the investigation of its cellular trafficking

14.30 Sonja Skopp, Fighting schistosoiasis in young children: The Pediatric Praziquantel Consortium

14.30 Holger Gohlke, Identification of a mechanism-of-action target exploiting similarities of chemo-types and signalling events, and biophysi-cal simulations

14.45 Finn Hansen, Plasmodium falcipa-rum histone deacety-lases (PfHDACs) as epigenetic drug targets

14.45 Christian Wischke, A polymeric multi-functional glaucoma implant

14.50 Steffen Lü-deke, Chirality in polyketide antibiotics: substrate-dependent inversion of stereoselectivity in Tyl-KR1-catalyzed reductions

14.45 Discussion

15.00-15.30 Coffee break

OSZ

Short talks, parallel sessions IV

15.30-17.00 OSZ H3 OSZ H4 OSZ H5

GPCR Medicinal Chemistry Chair: P. Gmeiner, U. Holzgrabe

Industrial Pharmacy Chair: A. Link, S. Schmidt

Biopharmaceutics and Phar-maceutical Technology Chair: P. Langguth, S. Yamashita

15.30 Armin Buschauer, Toward selective molecular tools for histamine receptor subtypes: conformational constraints, bioisosteric and bivalent approaches

15.30 Norbert Nagel, Biophysical characterization of pharmaceutical peptides

15.30 Heinrich Haas, Novel injectable RNA formula-tions for tumor immunothera-py

12

OSZ H3 OSZ H4 OSZ H5

16.00 Gerhard Wolber, Modulation of GPCR signaling: Understanding ligand binding effects

15.50 Carsten Olbrich, Subvisible particles in protein formulations

15.50 Herbert Wachtel, Automated testing of inhala-tion devices in early develop-ment phase

16.20 Michael Decker, Molecular combination of GPCR ligands: bivalent, hybrid and dualsteric compounds

16.10 Harry F. Abts, Dissecting pharmacodynamic action of compound mixtures by use of in vitro models

16.10 Peter Serno, Orodispersible dosage forms

16.40 Nuska Tschammer, Boronic acids as probes for exploration of allosteric regulation of the chemokine receptor CXCR3

16.30 Uwe Muenster, Current biopharmaceutics prediction tools - an overview

16.30 Mai Anh Nguyen, Pharmacokinetic drug neutraceutical interactions: A particular type of food - drug interaction

16.45 Thomas Nawroth, Gastro-intestinal simulator for in-vitro drug and nanoparticle tracing in oral drug develop-ment

Short talks, parallel sessions V

17.00-18.30 OSZ H3 OSZ H4 OSZ H5

Anticancer and Epigenetic Drugs Chair: C. Brandts, R. Marschalek

Evidence-based Medication Management Chair: K. Friedland, U. Jaehde

Optimizing Oral Drug Perfor-mance Chair: M. Brewster, J. Dressman

17.00 Tom Milne, Unraveling the aberrant epigenetic programming of MLL leukemias

17.00 Welcome and short introduc-tion

17.00 Marcus Brewster, O

improving oral bioavailability

17.30 Stefan Fröhling, Identifying therapeutic targets in MLL fusion-driven leukemia using functional genomics

17.10 Isabel Waltering, Susanne Koling, Georg Hempel, Evaluation of medication management in community pharmacies

17.30 Mathew Leigh, Assessing the gastrointestinal

the media

18.00 Rolf Marschalek, MLL leukemias and future treatment strategies

17.30 Kristina Friedland, Evidence-based medication management in psychiatric patients

17.50 Edmund Kostewicz, Will the parachute crash? transfer models for assessing performance of optimized formulations

18.15 Manfred Jung, Selective Sirt2-inhibition by ligand induced rearrangement of the active site

17.50 André Wilmer, Ulrich Jaehde, Evidence-based medication management in cancer pa-tients

18.10 Rodrigo Cristofoletti, Connecting oral formulation performance to therapeutic effect

18.10 Anna Laven, PHARMAGRIPS: Structured pharmaceutical counseling in the self-medication of the common cold. A randomised controlled study (RCT)

18.20 Discussion

19.30 Conference dinner



Friday, 26.9.2014

8.30-9.15

PL 5, Charlotte Kloft, Pharmacometrics for better therapies? OSZ H1+H2

9.15-10.00

PL 6, Ernst Wagner, Sequence-defined carriers for targeted intracellular drug and nucleic acid delivery OSZ H1+H2

10.00-10.30

Coffee break OSZ

Short talks, parallel sessions VI

10.30-12.00 OSZ H3 OSZ H4 OSZ H5

Multitarget Drugs Chair: T. Efferth, E. Proschak

Non-canonical GPCR-Signaling Chair: C. Hoffmann, K. Mohr

Biopharmaceuticals/ Biotech-nology Chair: O. Germershaus, E. Wagner

10.30 Jens-Uwe Peters, An introduction to polyphar-macology in drug discovery

10.30 Andreas Bock, Dynamic ligand binding

10.30 Michael Adler, Major trends and challenges in biotherapeutic product devel-opment: "polysorbate degra-dation" and "drug-device combination product devel-opment"

11.00 Thomas Efferth, Multifactorial activity of the naphthoquinone shikonin against cancer cells

10.55 Andreas Rinne, Voltage-dependent GPCR-activation

10.50 Eva-Maria Ruberg, Finding the right candidate integrated lead ID of next-generation molecules

11.15 Eugen Proschak, Polypharmacology: In silico recognition vs. rational design

11.20 Michael Mederos y Schnitzler, Mechanosensitivity of hista-mine H1 receptor

11.10 Carsten Rudolph, Non-immunogenic messenger RNA therapeutics

11.30 Samuel N. Okpanyi, Study of the anxiolytic ac-tions of Valeriana officinalis L., Melissa officinalis L., Passi-flora incarnata L. and their combination STW 32 in exper-imental models of anxiety

11.40 Annette Kaiser, Signaling in malaria parasites: A non-canonical G-protein from plasmodium falciparum

11.30 Leonard Kaysser, Divergent pathways for the biosynthesis of merochlorins, cyclic meroterpenoid antibiot-ics from a marine streptomy-cete

11.45 Olaf Kelber, Motility modulation beyond MCP: Mechanisms of action of a clinically proven herbal medicinal product, STW 5, in functional GI diseases

11.45 Arnold Grünweller, Combination strategies for targeting the oncogenic Pim1 kinase

12.00-13.00 Short Lunch break OSZ

13.00-13.45 PL 7, Zoltan Takats, London, Direct mass spectrometric characterization of biological tissues - from automated histology to DMPK studies

OSZ H1+H2

14.00-15.00 Closing ceremony OSZ H1+H2

Gründungsversammlung der DPhG-Arbeitsgemeinschaft Notfall- und Katastrophenpharmazie = AG KatPharm (OSZ H4)

14


Post-symposium Saturday, 27.9.2014

Tag der Offizinpharmazie, OSZ H2

Organizing Committee: Kathrin Müller, Annegret Birr, Erika Fink, Michael Hannig, Juliane Kresser

Moderation: Prof. Dr. Dieter Steinhilber Sprecher der Akademie für Pharmazeutische Fortbildung der LAK Hessen

Personalisierte Pharmakotherapie

14.30-15.30 Interaktionen Welche sind häufig und relevant, Dr. Nina Griese-Mammen

Zentrum für Arzneimittelinformation und Pharmazeutische Praxis (ZAPP) der ABDA, Berlin

15.30-16.00 Kaffeepause

16.00-17.00 Patientenorientierte Arzneimitteltherapie: Ein Starter Prof. Theo Dingermann

Institut für Pharmazeutische Biologie, Frankfurt am Main

17.00-18.00 Einfluss genetischer Variabilität auf die Wirkung von Arzneimitteln Prof. Dr. Manfred Schubert-Zsilavecz

Institut für Pharmazeutische Chemie, Frankfurt am Main

Ab 18.00 Mitgliederversammlung der Fachgruppe Allgemeinpharmazie


PLENARY LECTURES

16

New Disease relevant Functions of Ca2+‐activated Potassium Channels Title

Ruth, P.

Department of Pharmacology, Toxicology & Clinical Pharmacy, Institute of Pharmacy, University of Tübingen

The big conductance, Ca2+-activated K+ (BK) channel represents an important pathway for the outward flux of K+ ions from the intracellular compartment in responses to membrane depolarization and elevation in cytosolic free [Ca2+], which serves to drive cell membrane potential in the negative direction. BK channels are functionally expressed in a range of mammalian tissues (e.g., neurons, neurosensory cells, smooth muscles, tumor cells), where they can either enhance or dampen membrane excitability and thus Ca2+ influx into cells. BK channels can be regulated by several stimuli that allow them to work as integrators of cell signaling, excitability and metabolism. Genetic ablation of the BK channel in mice leads to several disease states (e.g., elevated blood pressure, hyperaldosteronism due to impaired K+ excretion, overactive bladder syndrome and cerebellar ataxia) but has also beneficial effects like suppressing breast cancer and preventing dietary obesity. Accordingly, BK channels are potential targets for therapeutic approaches. In the auditory and visual system they play a crucial role in the maintenance of neurosensory function. In the inner ear BK channels have an important role in the signal transduction process of cochlear inner and outer hair cells. Its genetic ablation leads to progressive hearing loss due to activation of apoptotic pathways. In addition, BK channel deletion led to a higher vulnerability towards noise-induced hearing loss, a global health hazard with considerable social consequences, suggesting that BK currents are involved in survival mechanisms of cochlear hair cells. Moreover, BK channels in inner hair cells are also crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy environment. In the visual system, BK channels modulate adequate visual responses at the bipolar cell level at dim light conditions as deduced from BK channel knockout mice. In pain sensing neurons, BK channels exert inhibitory control on sensory input in inflammatory states. These results suggest that the overall analysis of a protein´s function in a mammalian organism by gene knockout is able to predict adverse as well as desired effects of modulatory drugs acting on this protein target.

PL.01


Streamlining the development of oral drug product: Role of researchers in academia

Yamashita, S.

Setsunan University, College of Pharmacy 45-1 Nagaotoge-cho, Hirakata,Osaka 573-0101, Japan

Japanese pharmaceutical industries have developed many of innovative medicines so far such as pravastatin, tacroli-mus, that made Japan as one of the leading countries in the pharma-business. However, with the advent of paradigm shift in drug development, activity of pharmaceutical industry in Japan decreased gradually. Although pharmaceutical science and technology in Japan are still in the high level, rather small size of companies (compared with mega-companies in USA and EU) makes a new drug development difficult due to the smaller size of compound library, data-base and budget. As one of the possible ways to keep the high activity of drug development, Japanese companies should share the knowledge and technologies in some parts. “Consortium of Oral Drug Absorption Screening” with 25 Japanese pharmaceutical companies was started at 2001. Final goal of the consortium is to establish the effective strategy for development of oral drug product. Also, it aims to give a platform for mutual interaction of researchers working in different companies. During past 10 years, various projects were performed in the consortium relating to oral drug absorption and outcomes were shared with all member compa-nies. In the lecture, as an organizer, activity of the consortium will be presented with showing outcomes of 2 main pro-jects for “assessment of oral absorption of poorly-soluble drugs” and “BCS Classification to identify drug developability as oral products”. Governmental support is also essential to activate the industry. As a new strategy of drug development, the early phase clinical study (exploratory IND (eIND) study) including Microdose (MD) study was proposed to increase the success probability of clinical trials. In Japan, in order to promote the eIND study, the national project on MD clinical study was started at 2008 (supported by NEDO: New Energy and Industrial Technology Development Organization), entitled as “Establishment of Evolutional Drug Development with the Use of Microdosing Clinical Trial”. In the project, we performed more than 30 MD clinical studies with marketed drugs to validate the usefulness of this new strategy and to construct the system (both soft and hard) for conducting MD clinical study in Japan. Furthermore, we performed the molecular imaging study with PET to investigate the process of GI absorption and subsequent bio-distribution of orally administered drugs [1-4]. This is also a collaborative work with governmental research center “Riken”. As a second issue of the lecture, results of this innovative work will be highlighted with emphasizing an importance of integration of new technologies in drug development such as a molecular imaging technology.

Acknowledgments:

I would like to thank to the members of “Consortium of Oral Drug Absorption Screening” for their enthusiastic help to accomplish the projects in the consortium. Also, the study of molecular imaging was done as a part of “Research Project for Establishment of Evolutional Drug Development with the Use of Microdose Clinical Trial”, sponsored by the New Energy and Industrial Technology Development Organization (NEDO).

References:

1. Yamashita, S. et al.: J Nucl Med. 2011, 52(2): 249-256. 2. Kataoka, M. et al.: Pharm Res. 2012, 29(9): 2419-2431. 3. Shingaki, T. et al.: Clin Pharmacol Ther. 2012, 91(4): 653-659. 4. Takashima, T. et al.: Mol Pharm. 2013, 10(6): 2261-2269.

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18

Interference with bacterial quorum sensing - a novel anti-virulence strategy

Hartmann, R.W.1,2 1 Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany 2 Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany

The emergence and spread of bacteria resistant to current antibiotics are a serious and growing health problem world-wide. In pursuing our objective to develop antibacterials with novel modes of action we have focused our interest on the bacterial cell-to-cell communication. Among other infections, Pseudomonas aeruginosa causes severe pneumonia in patients suffering from cystic fibrosis. It is difficult to be eradicated, especially when present in biofilms. Biofilm formation and virulence factor production are regulated by intercellular signal molecules. A selective blockade of this so called quorum sensing system is a novel therapeutic strategy to limit pathogenicity and is considered to delay resistance development. Two proteins are targeted, PqsD, a key enzyme in the biosynthesis of the signal molecules PQS and HHQ and their receptor PqsR. Following different approaches, structure-based1 and ligand-based2-4 via transition states analogs of the enzymatic reaction3-4, the first PqsD inhibitors described so far were obtained. The nitrophenylmethanols were able to permeate the gram-negative bacterial cell wall and inhibited signal molecule production and biofilm formation. Regarding PqsR, SPR biosensor experiments led to the discovery of highly efficient binders with low molecular weights including antagonists5. Site-directed mutagenesis combined with isothermal titration calorimetry led to insights into the binding mode. The first highly potent antagonists have been developed by structural modification of the natural ligand6. After an unexpected functional inversion of these antagonists in P. aeruginosa had been revealed, second generation PqsR antagonists were rationally developed and able to efficiently reduce virulence factor formation7. One of these antagonists showed a strong reduction of P. aeruginosa pathogenicity in vivo. References:

1. Sahner et al.: J. Med.Chem. 2013: 8656-8664. 2. Weidel et al.: J. Med. Chem. 2013: 6146−6155. 3. Storz et al.: J. Am. Chem. Soc. 2012: 16143-16146. 4. Storz et al.: ACS Chem. Biol. 2013: 2794-2801. 5. Zender et al.: J. Med. Chem. 2013: 6761−6774. 6. Lu et al.: Chem. Biol. 2012: 381-390. 7. Lu et al.: Angewandte 2014: 1109 –1112.

PL.03


Recent Progress in Cooperative Asymmetric Catalysis

Shibasaki, M.

Institute of Microbial Chemistry, Tokyo (BIKAKEN), 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan

Our research focuses on the development of catalytic asymmetric C-C bond-forming reactions with particular emphasis on high atom economy and their application to the synthesis of biologically significant compounds. Thus, the concept of asymmetric cooperative catalysis such as Lewis acid-Brønsted base catalysis [1,2] and Lewis acid-Lewis base catalysis [3] plays the key role in our research paradigm. In this lecture, we report our recent progress in asymmetric Lewis acid-Brønsted base cooperative catalysis. In 1995, we developed the first example of a syn-selective catalytic asymmetric nitroaldol reaction using LLB (La-Li-BONOL) as catalyst. At this time the anti-selective reaction remained a longstanding problem. Finally, by changing the catalyst design to a Nd/Na heterobimetallic catalyst possessing a chiral amide ligand, we succeeded in developing an efficient and practical anti-selective catalytic asymmetric nitroaldol. In addition, the development of direct catalytic asymmetric aldol-type reaction of thioamides with aldehydes such as RCH2CHO was also considered to be impossible due to the low acidity of the a-proton. Recently we could overcome this inherent problem by identifying an asymmetric soft Lewis acid- hard Brønsted base cooperative catalyst. How to over-come this problem as well as application to an efficient and practical catalytic asymmetric synthesis of atorvastatin will also be discussed.

References:

1. Shibasaki, M. et al.: Acc. Chem. Res., 2009, 42: 1117-1127. 2. Kumagai, N. and Shibasaki, M.: Angew. Chem. Int. Ed., 2011, 50: 4760-4772. 3. Shibasaki, M. et al.: Synlett, 2005, 10: 1491-1508.

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20

Pharmacometrics for better therapies?

Kloft, C.

Freie Universitaet Berlin, Institute of Pharmacy, Dept. of Clinical Pharmacy & Biochemistry, Kelchstr. 31, 12169 Berlin, Germany

Pharmacometrics, a young but emerging computer-based science1, aims to understand and elucidate qualitatively, quantitatively and over time the relationships between drug intake – drug concentrations, preferentially at the site of action (pharmacokinetics) – resulting desired und undesired drug effects measured by e.g. biomarkers (pharmacody-namics) – and therapeutic outcome by influencing disease progression and exerting toxicity (benefit/ risk) considering knowledge of patient characteristics and their disease. The developed in silico models, pharmacometric models, attempt to incorporate data of multiple levels, namely on the molecular, cellular, tissue, organ and patient level and thus provide an increased quantitative understanding of dynamic complexity in time, space, and population diversity. Ultimately, a thorough understanding of the underlying mechanisms of drug disposition, target binding, drug-target complex signal transduction, e.g. inside the cell in various sub-cellular compartments and target dynamics, as well as the impact of patient, treatment and study characteristics shall be cov-ered in a coherent modelling framework. Hence, pharmacometrics creates a paradigm2 for enabling an integrated and higher level of understanding of drugs, (diseased) systems characteristics, and their interactions through mathematical models throughout the entire drug development process (model-based or model-integrated drug development, MBDD or MIDD)3 and for therapeutic usage (model-based or model-integrated patient care, MBPC or MIPC). Pharmacometrics is based on a transdisciplinary approach bridging concepts of biology, physiology, pharmacology, pharmacotherapy, clinical pharmacy and clinical pharmacology, medicine, mathematics and statistics. The advance-ments and success of pharmacometrics substantially increased the demand for scientists educated and trained to understand the science and underlying framework as well as to develop pharmacometric concepts for drugs and diseas-es. To this end, the Structured Graduate Research Training program PharMetrX (www.PharMetrX.de) has been launched to implement this field also in the academic environment. Pharmacometrics has already demonstrated an added value on, e.g., predictive (translational) pharmaco-kinetic/pharmacodynamics models and proved instrumental in decision-making in the drug development process and will hopefully in future contribute to better bridge the gap toward improving patient care.

References:

1. Barrett, J.S. et al.: J. Clin. Pharmacol. 2008, 48: 632-649. 2. Vlasakakis, G. et al.: Clin. Pharmacol. Ther.: Pharmacomet. Syst. Pharmacol. 2013, 2: e40. 3. Milligan, P.A. et al.: Clin. Pharmacol. Ther.. 2013, 93(6): 502-514.

PL.05


Sequence-defined carriers for targeted intracellular drug and nucleic acid delivery

Wagner, E.1,2 1 Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig–Maximilians-University, Butenandtstrasse 5-13, 81377 Munich, 2 Nanosystems Initiative Munich, Schellingstraße 4, 80799 Munich, Germany

Already five decades ago cationic polymers were used for transfection of nucleic acids [1]. Since then our understanding of polymers and their rational use in gene delivery has gradually increased. Biological challenges in extra- and intracellu-lar delivery without toxicity were recognized and resulted in breakthrough developments, including surface-shielded polyplexes, polymers with endosomal escape properties, and biodegradable polymers. Despite this, significant challeng-es remain and medical translation still has to be demonstrated. The therapeutic perspectives have widened from pDNA based gene therapy to application of various novel therapeutic nucleic acids including mRNA, siRNA and microRNA. High resolution microscopy strongly supported our insight on involved biological delivery mechanisms. Improved precision in macromolecular chemistry enables better design of polymeric carriers. In our recent research solid phase assisted synthesis was applied in the design of >700 precise oligomers [2-3] for targeted transfer of therapeutic nucleic acids. Such oligomers self-assemble with nucleic acids or are conjugated, which results in nanostructures resembling “synthetic viruses”. Like natural viruses, they contain functional subdomains for facilitating the various delivery steps, including improved packaging [4], facilitated intracellular release [5], and targeted cell uptake [5-7].

Recently we realized that such carriers can be quite useful for intracellular delivery of other therapeutic agents including recombinant proteins [8] or low-molecular weight drugs [9] as will be presented in this talk.

References:

1. Vaheri, A.; Pagano, J.S.: Virology 1965, 27: 434-436. 2. Schaffert, D. et al.: Angew. Chem. Int. Ed. Engl. 2011, 50: 8986-8989. 3. Scholz, C. et al.: Chem. Med. Chem. 2014, online. 4. Troiber, C. et al.: Biomaterials 2013, 34: 1624-1633. 5. Lächelt, U. et al.: Nanomedicine NBM 2014, 10: 35–44. 6. Dohmen, C. et al.: ACS Nano 2012, 6: 5198–5208. 7. Zhang, C.Y. et al.: J. Control. Release 2014, 180: 42–50. 8. Maier, K.; Wagner, E.: J. Am. Chem. Soc. 2012, 134: 10169-10173. 9. Lächelt, U. et al.: Mol. Pharmaceutics 2014, 11: 2631–2639.

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22

Direct mass spectrometric characterization of biological tissues - from automated histology to DMPK studies Takats, Z.

Direct mass spectrometric analysis of tissues has been developed in a parallel fashion with the development of desorp-tion ionization methods from the 1970’s. In spite of the success of early studies, the technology remained at the proof-of-concept stage until the advent of mass spectrometric imaging (MSI) by Matrix-assisted laser desorption ionization (MALDI) in the late 1990’s. MSI is a unique assay capable of providing spatial distribution information on hundreds or thousands of chemical spe-cies, which information is particularly valuable in case of biological tissues. Prior to the advent of MSI, chemical analysis of tissues started with an obligatory homogenization step, where any spatial resolution information was immediately lost. In order to obtain such information, histological approaches (e.g. immunohistochemistry) or autoradiography were employed, targeted at one or another chemical species. In contrast, imaging mass spectrometry provides semi-quantitative information on all detectable species in an untargeted fashion. This information can be interpreted in two, markedly different ways. On one hand, one can use the intensity distribution of individual species to gain concentration distribution-type information, which is particularly important in case of DMPK studies. Thanks to the untargeted nature of MSI, information on the distribution of drugs and their metabolites is obtained in a single experiment without radioactive (or fluorescent) labelling. On the other hand, the full spectral information (i.e. the entire mass spectrum belonging to single pixel) can also be used for the histological or histopathological classification of tissues using multivariate statistical tools. This type of data analysis itself has the potential of substituting classical morphology-based histological examina-tions and it can also facilitate the co-registration of histological and drug distribution information. The capabilities of MSI are demonstrated using data obtained by the more recently developed Desorption Electrospray Ionization (DESI) MSI technique. DESI – in contrast to MALDI – focuses solely on low molecular weight species (metab-olites, lipids), however the technique does not require any chemical modification of the sample, which makes it particu-larly useful for pharmacological studies. In case of DESI MSI the structural lipid composition of the tissue is used to establish the histological identity of the individual pixels, while the abundance of the molecular ions of drugs is associat-ed with their concentration distribution. The combined results give unique information on the histologically localised metabolism and accumulation of drugs and their metabolites.

PL.07


SCIENTIFIC SESSIONS

24

ANTIINFLAMMATORY DRUGS Resolvins, protectins and maresins as candidates to propagate resolution of inflammation in lesions of the central nervous system (CNS)

Schwab, J.1,2 1 Department of Neurology and Experimental Neurology, Clinical and Experimental Spinal Cord Injury Research (Neuroparaplegiology), Charite - Universitatsmedizin Berlin, D-10117 Berlin, Germany 2 Department of Neurology & Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University Medical Center, Columbus, OH 43210, USA

Inflammatory resolution is an active, highly regulated process already encoded at the onset of inflammation and required to prevent the transition into chronic inflammation associated with spreading of tissue injury, exacerbated scarring and pain. We introduce the concept of resolution of inflammation in CNS lesions and summarize emerging evidence for the bioactivity of specialized pro-resolving mediators (SPM) such as the resolvins, protectins and maresins to attenuate inflammation-associated neuropathogy. Leukocyte composition in the acute inflammatory milieu in CNS lesion differs from that after peripheral lesions as being an immune privileged site. PMN infiltration is not a conserved prominent feature of the CNS inflammatory milieu. The defective resolution of acute inflammation is a major hallmark of a dysfunc-tional immune response after experimental and human CNS injury. The persisting, chronified inflammation is composed mostly of microglia/macrophages and likely to participate in late degeneration and non-resolving inflammation. Tradition-al anti-inflammatory treatment strategies only might be insufficient since impaired resolution of inflammation is part of the underlying pathophysiology. Modelling neuroinflammatory phenotypes targeting impaired resolution remain underecog-nized to date. Given that the acute inflammatory response in central nervous system (CNS) lesions is late or non-selflimiting per se SPMs qualify as causal candidates to shape a maladaptive immune response. SPMs constitute a group of drug targets validated to excert robust bioactivity in CNS neuropathology.


Functional lipidomics reveals phosphatidylcholine‐bound arachidonic acid as regulator of protein kinase B Koeberle, A.1; Shindou, H.2; Koeberle, S.3; Laufer, S.4; Shimizu, T.2,5; Werz, O.1 1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, 07743 Jena, Germany 2 National Center for Global Health and Medicine, 162-8655 Tokyo, Japan

3 Leibniz Institute of Age Research - Fritz-Lipmann-Institute, 07745 Jena, Germany 4 Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Tübingen, 72076 Tübingen, Germany 5 Department of Lipidomics, Faculty of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan

Dysregulation of membrane lipids has been associated with disease (e.g., inflammation and cancer), though the underly-ing mechanisms are poorly defined. Functional lipidomics combines comprehensive lipid profiling with mechanistic and cell-biological studies to unravel the signalling mechanism of bioactive lipid mediators. We applied this approach to study the role of membrane lipids for the cell cycle-dependent regulation of protein kinase B (Akt) - a major kinase for cell proliferation, survival and innate immunity. Since Akt is recruited to membranes for activation, we speculated that its activity might be regulated by an oscillating membrane lipid component. By monitoring the lipid profile of synchronized mouse fibroblasts during the cell cycle by UPLC-MS/MS, we found an inverse correlation between the proportion of arachidonic acid-containing phosphatidylcholine (20:4-PC) and Akt activity (1). Increasing the ratio of 20:4-PC inhibited Akt membrane binding, Akt (S473) phosphorylation, Akt downstream signalling, S-phase transition and cell proliferation. In a lipidome-wide screen, the small molecule indirubin-3’-monoxime was identified to reprogram cells towards an accumulation of 20:4-PC thereby blocking Akt signaling and cell proliferation. The direct influence of 20:4-PC on Akt membrane binding together with the specificity by which 20:4-PC inhibits Akt activation is surprising. Akt activity is usually regulated through the level of phosphatidylinositol-3,4,5-trisphosphate (PIP3) - which anchors Akt to membranes – and not through the affinity of Akt for binding PIP3, and biological effects of 20:4-PC are ascribed in most studies to the release of arachidonic acid and biosynthesis of eicosanoids instead of to the phospholipid itself. We speculate that targeting the metabolism of polyunsaturated phospholipids might be a promising approach for the treatment of hyperpro-liferative and inflammatory diseases.

References:

1. Koeberle, A. et al.: PNAS 2013, 110: 2546.

26

Nitro lipids as novel regulators of leukotriene biosynthesis

Awwad, K.1,2; Steinbrink, S.D.3; Frömel, T.1,2; Lill, N.1,2; Isaak, J.1,2; Häfner, A.-K.3; Roos, J.3; Hofmann, B.3; Heide, H.4; Geisslinger, G.5; Steinhilber, D.3; Freeman, B.A. 6, Fleming, I.1; Maier, T.J. 3,7 1 Institute for Vascular Signaling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany. 2 DZHK (German Centre for Cardiovascular Research) partner site Rhine-Main, Frankfurt, Germany. 3 Institute of Pharmaceutical Chemistry, Goethe-University, Frankfurt, Germany. 4 Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe-University, Frankfurt am Main, Germany. 5 Pharmazentrum Frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany. 6 Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 7 Institute of Biomedicine, Pharmacology, Aarhus University, DK-8000 Aarhus C, Denmark

The reaction of nitric oxide generated during inflammatory processes with polyunsaturated fatty acids yields electrophilic nitro fatty acids (NO2-FA), which display anti-inflammatory properties [1]. Because the 5-lipoxygenase enzyme contains critical nucleophilic amino acids potentially sensitive to electrophilic attack [2], we investigated whether NO2-FA sup-press 5-LO enzyme activity in vitro and 5-LO-dependent inflammatory reactions in vivo. We were able to show that treatment of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO2-OA) or nitro-linoleic acid (NO2-LA) (but not the parent lipids) led to a concentration dependent and irreversible inhibition of 5-LO product formation. Suppressive effects were also observed in cell lysates and using the recombinant human 5-LO protein, indicating a direct reaction with 5-LO. Activity of the related enzymes 12-LO or 15-LO-1 as well as cellular prostaglandin E2 synthesis were not affected by NO2-FAs. Mechanistically, NO2-FA-induced inhibition of 5-LO was due to nitroalkylation of a specific cyste-ine residue (Cys418) via a Michael reaction, and the exchange of Cys418 to serine by mutation rendered 5-LO insensi-tive to NO2-FA. Notably, structurally related derivatives of NO2-OA containing a Michael-acceptor also caused direct and potent suppression of 5-LO enzyme activity. Systemic administration of NO2-OA to mice decreased neutrophil and monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5-hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. Administration of NO2-OA to 5-LO knockout mice had no effect on LPS-induced neutrophil or monocyte mobilization as well pulmonary inflammation. NO2-FAs are a novel type of endogenous 5-LO inhibitor directly and irreversibly suppressing 5-LO and contributing to resolution of inflamma-tion in vivo. We hypothesize that synthetic homologues of the NO2-FAs may represent an innovative strategy to treat inflammatory diseases and may represent a novel potential pharmacological option for 5-LO inhibition, which might have therapeutic implications for asthma.

The authors are indebted to Marie von Reutern, Isabella Schlöffel, and Sven George for their excellent technical support. This study was supported by the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF) and the Deutsche Forschungsgemeinschaft (Exzellenzcluster 147 ‘‘Cardio-Pulmonary Systems’’ and SFB 815/Z1). T.J.M. is the recipient of a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft.

References:

1. Baker, P.R., Schopfer, F.J., Freeman, B.A.: Free Radic Biol Med 2009, 46(8): 989-1003. 2. Hornig, M. et al.: Biochim Biophys Acta 2011, 1821(2): 279-286.


Ex vivo and in vivo siRNA delivery to activated T cells as novel anti-inflammatory asthma therapy Xie, Y.1; Kim, N.H.1; Nadithe, V.1; Thakur, A.1,2; Lum, L.G.1,2; Bassett, D.J.P.1; Merkel, O.M.1,2 1 Wayne State University, DETROIT, MI, United States of America 2 Karmanos Cancer Institute, DETROIT, MI, United States of America

Local, targeted, cell-specific RNA interference (RNAi)-based therapies could improve patients’ ability to control asthma. Allergen-induced airway dysfunction was shown to be prevented by downregulating the secretion of Th2 cytokines. However, T cells are hard to transfect cells and not easily accessible for RNAi-based therapies. We recently reported that activated T cells (ATCs) overexpress the transferrin receptor (TfR) which is an internalizing transmembrane receptor that mediates endocytosis of transferrin-bound iron and which is broadly exploited for targeted nucleic acid delivery.1, 2

Here we aim to therapeutically downregulate the Th2 transcription factor GATA-3 which is known to drive IL-4, IL-5, and IL-13 secretion in asthma to silence all its downstream inflammatory cascades.

T cells are isolated from full blood or by magnetic bead isolation from mouse spleens. TfR overexpression is measured by flow cytometry upon activation with anti-CD3. TfR-targeted nanocarriers are designed by optimizing the coupling chemistry of Tf and low molecular weight polyethylenimine (PEI) and are purified by FPLC and ultrafiltration. Fluores-cently labeled siRNA is delivered to ATCs, and the uptake is evaluated by flow cytometry. Gene knockdown in primary ATCs after siRNA delivery is quantified by qRT-PCR. Balb/c mice are sensitized and challenged with ovalbumin (OVA) or with NaCl as negative control. Mice are treated intratracheally with free fluorescent siRNA, targeted, or non-targeted nanocarriers. Their lung function is measured as changes in resistance to metacholine challenge.

A: Coupling of Tf to PEI, B: siRNA uptake in ATCs, C: Asthma model, D: siRNA uptake in CD4+ cells in OVA-challenged mice.

The coupling of Tf to PEI was optimized and the nanocarriers were shown to be smaller than 100 nm in size. ATCs selectively took up targeted nanocarriers, and 70% gene knockdown was achieved in primary ATCs. OVA-challenged mice showed specific uptake of fluorescently labeled siRNA in pulmonary T cells mediated by targeted nanocarriers. No other cell types (macrophages, epithelial cells, endothelial cells, eosinophils, B cells) took up significant amounts of siRNA. The lung function was not negatively affected by the Tf-shielded nanocarriers.

Wayne State Start-Up Grant, FRAP, BOOST and NanoIncubator grants to OMM are gratefully acknowledged.

References:

1. Kim, N.H. et al.: J Aerosol Med Pul Drug Del 2013, 26 (2): A46-A47. 2. Kim, N.H. et al.: J Drug Del Sci Tech 2013, 23 (1):17-21.

28

Rational protein-engineering yields a minimised innate immune inhibitor with unique targeting proper-ties

Harder, M.; Simmet, T.; Schmidt, C.Q.

Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany

Introduction: The complement system is an integral part of the human immune system and is well recognized for its contributions to host defense and tissue homeostasis [1]. While complement activation is necessary to protect and maintain self-tissue, prolonged and unrestricted activation is causative or associated with several illnesses [2]. The increasing evidence of complement involvement in many frequent chronic conditions has revived the surge for interven-ing options, but several challenges specific to the complement system have prevented the availability of an efficient and cost effective complement therapeutic. Three complement activation pathways exist. Especially the so called “alternative pathway” of complement activation is the underlying factor in several human disease conditions (e.g. age-related macu-lar degeneration). However, treatment options that specifically block the alternative pathway (AP), leaving other com-plement pathway uninhibited, are not available in the clinic [3].

Objectives: This study describes the rational engineering of the innate immune modulator “mini-FH”, which is based on the template glycoprotein “Factor H”, a natural complement regulator in human serum. The study also evalu-ates the biological functions and pharmacokinetic behaviour of mini-FH and mini-FH-derived second generation prod-ucts.

Results: Based on resent insights into the structure and function of the crucial complement AP-regulator Factor H [4-6], we rationally engineered a protein therapeutic (mini-FH) by directly joining selected N- and C-terminal domains of FH through a rationally optimized linker in such way that Factor H functionality is preserved. Importantly, the chosen design also attributes a novel function: mini-FH shows a strong preference over Factor H in binding complement inacti-vation products. This furnishes mini-FH with a unique triple targeting mechanism to facilitate simultaneous targeting to (i) complement activation/inactivation products and (ii) polyanionic host surface markers and (iii) to markers of oxidative stress. The engineered mini-FH protein was expressed in the methylotrophic host Pichia pastors, purified to homogenei-ty and submitted to several in vitro assays including an AP-mediated disease assay on patient-derived cells: mini-FH conferred efficient complement regulation at an IC50 of 60 nM. In a next step novel mini-FH derivatives with extended structures were generated to optimise efficacy and pharmacokinetic profiles. The resulting 2nd generation mini-FH class of proteins were tested in a panel of established interaction, functional and inhibition studies and exhibited improved activity when compared to the first generation. Finally, a first pharmacokinetic profile and the in-vivo activity of a selected mini-FH candidate-protein were assessed after in vivo administration into a wildtype mouse and transgenic mice mimick-ing a complement-mediated disorder, respectively.

Conclusions: By employing a rational engineering approach of the natural complement alternative pathway reg-ulator FH, we gained a set of very efficient complement regulators that largely exceed (about ten- to twenty-fold) the biological activity of the template molecule Factor H. Our data show that the rationally engineered mini-FH proteins are able to prevent disease-related complement activation by targeting sites of ongoing complement amplification on host cells; thus, the mini-FH class of engineered proteins has high therapeutic potential for a variety of complement-mediated diseases.

Acknowledgments: Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA, John D. Lambris & Daniel Ricklin.

References:

1. Ricklin, D. et al.: Nat Immunol. 2010, 11: 785-797. 2. Ricklin, D.; Lambris, J.D.: Nat Biotechnol. 2007, 25: 1265-1275. 3. Ricklin, D.; Lambris, J.D.: J Immunol. 2013, 190: 3839-3847. 4. Schmidt, C.Q. et al.: Clin Exp Immunol. 2008, 151: 14-24. 5. Schmidt, C.Q. et al.: J Immunol. 2008, 181: 2610-2619. 6. Morgan, H.P. et al.: Nat Struct Mol Biol. 2011, 18: 463-470.


NEURODEGENERATION New insights into Bid-mediated mitochondrial demise in neuronal cell death

Culmsee, C.

Institute of Pharmacology and Clinical Pharmacy, University of Marburg, 35032, Germany

Mitochondria are highly dynamic organelles with essential functions in the physiology of energy metabolism, controlled reactive oxygen species (ROS) formation and the regulation of intracellular Ca2+ homeostasis. In the nervous system, mitochondrial integrity is crucial for the maintenance and function of neurons. In fact, mitochondrial damage is a major feature of many neurological and neurodegenerative diseases, and cellular stress and death signaling pathways con-verge at the level of mitochondria. In particular, disturbed intracellular Ca2+ homeostasis, increased ROS formation, dysbalanced fusion and fission of the organelles, loss of the mitochondrial membrane potential, and the release of mitochondrial proteins such as apoptosis inducing factor (AIF) are prominent in many different paradigms of neuronal dysfunction and death.

The lecture will highlight novel insights into the molecular regulation of increased mitochondrial fission and associated damage in models of neuronal cell death induced by oxidative stress, glutamate toxicity and oxygen glucose deprivation. In particular, the role of Bid will be discussed as potential targets for therapeutic approaches in models of neuronal death in vitro and in vivo. The data demonstrate that Bid interacts with dynamin related protein-1 (Drp-1) and the voltage-dependent anion channel (VDAC1) in mediating mitochondrial damage and intrinsic pathways of cell death, while phar-macological inhibition or genetic deletion of each factor preserves mitochondrial functions, thereby providing neuropro-tective effects. In addition, further approaches of mitoprotection are discussed, including strategies of mitochondrial preconditioning mediated by inhibition of complex I, AIF depletion or inhibition of Cyclophilin A (CypA).

Overall, several lines of evidence expose mitochondria as key regulators in pathways of cellular stress with relevance to progressive neuronal dysfunction and death which is prominent in many neurodegenerative diseases and conditions of acute brain damage. Thus, novel concepts aiming at preserved mitochondrial integrity and function may provide effective neuroprotection.

30

Experimental stroke research: problems and opportunities

Klein, J.

Department of Pharmacology, FB 14, Goethe University, Frankfurt, Germany

A stroke is an acute incidence in humans and can hardly be foreseen. Accordingly, clinical stroke research is largely restricted to patients that have sustained a stroke hours or days earlier. In experimental stroke research, brain ischemia can be induced in rodents in a controlled manner, parameters such as cerebral blood flow and brain damage can be monitored in situ, and neurologic outcome can be determined for weeks afterwards. Nevertheless, many compounds that were identified as therapeutically effective in rodents did not work when tested in clinical studies. To improve the transla-tion of drugs from bench to bedside, we have therefore attempted to get a clearer picture of the metabolic status of ischemic brain tissue in rodents, using microdialysis and analytical approaches to quantify energy metabolites in ischem-ic and healthy tissue. In our experimental work, we presently focus on preventive measures which could be used to reduce ischemic damage in patients at risk. In one approach, we administered bilobalide, a neuroprotective compound isolated from Ginkgo biloba extracts which are known to be well tolerated by humans. At 1-10 mg/kg, bilobalide reduces ischemic brain damage up to three hours after stroke and also improves motor function in mice. Microdialysis studies showed that the dramatic, more than 50-fold increase of glutamate which is observed in untreated animals is reduced by 80% in treated mice. At the same time, mitochondrial function is preserved, especially in complex I of the electron transport chain (ETC). Protection of mito-chondria leads to higher energy (ATP) levels, reduced glutamate release and protects neurons from excitotoxicity and cell death. In a second approach, we used an anaplerotic diet in mice. Anaplerosis, the mechanism of refilling lost substrates in the citric acid cycle (CAC), is a new approach for neuroprotection. Using triheptanoin (glycerol-triheptanoate) as a dietary supplement in mice, we expected the formation of odd-chain fatty acids which can be metabolically transformed into succinate, a metabolite of the CAC which also fuels directly into complex II of the ETC. After 14 days of diet, stroke was induced and neurological deficits were determined by behavioral testing. Triheptanoin-fed mice showed a significant improvement in three behavioral tests whencompared to mice on control diet containing soybean oil. In microdialysis studies, blood flow and glucose consumption were similar between the two dietary groups, but glutamate release was reduced by 66% in triheptanoin-fed mice. What is more, the activities of complexes II and IV of the ETC were higher in the triheptanoin group, and ATP levels and mitochondrial membrane potentials were also better preserved. This data shows that the triheptanoin-rich diet produced a neuroprotective effect in ischemic stroke in mice. It should therefore be tested as a prophylactic treatment against brain ischemia in humans. In summary, experimental stroke research helps to identify potential prophylactic treatments that could be tested in clinical trials. It may be more promising to prevent ischemia-induce damage than to treat it after the damage has been done. Alternatively, these treatments could be used to improve regeneration after stroke. Experiments to that effect are currently in the planning stage.


Emerging options for pharmaceutical care in stroke patients

Hohmann, C.1,2; Neumann-Haefelin, T.2; Radziwill, R.1 1 Klinikum Fulda gAG, Department of Pharmacy, Pacelliallee 4, 36043 Fulda, Germany 2 Klinikum Fulda gAG, Department of Neurology, Pacelliallee 4, 36043 Fulda, Germany

Stroke is one of the leading causes of death in Europe and the major cause of disability in the elderly. Ischaemic stroke accounts for 85–90 % of all strokes and is characterized by the sudden occlusion of a cerebral artery resulting in re-duced blood flow and loss of neurological function. Ischaemic stroke is mainly caused by large-artery atherosclerosis, cardio embolism (e.g. atrial fibrillation), or small-vessel occlusion. Determing the cause of ischaemic stroke is very important for further therapy management, especially the secondary stroke prevention. The main risk factors for stroke are hypertension, atrial fibrillation, diabetes mellitus, high cholesterol and smoking. A consistent, safe, and effective secondary prevention after ischemic stroke is very important and contains the intake of acetylsalicylic acid in atheroscle-rotic stroke or an oral anticoagulant for the prevention of cardioembolic stroke as well as the treatment of the cardiovas-cular risk factors. The main issues involved with clinical pharmacy practice are (i) medication reconciliation, (ii) drug therapy optimiza-tion, and detection, resolution and prevention of drug-related problems (DRPs), (iii) consultation with the patient regard-ing new drugs including advice about indication, dosage, adverse events, and drug-interactions, (iv) giving detailed information on medication upon hospital discharge in the discharge letter by the clinical pharmacist. These different issues of the clinical pharmacy service in stroke patients will be presented [1-5]. In conclusion, clinical pharmacists can provide a valuable contribution in the multidisciplinary team by detecting and resolving DRPs that lead to an optimized and safe pharmacotherapy, especially regarding to antihypertensive medica-tion, secondary prevention, and statin therapy. Furthermore, they support the information transfer through medication review and medication reconciliation on hospital admission and hospital discharge that lead to an optimized transition care process. References:

1. Hohmann, C. et al.: Pharm World Sci. 2009, 31(5): 550-558. 2. Hohmann, C. et al.: Health Qual Life Outcomes. 2010, 8: 59. 3. Hohmann, C. et al.: Int J Clin Pharm 2012, 34(6): 828-831. 4. Hohmann, C. et al.: Stroke 2013, 44(2): 522-524. 5. Hohmann, C. et al.: J Clin Pharm Ther. 2014, 39(3): 286-291.

32

SK channel modulation attenuates mitochondrial dysfunction, neuroinflammation, and neuronal cell death

Dolga, A.M. 1; Terpolilli, N.2; Netter, M.3; Richter, M.4; Decher, N.3; Plesnila, N.2; Culmsee, C.1 1 Institute of Pharmacology and Clinical Pharmacy, University of Marburg, 35032, Germany 2 Department of Neurosurgery, Ludwig-Maximilians-Universität München, München, 81377, Germany 3 Institute of Physiology and Pathophysiology, Vegetative Physiologie, University of Marburg, 35037, Germany 4 Department of Neurology, University of Marburg, 35043, Germany

Potassium channels are a family of highly diverse transmembrane proteins with multiple functions in the physiology of excitable cells, and according dysfunctions have been linked to degeneration and death of neurons in various neurologi-cal diseases. According to current knowledge, small-conductance calcium-activated potassium (SK/KCa2) channels are located in close vicinity to synaptic NMDA receptors and control excitability and Ca2+ influx by reducing the amplitude of synaptic potentials. Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i), mitochondrial dysfunction, ER stress and inflammation are established hallmarks of neu-ronal cell death. Recently, we showed that pathological [Ca2+]i deregulation occurring after glutamate receptor stimula-tion is effectively modulated by SK channels. Activation of SK channels preserved SK expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo in a model of middle cere-bral artery occlusion. In addition, SK channel opening restores microglial activation, cytokine production and nitric oxide release.

Besides their plasma membrane localization we have demonstrated that functional SK2 channels are also expressed in the mitochondrial inner membrane and prevent glutamate-induced neuronal oxidative stress and mitochondrial dysfunc-tion by 80-90%. Activation of SK channels inhibits mitochondrial fragmentation, loss of mitochondrial membrane poten-tial, and translocation of apoptosis inducing factor (AIF) to the nucleus. Using a neuronal cell line devoid of NMDA receptors, we demonstrated that the neuroprotective effects were independent of direct interaction of SK channels with NMDA receptors and calcium influx, further supporting the functional activity of SK channels in mitochondria. Further-more, overexpression of SK channels in mitochondria using mitochondrial-targeted SK channels demonstrated substan-tial neuroprotective effects in a model of oxidative stress (induced by glutamate) and ER stress (induced by brefeldin A). SK channel activation altered the level of unfolded protein response proteins, i.e. further increased CHOP levels and reduced PERK levels compared to brefeldin A-treated cells. Moreover, activation of SK channels resulted in slight ATP depletion and reduced mitochondrial metabolic activity, as assessed by the Seahorse Bioscience XFe cell metabolism analyzer. These results expose a pre-conditioning effect as a mechanism for neuroprotection mediated by SK channel activation.

Our findings show a critical role for SK channels in excitotoxic neuronal cell death, mitochondrial dysfunction, ER stress-associated cell death and neuroinflammation, proposing their activation as potential therapeutic strategy for the treat-ment of acute and chronic neurodegenerative disorders.


PHARMACEUTICAL TECHNOLOGY AND DRUG DELIVERY

Formulation Strategies for Poorly Water Soluble Drugs Thommes, M.

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Duesseldorf, Germany

The low aqueous solubility of novel active pharmaceutical ingredients is one of the major challenges pharmacists have been facing for more than 35 years. Since then several formulation strategies have been developed, which all have advantages but also limitations. The presentation covers the physical reasons as there are particularly low crystal lattice energy as well as high lipophilic-ity as main reasons for low aqueous solubility. This requires different formulation strategies like “emulsifying systems”, “solid dispersions”, “nano particles” as well as “co-crystals” in order to increase the oral bioavailability. These concepts are discussed in detail while market products are mentioned and actual research trends are debated.

34

Valid cell culture models of the human cornea for drug transport investigations - where are we?

Reichl, S.; Hahne, M.; Verstraelen, J.; Kölln, C.

Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu Braunschweig, Mendelssohnstraße 1, 38106 Braun-schweig, Germany

Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treat-ment. Since the cornea is the main barrier for most topically applied drugs, such preclinical investigations on transcorne-al drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. Cell culture models of the human cornea can avoid several of the disadvantages of widely used animal experimental models, including ethical concerns and poor standardisation [1]. However, for widespread use, the con-temporary validation of existent systems is required. Based on SV40 immortalized human corneal epithelial cells and keratocytes we established a serum-free cultivated human hemicornea construct (HC) that exhibited high degree of equivalence to ex vivo tissue regarding histological characteristics and barrier properties [2]. In a next step, our standard operating procedures were transferred and the HC model was independently cultured in three different laboratories, and the intralaboratory and interlaboratory reproducibil-ity was analyzed and compared with animal corneas. This analysis showed that the HC has a barrier in the same range as excised animal corneas, although with a higher reproducibility and lower variability [3]. While transcorneal passive drug transport processes are well understood, only few is known about the expression of transporter proteins, in particular ABC transporters, and drug-metabolizing enzymes in both human and animal corneal tissue as well as human cornea cell culture models. A comparison of the expression of ABC transporters (MDR1, MRP 1-5 and BCRP) and phase I and II enzymes, in particular, cytochrome P450 enzymes (CYP) and glutathione transferas-es (GST), between HC and the most commonly used ex vivo models, namely, rabbit and porcine corneas, was conduct-ed. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors or activity assays in the case of GST and CYP. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas HC and porcine corneas did not show MDR1 expression. However, HC possessed MRP1, 3-5 and BCRP expression, whereas no functional expression of MRP 1-3, 5 and BCRP was found in porcine corneas and MRP 3, 4 and BCRP in rabbit corneas. Therefore, the transfer of data obtained from animal experiments to an in vivo situation in humans should be performed with caution. Even though the expression levels of drug-metabolizing enzymes in corneal tissue are low in comparison to liver or kidney, the expression of GSTO1, GSTP1 and CYP2D6 isoenzymes was detectable in HC, human and animal corneas on mRNA, protein and functionality level. GST activity was found to be higher in rabbit and porcine cornea compared to HC. In contrast, total CYP450 and CYP2D6 activity were detected to be similar in HC and excised corneas. The HC represents a promising in vitro alternative to the use of ex vivo tissue and offers a well-defined and standardized system for drug absorption studies. Acknowledgments: We are grateful to the German Federal Ministry of Education and Research (BMBF) and German Federal Institute for Risk Assessment (BfR), which funded this work under grant nos. 0315504E, 0315504B and 3-1328-30652054369.

References:

1. Pepić, I. et al.: Drug Discov Today 2014, 19: 31-44. 2. Hahne, M., Reichl S: Int J Pharm. 2011, 416: 268-279. 3. Hahne, M. et al.: J Pharm Sci. 2012, 101: 2976-2988.


Protein engineering of fibroblast growth factor 2 (FGF-2) for bioresponsive protein delivery

Lühmann, T.1; Jones, G.1; Memmel, E.2; Seibel, J.2; Meinel, L.1

1 Institute for Pharmacy and Food Chemistry, University of Würzburg, Germany 2 Institute for Organic Chemistry, University of Würzburg, Germany

Current needs in regenerative medicine include the design and development of sophisticated materials that integrate tissue specific growth factors. Immobilisation of growth factors to implant materials has been generally performed by (i) non-covalent physiochemical adsorption of the protein (ii) covalent random immobilization by e.g. primary amino-groups of the protein or (iii) enzymatic coupling approaches. Methods, which covalently link proteins non-specifically to surfaces, have certain limitations as the presentation and bioactivity of the protein can be severely diminished during the modifica-tion process. Although the bioactivity of growth factors can be retained by non-covalent physiochemical adsorption onto implant materials, desorption of the protein occurs uncontrolled and in a rapid manner, a bottleneck when e.g. a long-term release of the protein into the environment is desired. An alternative strategy deploys the genetic mechanisms in archaebacteria, which use a stop codon to encode for the 22nd amino acid pyrrolysine during translation [1]. By replacing pyrrolysine with its analogue propargyl-L-lysine (plk), genetic engineered proteins can be recombinantly expressed and modified in a site-directed fashion thereafter [2, 3].

This study was designed to elucidate the impact of site-directed immobilisation by copper catalyzed azide alkyne cy-cloaddition (CuAAC) of genetic engineered plk-FGF2 on cellular responses in vitro.

Wild-type murine FGF2 and murine plk-FGF2 were expressed in E.coli BL21 (DE3) and were purified using heparin based affinity chromatography as previously described [4]. Azide groups (azide-mannose) and control groups (mannose) were introduced on model surfaces and plk-FGF2 was linked via click reaction in comparison to wild-type FGF2 and to plk-FGF2 in the absence of copper (I). The potency of both soluble and immobilized FGF2 was determined by the proliferation of human MG-63 cells and by the investigation of ERK signalling, respectively.

Plk-FGF2 was successfully expressed in the presence of 3 mM plk and was purified by heparin chromatography in an analogue manner to wild-type FGF2. The soluble plk-FGF2 analogue was found to induce proliferation of MG-63 cells in the same magnitude compared to the wild-type protein. The correct incorporation of plk at position 8 at the N-terminus of FGF2 was confirmed by ESI-MS analysis and peptide mapping after trypsin digest. Functionality of the introduced alkyne-group of plk-FGF2 was demonstrated by Cy3-azide conjugation in the presence of copper (I) by monitoring fluorescence signals by SDS-PAGE and subsequent Coomassie protein staining. Plk-FGF2 was immobilized to azide-mannose decorated surfaces by CuAAC and immobilized FGF-2 on the surface was visualized by immunosttaining. Moreover, MG-63 cells were successfully stimulated by immobilized FGF2.

Site-directed immobilisation of growth factors might be a powerful tool to decorate implant surfaces and opens the possibility to tailor bioresponsive release mechanism in the future.

References:

1. Gaston, M.A. et al.: Curr Opin Microbiol 2011, 14: 342-349. 2. Eger, S. et al.: Methods Mol Biol 2012, 832: 589-596. 3. Nguyen, D.P. et al.: J Am Chem Socl 2009, 131: 8720-8721. 4. Zhao, H. et al.: Journal of structural biology 2014, 186(3): 420-430.

36

In vitro estimation of drug transfer from paclitaxel-coated balloon catheters

Seidlitz, A.1; Kempin, W.1; Reske, T.2; Kaule, S.2; Grabow, N.2; Petersen, S.2; Nagel, S.1; Weitschies, W.1

1 Ernst-Moritz-Arndt University of Greifswald, Institute of Pharmacy, C_DAT, Felix-Hausdorff-Straße 3, 17487 Greifswald, Germany 2 University of Rostock, Institute for Biomedical Engineering, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany

Drug-coated balloons (DCB) are an innovative approach to locally treat stenosis of coronary arteries for lesions in which a therapy with drug-eluting stents is considered impossible (for example due to small vessel size or length of lesion) or unpromising (e.g. after multiple stent implantations in the concerned vessel segment). In 2011 at least 5 different types of such medical devices had received the CE-mark in Europe [1]. The current drug of choice for this application is paclitaxel, a mitotic inhibitor which is intended to supress hyperproliferation and migration of smooth muscle cells which may cause a re-narrowing of the treated vessel portion. For this treatment, the drug-coated balloon is advanced through the vascular system to the generally previously angioplastically re-opened lesion and is expanded. During this expansion which typically allows for contact times of up to 60 s the drug is delivered to the vessel wall. Due to this short time frame available for drug transfer in combination with a challenging application through the vascular system, the coating mor-phology and the mechanical stability of the coating are of greatest importance.

To systematically evaluate the drug transfer from DCB in vitro, a previously established perfused model of the implanta-tion pathway including a guiding catheter and a simulated arterial passage was combined with a hydrogel cylinder [2]. Using this system drug loss during the passage to the site of application and drug transfer to the gel representing the vessel wall in this test setup were evaluated. The model coronary artery pathway (fig. 1, left) was perfused with PBS pH 7.4 at a flow rate of 35 mL/min for 60 s. During this time the DCB was rapidly advanced through the system until reach-ing the resting position (indicated by asterisk). After the perfusion time the balloon was further advanced until exiting the model. The DCB was inserted into the gel cylinder and expanded (inflation pressure 8 atm, contact time 60 s). After re-folding and removal of the balloon the drug content of the gel, the perfusion liquid, the residual amount on the balloon surface and potential residual drug in the pathway model were determined via hplc. Balloons coated with Paclitaxel in combination with different excipients (fabricated via micro-pipetting) and commercially available SeQuent® Please balloons were tested using this method.

The results of the drug transfer testing are depicted in figure 1 (right). It becomes evident, that the simulation of the implantation process had a great influence on drug transfer due to the occurrence of great losses of the coated drug. The best drug transfer rates were obtained for the coatings containing iopromide (SeQuent® Please) or PVP as an additive with 18 % and 6 % of the initial drug load. The coatings fabricated without additives except for the solvents showed very low drug transfer rates from 0.8 % to 2.6 %. Opposed to the other two purely solvent containing formula-tions which lost almost the entire drug load during the passage, the ethyl acetate coating possessed a very smooth glassy structure and 15 % of the drug remained on the balloon after the transfer experiment. These findings emphasize the necessity to design coating strategies for DCB which produce mechanically stable coatings that are able to withstand the implantation procedure while at the same time allowing for fast drug transfer at the site of application. The use of certain additives in the coating seems to be favourable over purely solvent-containing coating liquids. In addition, the solvent also seems to greatly influence coating morphology and adhesion to the balloon surface.

Acknowledgement: Financial Support by the German Federal Ministry of Education and Research (BMBF) within “REMEDIS” and by the European Social Fund (ESF) and European Regional Development Fund (ERDF) is gratefully acknowledged.

References:

1. Scheller, B. et al.: Kardiologe 2011, 5(6): 411-435. 2. Seidlitz, A. et al.: PLOS ONE 2013, 8(12): e83992.


Self-developed sensor membranes for etongue sensors

Pein, M.; Schneider, K.

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University Düsseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany, e-mail: [email protected], phone: +49 2118114225

Purpose: Sensor membranes for etongue sensors should be developed by applying solvent-casting. Their performance should be assessed regarding taste-masked oral formulations and the results compared with those of commercially available sensors.

Materials & Methods: Sensor membranes were prepared based on high molecular weight polyvinylchloride (PVC, Sigma-Aldrich), acetone (VWR), tetrahydrofuran (THF, AppliChem), isopropyl myristate (IPM, Cognis), tetradodecyl-ammonium bromide (TB, Sigma-Aldrich), trioctylmethyl-ammonium chloride (TC, Alfa Aesar), bis(2-ethylhexyl) phos-phate (BP, Sigma-Aldrich) and hydroxypropyl-ß-cyclodextrin (HP, Roquette). Solutions were casted, dried and adhered to PVC based sensor heads [1]. The sensors were filled with KClaq (3.33 M) in saturated AgClaq and equipped with an Ag/AgCl electrode. Sensor performance was compared to the commercially available astringency (SB2AAE) and bitter sensors (SB2AC0, SB2AN0, SB2BT0) utilizing the Insent taste sensing system TS-5000Z (Insent, Inc.) according to Woertz et al. [2], but with a stability criterion of 2 mV. Evaluated samples were A: oral films containing dimenhydrinate (DMH), B: oral films containing DMH and sweeteners, C: placebo oral films, D: placebo oral films containing sweetener, E: pure DMH, F: DMH + sweetener, G: pure sweetener [3]. 20 films of sample A-D were dissolved over 3 min in 100.0 ml of purified water at 37 °C and immediately filtered.

Results & Discussions: Principle component analyses (PCA) were performed based on the responses of the commer-cially available and of the self-developed sensors. The information of the DMH containing samples is influenced by the responses of the commercial bitter sensors SB2AC0, SB2AN0 and SB2BT0 (Figure a: Loading Scatter Plot) and thus displayed on the right side of the according PCA map (Figure A). A comparable result is seen for the self-developed sensors with the major influence of sensor TBHP (Figure B and b). In both cases, the information is mainly separated along principle component 1 (PC1: 98.8 % and 94.1 %). Regarding the similar location, DMH containing samples A, F and E are detected comparably. Data points of sample B are located in the direction of the (sweet) placebo samples C, D and G, indicating a slight taste-masking effect. Minor differences between Figure 1A and 1B can be seen regarding the location of the placebo film sample C, indicating that the self-developed sensors enable a discrimination between film based samples (A-D) and the pure substances (E-F), resulting in a slight separation along the y-axes.

Conclusion: Sensor membranes with comparable performance to commercially available ones were developed. Both sensor sets registered a taste-masking effect of sample B. The indicated benefit of the self-developed sensors – to discriminate between film based samples and pure substances – will be further investigated.

References:

1. Schneider, K.: Diploma Thesis: 2014, Ernst-Moritz-Arndt-Universitaet Greifswald. 2. Woertz, K. et al.: J. Pharm. Biomed. Anal. 2010, 51: 497-506. 3. Pein, M. et al.: Int. J. Pharm. 2014, 469: 228–237.


38

MEDICINAL CHEMISTRY (PSJ)

Paradigm Re-shift of Medicinal Chemistry in Japan

Tomioka, K.

Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts, Kodo Kyotanabe 610-0395 Japan

Drug discovery of small molecules is the main and only one aim of medicinal chemistry. Approach to this excellent goal had been influenced by the great progress of biological sciences. Especially many and distinct proteins provide us the targets for small molecules called as, for example, genom-based drug discovery. This trends expanded the criteria of drugs to antibody drugs. Recent trend on this line is the development of antibody-drug conjugate, again a revival of small molecules. These paradigm shifts in drug discovery of medicinal chemistry and recent progress of catalytic bond forming reactions which enable the efficient synthesis of target small molecules1 would be the subject of this lecture.

Reference:

1. Xinyu, H. et al.: Catalysis Science & Technology, 2011, 1(1): 62-64.


Design, synthesis and biological activity of lysine-specific demethylase (KDM) inhibitors

Miyata, N.1; Suzuki, T2; Nakagawa, H.1 1 Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi 467-8603, Japan 2 Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 603-8334, Japan

Methylation of histone lysine residues is reversibly controlled by histone lysine methyl transferases (KMTs) and histone lysine demethylases (KDMs) and plays an important role in the regulation of gene expression. Two classes of KDMs have been identified. One is lysine-specific demethylases (LSDs), which are flavin-dependent amine oxidase domain-containing enzymes, and the other is Jumonji domain-containing demethylases (JMJDs), which are Fe(II) and α-ketoglutarate-dependent enzymes. KDMs are associated with various disease states, and have emerged as attractive targets for the development of new therapeutic drugs. We designed and synthesized several selective KDM inhibitors (Figs. 1 & 2) and will discuss their potential as cancer therapeutic agents.

References:

1. Ueda, R. et al.: J. Am. Chem. Soc., 2009, 131(48): 17536-13537. 2. Ogasawara, D. et al.: Angew. Chem. Int. Ed., 2013, 52(33): 8620-8624. 3. Hamada, S. et al.: Bioorg. Med. Chem. Lett., 2009, 19(10): 2852-2855. 4. Hamada, S. et al.: J. Med. Chem., 2010, 53(15): 5629-5638. 5. Suzuki T. et al.: J. Med. Chem., 2013, 56(18): 7222-7231.

40

Synthesis of a novel opioid receptor agonist, SYK-146 with 1,3,5-trioxazatriquinane skeleton and its pharmacologies Nagase, H.2; Hirayama, S.1; Wada, N.1; Kuroda, N.1; Iwai, T.1; Fujii, H.1 1 School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan 2 International Institute for Integrative Sleep Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan

We designed and synthesized of 1,3,5-trioxazatriquinanes with m-hydroxyphenyl groups. The designed 1,3,5-trioxazatriquinanes include the phenethylamine structure within them, which is a common structure observed in morphinan derivatives like morphine. Among the synthesized compounds, SYK-146 (1) with two m-hydroxyphenyl groups selectively bound and exerted full agonist activity toward the κ opioid receptor (KOR).[1] Subcutaneously adminis-tered (1) exhibited significant antinociceptive effects via the KOR in a dose dependent manner. These results suggest the emergence of a novel class KOR agonist. We also report the pharmacological activities of the optically active SYK-146, and the o- and p-hydroxyphenyl deriva-tives.[2]

References:

1. Hirayama, S.; Nagase H. et al.: ACS Med. Chem. Lett. 2014. in press. 2. Hirayama, S.; Nagase H. et al.: Bioorg. Med. Chem. Lett. 2014. in press.


Gold-catalyzed annulations and their medicinal applications

Ohno, H.; Suzuki, Y.; Hou, Z.; Tokimizu, Y.; Oishi, S.; Fujii, N.

Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

The development of cascade reactions is an area of considerable interest in modern organic chemistry. Efficient cascade reactions realize the economical synthesis of complex target molecules through multiple bond formations in a single operation. Elementary reactions that form fewer waste products are desirable in terms of atom economy and to suppress side product formation in sequential processes. Recent advances in homogeneous gold catalysis have opened up further possibilities for cascade reactions. We are involved in development of gold-catalyzed atom-economical cascade reactions and their medicinal applications. (1) Synthesis of dihydropyrazoles by gold-catalyzed three-component annulation (1-1) Efficient synthesis of highly functionalized dihydropyrazoles1 A transition metal-catalyzed Mannich-type three-component coupling of alkynes, aldehydes and amines (A3 coupling) is an attractive reaction not only for facile preparation of propargylamines but also as an elementary reaction for a cascade process. We developed a gold-catalyzed A3 coupling to produce polysubstituted dihydropyrazoles 5, in which all the reaction components are incorporated in the newly-formed ring.

R1 +cat. [Au+]

R1

NR3

HN

[Au+]

R2 NN

R1

R3R4R4

1 2 3 4 5

HN

NH

R4R3R2 H

O+

R2

(1-2) Fused pyrazole syntheses and identification of novel CK2 inhibitors2,3

Using the gold-catalyzed three-component annulation, various benzo[g]indazole derivatives 8 and pyrazolo[4,3-b]indole derivatives 9 were prepared. Evaluation of their CK2 inhibitory activities revealed that benzo[g]indazole and pyrazolo[4,3-b]indole are appropriate scaffolds for potent CK2 inhibitors.

R1

6

NHN

R2

R1HO2CN3

7

RO2CNH

N

HN

HO2CR2

RO2C

8 9

cat. [Au+]or or

(2) Gold-catalyzed cascade cyclization of (azido)ynamides for the construction of indoloquinolines4

(Azido)ynamides 10 were efficiently converted into indoloquinolines 12/13 by the use of gold catalyst. While ynamides bearing an allylsilane gave terminal alkenes 12, ynamides bearing a simple alkene gave cyclopropanes 13. These reactions proceed through the formation of an α-amidino gold-carbenoid 11.

N3

N

Ts R

NH

NTs

N NTs

HR

13 (R = Ph or n-Bu)12 (R = CH2TMS)

cat. [Au+]or

N NTs

R

[Au+]

10 11 Acknowledgments: The authors wish to thank Prof. Tsujimoto, G. and Prof. Hirasawa, A. (Graduate School of Pharmaceutical Sciences, Kyoto University) for the CK2 inhibition assay; Professor Nakanishi, I. (Department of Pharmaceutical Sciences, Kinki University) for the docking simula-tion and valuable comments for the molecular design. This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists (A), as well as Platform for Drug Design, Discovery, and Development from the MEXT, Japan.

References:

1. Suzuki, Y. et al.: Org. Lett. 2012, 14(1): 326. 2. Suzuki, Y. et al.: Org. Biomol. Chem. 2012, 10(25): 4907. 3. Hou, Z. et al.: Org. Biomol. Chem. 2013, 11(20): 3288. 4. Tokimizu, Y. et al.: Org. Lett. 2014, 16(11): 3138.

42

BIOMARKER AND MODELING

Implementation of dosing algorithms of anticancer drugs based on pharmacological biomarkers

Joerger, M.

Kantonsspital St.Gallen, Switzerland

Anticancer drugs are typically given either as a flat-dose or based on the patient’s body-surface area (BSA). Both methods do not account for the wide interindividual variability in drug pharmacokinetics and pharmacodynamics. This may not be a substantial issue in drugs with a broad therapeutic window, but is a major concern in oncology. Modern technology such as drug immunoassays or population PKPD-modeling has enabled to elaborate more sophisticated dosing algorithms that implement therapeutic drug monitoring (TDM) with the aim to reduce interindividual PK variability, and improve the benefit-risk ratio of common anticancer drugs. Still the only anticancer drug were standard TDM is done in adult oncology is high-dose methotrexate (MTX), with the aim to avoid severe MTX-related toxicity by guiding leucovorin-rescue. At present, there is evidence from one randomized study on TDM-based administration of continous-infusional 5-fluorouracil (5FU), and there is mainly retrospective evidence for TDM-based administration of paclitaxel, docetaxel, busulfan and imatinib. Limited retrospective evidence is also available for other oral tyrosine kinase inhibitors. Pharmacogenetic markers of increased risk for toxicity are available for selected anticancer drugs, chiefly DYPD for the fluoropyrimidines, UGT1A1 for irinotecan and TPMT for 6-mercaptopurine. The clinical significance of CYP2D6 (2C19)-associated metabolism for tamoxifen is still controversial and results from prospective clinical studies are awaited. In the future, individualized dosing algorithms will become more ‘mainstream’ in oncology as an inherent part of personalized treatment.


Mathematical modeling of amyloid beta for the diagnosis and treatment of Alzheimer’s disease

Lehr, T.

Saarland University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany

Accumulation of amyloid-β (Aβ) peptide in the central nervous system (CNS) is believed to play a crucial role in the pathogenesis of Alzheimer’s disease (AD) [1]. In order to gain more insight in Aβ metabolism in healthy subjects as well as in AD patients Bateman et al. developed a method to quantify Aβ turnover in the cerebrospinal fluid (CSF) [2]. There-fore, the stable isotope labelled amino acid leucine is infused into the bloodstream, wherefrom it can be transported to the brain and incorporated into newly synthesized proteins such as the amyloid precursor protein (APP). Labelled APP is processed by the enzymes β- and γ-secretase to produce labelled Aβ. Both labelled and unlabelled Aβ are cleared through the CSF, where sampling and quantification occurs at several time points. This technique of stable isotope labelling kinetics (SILK) has been successfully used to measure endogenous Aβ production and degradation rates in human CSF [3]. An additional study comparing the fractional production and clearance rates between control and AD patients revealed 30% impairment in the clearance of both Aβ, while average production rates did not differ between the two groups [4]. Thus, altered clearance mechanisms may contribute to the Aβ accumulation in the CNS.

Based on the exciting data from various SILK studies several mathematical models were generated in order to describe Aβ biosynthesis and degradation in humans [5] and to link these processes to the Alzheimer disease status. Aim of this presentation is to outline the future use of these mathematical models to diagnose Alzheimer’s disease in humans and to potentially guide the treatment of Alzheimer’s disease.

References:

1. Karran, E. et al.: Nat Rev Drug Discov. 2011, 10(9): 698-712. 2. Bateman, R.J. et al.: J Am Soc Mass Spectrom. 2007, 8(6): 997-1006. 3. Bateman, R.J. et al.: Nat Med. 2006, 12(7): 856-861. 4. Mawuenyega K.G. et al.: Science 2010, 330(6012): 1774. 5. Haug, K.G. et al.: Journal of Clinical Pharma 2012, 7(53): 691–698.

44

Understanding Coagulation Biomarkers and Deriving Clinically-Relevant Surrogates by Use of an In-Silico Coagulation Model

Burghaus, R.1; Willmann, S.1; Siegmund, H.-U.2; Lippert, J.1 1 Bayer HealthCare Pharmaceuticals, Clinical Pharmacometrics, 42113 Wuppertal, Germany 2 Bayer Technology Services, Technology Development, 51368 Leverkusen, Germany

Objectives: Mechanisms of coagulation are being successfully investigated and characterized in the scientific communi-ty for decades. As a consequence, there is a large body of related knowledge, information and data in the public as well as proprietary R&D space. This evidence can be used to understand coagulation biomarkers in mechanistic detail and generate clinically relevant InSilico surrogates to assess expected clinical performance of novel pro- and anticoagulant drugs and treatment options. Methods: A mechanistically detailed systems pharmacology simulator called “Blood Coagulation Simulator” (BCS) was established integrating and summarizing community and proprietary information about blood coagulation. This tool is used to simulate conditions typical for standard blood coagulation assays as well as physiological conditions represent-ing clinically relevant scenarios. Results: The BCS is able to integrate biological structural knowledge and quantitative information about the dynamics of actual protein activation processes as well as processes on a cellular and blood vessel scale. Systemic properties of the simulator – e.g. platelet activation responses - are in line with experimental observations. The impact of anti-thrombotic agents on standard coagulation assays is simulated and found to be in accordance with ex-vivo data. Simulations of anti-coagulant treatments in physiologically motivated settings identify doses which show clinically favorable risk/benefit profiles. Discussion/Conclusions: Systems Pharmacology methods have been successfully applied to blood coagulation. Such an approach allows to understand systemic properties of coagulation assays as well as pharmacological interventions. Translatability into a clinical framework has been demonstrated by simulation work being in line with confirmatory clinical trials. Application of the technology allows for evaluating innovative treatment paradigms in terms of their expected clinical profile and streamlining development programs by early identification of reasonable dosing schemes. The application described can be seen as a prototype for applying Systems Pharmacology in combination with bi-omarker data to rationalize pharmaceutical research and streamline development.

References:

1. Burghaus, R. et al.: PLoS ONE 2011, 6(4): e17626. 2. Küpfer, L.; Lippert, J.; Eissing, Th.: Adv Exp Med Biol. 2012, 736: 543-561.


LIGAND BINDING ASSAYS

Biophysical techniques in fragment hit identification and lead optimization ‐ A change of perspective? Boeckler, F.M.1; Wilcken, R.1,2; Bauer, M.R.1,2; Cieslik, M.B.1; Rutherford, T.J.2; Fersht, A.R.2; Joerger, A.C.2

1 Laboratory for Molecular Design and Pharmaceutical Biophysics, Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. 2 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.

In recent years we have worked on various projects involving fragment hit identification and optimization toward lead structures using different biophysical approaches. We have identified lead structures for rescuing the p53-mutant Y220C [1-4] and identified lithocholic acid as an inhibitor of MDMX and MDM2 [5]. Experiences with the advantages and disad-vantages of using methods including NMR-based techniques, fluorescence-based techniques and calorimetric tech-niques for the different stages of the projects will be discussed. During the initial stages of the project there is a strong preference for techniques that help to avoid false positives and false negatives. However, the perspective can change significantly when the project evolves.

References:

1. Boeckler, F.M. et al.: Proc. Natl. Acad. Sci. U. S. A. 2008, 105(30): 10360-10365. 2. Wilcken, R. et al.: J. Am. Chem. Soc. 2012, 134(15): 6810–6818. 3. Wilcken, R. et al.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(34): 13584-13589. 4. Wang, G.; Fersht, A.R.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(34): 13590-13595. 5. Vogel, S. et al.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(42): 16906-16910.

46

The Impact of Experimental Uncertainty on Decision Making in Drug Design

Kramer, C.

Center for Molecular Biosciences; Institute for General, Inorganic, and Theoretical Chemistry; Leopold-Franzens University Innsbruck; Innrain 82; 6020 Innsbruck; Austria

All experimental results contain some experimental uncertainty. This is well appreciated in almost all engineering disci-plines, and good strategies for coping with the uncertainty have been developed there. In contrast, the analysis of experimental uncertainty in physicochemical and biochemical measurements is often worked out less stringent, and the impact of the amount of experimental uncertainty on SAR analysis and drug design decisions are not rigorously taken into account. Even more, the experimental uncertainty of the individual measurements itself is often not really known.

Figure 1: Independently measured pKi values for the same protein-ligand system from CHEMBL14 (8524 pairs). The diagonals indicate the 1:1 agreement and the thresholds where two measurements disagree by 2.5 log units.

Upon comparison of a large number of independent affinity measurements of pairs of protein-ligand systems, we found that the average experimental uncertainty of heterogeneous public Ki values is 0.54 log units (see Figure 1 below).[1] This is in sharp contrast to repeated measurements of the same assay in the same laboratory, where we found an experimental uncertainty of 0.2 log units.[2]

The amount of experimental uncertainty has a limiting effect on all rational techniques that help guiding drug design. For three different standard data analyses and affinity prediction techniques that are in daily use of drug designers, we will show how the ignorance of experimental uncertainty can lead to wrong conclusions: Without taking into account experi-mental uncertainty, pseudo activity cliffs will be detected in SAR analysis, the true predictive power of QSAR models will be underestimated, and Matched Molecular Pair Analysis will seem more fuzzy than it really is.[3] We thus need to either reduce the experimental uncertainty in the data, or develop a far better understanding of its magnitude and the impact on the daily drug design decisions.

References:

1. Kramer, C. et al.: J. Med. Chem. 2012, 55(11): 5165-5173. 2. Kalliokoski, T. et al.: PLoS One 2013, 8(4): e61007. 3. Kramer, C. et al.: J. Med. Chem. 2014, 57(9), 3786-3802.


Fluorescent probes to track GPCR binding and dimerization

Bonnet, D.; Hibert, M.

Laboratoire d’Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg, Labex Médalis, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France

G-protein-coupled receptors (GPCRs) represent the largest family of cell surface membrane proteins encoded by the human genome and more than 40% of all marketed therapeutics act on them. However, these drugs target only few members of the family (15%). So, there is an enormous potential to exploit the remaining family members, including the orphan receptors for which no ligands have so far been identified. Besides, in the last decade, homo- and hetero-oligomerization of GPCRs have been described as a new way to modulate receptor pharmacology and functional activi-ty. Thus, heteromer-targetted drug discovery opens new perspectives both in Academic pursuits and for the Pharmaceu-tical industry. In this context, we have set up innovative fluorescent-based assays in order to gain a better understanding of GPCR functional architecture but also to set up new receptor-selective high-throughput screening (HTS) assays for classical, orphan and heterodimeric GPCRs. Owing to their high sensitivity and to their reduced environmental safety risk, fluores-cent technologies represent a powerful molecular tool to study ligand-GPCR interactions [1]. However, the prerequisite to develop such methods is to design and to synthesize high affinity and selective fluorescent probes.

As we will illustrate, synthetic methods have been set up to facilitate the access to original fluorescent GPCR probes with potential applications in drug discovery. For instance, the first environment sensitive (“Turn-on”) probe was developed to detect and to monitor oxytocin GPCR at the surface of living cells [2]. We have also designed and synthesized fluores-cent compound-based libraries allowing the discovery by FRET of the first non-peptidic agonist of the apelin receptor [3]. Finally, selective fluorescent ligands were developed to detect vasopressin V1a-V2 heterodimers at the cell surface and to set up a novel TR-FRET assay to screen for heterodimers [4].

Acknowledgments: Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université de Strasbourg

References:

1. (a) Durroux, T. et al.: Nat. Chem. Biol. 2010, 6(8): 587-594; (b) Ilien, B. et al. : J. Med. Chem. 2012, 55(5): 2125–2143. 2. Karpenko, I. et al.:: ChemBiochem 2014, 3(15): 359-363. 3. (a) Bonnet, D. et al.: Chem. Eur. J. 2008, 14(20): 6247-6254; (b) Iturrioz, X. et al.: FASEB J. 2010, 24(5): 1506-1517; (c) Bonnet, D. et al.: J.

Med. Chem. 2014, 57(7): 2908−291. 4. Bonnet, D. et al.: J. Med. Chem. 2012, 55(20): 8588−8602; Patent PCT/EP2013/070837.

48

Development of triplex-forming oligonucleotide having artificial nucleoside analogues to inhibit the gene expression as an antigene strategy Taniguchi, Y.; Okamura, H.; Sasaki, S.

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, JAPAN

The sequence specific formed triplex DNA is one of the most important method for the inhibition of gene expression as an antigene strategy in living cell. Basically, the triplex DNA is formed by the interaction of triplex forming oligonucleo-tides (TFOs) with duplex DNA, which is stabilized by the hydrogen bonding between dG and GC or between dA and AT base pair. Thus, the TA and CG base pair within target duplex DNA hampered the stable triplex DNA. We have designed

and synthesized W-shaped nucleoside analogues (WNA) for the formation of stable triplex DNA, and the WNA-T

having thymine as a recognition base and WNA-C having cytosine as a recognition base showed a high selectivity for the TA and CG interrupting site, respectively (Table 1 and Figure 1).1 However, the recognition ability of WNA was dependent on the sequence context of TFOs. In order to overcome this limitation, we developed the several WNA analogues and evaluated the ability of triplex formation by the gel-shift assay. Consequently, the combination use of

WNA-T derivatives showed the recognition of the TA base pair at any different sequences.2 Furthermore, we applied

the WNA-T for the antigene strategy for targeting the survivin oncogene. The antigene TFOs having WNA-T showed the effective anti-proliferative effect for the living cell by the inhibition of the survivin expression (Figure 2).3

Recently, we have found the novel nucleoside unit, isocytidine skeleton, for the selective recognition of CG interrupting site. It was revealed that TFOs containing 5-methylisocytidine with guanidinoethyl group or aminopyridine methyl group showed the selective and stable triplex DNA including the CG site without sequence dependency (Table2, Figure 3).4,5

In conclusion, we have developed the artificial nucleoside analogues to recognize the TA or CG interrupting sites for the formation of stable triplex DNA. And we have demonstrated that the antigene TFOs containing WNA analogues showed the effective anti-proliferative effect in compari-son to the effect of the correspond-ing natural antigene TFOs.

References:

1. Sasaki, S. et al.: J. Am. Chem. Soc. 2004, 126: 516-528. 2. Taniguchi, Y. et al.: J. Org. Chem. 2006, 71: 2115-2122. 3. Taniguchi, Y.; Sasaki, S.: Org. Biomol. Chem.: 2012, 10: 8336-8341. 4. Okamura, H.; Taniguchi, Y.; Sasaki, S.: Org. Biomol. Chem.: 2013, 11: 3918-3924. 5. Okamura, H.; Taniguchi, Y.; Sasaki, S.: ChemBioChem: 2014, in press.


COMPUTATIONAL CHEMISTRY AND MOLECULAR DESIGN

Integrating Chemical and Biological Data for Drug Design and Mode-of-Action Analysis

Bender, A.

Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.

More and more chemical and biological information is becoming available, both in public databases as well as in compa-ny repositories. However, how to make use of this information in chemical biology and drug discovery settings is much less clear. In this work, we will discuss how chemical and biological information from different domains – such as com-pound bioactivity data, pathway annotations from the bioinformatics domain, and gene expression data – can be used for a variety of purposes, such as the mode-of-action analysis from phenotypic readouts,[1,2] anticipating compound toxicities in early discovery, and for designing and selecting compound with the desired bioactivities.[3,4] We will show that cheminformatics algorithms trained on large chemogenomics databases can be employed to support target decon-volution in high-content screening as well as organism-based screens using e.g. Xenopus laevis[2] as well as phenotypic data obtained from rat models. When anticipating compound adverse compound properties early on, we will show than gene expression data can be used for this purpose; however, how to generate and analyze data is very much case-dependent. Relating to compound design and selection, we can employ both bioactivity-driven approaches as well as gene expression based resources, and examples of both will be presented. Hence, overall, while the chemical and biological data available currently is very diverse, we are able to show that it can already be used successfully for under-standing the mode of action of compounds, anticipating their toxicities early on in discovery, and designing and selecting novel chemical matter to modulate biology.

References:

1. Koutsoukas, A. et al.:J. Proteomics 2011, 74(12): 2554-2574. 2. Liggi, S. et al.: Mol. Inf. 2013, 32(11-12): 1009-1025. 3. Van Westen, G.J.P. et al.: MedChemComm 2011, 2(16): 16-30. 4. Van Westen, G.J.P. et al.: PLoS Comp. Biol. 2013, 9(2): e1002899.

50

A Knowledge-Based Approach to Assessing Propensity for Polymorphism in the Pharmaceutical Crystalline Solid Form

Maginn, S.J.; Feeder, N.

Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, United Kingdom

Uncontrolled crystal form polymorphism can have a critical impact on pharmaceutical or agrochemical product robust-ness. This has been particularly exemplified by the pharmaceutical cases of Norvir™ [1] and Neupro™ [2], which were withdrawn from the market after the unexpected appearance of a more stable polymorph. The Norvir™ example illus-trates how such polymorphism can be driven by a stronger set of hydrogen bonds in the stable form. At the CCDC we are developing structural informatics approaches that can mitigate solid form risk and move us towards the notion of solid form by design. Here the vast knowledge base of 700,000+ crystal structures of the CSD and the millions of discrete data points on the geometry of intermolecular interactions contained therein are mined to reveal the underlying rules that control crystal packing. Such an approach compliments more brute force ab-initio energy calcula-tions yet offers the advantage of being applicable across all solid form types and accessible to solid state scientists rather than just computational specialists. For example, we have developed a CSD based Hydrogen-Bond Propensity tool which would have clearly predicted the likely existence of the more stable polymorph of ritonavir (Norvir™)[3]. The principles behind these approaches and their practice will be described, and a view to the future will be provided.

References:

1. Bauer, J. et al.: Pharm. Res. 2001, 18: 859-866. 2. Cajigal, S.: Neurology Today 2008, 8: 1 & 8. 3. Galek, P.T.A. et al.: CrystEngComm 2009, 11: 2634-2639.


Direct Integration of Ligand-Based NMR Data into Protein-Ligand Docking

Exner, T.E.1,2; Onila, I.2; Fredriksson, K.2; Codutti, L.3; Mazur, A.4; Möller, H.M.2; Carlomagno, T.3; Griesinger, C.4 1 Institute of Pharmacy, Eberhard Karls University of Tübingen, Tübingen, Germany 2 Department of Chemistry, University of Konstanz, Konstanz, Germany 3 European Molecular Biology Laboratory, Heidelberg, Germany 4 Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

In structure-based drug design, the experimental elucidation of protein-ligand complexes plays a central role in the design of high-affinity drug candidates from weakly bound lead compounds. As an alternative to X-ray crystallography, NMR techniques can be applied to obtain information on the bound ligand. STD [1] (saturation transfer difference) is often used to find the epitope in the ligand by determining the distance between a specific part of the ligand to the surface of the protein. In contrast, the relative new INPHARMA [2,3] (Internuclear NOEs for Pharmacophore Mapping) approach, determines the relative orientation of two competitive ligands in the receptor binding pocket. It is based on the observation of interligand transferred NOEs mediated by spin diffusion through protons of the protein and is, therefore, sensitive to the specific interactions of each of the two ligands with the protein. The docking program PLANTS [4,5] (Protein-Ligand ANT System) developed in our group was extended to directly include the experimental information. The standard scoring function ChemPLP [5] is augmented with STD and/or INPHARMA scores, which describes the agreement between the experimental spectrum and a back-calculated spectrum by defining distance constraints or using the full relaxation matrix approach. We will show that this integration is more beneficial and efficient than a two-step procedure generating first trial poses with a docking program and then identifica-tion of the correct pose by a rescoring with the experimental data as used in [6]. We will also demonstrate that, besides this improved scoring function, the careful preparation of the input structures and the docking setup including protonation states is essential.

References:

1. Mayer, M., Meyer, B.: J. Am. Chem. Soc. 2001, 123: 6108 – 6117. 2. Sanchez-Pedregal, V.M. et al.: Angew. Chem. Int. Edit. 2005, 44(27): 4172 – 4175. 3. Orts, J. et al.: Angew. Chem. Int. Edit. 2008, 47(40): 7736 – 7740. 4. Korb, O.; Stützle, T.; Exner, T.E.: Swarm Intell. 2007, 1: 115 – 134. 5. Korb, O.; Stützle, T.; Exner, T.E.: J. Chem. Inf. Model. 2009, 49: 84 – 96. 6. Skjaerven, L. et al.: J. Am. Chem. Soc. 2013, 135(15), 5819-27.

52

Screening reaction pathway-driven very large chemical space: Discovery of potent mdm2-p53 antago-nist

Dömling, A.1; Camacho, C.2; Holak, T.3; Koes, D.2; Neochoritis, C.1; Khoury, K.4 1 Dept. Drug Design, RUG, Netherlands, 2 University of Pittsburgh, USA, 3 Jagiellonian University, Krakow, Poland, 4 Carmolex Inc, Pittsburgh, USA

Current industrial screening paradigm is HTS: Most medicinal chemistry program start with hits from high throughput screening. Virtual screening of very large chemistry space combined with structure-based drug discovery offers a valua-ble alternative. However, until recently, most virtual screening exercises used rather small libraries of limited chemical diversity. And, although, identified compounds are often commercially available for validation, such as in ZINC, PUB-CHEM or ChEMBL databases, follow-up SAR is often challenging and expensive. For instance, GB-11 (what is GB11) is very large and hard to test due to the lack of efficient synthetic access to hit compounds for validation. We have introduced ANCHOR.QUERY, a web-based and google-like technology for the structure-based mining of billions of small molecules (anchorquery.csb.pitt.edu/).1 The compound database is based on >20 diverse scaffolds with a defined reaction pathway based on efficient and rapid multicomponent reaction chemistry (MCR).2 The virtual com-pound library is design for a high level of confidence in synthetic feasibility and speed: Every hit compound can be rapidly accessed from commercial starting materials in less than four chemical steps. Protocols for the synthesis are provided online. To increase virtual screening hit rates the compound library is biased towards deeply buried anchor residues which play a key role in molecular recognition, e.g. deeply buried amino acid side chains in PPIs. We have validated ANCHOR.QUERY with the discovery of more than 10 different compound classes able to antagonize the protein protein interaction p53-Mdm2-Mdm4.3-6 Several of the predicted compounds could be validated by cocrystal structure analysis and compared with the predicted binding poses (Figure). Some scaffolds were optimized towards low nM compounds highly active in cancer cells and a xenograft.

Acknowledgments: The Dömling laboratory is generously funded by the University of Groningen, the Innovative Medicines Initiative (grant agree-ment n° 115489), Qatar National Science Foundation (NPRP 6 - 065 - 3 - 012), the National Institute of Health (1R01GM097082-01) and Carmolex Inc.

References:

1. Koes, D. et al.. PLoS One 2012, 7: e32839. 2. Dömling, A.; Wang, K.; Wang, K. : Chem. Rev. 2012, 112: 3083-3135. 3. Czarna, A. et al.. Angew. Chem. Intl. Ed. 2010, 48: 5352-5356. 4. Bista, M. et al.. Structure 2013, 21: 2143-2151. 5. Huang, Y. et al. ACS Chem. Biol. 2014.

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Identification of a mechanism-of-action target exploiting similarities of chemotypes and signalling events, and biophysical simulations

Gohlke, H.1; Schmitz, B.1; Bonus, M.1; Sommerfeld, A.2; Reinehr, R.2; Häussinger, D.2

1 Institute for Pharmaceutical and Medicinal Chemistry, Department of Mathematics and Natural Sciences, Heinrich-Heine-Universität, Düsseldorf, Germany 2 Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-Universität, Düsseldorf, Germany

Recent studies reported that for about 7% of approved drugs, a primary target is unknown.[1] One such drug has been ursodeoxycholic acid, which is in vivo converted to tauroursodeoxycholic acid (TUDC). This bile acid is a mainstay for

the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in an 51 integrin-mediated way.[2, 3] However, an intracellular receptor/sensor specific for TUDC that initiates the signaling has remained unknown.

Exploiting that I) TUDC-induced signaling events strongly resemble those when mechano/swelling-sensitive 51 integ-

rin become activated, II) TUDC-induced signaling is inhibited in the presence of an 51 integrin inhibitory peptide, and

III) TUDC bears structural similarity with tirofiban, an IIb3 integrin inhibitor, both in terms of molecular shape and

molecular recognition properties, we hypothesized that 51 integrins act as sensors for TUDC in hepatocytes.

The hypothesis was verified by immunofluorescence staining experiments showing direct integrin activation by TUDC, by Western blot analyses showing the initiation of integrin signaling involving downstream kinases, and by integrin knock-down abolishing TUDC signaling. TUDC-induced integrin activation occurs predominantly inside the hepatocyte and requires TUDC uptake via the Na+/taurocholate cotransporting peptide. Furthermore, molecular dynamics simulations of

a model of the 51 integrin ectodomain with TUDC revealed that TUDC induces pronounced allosteric conformational changes known to be associated with integrin activation.

This finding of a long-sought intracellular sensor of TUDC is key for understanding the choleretic and cytoprotective effects as well as the hepatocyte-specificity of TUDC at a molecular level.[4] Moreover, our results yield the first structur-

al model of a bile acid binding to 51 integrin, which is expected to foster the development of novel small-molecule integrin agonists. Finally, our results suggest that exploiting similarities of signaling events and biophysical simulations, in addition to chemical similarity as done previously,[5] can lead to highly accurate predictions of drug-target associa-tions, which is of high interest currently.

Acknowledgments: We acknowledge support by the Deutsche Forschungsgemeinschaft through the Collaborative Research Centers SFB 575 and SFB 974 and the Clinical Research Group KFO 217, and by the initiative ‘‘Fit for Excellence’’ at the Heinrich-Heine-Universität.

References:

1. Drews, J.: Science 2000, 287: 1960-1964. 2. Häussinger, D. et al.: Gastroenterology 2003, 124: 1476-1487. 3. Schliess, F. et al.: Gastroenterology 1997, 113: 1306-1314. 4. Gohlke, H. et al.: Hepatology 2013, 57: 1117–1129. 5. Gregori-Puigjane, E. et al.: Proc. Natl. Acad. Sci. 2012, 109: 11178-11183.

54

NATURAL COMPOUNDS

Neolignans: from PPAR to RXR

Dirsch, V.M.

Department of Pharmacognosy, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria

Peroxisome proliferator-activated receptor gamma (PPAR) agonists have been and are still used to treat type 2 diabe-tes and the metabolic syndrome due to their ability to increase “safe” free fatty acid (FFA) storage in adipocytes thereby

lowering FFA-mediated lipotoxicity and increasing insulin sensitivity, among others [1]. Most full PPAR agonists, how-ever, display serious side effects, which led to great interest in novel ligands with more favorable properties. Thus, one

aim of our group is to identify new PPAR agonists. Using pharmacophore-based virtual screening of 3D natural product

libraries we discovered several neolignans (dieugenol, tetrahydrodieugenol, and magnolol) as partial PPAR agonists

[2]: These neolignans bound to the PPAR ligand binding domain with Ki values in the nano- to low micomolar range. In

intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPAR-mediated luciferase reporter gene expression with EC50 values comparable to pioglitazone (pioglitazone: 0.26 µM; dieugenol: 0.63 µM; tetrahydrodieuge-nol: 0.33 µM) but with a considerable lower maximal response suggesting partial agonism. All three compounds promot-

ed 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPAR expression [2]. Interestingly, the structurally closely related neolignan, honokiol did not induce adipogenesis under the same conditions [3]. Nevertheless, as pioglitazone it led to an increase in glucose uptake in adipocytes. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain [3]. Additional studies by other groups

characterized honokiol as specific partial retinoid X receptor alpha (RXR agonist [4], and magnolol as dual agonist of

RXR and PPAR [5]. Based on these findings we screened a library of 53 (semi)synthetic neolignan derivatives for

their activity against PPAR and RXR in respective luciferase reporter models. As a result, we identified several new

RXR agonists with EC50 levels below 1 µM. Further studies will characterize these compounds in functional models for adipogenicity, glucose uptake in adipocytes, cholesterol efflux from human THP-1 macrophages and lipid accumulation in HepG2 hepatocytes.

Acknowledgements: This collaborative study was conducted by groups from the Universities of Vienna, Innsbruck and Graz, as well as by the China Academy of Chinese Medical Sciences. The study was mainly funded by the Austrian Science Fund (FWF): S107 (NFN: Drugs from Nature Targeting Inflammation)

References:

1. Ahmadian, M. et al.: Nat Med. 2013, 19(5): 557-566. 2. Fakhrudin, N. et al.: Mol. Pharmacol. 2010, 77(4): 559-566. 3. Atanasov, A.G. et al.: Biochim. Biophys. Acta 2013, 1830(10): 4813-4819. 4. Kotani, H. et al.: J. Nat. Prod. 2010, 73(8): 1332-1336. 5. Zhang, H. et al.: PLOS one 2011, 6(11): e28253.


Waking up biosynthetic gene clusters in a row Bechthold, A.

Department of Pharmaceutical Biology and Biotechnology, Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany

Natural products are a rich source of commercial products for the pharmaceutical and other industries. An enormous number of such compounds have derived from microorganisms colonizing various habitats. However, industrial interest in metabolites research from microorganisms has declined significantly in the past years because of the rediscovery of the same bioactive compounds and redundancy of the sample strains. In the last few years, microbial natural product research has been revolutionized by genomic technologies. Complete sequences of microbial genomes revealed a remarkable number of gene clusters encoding enzymes involved in the production of undetected and unknown secondary metabolites. Different strategies have been pursued to wake up and express these ‘cryptic’ gene clusters including culture manipula-tion, the genetic manipulation of pathway specific regulatory genes and the use of heterologous expression techniques. In my talk I will give examples for each of these strategies and I will introduce an unusual way of activating “cryptic” gene clusters resulting in the isolation of novel undiscovered natural products.

56

Chondramides: setting the stage for actin binding compounds in cancer therapy Herrmann, J.1; Förster, F.2; Hüttel, S.1; Müller, R.1; Vollmar, A.M.2 1 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Campus C2.3, Saarland University, 66123 Saarbrücken, Germany 2 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377 Munich, Germany

Myxobacteria have been established as a rich source of bioactive compounds with unique structures.[1,2] We routinely screen crude extracts of novel myxobacterial isolates by LC/hrMS and Chondromyces sp., strain MSr9030, was found to produce the already described chondramides A-D. These cyclodepsipetides were originally isolated from Chondromyces crocatus due to their nanomolar cytotoxic activity on cancer cell lines, which originates from a stabilizing effect on actin filaments[3].

Of note the mode of cytotoxicity and moreover the molecular mechanism of action linked to actin have not been exam-ined yet. We report here that chondramide A disrupts the actin cytoskeleton and forms bundles of amorphous actin aggregates (FRAP analysis and confocal microscopy). Chondramide induces apoptosis (Annexin-V/PI-costaining, Parp cleavage, Caspase 9 activation) via a collapse of the mitochondrial membrane potential (decrease of mitochondrial membrane potential and Cytochrome C release) and affecting the mitochondrial permeability transition pore (mPTP) (translocation of Hexokinase II to the cytosol and Bad dephosphorylation). In search of a link between effects of chon-dramide on actin cytoskeleton and apoptosis induction the oncogene PKCε gained attention as this kinase posseses an actin-binding site and is involved in the control of the mPTP. In fact, a colocalization of PKCε and chondramide A in-duced actin bundles was shown by confocal microscopy and Westernblot analysis of cytoskeletal fractions. Furthermore, PKCε membrane translocation after phorbol ester stimulation was decreased upon chondramide A treatment indicating reduced activity of PKCε. Finally, PKCε overexpression was able to significantly reduce cell death induced by chon-dramide A proving our hypothesis. Of special importance the mechanism of trapping PKCε in actin bundles could also be found in in vivo tumor samples, which showed a decreased tumor volume compared to control tumors. These results set the stage for actin binding compounds as innovative leads for future tumor therapies.

Along this line the MSr9030 crude extract was analyzed in more detail as the chondramides A-D might mask the pres-ence of further interesting bioactive metabolites. For bioactivity-guided isolation of new natural products we applied high-content-screening (HCS) and could finally detect more than 30 novel chondramide derivatives, some of which were present in minute amounts only and which would have not been detected in conventional cytotoxicity assays. Initial biological profiling of 11 new natural derivatives in comparison to the chondramides A-C showed that brominated vari-ants were the most active (GI50 in the low nanomolar range) on a panel of human cancer cell lines. Given the fact that these analogs were also by factor 2-4 less potent on non-cancerous human cells in comparison to average values on cancer cell lines, our results aid the further SAR-guided development of chondramides via chemical syntheses.[4]

Financial support by the Deutsche Forschungsgemeinschaft (DFG; FOR1406) is gratefully acknowledged.

References:

1. Weissman, K.J., Müller, R.: Nat. Prod. Rep. 2010, 27(9): 1276-1295. 2. Krug, D., Müller, R.: Nat. Prod. Rep. 2014, 31(6): 768-783. 3. Sasse, F. et al.: J. Natl. Cancer Inst. 1998, 90(20): 1559-1563. 4. Herrmann, J., Hüttel, S., Müller, R.: Chembiochem 2013, 14(13): 1573-1580.


Cdk5 inhibition potentiates Imatinib responsiveness of Philadelphia chromosome positive chronic myeloid leukemia cells Mandl, M.; Vollmar, A.M.; Liebl, J.

Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, Munich, Germany

Chronic myeloid leukemia (CML) is a malignancy that arises from transformation of the hematopoietic stem cell. The hallmark of CML is the Philadelphia chromosome, a reciprocal translocation of chromosomes 9 and 22, which generates the BCR/ABL fusion gene encoding a constitutively active tyrosine kinase. BCR/ABL tyrosine kinase exerts oncogenic function by activating various intracellular signaling pathways leading to increased cell survival and proliferation as well as abrogated dependency on growth factors. The ATP-competitive BCR/ABL tyrosine kinase inhibitor (TKI) Imatinib has revolutionized CML therapy. However, resistance to imatinib treatment due to mutations in BCR/ABL that impair the ability of imatinib to interact with the tyrosine kinase occurred as major problem. Moreover, Philadelphia chromosome positive CML stem cells can be intrinsically insensitive to imatinib. Second generation inhibitors targeting also imatinib insensitive BCR/ABL mutants show improved effectiveness. Nevertheless, resistance to these second generation TKIs has been described. Therefore, new therapeutic strategies to improve CML treatment are required. Novel approaches addressing alternative targets in combination with TKIs might be more effective to treat resistant CML cells. Our present study provides evidence for Cyclin dependent kinase 5 (Cdk5) as drugable target to improve the therapeutic response of Philadelphia chromosome positive CML cells to TKI treatment. Cyclin dependent kinase 5 (Cdk5) is a serine/threonine kinase with essential functions in neuronal development, func-tion, and disease. Regulation and downstream signalling of neuronal Cdk5 is well-established whereas knowledge about Cdk5 in peripheral tissues - particularly in cancer - is limited. We recently demonstrated that Cdk5 exerts important functions in the endothelium and angiogenesis and elucidated Cdk5 as drugable target for treatment of hepatocellular carcinoma (HCC). In neurons, Cdk5 gets phosphorylated and activated by the Abelson tyrosine kinase (c-Abl). Along this line, we hypothe-sized that Cdk5 is a downstream target of BCR/ABL in CML. In fact, Cdk5 phosphorylation and activity was increased in the Philadelphia chromosome positive CML cell line K562 in comparison to normal CML and T-ALL cells. Moreover, imatinib decreased Cdk5 phosphorylation and activity. Importantly, combined Cdk5 inhibition and imatinib treatment resulted in increased apoptosis and reduced proliferation of BCR/ABL CML cells. Therefore, the present study provides first evidence for the combination of Cdk5 inhibition and TKI as promising novel alternative approach for Philadelphia chromosome positive CML therapy.

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Plasmodium falciparum histone deacetylases (PfHDACs) as epigenetic drug targets Hansen, F.K.1; Sumanadasa, S.D.M.2; Stenzel, K.1; Duffy, S.2; Marek, L.1; Meister, S.3; Skinner-Adams, T.S.2; Held, J.4; Schmetter, R.1; Mordmüller, B.4; Hamacher, A.1; Kassack, M.U.1; Winzeler, E.A.3; Avery, V.M.2; Andrews, K.T.2; Kurz, T.1 1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany 2 Eskitis Institute for Drug Discovery, Don Young Road, Nathan Campus, Griffith University, QLD 4111, Australia 3 Department of Pediatrics, University of California, San Diego, School of Medicine, 9500 Gilman Drive 0741, La Jolla, CA 92093, USA 4 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany

Histone deacetylases (HDACs) play a key role in the epigenetic modulation of gene expression by altering chromatin structure and HDAC inhibitors (HDACi) are widely studied and developed as effective treatments for human cancers. Interestingly, HDAC gene homologues have been discovered in all Plasmodium species that can infect humans and five HDAC encoding genes have been identified in the P. falciparum genome.[1] Three of these Plasmodium falciparum histone deacetylases (PfHDACs) show homology to human class I (PfHDAC1) or class II HDACs (PfHDAC2,3), whereas two genes are class III homologues (PfSir2A and B). The enzyme PfHDAC1 is considered as an emerging target for malaria intervention strategies.[1]

Recently, we demonstrated for the first time that HDACi cause death and histone hyperacetylation in Plasmodium falciparum gametocytes.[2] Moreover, the transcription of all five PfHDACs in early gametocytes (stage III) and late stage gametocytes (stage V) was confirmed by diagnostic RT-PCR.[2] Based on these results, it was our aim to discover novel HDACi with activity against multiple malaria life cycle stages using LMK235, a HDACi with a unique preference for human HDAC4 and HDAC5,[3] as starting point. Thus, we synthesized two series of HDACi containing novel connection-unit linker regions.[4,5] An extensive biological evaluation disclosed that some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages), while other compounds revealed increased parasite selectivity in combination with at least dual-stage activity.[5] Mode of action studies with representative compounds showed that our HDACi caused hyperacetylation of P. falciparum histones and inhibited deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts.[4,5]

In summary, our data identify HDACi as being among a limited number of compounds that target asexual, exo-erythrocytic and gametocyte stage Plasmodium parasites, making them a potential new starting point for future devel-opment of antimalarial drug leads with multistage activity.

References:

1. Andrews, K.T.; Tran, T.N.; Fairlie, D.P.: Curr. Pharm. Des. 2012, 18(24): 3467–3479. 2. Trenholme, K. et al.: Antimicrob. Agents Chemother. 2014, 58(7): 3666–3678. 3. Marek, L.; Hamacher, A. et al.: J. Med. Chem. 2013, 56(2): 427–436. 4. Hansen, F.K. et al.: ChemMedChem 2014, 9(3): 665–670. 5. Hansen, F.K. et al.: Eur. J. Med. Chem. 2014, 82: 204–213.


ANALYTICS

Analysis of vitamin D metabolic markers by mass spectrometry: advantages and limitations of the gold standard method Volmer, D.A.

Institute of Bioanalytical Chemistry, Saarland University, 66123 Saarbrücken, Germany

Vitamin D compounds are secosteroids, which are found naturally as vitamin D3 in mammals and D2 in plants. Vitamin D is essential for bone health; recent studies, however, have shown involvement of vitamin D in the pathologies of a much wider range of diseases such as cancer, diabetes, autoimmune, neurodegenerative, mental and cardiovascular diseas-es. Vitamin D is synthesized in the human skin under the influence of UVB radiation, and subsequent hepatic and renal metabolism generates a number of transformation products over a large dynamic range from picomolar to nanomolar levels.

Vitamin D3 and most significant metabolites, 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3).

Capturing vitamin D metabolite levels requires sensitive and selective analytical methods that permit quantitative deter-mination of the low concentration levels of vitamin D compounds in relevant tissues such as blood. Ideally, vitamin D assessment would be performed by a standardized reference method, available to nutritional and clinical laboratories, that provides reliable, precise and accurate quantitative results for all relevant vitamin D metabolites with sufficiently high throughput. Unfortunately, no such method exists at the present time. Currently, LC-MS/MS assays are the most promis-ing assay techniques for vitamin D. This presentation focuses on developments in recent mass spectrometry methodolo-gies for vitamin D and its metabolites, and the experimental approaches chosen in our laboratory for determining finger-prints (“chemotypes”) of relevant vitamin D metabolites. It will highlight detrimental influences of the biological matrix, epimer contributions, problems with specific mass spectrometry data acquisition routines (in particular, multiple reaction monitoring, MRM), ionization efficiency, chemical derivatization reactions to improve detectability, gas-phase separation techniques (ion mobility spectrometry) for removing chemical noise, and accuracy issues and inter-laboratory compari-sons.

25-hydroxylase

liver

1-hydroxylase

kidney

D3 25(OH)D3 1,25(OH)2D3

60

High Resolution MALDI Imaging: Reliable Molecular Identification at Cellular Resolution Römpp, A.; Bhandari, D.; Schober, Y.; Guenther, S.; Spengler, B.

Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Schubertstrasse 60, D-35392 Giessen, Germany

Mass spectrometry imaging (MS imaging) is the method of scanning a sample of interest and generating an image of the intensity distribution of a specific analyte ion. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate (amino acid) modifications and does not require labelling of compounds. Our work is focused on obtain-ing reliable chemical information and on increasing the spatial resolution in order to detect (sub)cellular features. Here we present a number of improvements in instrumentation, sample preparation, measurement parameters and data processing. MS imaging experiments were performed with a high resolution atmospheric-pressure imaging source (AP-SMALDI10, TransMIT GmbH, Giessen) attached to ‘LTQ Orbitrap’, ‘Exactive Orbitrap’ or ‘Q Exactive’ mass spectrometers (Thermo Scientific GmbH, Bremen) [1,2]. Pixel size was between 2 and 10 µm. Mass accuracy was better than 2 ppm (root mean square error) under imaging conditions. Tentative identification based on accurate mass was confirmed by on-tissue MS/MS experiments. Phospholipids were analysed in a wide range of tissue types in order to characterize and differentiate cell types. This includes investigation of intra-tumor heterogeneity in a human biopsy of gastric cancer at 10 µm pixel size and mouse model tissue at 2 µm pixel size. The spatial distribution of the tyrosine-kinase inhibitor Imatinib was imaged in mouse kidney at 10 mm pixel size [3]. These measurements revealed the detailed distribution of the compound within the mouse organ. At the same time our method provides detailed information on histological features based on the distribution of phospholipids and other endogenous compounds. Correlation of these different images allows for fast and easy interpretation of the drug com-pound distribution and areas of accumulation can be directly linked to certain tissue types. Additional examples of drug compound include the analysis of whole-body rat sections. In an effort to investigate metabolites in cell cultures, a dedicated sample preparation protocol was established for the analysis of single cells. A range of metabolites including nucleic acids, cholesterol and phospholipids were imaged in single cells at 7 µm pixel size [4]. MS image analysis for all these experiments showed excellent agreement with histological staining evaluation. In addi-tion it provided highly specific molecular information. In many cases signals with very similar mass (∆m/z<0.1) showed distinctly different distributions, which demonstrates the need for high mass resolution in order to obtain reliable infor-mation from MS imaging experiments of complex biological samples. General trends and developments in the field of mass spectrometry will be briefly discussed. This includes strategies for flexible data analysis on the basis of the data format imzML (www.imzml.org) and activities in the framework of COST action (European Cooperation in Science and Technology) „Mass Spectrometry Imaging: New Tools for Healthcare Research” (BM1104).

References:

1. Römpp, A. et al.: Angew. Chem. Int. Ed. 2010, 49(22):3834-3838. 2. Römpp, A.; Spengler, B.: Histochem. Cell Biol. 2013, 139(6): 759-783. 3. Römpp, A. et al.,: Anal Bioanal Chem 2011, 401(1):65-73. 4. Schober, Y. et al.: Anal Chem 2012, 84(15):6293-6297.


Mass Spectrometric Characterization of Biopharmaceuticals - Possibilities, Challenges and Limitations Scheffler, K.

Thermo Fisher Scientific, Dreieich, Germany

Biopharmaceuticals are in most cases challenging molecules that require a variety of different techniques to perform full characterization including all qualitative and quantitative aspects and the analysis of modifications on the protein and peptide level. Monoclonal antibodies (mAbs) play a major role in the treatment of a variety of conditions such as cancer, infectious diseases, allergies, inflammation, and auto-immune diseases. Because mAbs can exhibit significant heteroge-neity, extensive analytical characterization is required to obtain approval for a new mAb as a therapeutic product. Mass spectrometry has become an essential tool in the characterization of mAbs, providing molecular weight determinations of intact proteins as well as separated light and heavy chains, elucidation of glycosylation and glycan structures, confirma-tion of correct amino acid sequences, and identification of impurities such as host cell proteins (HCP) inherent to the production process. The analysis of intact proteins and especially large proteins such as intact antibodies and protein complexes on the OrbitrapTM platform have steadily advanced on a technical level ever since the Orbitrap mass analyzer became commer-cially available in the year 2005, only been made possible due to several technological advancements we implemented in newer generations instruments. One of the newer generation instrument platforms is the Thermo Scientific Q Exactive benchtop Orbitrap mass spectrometer, an instrument we introduced into the market in the year 2011. This presentation is focused on qualitative and quantitative LC-MS workflows applicable to the Orbitrap platform aiming at the full characterization of biopharmaceuticals. During the course of the presentation many examples of application data obtained from these workflows will be discussed.

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Fluorescent oxaliplatin analogue as a model for the anticancer drug oxaliplatin for the investigation of its cellular trafficking Kalayda, G.V.1; Gollos, S.2; Kullmann, M.1; Metzger, S.3; Jaehde, U.1 1 Department of Clinical Pharmacy, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 3 Cologne Biocenter, University of Cologne, Züplicher Str. 47b, 50674 Cologne, Germany

Oxaliplatin (Eloxatin®) is a platinum-based anticancer drug with a better safety profile and the lack of cross-resistance with cisplatin. It is used as a standard treatment of advanced colorectal cancer. Nevertheless, tumour cells can develop resistance to oxaliplatin as well [1]. In contrast to the mechanisms of cisplatin resistance, resistance to oxaliplatin is less well investigated. As in the case of the other platinum drugs, cytotoxic action of oxaliplatin is assumed to result from the binding of the drug to genomic DNA, which subsequently triggers apoptosis in cancer cells. However, only a small fraction of oxaliplatin, which enters the cell, reaches genomic DNA. The fate of the rest of intracellular oxaliplatin and its relevance for oxaliplatin sensitivity of tumour cells and for resistance to the drug remains unclear.

Investigation of the intracellular trafficking of oxaliplatin represents a challenge, as the number of methods allowing detection of platinum drugs inside the cell is very limited. Moreover, many procedures require tiresome cell fractionation, which often do not provide entirely pure cellular organelles.

For these reasons, there is a need for model compounds, which are easy to detect in the cells on one hand, and still reflect biological properties of the parent drug on the other hand. Several fluorescent cisplatin analogues were developed and one of them was shown to mimic biological behaviour of cisplatin with respect to cisplatin resistance [2,3]. Fluores-cence allows monitoring the processing of the compound in living cells. Furthermore, it enables detection of binding partners of platinum drugs.

The aim of our work was to develop a fluorescent oxaliplatin analogue and to evaluate it as a model compound for the studies of cellular trafficking of oxaliplatin. An oxaliplatin derivative labelled with carboxyfluorescein diacetate (CFDA-oxPt, the structure is shown below) has been synthesized and fully characterized. The label is introduced into the cyclo-hexane ring, as the oxalate ligand is readily exchanged upon interaction of oxaliplatin with (cellular) nucleophiles. The cytotoxicity of CFDA-oxPt in HCT-8 human ileocecal colorectal adenocarcinoma cell line and its oxaliplatin-resistant derivative HCT-8ox was studied using a MTT-based assay. Cellular processing of the model complex in both cell lines was investigated by fluorescence microscopy. Based on the results, the suitability of CFDA-oxPt as a model for investi-gation of the cellular trafficking and intracellular interactions of oxaliplatin is discussed.

NH2

Pt

H2

N

O

OHN

O

O

O

O

O

O

O

O

O

O

NH2

Pt

H2

N

O

OO

O

oxaliplatin

CFDA-oxPt

The authors acknowledge the financial support by the Deutsche Forschungsgemeinschaft (grant JA 817/4-1). The authors are grateful to Prof. Christa E. Müller (Institute of Pharmacy, University of Bonn) for providing laboratory facilities for synthetic chemistry.

References:

1. Mishima, M. et al.: Eur. J. Cancer 2002, 38(10): 1405-1412. 2. Kalayda, G.V. et al.: BMC Cancer 2008, 8: 175. 3. Kalayda, G.V.; Wagner, C.H.; Jaehde, U.: J. Inorg. Biochem. 2012, 116: 1-10.


A polymeric multifunctional glaucoma implant

Wischke, C.1; Löbler, M.2; Neffe, A.T.1; Hanh, B.D.1; Sternberg, K.2; Stachs, O.3; Guthoff, R.3; Lendlein, A.1 1 Institute of Biomaterial Science and Berlin-Brandenburg Center of Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Kantstr. 55, 14513 Teltow, Germany 2 Institute for Biomedical Engineering, University of Rostock, F.-Barnewitz-Str. 4, 18119 Rostock, Germany 3 Department of Ophthalmology, University of Rostock, Doberaner Str. 140, 18057 Rostock, Germany

Glaucomas are eye diseases that can lead to blindness due to ocular and neuronal tissue damage by elevated intraocu-lar pressure. If topical medication fails, surgical intervention such as implantation of aqueous drainage devices is advised in open angle glaucoma. Still, due to deficiencies of existing systems that can lead to hypotony, fibrosis, long-term failure, and damage of adjacent tissues, alternative polymer-based implants should be developed.

This study presents a degradable copolyester blend with remarkable mechanical properties and the development of multilayered, drug releasing glaucoma microstents for suprachoroidal implantation based on these materials [1, 2]. The degradation of poly(ε-caprolactone) [PCL] could be accelerated in a controlled manner by introduction of 8 wt.% gly-colide units leading to poly[(ε-caprolactone)-co-glycolide] (PCG), which, however, exhibited poor mechanical properties. By blending of PCG with PCL (50/50 w/w), the advantageous individual properties of both components could be com-bined, i.e. elastic properties comparable to pure PCL and a water uptake during degradation similar to pure PCG [1]. At the same time, processing by hot melt extrusion to tubes with internal diameters as low as 50 µm was realized. In this continuous process that did not involve solvents for drug loading, diclofenac sodium could be incorporated as a model drug. In addition to microstents with a wall from one polymer layer, also bilayer microstents could be obtained. By using a drug free internal polymer layer, drug diffusion was directed to the outer side of the microstent that would be in contact with the tissue. The apparent diffusion coefficients in the different polymer layers could be determined in systematic diffusion experiments. Furthermore, comprehensive degradation and mechanical studies, sterilization experiments, and analysis of in vitro biocompatibility e.g. with human fibroblasts were conducted. Finally, an in vivo study in rabbits with a 100 day follow-up examination illustrated the general suitability of the microstents for ocular implantation [2].

Scheme of multifunctional microstent and ocular implantation for suprachoroidal drainage, reprinted from [2] with permission.

Overall, this study found that blending of the two (co)polyester components allowed combining interesting elastic proper-ties with a desired accelerated degradation. Ocular single and bilayer microstents have been prepared as thin diameter, sterilizable candidate devices for aqueous drainage in glaucoma. These multifunctional systems combine i) a drainage function with adjustable outflow rates by their tailorable diameters in the micrometer range, ii) a mechanical support function, iii) hydrolytic degradability for a regenerative medicine approach, iv) biocompatibility, and v) spatially directed controlled drug release.

In the future, the incorporation of antiproliferative drugs and the in vivo functional analysis of long-term performance should be addressed.

References:

1. Wischke, C. et al.: Macromol. Sympos. 2011, 309: 59-67. 2. Wischke, C. et al.: J. Controlled Release 2013, 172: 1002-1010.

64

CASE STUDIES FROM PHARMACEUTICAL RESEARCH AND DEVELOPMENT

Dendritic cell-targeting cancer vaccine formulations for pulmonary and peroral delivery

Hanefeld, A.1; Schiller, S.1; Wolf, M.1; Weigandt, M.1; Scherließ, R.2; Diedrich, A.2; Janke, J.2; Knolle, P.3, Schröder, M.4; Walden, P.5; Baleerio, R. 5; Lehr, C.-M.6; Rietscher, R.6; Schneider, M.7 1 Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany 2 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9, 24118 Kiel, Germany 3 Klinikum rechts der Isar, Technische Universität München, Schneckenburgerstr. 8, 81675 München, Germany 4 BioMedX, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany 5 Department of Dermatology, Venerology and Allergy, Charité, Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany 6 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus, Geb. A4.1, 66123 Saarbruecken, Germany 7Institute for Pharmaceutics and Biopharmacy, Philipps University Marburg, Ketzerbach 63, 35037 Marburg, Germany

Dendritic cells (DCs) play a crucial role in initiating anti-tumor immunity. We designed nanoparticles for passive dendritic cell-targeting with the aim to trigger potent and specific anti-tumor T cell responses. Activated cytotoxic T lymphocytes (CTLs) are needed to combat tumor cells. Cross-presentation of antigen via the MHC Class I pathway is necessary to effectively induce a CTL response. Current adjuvants are optimized for Th2-biased responses and high antibody serum titers. Ideally, a tumor vaccine formulation would combine direct CTL activation (MHC Class I) with indirect Th1/2 mediated support. We studied a variety of nano-particles (NPs) on their ability to do so and ways to formulate those antigen carriers into dosage forms for mucosal administration.

We could elucidate the routing of different NPs in human dendritic cells. The particle fate seems to be determined by its composition. In vitro assays showed that the immunological response to the model antigen ovalbumin (OVA) could be increased by factor 10 to 30 in comparison to soluble OVA depending on the NP type. OVA-loaded NPs could be successfully formulated into inhalable or enteric microparticles for pulmonary or peroral delivery.

Acknowledgments: BMBF (Förderkennzeichen 13N11455)


Selection of solid state forms for New Chemical Entities: Challenges, opportunities, adventures and lessons learned

Saal, C.

Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany

Selection of solid state forms including pharmaceutical salts, polymorphs and co-crystals has drawn increasing interest since the coincidental discovery of Ritonavir's stable polymorph which represented a major challenge for one of the first anti-HIV drugs. The so called "Ritonavir case" together with the advent of New Chemical Entities which became more and more demanding with regards to physico-chemical properties stimulated the systematic assessment and develop-ment of solid state forms for clinical development. The presentation will provide an overview on selection of solid state forms to overcome hurdles for New Chemical entities including experiences made during the last decade and new technological approaches.

We are greatful to Axel Becker and Michael Lange for fruitful discussion and strong support.

References:

1. Paulekuhn, S.; Dressman, J.; Saal, C.: J. Med. Chem., 2007, 50(26): 6665-6672. 2. Saal, C.; Becker, A.: Eur. J. Pharm. Sci., 2013, 49(4): 614-623. 3. Paulekuhn, S.; Dressmann, J.; Saal, C.: Die Pharmazie, 2013, 68: 555-564.

66

Fast track formulation development for biotherapeutics Winzer, M.


Fighting Schistosomiasis in Young Children: The Pediatric Praziquantel Consortium

Skopp, S.

Merck KGaA, R&D Chemical and Pharmaceutical Development, Frankfurter Str. 250, 64293 Darmstadt, Germany

The current gold standard treatment of schistosomiasis employs annual single oral dose of the drug praziquantel (PZQ) 600 mg tablets, jointly developed by Bayer and Merck in the 1970ies. The tablet is available as an oral immediate release tablet for adults and school-aged children1. The available formulation of PZQ is not suitable for pediatric use and cannot be readily administered to children especial-ly to preschool-age children and infants2. The drug product consisting of the two enantiomers, levopraziquantel (L-PZQ) and dextropraziquantel (D-PZQ), has a severe bitter taste, which can lead to gagging or vomiting if tablets are chewed. To date traditional methods of taste masking have been ineffective for PZQ. The described age groups of children have difficulties swallowing the medication due to the large size of the tablet. Important quality and efficacy issues can be raised3. Morever, the tablets are not registered for pediatric use in pre-school aged children and lack adequate clinical data in this population. In order to tackle this important public health problem, a consortium was formed in July 2012 under the leadership of Merck KGaA with the goal of developing a suitable pediatric praziquantel formulation appropriate for children from the age of 3 months to 6 years and register its use in schistosomiasis. The new formulation will preferably contain the L-PZQ enantiopure active pharmaceutical ingredient (API), yet formulation of the racemate PZQ is also considered as a parallel development. The project has completed the pre-clinical phase and will enter Phase I clinical trials in the last quarter of 2014.

References:

1. Preventive chemotherapy in human helminthiasis: coordinated use of anthelminthic drugs in control interventions: a manual for health profes-sionals and programme managers (Geneva: World Health Organization) 2006.

2. Ekpo, U.F. et al.: Parasitology. 2012, 139: 835-841. 3. Richey, R.H. et. al.: BMC Pediatr. 2013, 13:81.

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Chirality in polyketide antibiotics: substrate-dependent inversion of stereoselectivity in Tyl-KR1-catalyzed reductions Häckh, M.; Lucas, X.; Müller, M.; Günther, S.; Lüdeke, S.

Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany

Polyketide synthases (PKS) are mega-enzyme clusters that assemble acetate and propionate subunits for the biosyn-thesis of numerous antibiotics. During this process ketoreductases (KR) inside of the PKS are responsible for the stere-oselective introduction of chiral secondary alcohols that are characteristic for polyketide scaffolds. The absolute configu-ration of their reduction products correlates strongly with conserved amino acid motifs within the involved KR domain.1 Recently, we found a significant dependence of stereoselectivity and –specificity of recombinant PKS ketoreductase Tyl-KR1 from Streptomyces fradiae on both the chain length and the kind of ester used for different non-natural keto-ester substrates.2 This enzyme exhibits high (S,S)-selectivity for a number of artificial substrates despite of its physiological (R,R)-selectivity.

On the basis of modeling studies using the crystal structure of Tyl-KR1,3 and 2-methyl-3-oxovaleric acid N-acetylcysteamine thioester, a surrogate for the physiological substrate,4 we identified molecular features that may be crucial for (R,R)-selectivity and –specificity. We synthesized different analogs, for which at least one of these interactions would be interrupted (Scheme 1). The stereochemical outcome of the Tyl-KR1-catalyzed reduction of these analogs was studied qualitatively and quantitatively by vibrational circular dichroism and chiral phase gas chromatography. This study does not only provide new insights into the understanding of stereoselectivity in the biosynthesis of polyketide antibiotics but also provides a basis for the utilization of Tyl-KR1 or related enzymes in the biocatalytic synthesis of valuable chiral compounds.

Scheme 1

O

S

OHN

O

O

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OHHN

O

Tyl-KR1NADPH

2R,3R84% ee

O

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O O

S

OHTyl-KR1NADPH

2S,3S18% ee

References:

1. Caffrey, P.: ChemBioChem 2003, 4(7): 654–657. 2. Häckh, M.; Müller, M.; Lüdeke, S.: Chem. Eur. J. 2013, 19(27): 8922–8928. 3. Keatinge-Clay, A.T.: Chem. Biol. 2007, 14(8): 898–908. 4. Siskos, A.P. et al.: Chem. Biol. 2005, 12(10): 1145–1153.


GPCR MEDICINAL CHEMISTRY

Toward selective molecular tools for histamine receptor subtypes: conformational constraints, bioiso-steric and bivalent approaches Buschauer, A.

Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry II, University of Regensburg, D-93040 Regensburg, Germany

Four histamine receptor subtypes, designated H1 (H1R), H2 (H2R), H3 (H3R) and H4 receptors (H4R), are encoded in the human (h) genome. Antagonists for hH1R and hH2R are well established drugs for the treatment of allergic conditions and gastroduodenal diseases, respectively, whereas pitolisant, the first clinically available hH3R antagonist, has obtained orphan drug status for the treatment of narcolepsy. H4R antagonists are not yet available as approved drugs, but are considered to be of potential value for the pharmacotherapy of inflammatory diseases and pruritus. With the discovery of the H3R and, in particular, the H4R, the field became considerably more complex. Numerous compounds developed as subtype-selective ligands for H1R and H2R decades ago, have been proven to possess much higher affinity to H3R and H4R [1]. This holds, e. g., for imidazole-type ligands such as the potent H4R agonist 5-methylhistamine, which was initially described as the first selective H2R agonist, as well as for guanidines derived from impromidine.

Aiming at subtype-selective, radiolabeled and fluorescent hHxR ligands as pharmacological tools, bioisosteric and bivalent approaches were explored in our laboratory, starting from guanidine-type H2R agonists or piperidinomethyl-phenoxypropylamine-type H2R antagonists, respectively. Modification of the latter gave tritiated (([3H]UR-DE257) and fluorescent ligands for the H2R and paved the way to fluorescent H3R antagonists. In the guanidine series, the replace-ment of the imidazole ring by 2-aminothiazole in combination with an acyl- or carbamoylguanidine moiety resulted in highly potent and selective H2R agonists, including bivalent agonists [2,3]. With regard to H4R selectivity, the suitability of guanidine replacements, various heterocycles and conformationally constrained linkers was explored, and the substitu-tion pattern of acylguanidines [4] and cyanoguanidines [5] was varied, resulting, e. g., in the high-affinity H3/4R radiolig-and [3H]UR-PI294 [6] and the potent H4R agonists UR-PI376 [5] and trans-(+)-(S,S)-UR-RG98.

Selectivity for H4R over H3R is especially challenging. Beyond HR subtype selectivity, activities of many ligands differ significantly from those at the human H4R, especially at rodent H4Rs, in terms of ligand efficacies, potencies and affini-ties [7, 8]. Such differences were extremely pronounced in case of proximal readouts ([32P]GTPase, [35S]GTPγS assay). Moreover, in contrast to the mouse and rat H4R, the hH4R shows a high degree of constitutive activity [9].

In conclusion, agonists, antagonists as well as labeled ligands are required as molecular tools for the investigation of HR subtypes of various species. With respect to predictivity of in vitro studies and translational animal models, ortholog- and assay-dependent activity profiles have to be considered, e. g. binding and functional data (GPTase, GTPγS assay, reporter gene, arrestin and label-free assays) using recombinant human, murine and rat H4Rs including mutants as well as native cells.

Acknowledgments: This work was supported by the Graduate Training Programmes GRK 760 and GRK 1910 of the DFG and by the European Cooperation in Science and Technology, COST Action BM0806

References:

1. Seifert, R. et al.: Trends Pharmacol. Sci. 2013, 34: 33-58. 2. Kraus, A. et al.: ChemMedChem 2009, 4: 232-240. 3. Birnkammer, T. et al.: J. Med. Chem. 2012, 55: 1147-1160. 4. Igel, P. et al.: J. Med. Chem. 2009, 52: 2623-2627. 5. Igel, P. et al.: J. Med. Chem. 2009, 52: 6297-6313. 6. Igel, P. et al.: ChemMedChem 2009, 4: 225-231. 7. Schnell, D. et al.: Naunyn. Schmiedebergs Arch. Pharmacol. 2011, 383: 457-470. 8. Nordemann, U. et al.: PLoS ONE 2013, 8: e73961. 9. Wifling, D. et al.: Br. J. Pharmacol. 2014, in press, doi: 10.1111/bph.12801.

70

Modulation of GPCR signaling: Understanding ligand binding effects Bermudez, M.; Wolber, G.*

Computer-Aided Drug Design, Institute of Pharmacy, Department Pharmaceutical Chemistry, Freie Universität Berlin, 14195 Berlin E-Mail: [email protected]

G-Protein coupled receptors (GPCRs) have played a key role for drug development over the last decades. Each class of GPCRs can trigger different functions by adopting specific conformations upon ligand binding. Recent advances in X-ray protein crystallography now provide us with new fundamental structural data on GPCRs with co-complexed ligands [1]. These new insights give us the opportunity to develop structure-based binding models by computer simulation in order to rationally develop drug candidates that selectively bind to a specific conformation. Two case studies for such models will be presented: The first model, based on the recently published crystal structure of the M2 muscarinic acetylcholine receptor (PDB: 3UON [2]) provides the possibility to rationalize and understand the binding of ligands, explain their subtype preference and give insights into the flexibility of their binding pockets. Since allosteric, orthosteric and dualsteric ligands are availa-ble, we were able to connect multiple signaling roles with conformational states [3-4]. Extensive molecular dynamics (MD) simulations in combination with molecular docking and 3D-pharmacophore analyses of known ligands and related structures were used to understand and explain this relation. The second model describes the binding of two recently discovered ligands (JSM10292 and MEN16132) of the bradykin-in B2 receptor. Based on the crystal structure of CXCR3 receptor, a homology model could be developed that explains specific conformational changes and provides a basis for further rationally designing optimized ligands [5].

Fig. 1.: Conformation of the atropin-based hybrid ligand atr-6-phth complexed with the M2 receptor

References:

1. Venkatakrishnan, A.J. et al., Nature 2013, 494(7436): 185–194. 2. Haga, K., et al.: Nature 2012, 482(7386): 547–551. 3. Antony, J., et al.: Faseb Journal 2009, 23(2): 442-450. 4. Holzgrabe, U.; Mohr, K.: Drug Discov Today 1998, 3(5): 214-222. 5. Schmitz, J. et al.: J. Med. Chem. 2014, 57(15):6739-6750. 5. Leschner, J. et al.: J Pharmacol Exp Ther 2013, 1344(1):85-95.


Molecular combination of GPCR ligands: bivalent, hybrid and dualsteric compounds Decker, M.

Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

The development of GPCR ligands with high affinity and selectivity is one of the major success stories in medicinal chemistry, pharmacy and medicine. An array of methods has been developed to find such selective compounds starting from suitable lead structures. Nevertheless, many problems based on receptor function are yet unsolved. It is still un-clear how GPCR move spatially and temporally to mediate their intracellular activity. Also the role of receptor aggrega-tion and/or dimer formation for mediation of effects and receptor internalization is not understood. Based solely on the ligand structure, there is no general knowledge - although there is numerous empirical data – about what determines the intrinsic activity of a chemical compound. Molecular tools are needed to help and enable pharmacologists to investigate these effects [1]. Furthermore, pharmaceutical companies face the demanding task to identify lead structures. Improving a lead’s binding profile can be achieved as mentioned above. Nevertheless, the efforts to find a lead, like screening compound libraries, are difficult to perform and cost-intensive. Another unmet problem lies in the fact that several dis-eases seem to be multifactorial in nature, such as neurodegenerative disorders. Even the best (in terms of affinity and selectivity) GPCR ligand will most of the time only target one aspect of combating the disease, but will never be able to target a larger spectrum of biological effects. Out of these reasons, in the last years several approaches have emerged and successfully applied, in which known GPCR ligands are connected or merged with other biologically active molecules: either combination of identical or related ligands to bivalent ones [1], combination of orthosteric with allosteric ligands [2], sometimes leading to dualsteric (or bitopic) ones, and finally merging chemical structures to dual-acting or multifunctional compounds. In this talk some examples for the above approaches at different GPCRs and their subtypes will be presented showing the versatility of this approach. This includes the synthesis and characterization of bivalent ligands at opioid (OP) [3] and both cannabinoid (CB) 1 and CB2 receptors [4] and some aspects to consider when designing bivalent ligands will be discussed such as an alteration of intrinsic activity. Tasks emerging when designing dualsteric ligands will be presented with regard to muscarinic acetylcholine 1 (M1) receptor ligands [5,6], here it is of major importance to determine whether dualsteric ligands are actually formed. Finally, developing a low-molecular-weight dual active compound acting at the histamine (H) 3 receptor as an antagonist and inhibiting the enzyme and Alzheimer’s disease drug target acetylcholines-terase (AChE) at the same time will be presented [7]. Such a compound might represent an approach to combat multi-factorial diseases like Alzheimer’s disease. All these methods offer unique chances for developing novel GPCR ligands, but also challenge the medicinal chemist when applying them in terms of design, synthetic approaches, and pharmaco-logical characterization.

Financial support by the German Research Foundation is gratefully acknowledged (DFG DE1546/4-1)

References:

1. Hiller, C.; Kühhorn, J.; Gmeiner, .P: J. Med. Chem. 2013, 56(17): 6542-6559. 2. Mohr, K. et al.: Angew. Chem. Int. Edit. 2013, 52(2): 508-516. 3. Decker, M. et al.: J. Med. Chem. 2010, 53(1): 402-418. 4. Nimczick, M. et al.: Bioorg. Med. Chem. 2014, 22(15): 3938-3946. 5. Fang, L.. et al.: J. Med. Chem. 2010, 53(5): 2094-2103. 6. Decker, M., Holzgrabe, U.: Med. Chem. Commun. 2012, 3(7): 752-762. 7. Darras, F.H. et al.: ACS Chem. Neurosci. 2014, 5(3): 225-242.

72

Boronic acids as probes for exploration of allosteric regulation of the chemokine receptor CXCR3 Bernat, V.; Admas, T.H.; Brox, R.; Tschammer, N.

Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstraße 19, 91052 Erlangen, Germany

The G protein coupled chemokine receptor CXCR3 is associated with numerous pathologies including autoimmune diseases, cancer, vascular disease and transplant rejection. To facilitate the understanding of complex allosteric regula-tion of CXCR3, we combined computational modelling with the synthesis of novel chemical tools containing boronic acid moieties, site-directed mutagenesis and detailed functional characterization followed by the analysis of biased signalling. The design of boronic acid derivatives was based on the predictions from homology modelling and docking. Boronic acids derivatives are scarcely used as GPCR ligands. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target bio-molecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduc-tion of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp1092.60 and Lys3007.35 inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore we report the boronic acid derivative as the first biased negative allosteric modulator of the receptor that preferentially inhibits the recruitment of β-arrestin 2 over G protein activation. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signalling.

N.T., T.H.A. and R.B. were financially supported by Graduate Training Schools GRK1910 and GRK1962, and grant TS287/2-1 of German Re-search Foundation (DFG). N.T. participates in the European COST Action CM1207 (GLISTEN: GPCR-Ligand Interactions, Structures, and Trans-membrane Signalling: a European Research Network).


INDUSTRIAL PHARMACY

Biophysical Characterization of Pharmaceutical Peptides Nagel, N.

Sanofi-Aventis Deutschland GmbH, R&D LGCR / Analytical Sciences, D-65926 Frankfurt am Main

Physical characterization of pharmaceutical peptides involves not only secondary, tertiary and quaternary structure investigations, but also exploring the stability of peptides with respect to non-specific aggregation or amyloid fibril for-mation. Recent examples from sanofi development Frankfurt illuminate potentials and limitations of up to date analytical methods.

74

Subvisible Particles in Protein Formulations Olbrich, C.

Bayer Pharma AG, Building 230, 42117 Wuppertal

Particles are a challenge in parenteral formulations. Specific attention is paid to particle formation in protein formulations, as they can be immunogenic and can have severe side effects. For a long time, particles could only be monitored in the range of visible particles and with respect to subvisible particles in the size range of ≥ 10 and ≥ 25 µm (particulate matter; USP monograph). Besides this, size exclusion chromatography was available for the determination of Dimers and Oligomers in the lower nm range. Due to the lack of suitable analytical methods, the range of particles between 100 m and 10µm was not accessible to analytical charav´cterisation, especially the quantification of particles.. Recent developments in analytical equipment opens now the perspective for monitoring protein particles in the size range mentioned allowing the optimisation of formulation and manufacturing processes to minimise particle formation and monitor consitencey over manufacturing changes, which is an important aspect in comparability excersises for protein drugs. Mikroflow Imaging opens the perspectzive of counting and characterising particles between 1 µm and 10 µm and resonant mass measurement is available to to so with particles from 50 nm to 5 µm. This allows to cover the interesting size range for protein particles and to study mechanistically protein particle formation as a consequence of stress. These new analytical methods allow a more reliable development of protein formulations and to conecutively fulfill the upcoming requirements of limitations of partaicle formation and occurance in protein therapeutics, e.g. as a consequence of the FDA Immunogenicity guidline. Examples are shown of stress factors leading to the formation of protein aggregates, as well as to mitigation strategies to avoid or minimise these effects.


Dissecting pharmacodynamic action of compound mixtures by use of in vitro models

Hengl, T.1; Riegel, K.1; Schlinzig, K.1; Ansari, N.²; Stelzer, E.²; Abts, H.F.1 1 Merz Pharmaceuticals, 60318 Frankfurt, Germany ² Buchmann Institute for Molecular Life Science (BMLS), Goethe University, 60438 Frankfurt, Germany

Established use of traditional or “grandfather” products are often based mainly on clinical effects while detailed knowledge of the underlying pharmacodynamics might not be available. In particular information on the detailed mode of action on the target cell would be of interest in order to further support current use but also allow further development. For obtaining such information on pharmacodynamics appropriate cell based in vitro assay and molecular identification of potential functional pathways could be used. In vitro cultured human keratinocytes are a well-established tool for investigating general aspects of skin and hair physi-ology. Within the epidermis normal human epidermal keratinocytes (NHEK) generate the multi-layered skin barrier by a distinct differentiation process. The major part of the hair follicle is build up by hair follicle associated keratinocytes (HHFK) that form as a result of another differentiation program the hair-shaft. General aspects of keratinocyte physiology could be investigated in both cells, while examination of hair-specific processes requires the analysis in HHFKs. Cultiva-tion of keratinocytes in a minimal growth medium (MGM) was used as an in vitro model for undersupplied keratinocytes, mimicking the situation of diminished hair growth for analyzing the effect of a hair growth promoting formulation, Pan-to(vi)gar. To investigate the genes that are modulated during Panto(vi)gar treatment we performed an Agilent whole genome array expression analysis using HHFK and NHEK cultivated either in a minimal growth medium (MGM) alone or supplemented with a Panto(vi)gar in vitro correlate (P-IC). In accordance with the P-IC induced cellular phenotype P-IC modulated genes were identified that are involved in cell cycle, proliferation and metabolic processes in NHEK and HHFK. Furthermore P-IC appears to regulate genes associated with cell death, extracellular matrix, stress responses and hair follicle physiology. Two-dimensional monolayer cell culture models are thus successfully used for understanding physiological pathways on a cellular level. However, physiological properties and differentiation processes are in vivo often also the result of three-dimensional interactions within organs or mini-organs such as the hair-follicle. Associated processes like differentiation or morphogenesis can be simulated in vitro by using 3-D culture models. As second example for the use of in vitro models we thus present a 3-D culture system mimicking more closely the in vivo situation of the human hair follicle. This 3-D model was used to further investigate the mode of action of hair-growth promoting formulations. As 3-D hair follicle model heterotypic spheroids were established from human dermal papilla cells (DP) and hair-follicle-associated keratinocytes (HHFK). The analysis of the initial spheroids formation process was performed by observing cell type specific migration/distribution patterns by automated live cell fluorescence microscopy. Spheroid formation process was shown to be fully functional in P-IC whereas cultivation in MGM only resulted in only reduced spheroid quality and stability. Data indicate that the tested actives could thus deliver a significant contribution to the formation and preservation of the hair-follicle structure. In conclusion use of 2-D and 3-D cell based models allow the molecular dissection of the functional aspects of pharma-cologic active ingredients. Such knowledge not only uncovers the mode of action but could form also the basis of alter-native and improved drugs.

76

Current Biopharmaceutics Prediction Tools - An Overview Muenster, U.

Bayer Pharma AG, Research Center Aprath, 42096 Wuppertal, Germany

In order to bring innovative drug products to the patient in need in good time, it is key within Pharmaceutical Develop-ment to focus on the right formulation strategy early-on. The type of formulation needed (e.g. immediate release (IR) with or without solubilization technology, or e.g. slow release (SR) tablet) depends on the pharmacokinetic profile as well as the physicochemical properties of the API. Choosing the right formulation and making an acceptable prediction of its biopharmaceutics performance has been more and more supported over the last two to three decades by the generation of various biopharmaceutics prediction tools. These include physiologically based pharmacokinetic (PBPK) in silico softwares, in vitro experimental set-ups (e.g. mimicking gastrointestinal compartments) and respective in vivo animal models. The talk will give an overview on the current status and most often used biopharmaceutics prediction tools within Pharmaceutical Industry.


BIOPHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY

Novel injectable RNA formulations for tumor immunotherapy Haas, H.1; Fritz, D.1; Meng, M.1; Popa, A.-L.1; Esparza, I.1; Kranz, L.M.3; Reuter, K.C.2; Diken, M.2; Kreiter, S.2; Funari, S.4; Pawlowska, D.5; Brezesinski, G.5; Sahin, U.1,2,3 1 BioNTech RNA Pharmaceuticals GmbH, An der Goldgrube 12, 55131 Mainz, Germany 2 TRON gGmbH, Building 708, Langenbeckstrasse 1, 55131 Mainz, Germany 3 Universität Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany 4 HASYLAB, DESY, Notkestrasse 85, 26607 Hamburg, Germany 5 Max-Planck-Institute for Colloids and Interfaces, Am Mühlenberg 1, 14476 Golm, Germany

Novel nanoparticulate lipoplex formulations to deliver mRNA to Antigen Presenting Cells (APCs) are presented. After intravenous (i.v.) injection of the lipoplex formulations, expression of mRNA in splenic Antigen Presenting Cells (APCs) with high selectivity and efficacy can be obtained. For targeting certain physicochemical parameters of the lipoplex nanoparticles are decisive, and no ligands for specific binding tot the target membrane, such as mannose, are required. With this type of formulations, tumor immunotherapy based on expression of mRNA coding for tumor antigens in APCs may be substantially facilitated and provided to high numbers of patients under daily routine conditions.

Similar to the well-known approaches for non-viral gene delivery with DNA, mRNA lipoplexes were formed by incubation of RNA to cationic (positively charged) liposomes. Ionic conditions, lipid composition, lipid-to-RNA ratio, and further physicochemical parameters were varied for formation of the lipoplex formulations. Particle size, colloidal stability and other parameters that are relevant for pharmaceutical products for parenteral administration were investigated. Formula-tions consisting of positively charged or negatively charged nanoparticles, which were stable in the liquid phase for several days, and where the RNA was protected with respect to interactions with serum components and degradation were identified.

After intravenous injection of such lipoplex formulations from mRNA, coding for the reporter gene luciferase into mice, expression of luciferase was measured in various target organs, dependent on the particle properties. While with posi-tively charged (cationic) particles, such as known from classical gene delivery, preferentially luciferase expression in the lung was observed, negatively charged lipoplex nanoparticles lead to luciferase expression almost exclusively in Dendrit-ic Cells (DCs) in the spleen. It appears that the charge of the particles was a key factor for the targeting selectivity. Different mechanisms of cellular uptake in DCs and other cells may play a role for the observed selectivity.

The finding, that high targeting selectivity can be achieved without using specific ligands or complex nanoparticulate architecture makes formulations assembled by the described approach promising for pharmaceutical development towards products for application in patients. Robust, straightforward and scalable manufacturing processes can be realized, using standard lipids which are compliant with the requirements of products for parenteral administration.

This will help to bring novel therapeutic strategies based on selective expression of mRNA in Antigen Presenting Cells (APCs) readily into clinical practice. Products for tumor immunotherapy by selective expression of tumor antigens in APCs can be administered systemically (intravenously) on a regular basis.

Acknowledgments: We would like to thank The innovation and technology program by the government of Rhineland Palatinate within the InnoTop Project “Innovative therapeutic RNA formulations for systemic application of biologicals in humans for financial support. Wealso would like to acknowledge financial support for the project “Nanopartikuläre Ribopharmaka zur individualisierten Tumortherapie –Ph I im Mammakarzinom, within the ” CI3 Cluster für Individualisierte ImmunIntervention”.

78

Automated testing of inhalation devices in early development phases Wachtel, H.; Jung, A.; Richter, F.

Respiratory Drug Delivery Department, Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Str. 173, 55216 Ingelheim, Germany

Advanced testing of quality aspects of the Respimat® Soft Mist™ Inhaler (SMI) is discussed where the required release tests and one-time studies were complemented by automated tests. The automated testing was established as a volun-tary additional effort because it allowed to significantly increase the sample size and thereby to improve the understand-ing of this device in the early development phase.

Background: The manufacture of inhalers and the applicable testing methods are strictly regulated. According to the classification of the inhalers they are either medicinal products or medical devices or combination products, depending on different national legislations. In case of international development programs, the regulations of the importing coun-tries must already be considered during development, where e.g. a design history file is required. The regulations strong-ly influence the testing strategies applied e.g. for release.

Requirements: During the development phase of a multi-dose inhaler like the Respimat SMI, the reliable dosing is key and must be checked with high statistical power, increasing the number of test samples in a way that manual testing according to Pharm. Eur. (0671) is not feasible. In addition, the innovative technology of spray generation motivates paying special attention to the particle size distribution which by convention previously was checked using cascade impactors according to Pharm. Eur. 2.9.18. Handling e.g. the torque for preparing the Respimat SMI for use or the force to press the dose release button should also be recorded thus taking into account a recent initiative towards human factor engineering which puts emphasis on usability.

Technical solution: Boehringer Ingelheim built a fully automated test system which comprises a six-axis robotic arm for device transfer between individual stations which are dedicated to the following tasks: 1. Storage of 300 inhalers, 2. Checking handling parameters, e.g. torque to prepare the inhaler and force to press the release button, 3. Sound detec-tion, 4. Measurement of the particle size distribution (laser diffraction), 5. Detection of the plume geometry, 6. Measure-ment of spray duration, 7. Check of consistent dosing by determining delivered mass / metered mass. The system was designed for 1000 actuations within 24h and the large number as well as the high reproducibility (low standard deviation) help in making rational design decisions. The system is operated in a non-GMP environment because during early development the flexible adaptation to the changing inhaler features is important. For the same reason there is no full formal compliance with 21 CFR Part 11 as the complexity of the heterogeneous multi-processor system (see Fig. 1) prohibits fast revalidation cycles. The accuracy of the measured data however is guaranteed by regular checks of the individual sensors. All submission data are generated by conventional manually operated tests in the appropriately regulated environment.

Figure 1: IT structure of the automated system allowing for flexibility on a local level while interfacing to a strictly regulat-ed company-wide IT structure and the Oracle database.

Summary: The Respimat® Soft Mist™ inhaler represents a novel and unique principle for atomizing pharmaceutical aerosols. Even in early development, the advantage of automated testing of this inhaler is apparent. In early develop-ment, the flexibility of an automated system is key and outweighs full compliance (i.e. the system is operated at a non-GMP status). The large number of devices and repeated actuations automatically investigated leads to a better under-standing and consequently to high reliability of the finished product.


Orodispersible Dosage Forms Serno, P.

Bayer Pharma AG, Building D303, 51368 Leverkusen, Germany

Orodispersible dosage forms passed through significant steps of evolution during the last two decades and represent a well accepted formulation option for dysphagic patients, poorly cooperating patients, medication to be taken during travelling and other instances. Three formulation technologies are being used: Oral lyophilisates, orodispersible tablets and orodispersible films. Oral lyophilisates require highly specialized and expensive manufacturing technology. They exhibit limited mechanical strength but still offer the shortest disintegration time and lead to pleasant mouthfeel upon administration. Orodispersible tablets have initially been manufactured by highly complex technologies involving spinning of flosses, compression of fine particular sugars, curing steps and others. Nevertheless, mechanical properties of early orodispersible tablets had often been poor. Meanwhile a large number of coprocessed excipients has become available for easy formulation of orodispersible tablets. The “coprocessing” usually comprises spray drying of one or more polyols and addition of a superdisintegrant like crospovidone. Orodispersible tablets are subsequently manufactured in a direct compression process with the tablet compression force as a key critical process parameter. Alternatively, orodispersible tablets may be formulated by addition of silicates and superdisintegrants in high concentrations. Orodispersible films are manufactured by casting thin layers of organic polymer solutions and subsequent drying in long drying tunnels. The biopharmaceutical performance of orodispersible dosage forms ranges from bioequivalence to substantially enhanced bioavailability in comparison to conventional swallow – tablets. Several case studies suggest that “faster action”, i.e. faster occurrence of therapeutically relevant plasma concentrations, is usually not achieved.

80

Pharmacokinetic Drug-Nutraceutical Interactions: A Particular Type of Food-Drug Interaction Nguyen, M.A.; Langguth, P.

Institute of Pharmacy Institute of Pharmacy, Johannes Gutenberg University, Staudingerweg 5, 55099 Mainz, Germany

In addition to the daily diet, components present in food and beverages are increasingly consumed in dietary supple-ments due to their presumed health promoting effects, for example anti-oxidative and anti-inflammatory activities. These nutraceuticals represent a relevant source of interaction with drug pharmacokinetics for two main reasons: Specific components are by several magnitudes higher dosed compared to the normal dietary intake and unlike prescribed drugs, their consumption is usually not reported to the physician upon visit. Our data presented here focus on the modulation of drug transport by selected flavonoids in humans. Although quercetin remarkably inhibits P-glycoprotein (P-gp)-mediated drug efflux in vitro1, the bioavailability of talinolol, a substrate of intestinal P-gp, was reduced following oral co-administration with the nutraceutical.2 This observation indicates that inhibition of intestinal uptake by organic anion-transporting polypeptides (OATPs)3 rather than P-gp inhibition dominates when talinolol and quercetin are co-administered in humans. On the other hand, naringin which had been reported to be the causative ingredient for the inhibition of intestinal OATPs by grapefruit juice3 did not alter the pharmacokinetics of talinolol, a substrate of intestinal OATP1A2 and OATP2B1, after 7-day supplementation with naringin capsules. Possible explanations include the higher dose and the solid state which is different from the common intake of naringin in grape-fruit juice. In the proximal parts of the small intestine, the main site of talinolol absorption, the concentration of dissolved naringin may not be high enough for efficient inhibition of OATP-mediated uptake, whereas in more distal parts, naringin and its aglycone naringenin may reach higher concentrations provoking P-gp inhibition which counteracts the uptake inhibition.

Like food, nutraceuticals can interfere with a variety of processes during drug absorption, distribution, metabolism and elimination. Due to their different dose and dosage forms, however, their effect on drug pharmacokinetics may deviate from that observed when administered as food components. Better understanding of their pharmacokinetics will help reducing the risk of uncontrolled modulation of drugs’ efficacy and adverse effects.

The research work discussed here was supported by the German Research Foundation (DFG).

References:

1. Ofer, M. et al.: Eur. J. Pharm. Sci. 2005, 25: 263-271. 2. Nguyen, M.A. et al.: Eur. J. Pharm. Sci. 2014, 61: 54-60. 3. Shirasaka, Y. et al.: J. Pharmacol. Exp. Ther. 2010, 332: 181-189. 4. Bailey, D.G. et al.: Clin. Pharmacol. Ther. 2007, 81: 495-502.


Gastro-Intestinal Simulator for in vitro Drug and Nanoparticle Tracing in Oral Drug Development Nawroth, T.1; Khoshakhlagh, P.1; Kindgen, S.1; Krebs, L.1; Johnson, R.1; Langguth, P.1; Schweins, R.2; Szekely, N.3 1 Gutenberg-University, Inst. Pharmacy and Biochemistry, Pharmaceutical Technology Department, Staudingerweg 5, D-55099 Mainz, Germany 2 Institut Laue Langevin ILL, DS / LSS, 71 Avenue des Martyrs, F-38042 Grenoble CEDEX 9, France 3 JCNS-FRM-II outstation at the Mayer-Leibnitz Zentrum MLZ, Lichtenbergstr.1, D-85747 Garching, Germany

Intestinal modelling and model fluids are tools for the in vitro investigation of drug formulations for oral administration. In vitro solubility and permeability studies at nearly physiological conditions are the key for an improvement of the bioavail-ability of drugs, which is difficult for hydrophobic materials, i.e. drugs of the classes II and IV of the Biopharmaceutics Classification System BCS [1]. These form nanoparticles with amphiphilic materials, e.g. bile and food, which can medi-ate a drug solubilisation and uptake. The structure of lipidic nanoparticles in solution can be estimated by dynamic light scattering DLS and small angle scattering, with best contrast using neutrons, i.e. neutron small angle scattering SANS [2].

We develop a gastro-intestinal simulator system for the in vitro estimation of drug and formulation development in a modelled human digestion system (fig.1). The flow-through modules for the sections of the GI-system are designed for a tracing of formulation disintegration, drug dissolution and structure analysis of intermediate nanoparticles, e.g. from bile-drug-formulation interaction. The transport can be analyzed by local sampling. The nano-structure is observed in situ through flat quartz windows with local and time resolution with a computer controlled (CNC) continuous flow (CF) + stopped flow (SF) system with a projecting DLS device, and for the most important samples with neutron scattering SANS at large science facilities [2,3].

Fig.1: Concept of the gastro-intestinal simulator for in vitro drug and nanoparticle tracing of oral drug formulations. Formation and decay of intermediate nanoparticles from bile, drug, fluid and food are analyzed in gut module flow cells with DLS and SANS with continuous flow (CF) and stopped flow (SF) technology.

This work was contributed to the OrBiTo project (http://www.imi.europa.eu/content/) as sideground.

References:

1. Amidon, G. et al.: Pharm. Res.1995, 12: 413-420. 2. Nawroth, T. et al.: Mol. Pharm. 2011, 8: 2162-2172. 3. Khoshakhlagh, P.: et al. Eur.J.Lipid Sci. Technol 2014 submitted. 4. Johnson, R. et al.: Eur.J.Lipid Sci. Technol 2014 in press.

http://www.imi.europa.eu/content/

82

ANTICANCER AND EPIGENETIC DRUGS

Unraveling the aberrant epigenetic programming of MLL leukaemias Milne, T.

There has been progress in treating human cancers, especially leukaemias, but many remain resistant to treatment. A potentially exciting approach is the development of small molecule inhibitors that specifically target aberrant processes in cancer cells but leave normal cells unharmed. In order to be successful, such an approach requires highly detailed information about normal and aberrant cellular processes on the molecular level. Understanding the function of key proteins on a molecular level requires the use of model systems that are amenable to experimental manipulation. The Mixed Lineage Leukaemia protein (MLL) has provided a very useful model system for exploring general mechanisms in leukaemia. MLL is an important epigenetic regulator normally required for the maintenance of gene activation during development. Chromosome translocations that fuse the MLL gene to over 75 different partner genes produce novel fusion proteins that are a major cause of therapy resistant acute leukaemias in children and adults. Relative to other acute leukaemias, MLL leukaemias have very few cooperating mutations. MLL fusion proteins are thought to promote leukaemogenesis through the epigenetic activation and maintenance of important gene targets that control cellular growth and proliferation pathways. Studying MLL leukaemias can aid in the identification of new drug targets that could also potentially impact a wider range of different cancers.


Identifying Therapeutic Targets in MLL Fusion-Driven Leukemia Using Functional Genomics Fröhling, S.

Department of Translational Oncology, German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT), Heidelberg

Chromosomal rearrangements involving the H3K4 methyltransferase MLL trigger aberrant gene expression in hemato-poietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies, because MLL is difficult to target directly and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by the MLL-AF9 fusion are excep-tionally reliant on the cell cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia, and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by palbociclib, a small-molecule CDK6 inhibitor currently in clinical develop-ment. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell cycle regulators may have distinct, non-canonical and non-redundant functions in different contexts. Based on this preclinical rationale, we are currently preparing a phase I/II clinical trial to evaluate the efficacy of palbociclib in patients with newly diagnosed, relapsed, or refractory MLL-rearranged leukemia who are not candidates for intensive chemotherapy and hematopoietic stem cell transplantation.

84

MLL leukemias and future treatment strategies Marschalek, R.

Inst. Pharm Biology, Goethe-University, Biocenter, Max-von-Laue-Str. 9, 60438 Frankfurt/Main

Childhood cancer affects about 1.800 children per year in Germany of which about 620 cases develop acute leukemia (~35%). This is different in adults, because of the 430.000 newly diagnosed cancer cases per year only about 9.300 cases suffer from acute leukemia (2%). Of all those leukemia cases (childhood and adult), MLL-rearranged leukemia cases play an important role. Generally, MLL-r leukemia patients are hard-to-treat and the overall survival of these patients is very poor. Moreover, a large portion of these leukemias is diagnosed in infants after birth, indicating that the onset of this type of leukemia already occured in utero. The human MLL protein - encoded by the MLL gene - plays a pivotal role in growth and differentiation processes. The MLL protein exerts a histone H3 lysine-4 (H3K4) tri-methylation activity. This particular histone modification represents a key signature of our "epigenetic programming system" which is necessary for developmental process, but also responsi-ble for "transcriptional memory" in differentiated cells. The MLL gene becomes disrupted in a process termed "chromosomal translocation". Basically, this process describes how 2 different chromosomes are recombined after a DNA damage situation. During this event, the MLL gene becomes disrupted and fused reciprocally to one of its many translocation partner genes. So far, 80 different translocation partner genes have been described. Consequently, all these events result in the expression of MLL fusion proteins, and subse-quently, in the development of acute leukemia [ALL (acute lymphoblastic leukemia) or AML (acute myeloid leukemia)]. Interestingly, only 4 out of 80 gene fusions are responsible for the majority of cancer cases (90% of patients in ALL, 50% of patients in AML). These translocation partner genes are AF4, AF9, ENL and AF10, respectively. Moreover, all 4 encoded proteins are part of a multiprotein complex that steers "transcriptional elongation". Thus, epigenetic imprinting as well as transcriptional processes are affected in a genome-wide fashion in the leukemic blasts of these leukemia patients. Consequently, novel drugs have been developed in the past years that affect key functions deriving from these MLL fusion proteins. This talk will summarize our knowledge about MLL-r leukemia and the different strategies to inhibit functions of these MLL fusion proteins by using molecules. This study is supported by grant DKS 2011.09 from the German Children Cancer Aid to RM, and by research grants Ma1876/10-1, Ma1876/11-1 from the DFG to RM. RM is PI within the CEF on Macromolecular Complexes funded by DFG grant EXC 115.


Selective Sirt2-inhibition by ligand induced rearrangement of the active site Jung, M.1; Rumpf, T.1; Schiede, M.l.1; Karaman, B.2; Roessler, C.3; North, N.J.4; Ladwein, K.I.1; Gajer, M.1; Pannek, M.5; Steegborn, C.5; Sinclair, D.A.4; Gerhardt, S.6; Schutkowski, M.3; Sippl, W.2; Einsle, O.6 1 Albert-Ludwigs-University, Institute of Pharmaceutical Sciences, Albertstr. 25, 79104 Freiburg, Germany 2 Martin-Luther-University, Institute for Pharmacy, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany 3 Martin-Luther-University Institute for Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany 4 Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, Boston, MA 02115 USA 5 University of Bayreuth, Department of Biochemistry, Universitätsstr. 30, 95445 Bayreuth, Germany 6 Albert-Ludwigs-University, Institute for Biochemistry and BIOSS, Albertstr. 21, 79104 Freiburg, Germany

Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been impli-cated in the pathogenesis of cancer, inflammation and neurodegenerative diseases which makes a modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2-inhibitors is lacking so far. Here, we present the first high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2-selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a cryptic binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2-inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

The studies have been supported by the Deutsche Forschungsgemeinschaft (Inhibitors: Ju295/8-1, Si868/6-1 structural work: SFB992 Medical Epigenetics, Project Z02) and the EU (cellular studies: SEtTReND, Nr. 241865, FP7 Health). D.A.S. was supported by grants from NIH/NIA (R01 AG028730 and R01 AG019719), the Glenn Foundation for Medical Research, the United Mitochondrial Disease Foundation, The Juvenile Diabetes foundation, and a gift from the Schulak family. B.J.N was supported by BIDMC/Harvard Translational Research in Aging Training Program (T32 AG023480). We also thank C. Kambach (University of Bayreuth, Department of Biochemistry, Universitätsstr. 30, 95445 Bayreuth, Germany) for providing Sirt6.

References:

1. Finnin, M.S. et al.: Nat. Struct. Biol. 2001, 8: 621–625. 2. Trapp, J. et al.: J. Med. Chem. 2006, 49: 7307–7316. 3. Outeiro, T.F. et al.: Science 2007, 317: 516–519. 4. Schemies, J. et al.: Cancer Lett. 2009, 280: 222-232. 5. Suzuki, T. et al.: J. Med. Chem. 2012, 55: 5760–5773. 6. Moniot, S. et al.: J. Struct. Biol. 2013,182: 136–143. 7. Yamagata, K. et al.: Structure 2014, 22: 345–352. 8. North, B.J. et al.: EMBO J. 2014: in press.

86

EVIDENCE-BASED MEDICATION MANAGEMENT

Evaluation of medication management in community pharmacies

Waltering, I.; Koling, S.; Hempel, G. Institute of Pharmaceutical and Medicinal Chemistry, Clinical Pharmacy, University of Muenster, Germany

Patients in community pharmacy – How to improve medication reviews Medication therapy is a well-known risk. Medication errors, adverse drug events and medication discrepancies are common, costly and preventable. A possibility to reduce the risk is a medication review conducted in community phar-macies [1-5]. With the Apo-AMTS-Concept a training-program was developed to teach pharmacist and pre-registration students how to conduct such a review on an intermediate level (PCNE-classification)[6]. After implementation of the program it was necessary to assess the ability of the participants to correctly and completely identify drug-related prob-lems (DRPs). This cross-sectional study with 76 patients from a convenience sample was performed within the Apo-AMTS-course. During the Apo-AMTS course pharmacists and pre-registration students received training in medication reviews consist-ing of a 4h basic-course and 3-day advanced-course. At the end of the course each participant had to turn in 5 medica-tion reviews conducted in their pharmacy. A checklist to document the detected problems and a documentation template to record the interventions were provided for this purpose. The results noted in the checklist were reviewed by a clinical pharmacist with experience in medication reviews and a physician. The number and types of DRPs found by the phar-macists/students were registered in an excel datasheet. Classification of the DRPs was deviated from the MAI Score[7]. The expert reviewers completed the different potential DRPs by reviewing all available information. The ATC-code was used to assess what drugs caused the main problems and which drugs are responsible for the most overlooked DRPs. The results showed that pharmacists and students detected interactions, side-effects, problems with time of intake and adherence problems almost completely. Double medication, duration of intake and dosage intervals were the most overlooked or not assessed problems. It could also be seen that many issues were connected with PRN (as needed) medication and with drugs prescribed from the ATC-class “A”. In conclusion, this study could show that with medication reviews in community pharmacies on an intermediate level, after training, DRPs that could be detected in the pharmacy. Most of these problems could be solved together with patients and pharmacists and improve medication and patient safety. DRPs like dosage and double-medication, where more information and clinical judgment could be necessary or a discussion with the prescriber was necessary were more often not detected. Here is more support and training necessary. Because we did not compared results of medication reviews from untrained pharmacists, the correct effect of the training could not be detected. Also further studies of the clinical importance of the detected and non-detected problems are necessary.

References:

1. Joint Commission on Accreditation of Healthcare Organizations, U., Using medication reconcilitation to prevent errors. Sentinel Event Alert., 2006, Jan 23(35): 1-4.

2. Gandhi, T.K. et al.: N Engl J Med, 2003, 348(16): 1556-1564. 3. Bryant, L.J. et al.: Int J Pharm Pract, 2011, 19(2): 94-105. 4. Lenaghan, E.; Holland, R.; and Brooks, A.: Age Ageing, 2007, 36(3): 292-297. 5. Sorensen, L. et al.: Br J Clin Pharmacol, 2004, 58(6): 648-664. 6. van Mil F, G.N., The PCNE guideline for medication review, in PCNE Working Symposium on Medication Review. 2011: Manchester. 7. Hanlon, J.T. and Schmader, K.E.: Drugs Aging, 2013, 30(11): 893-900.


Evidence-based medication management in psychiatric patients Pauly, A.1; Wolf, C.1; Kornhuber, J.2; Friedland, K.2

1 Molecular and Clinical Pharmacy, Department of Chemistry and Pharmacy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany 2 Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany

Low adherence and Drug-Related Problems (DRP) are important issues, which limit the clinical outcome in psychiatric patients. To address these problems we designed a clinical trial, which was conducted in the psychiatric university hospital in Erlangen. Patients aged >18 years, taking medications and who gave written consent were allocated to control or intervention group depending on the date of their admission. Control patients (09/2012-03/2013) received usual care whereas patients in the intervention group (05/2013-12/2013) benefited from a structured extended medica-tion management program. The medication management program consisted of two different components. Firstly, individualized patient counselling by two pharmacists was conducted regarding the pathophysiology of the psychiatric disease, mechanism of action of the psychoactive drugs, and information about the clinical relevance of side effects in two patient consultation during hospital stay. Secondly, DRP were identified in both groups in the course of structured clinical reviews of patients’ charts at admission, weekly during the hospital stay, at discharge as well as three months after discharge including a comprehen-sive patient interview, the assessment of laboratory parameters and adverse drug events by the two pharmacists. The detected DRP were classified according to a validated classification system with domains to classify the problem, the intervention and the outcome (1). Causing drugs were documented and the relevance of every DRP was estimated as minor, moderate or high in concordance with criteria used in other pharmaceutical care studies in psychiatry (2). Besides DRP, adherence was investigated during the patients hospital stay and for three months after discharge. Adherence was measured by the self-report questionnaires “Medication Adherence Report Scale” (MARS) and “Drug Attitude Inventory” (DAI) at admission, discharge and 3 months after discharge. The primary outcome measures were the total number of DRP per patient and the differences of change in MARS and DAI. Results were adjusted for age, gender, comorbidities and baseline values for MARS and DAI and additionally for length of stay and number of drugs at admission for DRP. Depending on their time of admission 269 patients were allocated to receive either usual psychiatric care (control) or the structured medication management program (intervention). 419 and 396 DRP were identified within the hospital stay corresponding with a median of 3 (IQR = 1-5) and 2 (IQR = 1-4) DRP per patient in control and intervention group. Differences were not statistically significant (p = 0.487). After the entire study period 303 DRP (median = 2 unsolved DRP per patient, IQR = 1-3) remained unsolved in control patients in contrast to 50 (median = 0, IQR = 0) in intervention group. The adjusted number of unsolved DRP in intervention group was 1.82 (95% CI: 1.52 – 2.14) less compared to usual care. Regarding adherence, the mean MARS score of control patients increased during hospitalization from 22.23 (2.87) by 4.84% to 23.44 (2.34) at discharge. Three months after discharge, the MARS score decreased by 3.9% near to his original level, 22.47 (2.99). In contrast, the mean MARS score improved in the intervention group from 22.02 (3.42) at admission by 11.60% to 24.34 (1.61) at discharge. This value declined by 2.36% to 23.75 (2.08) at the follow-up. Taking into account the improvement of control group, the adjusted change in the MARS score for intervention patients was 1.33 points (6.65%, 95% confidence interval 0.73 – 1.93, P < 0.001 ), significantly higher than in control patients. 53.4% of intervention group patients in comparison to 31.1% of patients in the control group achieved the possible maximum value of 25 points in the MARS indicating a perfect adherent behavior three months after discharge. In conclusion, our study provides first evidence that a structured medication management for psychiatric patients may be effective to reduce DRP and to improve patient adherence.

References:

1. Ganso, M. et al.: Krankenhauspharmazie 2009, 30(7): 349-362. 2. Campell, A.R. et al.: Am J Pharm Educ. 2011, 10;75(1): 8.

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Evidence-based medication management in cancer patients Wilmer, A.1; Tasar, A.2; Fleckenstein, K.3; Hack, C.3; Ruberg, K.2; Ko, Y.D.3; Jaehde, U.1 1 Institute of Pharmacy, Clinical Pharmacy, University of Bonn, Germany 2 Kronen Pharmacy Marxen, Wesseling, Germany 3 Department of Internal Medicine, Johanniter Hospital, Bonn, Germany

Many studies have shown that there is a strong need for specific interventions assuring medication safety, especially in patients receiving complex drug treatments such as cancer patients. In order to establish risk-minimizing interventions, it is crucial to generate evidence for their effectiveness in appropriately designed studies. We implemented a multiprofessional best-practice model at the oncology outpatient ward of the Johanniter Hospital in Bonn providing a structured and standardized medication management for cancer patients. It was the aim of this study to provide first data on the effectiveness of this intervention to enhance patient safety. The model was developed in a multiprofessional quality circle to define ‘best practice’. Care modules were developed for medication review and interaction check (basic module), malnutrition, and for the management of four common adverse events: nausea/emesis, mucositis, fatigue, and pain (supplementary modules). All modules include evidence-based recommendations for supportive care, written patient information, and an algorithm illustrating the care process. They can be applied individually for each patient according to the anticipated toxicity. After implementation, the feasibility of the model was evaluated in a pilot study. Among others, the effectiveness was evaluated using the patient-reported outcome (PRO) version of the CTCAE criteria [1]. The best-practice model was evaluated in a randomized two-arm interventional study. 51 outpatients with solid tumors were randomized either to the control group or the intervention group with multiprofessional and modular care, and monitored for a maximum of 5 cycle of chemotherapy. Primary endpoint of the study was the time to first occurrence of severe symptoms (PRO-CTCAE grade 3 to 4). Secondary endpoints were health-related quality of life and patient satisfaction with information received. The results suggest that the best-practice model may delay the time to first occurrence of grade 3 to 4 symptoms. However, the effect was not statistically significant due to the small sample size. It is remarkable that the delaying effect was especially observed for symptoms for which the supplementary care modules were developed (mucositis, nausea and vomiting, and fatigue). There was no difference between the two groups regarding the secondary endpoints health-related quality of life and patient satisfaction. In conclusion, our study provides first evidence that a structured and standardized medication management for cancer patients may be effective to enhance patient safety. However, a larger number of patients is needed in order to prove its effectiveness.

References:

1. US Department of Health and Human Services. National Cancer Institute: Development of the Patient-Reported Outcomes Version of the CTCAE. Available from: https://wiki.nci.nih.gov/x/cKul. Last access: 29 August 2014


PHARMAGRIPS: Structured pharmaceutical counseling in the self-medication of the common cold. A randomised controlled study (RCT)

Laven, A.1; Schäfer, J.2; Läer, S.3

1,2,3 Institut für Klinische Pharmazie und Pharmakotherapie, Heinrich-Heine-Universität Düsseldorf

Background: Many minor ailments are treated in Germany by self-medication. Most drugs dispensed by pharmacy staff are those for the common cold, general pain and gastrointestinal disorders. Whilst pharmacists express their need for further training in counseling on side effects, interactions and contraindications, they tend to receive feedback from patients to the effect that the drugs used have not worked.

Methods: From July to October 2013 we carried out a prospective, single-blind, quasi-randomised controlled study on the effect of training on structured pharmaceutical advice in self-medication of the common cold (PHARMAGRIPS Study). Using a controlled, interventional study design we investigated whether it is possible to improve the pharmaceu-tical counseling in self-medication within a short time, by using an appropriate teaching method. The counseling should be made in a systematic way and refer to evidence-based content in order to avoid incorrect advice. We enrolled 86 pharmacists and assigned them randomly into the study protocol. Of those, 56 completed the study as planned and were analysed.

In this study, we reviewed the structure of the average pharmaceutical consultation and added evidence-based content from the Cochrane Reviews on common cold. We then integrated this structured consultation in a methodical modern training program consisting of e-learning and live classes which we evaluated scientifically. For this purpose, we con-ducted telephone interviews with the participating pharmacists by using standardized case report forms. The case report forms contained the questions that the participants were supposed to ask. For every question asked, the participant received a certain amount of points, 18 in total.

The training was stated to be successful at the primary endpoint when an improvement of at least 3.5 out of 18 points was achieved on average. The secondary endpoints were related to various aspects of the interview process (medical history, limits of self-medication, evidence-based drug selection and integration of customer input in the consultation interaction).

Results: The training group improved in the primary endpoint by an average of 5,93 points (p < 0,001) and compared to the control group significantly in all secondary endpoints, with one participant managing to achieve the full score.

The participants recognized the importance and practical relevance of the exercise in a short time and were able to implement even integrate complex content in their consultations and to give the customer appropriate advice.

References: 1. Laven, A.; Läer, S.: Med Monatsschr Pharm. 2013, 36: 102-110. 2. Laven, A.; Schäfer, J.; Läer, S.: PHARMAGRIPS: Med Monatsschr Pharm. 2014, 37: 209-220.

90

OPTIMIZING ORAL DRUG PERFORMANCE

Of Springs and Parachutes – Improving Oral Bioavailability

Brewster, M.E.

Janssen Research and Development, Johnson & Johnson, Turnhoutseweg 30, Beerse 2340, BELGIUM

Contemporary drug pipelines are increasingly populated with difficult-to-formulate drug substances. The genesis of this heightened complexity is rooted in three confluent trends including (1) the reliance of the drug discovery process on high throughput screening, (2) growing issues with drug form and (3) the nature of current drug targets which are often associated with structure-activity relationships (SAR) which deviate or disconnect from the chemical space associated with good oral bioavailability and acceptable biopharmaceutical fitness. Taken in aggregate, this evolution in dosage form development challenges has forced formulators to innovate with a variety of novel approaches promulgated in the last few years. Enabling formulation strategies including the use of nanotechnology, lipid-based dosage forms giving rise to self-micro or nano-emulsifying drug delivery systems (S (M, N) EDDS) and amorphous solid dispersions in which a drug is rendered non-crystalline in a glassy matrix [1]. In many of these dosage forms, a postulated element critical to their biopharmaceutical success is the ability of the formulation to generate a supersaturated solution in the gastric or intestinal compartment [2]. This so-call “spring” effect (that is pushing concentrations of the drug, in excess of their thermodynamic solubility, into the media) is then modified with a formulation component which hinders precipitation or crystallization of the supersaturated, metastable solution (i.e., a parachute). In the case of an amorphous solid disper-sion, the glassy dispersant may also serve the dual role of acting as a parachute. In both designing and producing amorphous solid dispersions, appropriate assays are requires that can assess the effectiveness of an excipient in generating a stable dispersion, of generating a supersaturated solution in an appropriate media and the stability profile of the formed metastable system [3]. Such in vitro approaches include methodologies designed around solvent-shift approaches as well as non-crystalline solid approaches where supersaturation is induced, respectively, by adding a drug in a water-miscible solvent to an aqueous matrix or by adding the amorphous phase directly to the aqueous system. These approaches, while useful, can underestimate supersaturation stability in vivo [4]. This may be due to ignoring drug absorption which can impact the supersaturation ratio. Dissolution apparatus with an absorption compartment can be useful from that standpoint [5]. In vivo assessments of supersaturation rely on drug formulation administration fol-lowed by gastric and intestinal sampling through the use of specialized catheters with simultaneous assessments of bioavailability and pharmacokinetic parameters. Comparative assessments of in vivo and in vitro tools can be helpful in optimizing IVIVC and dosage form design. A number of amorphous solid dispersions have reached the market. These include dosage forms in which the drug is rendered amorphous and mixed with various excipients/carriers through the use of a number of processing techniques such as melt extrusion, melt blending, bead coating, solvent processing, spray-drying and antisolvent precipitation (i.e., the microprecipitated bulk powder approach). In all cases, post-process manipulation is needed to generate the final dosage form whether that is a tablet or capsule. Based on the traditional uncertainty of translate-ability of animal models, dosage form testing in man is often completed to ensure that the best concept is developed and forwarded to the market [2]. In these studies, indirect and increasingly, direct methods are being applied to better understand the applied supersaturating drug delivery system and its mechanism for augmenting oral bioavailability [3]. As was suggested, presenting the drug in a physically stable amorphous format meets only part of the challenges associated in the development of these systems. An appropriate processing approach also needs to be identified, confirmed and implemented and biorelevant approaches which translate to man are a sine qua non for successful drug development. References:

1. Branchu, S. et al.: Eur. J. Pharm. Sci. 2007, 22: 128-139. 2. Brouwers, J. et al.: J. Pharm. Sci. 2009, 98: 2549-2572. 3. Bevernage, J. et al.: Int. J. Pharm. 2013, 453: 25-35. 4. Psachoulias, D. et al.: Pharm. Res. 2011, 28: 3145-3158. 5. Bevernage, J. et al.: Eur. J. Pharm. Biopharm. 2012, 82: 424-428.


Assessing the gastrointestinal „Spring“ effect – the media Leigh, M.

92

Will the parachute crash? – transfer models for assessing performance of optimized formulations Kostewicz, E.S.; Ruff, A.

Institute for Pharmaceutical Technology, Goethe University, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

Given the greater prevalence of poorly-soluble compounds in contemporary drug discovery pipelines, formulation ap-proaches that enhance supersaturation or minimize gastrointestinal (GI) precipitation within the GI environment are becoming more widely adopted as a strategy for enhancing bioavailability. As the options for delivering poorly soluble drugs have become more innovative, predictive and reliable in vitro tools for appropriate evaluation of formulation behav-iour are needed.

Due to the complexity of the supersaturation and precipitation process occurring along the GI lumen, there are a multi-tude of factors that need to be considered when evaluating these parameters in vitro. For supersaturation and precipita-tion, luminal concentrations may be influenced not only by gastric emptying, permeability, ionization characteristics of the API, solubilisation by bile acid micelles, the dissolution characteristics of the formulation used, but also by the sharp pH change observed between stomach and intestine, prevailing composition and volume of the luminal fluids, dilution of the formulation with GI luminal fluids, digestion of solubilizing excipients and the nature of excipients used in the formulation, to name just a few (1).

The critical steps that need to be considered in an in vitro model therefore include the assessment of the formulations’ rapidly dissolving supersaturation “spring” with a precipitation inhibition or retarding “parachute” that can occur in vivo along the GI tract. The transfer model is an in vitro method that can be used for assessing the performance of such optimized formulations. This model utilizes an USP II dissolution apparatus containing two compartments simulating the stomach and intestine, respectively. To simulate the transfer of drug out of the stomach and into the intestine, the drug in the simulated gastric fluid compartment is pumped into a simulated intestinal fluid compartment in which supersaturation and/or precipitation is evaluated by concentration versus time measurements. In this model, the influence of transfer rate, bile salt concentration, pH and compartmental volumes on the supersaturation and precipitation behaviour of the formulation can be evaluated.

As part of this presentation, I will provide an overview of the current status of the transfer model, present a few examples utilizing this method for evaluating the supersaturation and precipitation behaviour of optimized formulations and discuss the in vivo relevance of this tool.

Reference:

1. Kostewicz, E.S. et al.: Eur. J. Pharm. Sci. 2014, 57: 342-366.


Connecting oral formulation performance to therapeutic effect Cristofoletti, R.

94

MULTITARGET DRUGS

An introduction to polypharmacology in drug discovery Peters, J.-U.

Roche Pharmaceutical Research and Early Development, Discovery Chemistry, Innovation Center Basel, Building 92 / 3.64C, CH-4070 Basel, Switzerland

Polypharmacology has been receiving an ever-increasing interest over the last 10 years, and has been proposed as the “next paradigm in drug discovery”.1 This is mainly due to the advent of modern polypharmacological drugs,2 but also to the recognition of unintended polypharmacology as a source of adverse drug effects.3 This talk gives an introduction to polypharmacology, its opportunities and risks, and its role in modern and historic drug discovery. Topics include: (1) the prevalence of polypharmacology in drug-like compounds; (2) the significance of polypharmacology for safety; (3) how to recognize polypharmacological compounds early; (4) why polypharmacological drugs may be more efficacious; (5) opportunities of polypharmacology: historic and modern examples; (6) strategies for the discovery of polypharmacologi-cal drugs. References:

1. Hopkins, A.L.: Nat. Chem. Biol. 2008, 4(11): 682–690. 2. Peters, J.-U.: J. Med. Chem. 2013, 56(22): 8955–8971. 3. Peters, J.-U. et al.: Drug Discovery Today 2012, 17(7–8): 325–335.


Multifactorial activity of the naphthoquinone shikonin against cancer cells

Efferth, T.

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz; E-mail: [email protected]

Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. In a screen of 40 phytochemicals derived from medicinal plants used in Japanese Kampo medicine, shikonin was the most cytotoxic compound [1]. This is a naphthoquinone derived from Lithospermum erythrorhizon, which is used in traditional Asian medicines for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca2+ and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimate-ly induction of apoptosis [2]. Next, we included micro-RNA microarrays and stable-isotope dimethyl labeling for quantitative proteomics. The integra-tion of bioinformatics and the three "-omics" assays showed that the PI3K-Akt-mTOR pathway was affected by shikonin. Deregulations of this pathway are frequently associated with cancerogenesis, especially in a wide range of hematologi-cal malignancies. The effect on the PI3K-Akt-mTOR axis was validated by demonstrating a decreased phosphorylation of Akt and a direct inhibition of the IGF1R kinase activity after shikonin treatment. Our results indicate that inhibiting the IGF1R-Akt-mTOR signaling cascade is a new cellular mechanism of shikonin strengthening its potential for the treatment of hematological malignancies [3].

References:

1. Efferth, T. et al.: Cancer Genomics Proteomics 2007, 4: 81-91. 2. Wiench, B. et al.: eCAM 2012: 726025. 3. Wiench, B. et al.: eCAM 2013: 818709.

96

Polypharmacology: In silico recognition vs. rational design Proschak, E.

Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue Str. 9, D-60438,Frankfurt a.M., Germany

The „one drug – one target – one disease“ paradigm in drug discovery has been reconsidered during the last decade. This paradigm change was mainly caused by high attrition rates in drug approvals due to toxicity and lack of efficacy. On top of that, the results of post-genomic and network biology showed that putative drug targets rarely act within isolated systems but rather as a part of a highly connected network.[1] Furthermore, the efficacy of several approved drugs has been traced back to the interaction with multiple targets.[2] Computational techniques play an important role in prediction and recognition of novel targets for approved drugs. We will discuss two machine learning approaches – self organizing maps and inverse distance weighting – for polypharmacological profiling of bioactive compounds, exemplified by two prospective studies [3,4]. While the recognition of occasional polypharmacological behavior is an established task, the rational design of multitar-get ligands remains challenging. Dual or multi-target ligands have several advantages compared with selective com-pounds, including improved efficacy and more simple pharmacokinetic and pharmacodynamic properties in comparison to the combination of several drugs [5]. In this context we present three methodic approaches to design dual inhibitors of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). In our first study we connected previously pubished 5-LO and sEH pharmacophores, an imidazo-[1,2a]-pyridine core with an aryl urea moiety via flexible propyl linker [6]. The second study contains the discovery of a benzimidazole-based dual 5-LO/sEH inhibitor by means of in silico screening [7]. The strategy of the virtual screening protocol was an exhaustive pairwise evaluation of pharmacophore models for both targets to obtain a dual-target pharmacophore model. Our last study deals with the development of a fragment based strategy for dual-target drug discovery. Here, we applied a modified self-organizing map algorithm for in silico recognition of molecular fragments binding both targets. The predicted properties were confirmed by complementary screening techniques: STD-NMR and enzyme assay. The enlargement of the fragment hit led to submicromolar dual target inhibitor of sEH and 5-LO.[8] References: 1. Jeong, H. et al.: Nature 2001, 411: 41-42. 2. Zimmermann, G.R.; Lehár, J.; Keith, C.T. : Drug. Discov. Today 2007, 12 : 34-42. 3. Paulke, A. et al.: J. Ethnopharmacol. 2013, 148: 492-497. 4. Steri, R.: Biochem Pharmacol. 2012, 83: 1674-1681. 5. Morphy, R.; Rankovic, Z.: J. Med. Chem. 2005, 48: 6523-6543. 6. Meirer, K. et al.: J. Med. Chem. 2013, 56: 1777-1781. 7. Moser, D. et al.: ACS Med. Chem. Lett. 2012, 3: 155-158.

8. Achenbach, J. et al.: ACS Med Chem Lett 2013, : 1169-1172.


Study of the anxiolytic actions of Valeriana officinalis L., Melissa officinalis L., Passiflora incarnata L. and their combination STW 32 in experimental models of anxiety Okpanyi, S.N.1; Kelber, O.1, Weischer, M.-L.2 1 Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany 2 Institute for Pharmacology and Toxicology, University of Münster, Germany

Several well established psychotropic herbal medicinal preparations, such as of Melissae folium, Passiflorae herba and Valerianae radix, are recognised and recommended officially both in the German Commission E and in the ESCOP Monographs for the treatment of tenseness, restlessness and irritability, with difficulty in falling asleep. Being that these phytomedicines do not negatively influence vigilance and reaction time, the primary mode of their calmative action is more likely to be anxiolytic and not sedation like benzodiazepines and barbituric acid derivatives.

The anxiolytic actions of the three hydroethanolic extracts and their combination were investigated in elevated plus maze (EPM) and social interaction of the mouse. Influence on exploratory behaviour (vigilance, rearing and locomotory activity) was tested. Diazepam 1.0 mg/kg b.w. was used as a standard anxiolytic agent.

Significant results of the EPM test were as follows: Diazepam increased no. of entry 2 0.4 (control) to 9 3.7 (p

0.01), duration of stay 13 3.1 to 49 14.3 (p 0.05). Passiflora (P) 350 mg/kg b.w. increased no. of entry 1.8 0.8

(control) to 5.0 0.6 (p 0.01), duration of stay 25 7.6 (control) to 91 19.3 (p 0.01). Valeriana (V) 1040 mg/kg

b.w. increased entry 2 0.6 (control) to 4 1.2 (p 0.05), duration of stay 20 7.6 (control) to 57.7 (p 0.01). The effect of the combination STW 32 (P 40 %, V 20 %, M 40 % of fluid extract) was very pronounced at a low dose: 60

mg/kg b.w. increased entry from 1.5 0.4 to 3.3 0.5 (p 0.05), duration from 11 4.8 to 29 6.0 (p 0.05). The higher doses 120 mg and 240 mg/kg b.w. also significantly increased all effects.

The test plant extracts and their combination did significantly increase social interactions to as well as vigilance and locomotory activity. A clear synergistic action was manifest in the combination.

These results bear evidence that the calmative and sleep inducing effects of the herbal extracts and their combination STW 32 are due to an anxiolytic effect.

98

Motility modulation beyond MCP: Mechanisms of action of a clinically proven herbal medicinal prod-uct, STW 5, in functional GI diseases Kelber, O.1; Hoser, S.2; Abdel-Aziz, H.1; Okpanyi, S.N.1; Nieber, K.2 1 Scientific Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany 2 Pharmaceutical Institute, Leipzig University, Leipzig, Germany

INTRODUCTION: Motility-modulating drugs have been an important therapeutic approach in functional GI diseases. This approach has been questioned by the withdrawal of the prokinetic drug cisapride from the market due to cardiac side effects and now again by the referral for metoclopramide (MCP) due to neurological side effects leading to the omission of functional dyspepsia and gastrooesophageal reflux disease from its therapeutic indications.

Therefore it is of increasing importance to identify other treatment options with proven clinical efficacy but a more favour-able safety profile. As has been recently shown by a clinical review publication (1), an herbal medicinal product, STW 5, is a safe and therapeutically effective treatment option in these indications, as has been shown by randomized controlled clinical trials in functional dyspepsia (FD) as well as in irritable bowel syndrome (IBS).

AIMS & METHODS: For identifying the mechanisms of action underlying its clinical effect, a systematic database search regarding its effect on GI motility was conducted in accordance to the PRISMA statement and checked for completeness by means of hand searching and cross referencing.

RESULTS: A large number of publications on STW 5 [2] and on the components of the product were identified. Already the first mechanistic studies on the combination [2, 3] suggested a dual mechanism of action on motility, with a spas-molytic effect in acetylcholine induced contractions and a tonicising effect in the relaxed state. This has been confirmed [4, 5] in human isolated intestinal segments [6] and in inflamed intestinal tissue in vitro and in vivo [7-10]. In the stomach, a region specific action was described in vitro, based on an inhibition of Ca influx via SOC channels in the gastric fundus and on a stimulation of Ca influx via L type Ca channels in the antrum [11]. This region specific action has been con-firmed in a human study in vivo [12]. In the lower esophageal sphincter, a tonicising action mediated by L type Ca channels has been shown in vitro [6]. The components of the herbal components have been shown to act synergistically by pharmacological studies [13].

CONCLUSION: The in vitro-, in vivo- and human studies showed spasmolytic as well as tonicising-prokinetic effects possibly relevant for the well documented clinical effect of STW 5 in functional GI diseases, so supporting the use of this medicine in these diseases, which previously have been treated with MCP or cisapride.

References:

1. Ottillinger et al.: WMW 2013, 163:65. 2. Brierley, Kelber: Curr Opin Pharmacol, 2011, 11: 604. 3. Okpanyi et al.: Acta Hort. 1993, 332: 227. 4. Ammon et al.: Phytomed 2006, 13: SV: 67. 5. Heinle et al.: Phytomed 2006, 13: SV: 75. 6. Kelm et al.: ZPT 2013, 34(S1): S31. 7. Schemann et al.: ZGastroenterol 2008, 46: 1039. 8. Michael et al.: Phytomed 2009, 16: 161. 9. Sibaev et al.: ZPT 2013, 34(S1): S31. 10. Wadie et al.: Int J Colorectal Dis 2012, 27: 1445. 11. Hohenester et al.: Neurog Motil 2004, 16: 765. 12. Pilichiewicz et al.: Am J. Gastroenterol 2007, 102: 1. 13. Hoser, S. et al.: Planta Med 2013, 79: 1258.


NON-CANONICAL GPCR-SIGNALING

Dynamic ligand binding Bock, A.

100

Voltage-dependent GPCR-activation Rinne, A.


Mechanosensitivity of histamine H1 receptor Mederos y Schnitzler, M.

102

Signaling in malaria parasites: A non- canonical G-protein from plasmodium falciparum

Kaiser, A.*; Langer, B.; Kersting, D.; Krüger, M.

Institute for Pharmacogenetics, University of Duisburg-Essen, Hufelandstrasse 55, 45177 Essen, Germany

During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts like the blood stream, the human liver and the midgut of the mosquito. Key cellular mechanisms involving cAMP or cGMP dependent signal transduction chains [1] are assumed to act at these interfaces. Previous findings showed that the parasite uses the G-protein from the human host for invasion [2]. We here describe the first cloning and expression of a guanine-nucleotide-binding protein (G-protein) from Plasmodium. The protein reveals an open reading frame of 2733 bp encoding a protein of 911 amino acids and has a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript formation and expression are significantly increased in the late developmental stages during schizont and gametocyte formation suggesting its role in stage conversion and transmis-sion. Most notably, the G-protein has GTP binding capacity and Gtpase activity due to an EngA domain which is also present in small Ras-like GTpases in a variety of Bacillus species and Mycobacteria. By contrast, P. falciparum G-protein is divergent from any human alpha-subunit [3]. The G-protein was expressed in E. coli as a histidine-tagged fusion protein with a short half life of 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chro-matography and separation by Blue Native page gel electrophoresis. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5’O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan [4] stimulated GTP binding in the presence of Mg2+. The determined GTpase activity of the human paralogue was 50% higher than the activity of the parasitic enzyme. In light of these significant results a non-canonical cGMP-regulated, rudimentary signaling pathway seems to be present in Plasmodium with a functional, non-heterotrimeric G-protein. A current research for the more prevalent receptor will delineate this pathway with respect to transmission and relevance to antimalarial chemotherapy.

References:

1. Baker, D.A.: Cell Microbiol. 2011, 13:331-339. 2. Murphy, S.C et al.:PLoS Med 2006, 3: e528. 3. Kimple, A.J. et al.: Pharmacol Rev.2011, 63:728-749. 4. Higashijima, T.; Burnier, J.; Ross, E.M.: J Biol Chem, 24: 14176-14186.

http://www.ncbi.nlm.nih.gov/pubmed?term=Murphy%20SC%5BAuthor%5D&cauthor=true&cauthor_uid=17194200


BIOPHARMACEUTICALS/BIOTECHNOLOGY

Major trends and challenges in biotherapeutic product development: "polysorbate degradation" and "drug-device combination product development" Adler, M.

104

Finding the right candidate – integrated lead ID of next-generation molecules Ruberg, E.-M.


Non‐immunogenic messenger RNA therapeutics Rudolph, C.

ethris GmbH, Lochhamer Str. 11, 82152 Martinsried

Replication-deficient viruses have been used successfully in the field of gene therapy because of their high transfection efficiency. However, the risk of insertional mutagenesis and induction of undesired immune responses remain critical obstacles for their safe medical application. On the other hand, nonviral vectors have been intensively investigated for plasmid DNA (pDNA) delivery as a safer alternative, although their gene transfer efficiency is still many folds lower than for viral vectors, which has been predominately attributed to the insufficient transport of pDNA into the nucleus. Instead of pDNA, messenger RNA (mRNA) has recently emerged as an attractive and promising alternative in the field of nonviral gene delivery1. This strategy includes several advantages compared to the use of pDNA: i) the nuclear mem-brane, which is a major obstacle for pDNA, can be avoided because mRNA exerts its function in the cytoplasm; ii) the risk of insertional mutagenesis can be excluded; iii) the determination and use of an efficient promoter is omitted; iv) repeated application is possible; v) mRNA is also effective in non-dividing cells, and vi) vector-induced immunogenicity may be avoidable. In particular, immunogenicity concerns have been successfully solved by inclusion of chemically modified nucleotides in the mRNA molecule during in vitro transcription. In our approach, replacement of 25% of uridine and cytidine by 2-thiouridin and 5-methylcytidin, respectively, largely reduced mRNA binding to sensors of the innate immune system, including Toll-like receptors and RIG-I, which largely reduced mRNA immunogenicity in vivo after intravenous and intrapulmonary application2. According to these characteristics, we termed these chemically modified mRNA stabilized non-immunogenic messenger (SNIM®) RNA which are now commercialized by the company ethris GmbH, for development of “Transcript therapies” for the treatment of various inherited diseases and application in regenerative medicine. The potential range of programs to which the technology can be applied is very broad spanning metabolic or hereditary monogenetic disorders to regenerative medicine. We have successfully developed technologies for the pulmonary delivery of SNIM® RNA, thus demonstrating SNIM® RNA products can be delivered conveniently and efficiently by a number of routes that will support patient compliance and quality of life. References:

1. Yamamoto, A. et al.: Eur J Pharm Biopharm. 2009, 71(3): 484-489. 2. Kormann, M.S. et al.: Nat Biotechnol. 2011, 29(2): 154-157.

106

Divergent Pathways for the Biosynthesis of Merochlorins, Cyclic Meroterpenoid Antibiotics from a Marine Streptomycete Kaysser, L.1,2; Bernhardt, P.2; Nam, S.-J.2; Teufel, R.2; Diethelm, S.2; Loesgen, S.2; Fenical, W.2; Moore, B.S.2,3 1 German Centre for Infection Research (DZIF) at the Department of Pharmaceutical Biology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany. Email: [email protected] 2 Scripps Institution of Oceanography and 3 Skaggs School of Pharmacy, University of California San Diego, 9500 Gilman Drive, La Jolla 92037, CA, USA

Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Four new meroterpenoid antibiotics, merochlorins A–D, have been isolated from the marine bacterium Streptomyces sp. strain CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents.[1,2] A distinctively cyclized sesquilavandulyl side chain in the merochlorins A, B and C indicated a sophisticated biosynthetic machinery for the formation, transfer and the conversion of this moiety. Draft genome sequencing, mutagenesis and heterologous expres-sion provided the merochlorin biosynthetic gene cluster, encoding two rare bacterial vanadium-dependent haloperoxi-dases (VHPO).[2] Genetic engineering and in vitro biochemistry gave detailed insights into assembly of the merochlorins including the divergence of the biosynthetic pathways and the complex cyclization mechanisms.[2-5]

References:

1. Sakoulas, G. et al..: PLoS ONE 2012, 7: e29439. 2. Kaysser L. et al.: J. Am. Chem. Soc. 2012, 134(29): 11988–11991. 3. Teufel, R. et al.: Angew. Chem. Int. Ed. 2014 (submitted). 4. Diethelm, S. et al.: Angew. Chem. Int. Ed. 2014 (submitted). 5. Kaysser, L. et al.: (in preparation).


Combination strategies for targeting the oncogenic Pim1 kinase Grünweller, A.1; Lange-Grünweller, K.1; Weißer, A.1; Weirauch, U.2; Aigner, A.2; Hartmann, R.K.1 1 Institut für Pharmazeutische Chemie, Pharmazie, Philipps-Universität Marburg, 35032 Marburg, Germany 2 Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Klinische Pharmakologie, Universität Leipzig, 04107 Leipzig, Germany

Pim1, a constitutively active serine/threonine kinase, is overexpressed in various aggressive solid tumors and lympho-mas. Pim1 is an effective inhibitor of apoptosis and an activator of cell proliferation, and its overexpression correlates with a bad prognosis for patients. Knockout of the Pim kinase family (Pim1, 2 and 3) resulted in fertile mice with only mild phenotypic changes, thus Pim kinases seem to have no essential function in healthy cells and adult tissues. Interesting-ly, the 3´-untranslated region (3´-UTR) of the Pim1 mRNA has several conserved putative binding sites for microRNAs (miRNAs). We have recently shown that Pim1 is a target of miRNA regulation by miR-33a and miR-15b [1-3]. Further-more, Pim1 is an epigenetic regulator by phosphorylating histone H3 at serine 10 to activate transcription in a c-Myc-dependent manner and by phosphorylation of heterochromatin protein HP1γ.

We have applied several RNA-based strategies to explore Pim1 as a new tumor target in mouse xenografts of colon carcinoma and glioblastoma. Delivery of miR-33a or Pim1-specific siRNAs into tumors was achieved by forming nano-plexes of RNA with branched low molecular weight polyethylenimine (PEI). This approach was also used to explore a new antisense strategy in vivo which is called U1-Interference (U1i) [4]. Our RNA-based strategies against Pim1 reduced tumor growth substantially without changing liver enzyme activities or inducing unwanted immune responses.

Efficient and successful antitumor strategies require combinatorial approaches to reduce the risk of chemoresistance and to lower unwanted side effects. The development of well-tolerated new combination strategies to target oncogenes hold great promise for the clinic. We are currently exploring combinations of RNA-Interference, statins, natural com-pounds and 5-FU to lower the respective doses of these compounds for Pim1 inhibition. Statins, which block the meva-lonate pathway by inhibition of HMG-CoA-reductase, have pleiotropic antitumor effects at low micromolar concentrations. However, strategies combining statins with siRNA or other Pim1-inhibitors improve statin-dependent effects on Pim1, thus lowering the statin concentrations for Pim1 inhibition towards the sub-micromolar range. We will now further evalu-ate statin effects on Pim1 in mouse xenografts of colon carcinoma and glioblastoma.

References:

1. Thomas, M. et al.: Oncogene 2012, 31(7): 918-928. 2. Ibrahim, A.F. et al.: Cancer Research 2011, 71(15): 5214-5224. 3. Weirauch, U.: Neoplasia 2013, 15(7): 783-794. 4. Weirauch, U. et al.: Nucleic Acids Therapeutics 2013, 23(4): 264-272.

108

SHORT LECTURES


Design, synthesis and biological evaluation of a stabilized RvE2 analog Fukuda, H.; Takakura, Y.; Ishimura, K.; Hirao, T.; Muromoto, R.; Matsuda, T.; Arisawa, M.; Shuto, S.

Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

Resolvins are lipidic chemical mediators that are metabolites of -3 fatty acids EPA or DHA. Because of the highly potent anti-inflammatory activities, they can be leads to develop novel anti-inflammatory drugs. However, resolvins have some problems for their use as clinical drugs that they are unstable to oxygen and to light due to the polyunsaturated bonds, and also that they are gained only a trace from mammals.

We addressed a challenge to develop stabilized derivatives of resolvins. Therefore, we expected that resolvins can be stabilized without losing the biological activity by replacing the Z-olefin of resolvin structure with a chiral cis-cyclopropane to reduce number of the unsaturated bonds.

Firstly, we achieved a total synthesis of resolvin E2 (RvE2) in short steps, and then succeeded to synthesize a cyclopro-pane analog of RvE2 (CP-RvE2), in which the C11-C12 Z-olefin moiety of RvE2 was replaced with a chiral cis-cyclopropane structrure.

The stability and anti-inflammatory activity of the analog will be reported.

SL.01

110

Biosynthetic studies on fungal meroterpenoids and their fascinating chemistry Matsuda, Y.; Akakawa, T. ; Mori, T. ; Wakimoto, T. ; Abe, I.

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan

Fungal natural products, represented by penicillin, lovastatin, and ciclosporin, have been a rich source of biologically active substances, and are still promising leads for future drug discovery and development. Fungal metabolites classified as “meroterpenoids” exhibits a wide range of biological activities; mycophenolic acid is used as an immunosuppressant agent, and pyripyropenes and ascofuranone have been intensively studied to develop as cholesterol-lowering and anti-trypanosomal drugs, respectively.

Besides their biological activities, diverse chemical structures are also important characteristics of fungal meroterpe-noids, and those derived from 3,5-dimethylorsellinic acid (DMOA) include an especially large variety of molecules often with complex carbon skeletons,1 as exemplified by the spiro-lactone system of austinol and the unique bridged-ring of anditomin. Understanding the molecular basis for their biosynthetic systems would not only facilitate the discovery of enzymes catalyzing an intriguing reaction but also provide genetic building blocks that can be used to obtain novel attracting compounds by metabolic engineering or synthetic biology approaches.

We established the biosynthetic pathways of austinol and anditomin by reconstituting their pathways using heterologous expression systems in Aspergillus oryzae and by in vitro assays with selected enzymes.2 In the course of our study, we identified two unique enzymes that catalyze intriguing reactions in both the austinol and anditomin pathways. In the austinol biosynthesis, AusE, which is a Fe2+ and α-ketoglutarate-dependent dioxygenase, was found to convert preausti-noid A1 into preaustinoid A3 by oxidative rearrangement to generate spiro-lactone structure of austinol. The anditomin also involves a dioxygenase AndA with a distinct activity. AndA utilizes preandiloid B as its substrate and yields the bicyclo[2.2.2]octane system of andiconin with an unprecedented structural reconstruction.

In summary, our study provides insight into how structurally diverse metabolites are generated by enzymes with a similar activity. Investigation of the biosyntheses of other related natural products as well as the construction of unnatural metabolisms by mixing the obtained genetic components will be performed in our future studies.

References:

1. Geris, R.; Simpson, T.: Nat. Prod. Rep. 2009, 26(8): 1063-1094. 2. Matsuda, Y. et al.: J. Am. Chem. Soc. 2013, 135(30): 10962-10965.

SL.02


Destruxin E, a Potent Negative Regulator of Osteoclast Morphology: Solid-Phase Library Synthesis and Biological Evaluation

Yoshida, M.1; Ishida, Y.1; Sato, H.1,2; Murase, H.3; Nakagawa, H.3; Doi, T.1 1 Graduate School of Pharmaceutical Sciences, Tohoku Uniniversity, 6-3 Aza-Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan 2 Mitsubishi Tanabe Pharm Corporation, 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan 3 Department of Applied Biological Chemistry, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan

Destruxin E (1), isolated from Metarhizium anisopliae, is a 19-membered cyclodepsipeptide consisting of five amino

acids (-alanine, L-NMe-alanine, L-NMe-valine, L-isoleucine and L-proline) and -hydroxy acid derivative [1]. The struc-tural features of destruxin E (1) are a 19-membered macrolactone and a terminal epoxide in the side chain, and the stereochemistry of the epoxide has not been elucidated until our total synthesis [2]. To date, several kinds of destruxin

derivatives have been isolated, destruxin E (1) exhibits the most potent V-ATPase inhibitory activity (IC50 0.4 M) among the destruxin family [3]. Because of the unique structural features and biological activity, we became interested in the total synthesis of 1 and its derivatives. We have recently achieved the solid-phase total synthesis and structure determi-nation of 1, and we also found that destruxin E (1) exhibits 10-fold stronger V-ATPase inhivitory activity than the stereoi-somer of the epoxide epi-1 [2].

Scheme 1. Solid-Phase Total Synthesis of Destruxin E (1)

Recently, it has also been reported that destruxin E (1) intriguingly induces morphological changes in osteoclasts-like multi nuclear cells (OCLs) at low concentration without affecting the V-ATPase activity of the OCLs [4]. Therefore, destruxin E (1) would be a good candidate for a new type of antiresortive agent. In order to elucidate the mode of action in OCLs, we next attempted the library synthesis of destruxin E (1) and elucidation of the structure-activity relationships. We designed 18 derivatives based on the structure of 1, and the library synthesis was performed by solid-phase synthe-sis using split & pool method. 19-membered macrolactone was formed by MNBA-mediated macrolactonization [5], and formation of the epoxide in parallel furnished the desired destruxin derivatives. Details of the synthesis and biological evaluation of destruxin derivatives will be presented.

Acknowledgement:

This work was supported by a Grant-in-Aid for Young Scinentists (B) (M.Y.) from Japan Society for the Promotion of Science and was also partially supported by Protein Research Foundation (M.Y.).

References:

1. Päis, M. et. al.: Phytochemistry 1981, 20(4): 715–723. 2. Yoshida, M. et. al.: Org. Lett. 2010, 12(17): 3792–3795. 3. Vázquez, M.J. et. al.: Chem. Biodiversity, 2005, 2(1): 123–130. 4. Nakagawa, H. et. al.: Bone 2003, 33(3): 443–455. 5. Shiina, I. et. al.: Chem. –Eur. J. 2005, 11(22): 6601–6608.

Destruxin E (1) (1gS)epi-Destruxin E epi-(1) (1gR)

NH

N

N

N

O

HN

OO

OO

O**

O

O1) SPPS

2) Cleavage1g

H2N

b-Ala

Polymer-support

O O

NH

NFmoc

NFmoc

O

O

O

OH

OH

HO

MeAla

MeVal

Ile

NH

N

N

N

O

HN

OO

OO

O**

O

OO

PyBroP

PyBroPN

TBSO

OO

**O

O

HO

PyBroP

1) H3O+

2) TsCl

3) K2CO3

DIC

Destruxin E (1) exhibits 10-fold V-ATPase inhibitory activity than epi-destruxin E epi-(1).

HA-Pro-OH 24

Fmoc

NH

N

N

N

OH

HN

OO

OO

O**

OO

3

O OH

MNBA

DMAPO

Macrolactonization

SL.03

112

Design and synthesis of NF-κB inhibitors carring epoxyquinol moiety

Saitoh, T.1,2; Ohta, E.2; Umezawa, K.3; Nishiyama, S.2 1 International Institute for Integrative Sleep Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba 305-8577, JAPAN 2 Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, JAPAN

3 Department of Molecular Target Medicine Screening, School of Medicine, Aichi Medical University, Nagakute, Aichi 408-1195, JAPAN

The NF-κB signaling pathway plays a central role not only in inflammation but also in the development of cancer. There-fore, NF-κB inhibitors are expected to be novel candidates as chemotherapeutic agents for inflammatory and cancer diseases, as well as bioprobes for the characterization of intracellular biological responses and cell function.1 Over the past decade, a number of structurally diverse small molecules that block the NF-κB signaling pathway, have been identified. Especially, the epoxyquinol class NF-κB inhibitors, such as DHMEQ (1),2 cycloepoxydon (2),3 and panepoxy-don (3),4 were found to exhibit remarkable inhibitory activity against NF-κB activation (Fig. 1). Despite their structural similarity (Fig. 1), all of them showed completely different modes of action in the inhibition of NF-κB activation. Whereas DHMEQ (1) exhibits inhibitory activity by covalently binding to the NF-κB components, panepoxydon (2) and cycloe-poxydon (3) show inhibition by interfering with the degradation of IκB-α and activation of IκB kinase (IKK).3, 4 In this study, we synthesized several epoxyquinol and non-epoxyquinol derivatives based on the structure of DHMEQ (1) to examine its structure–activity relationship.

Figure 1 We synthesized epoxyquinol and salicylic amide analogs carring the partial structure of DHMEQ (1) and evaluated these biological activities (Fig. 2).5, 6 Although some epoxyquinol analogs, parasitenone (4) and amino epoxyquinols (5, 6) showed the potent NF-κB inhibitory activity, salicylic amide analogs (7) did not inhibit the activitation of NF-κB. The results of the biological investigation of epoxyquinol analogs (4-6) indicated that these modes of action were slitely different from each other and it depended on the structure of side chain unit and syn or anti relative configuration. The presentation will provide an overview of synthesis and biological activity of these analogs.

Figure 2

References:

1. Kataoka, T.: J. Antibiot. 2009, 62: 655. 2. Ariga, A. et al.: J. Biol. Chem. 2002, 277: 24625. 3. Gehrt, A. et al.: J. Antibiot. 1998, 51: 455. 4. Erkel, G.; Anke, T.; Sterner, O.: Biochem. Biophys. Res. Commun. 1996, 226, 214. 5. Saitoh, T. et al.: Bioorg. Med. Chem. Lett. 2009, 19: 5383. 6. Saitoh, T. et al.: Bioorg. Med. Chem. Lett. 2010, 20: 5638.

OOH

O NH

O

HO

OOH

O

HO

OOH

O

O

OH

DHMEQ (1) Panepoxydon (2) Cycloepoxydon (3)

Inhibition of phosphorylation of IkBInhibition of DNA binding of NF-kB

TAK1

TAB1TAB2

Receptor

IKKg

IKKa IKKb

IkBa

p50 p65

p50 p65

Phosphorylation

Phosphorylation

Degradation

DNA bindingp50 p65

PanepoxydonCycloepoxydon

DHMEQ

O

OH

O

HN

O OH

Salicylic acid unit

Epoxyquinol unit

O

OH

O

OH

Parasitenone (4)

O

OH

O

HN

R

R = Ac: NAcEQ (5)R = Alloc: NAllocEQ (6)

HN

O OH

Salicyl amides (7)

R

DHMEQ (1)

SL.04


Structure-activity relationship studies on small molecule Bid-inhibitors

Barho, M.T.1; Oppermann, S.2; Schrader, F.C.1; Degenhardt, I.1; Elsässer, K.2; Wegscheid-Gerlach, C.1; Culmsee, C.2; Schlitzer, M.1 1 Institut für pharmazeutische Chemie, Philipps-Universität Marburg,, Marbacher Weg 6, 35032 Marburg, Germany 2 Institut für Pharmakologie und klinische Pharmazie, Philipps-Universität Marburg,, Karl-von-Frisch Str. 1, 35033 Marburg, Germany

Apoptosis is the underlying pathomechanism of several diseases affecting the central nervous system like Alzheimer's or Parkinson's disease and also plays an important role after traumatic injuries of the brain or injuries after an ischemic stroke [1,2]. In this context, induction of apoptosis, triggered by several mechanisms, will lead to an irreversible loss of neuronal tissue. Especially, mitochondrial demise is considered as the “point of no return” in programmed cell-death in neurons [3]. The BH3-only protein Bid has been established as an important regulator of mitochondrial integrity, thereby presenting Bid as a promising target for the development of neuroprotective substances [4,5]. In order to gain a deeper insight into SAR and to identify novel neuroprotective compounds, a known Bid-inhibitor, which resulted from a fragment based NMR approach [6,7], has been critically evaluated and was used as template. Supported by molecular docking into the NMR structure of murine Bid [8], moieties have been exchanged, respectively modified by addition or removal of functional groups. Also combinations of new substructures have been prepared, to screen for additive effects. All com-pounds have been tested for their ability to protect cultured neurons from glutamate induced apoptosis using the MTT Assay. The best compounds displayed significant neuroprotection in concentrations as low as 1 µM [9].

Scaffold of investigated compounds

References:

1. Mattson, M.P. et al.: Apoptosis. 2001, 6: 69-81. 2. Culmsee, C.; Landshamer, S.: Curr. Alzheimer Res. 2006, 3: 269-283. 3. Landshamer, S. et al.: Cell Death Differ. 2008, 15: 1553-1563. 4. Grohm, J.; Plesnila, N.; Culmsee, C.: Brain Behav. Immun. 2010, 24: 831-838. 5. Culmsee, C.; Plesnila, N.: Biochem. Soc. Trans. 2006, 34: 1334-1340. 6. Becattini, B. et al.: Chem. Biol. 2004, 11(8): 1107-1117. 7. Becattini, B. et al.: Proc. Natl. Acad. Sci. U.S.A 2006, 103(33): 12602-12606. 8. McDonnell, J.M. et al.: Cell 1999, 96: 625-634. 9. Barho, M.T. et al.: ChemMedChem 2014, (accepted).

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114

Synthesis and Structure Affinity Relationships of Dual Chemokine Receptor 2 and Chemokine Receptor 5 Antagonists and Development of a Selective, Fluorinated CCR2 Ligand for PET Studies Junker, A.1; Schepmann, D.1; Yamaguchi, J.2; Itami, K.2,3

; Faust, A.4; Wagner, S.5; Wünsch, B.1

1 Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstr. 48, D-48149 Münster, Germany Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: [email protected] 2 Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan 3 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa-ku, Nagoya 464-8602 Japan 4 European Institute for Molecular Imaging (EIMI), Mendelstr. 11, D-48149 Münster, Germany 5 Klinik für Nuklearmedizin, Albert-Schweitzer-Campus 1, Gebäude A1, D-48149 Münster, Germany

The chemokine receptor subtypes 2 (CCR2) and 5 (CCR5) are two G-protein coupled CC chemokine receptors, that play a crucial role in the trafficking of monocytes, macrophages and in the functions of other cell types relevant for the devel-opment and progression of several diseases, such as rheumatoid arthritis (RA) [1], atherosclerosis [2], asthma [3], and HIV-1 (AIDS) [4]. Strong preclinical evidence indicates greater efficacy for dual targeting of CCR2 and CCR5, than targeting CCR2 or CCR5 alone [5].

Herein, we present the design, synthesis and evaluation of biological activities of highly potent, benzo[7]annulene-based, dual CCR2 and CCR5 antagonists and the development of a highly selective, [18F]-labeled CCR2 receptor radioligand as a new diagnostic tool for positron emission tomography (PET) [6].

Scheme 1: Synthesis of various benzo[7]annulen-based, dual CCR2-CCR5 antagonists 6a-u. (a) Suzuki-Miyaura cross-coupling reaction (b) Amide coupling reaction.

In order to introduce broad diversification in the last step of the synthesis, two different strategies (A and B) were devel-oped. Strategy A allowed the variation of the amino part R1 of the benzo[7]annulen-8-carboxamides 6a-u, by amide coupling reactions of acid 4 with different amines. Whereas, strategy B provided a broad exploration of the aryl part Ar by Suzuki-Miyaura cross-coupling reaction of the aryl bromide 5 with various arylboronic acids.

The CCR2 and CCR5 affinities of the novel compounds were determined in competitive radioligand receptor binding assays ([3H]TAK-779 (CCR5), [125I]-CCL2 (CCR2)). Additionally, CCR2 receptor potencies were recorded using the intracellular Ca2+ mobilization assay (hCCR2) and the β-arrestin recruitment assay (mCCR2). A highly potent and selec-tive CCR2 antagonist was further converted into a fluorinated PET ligand [18F]7b.

References: 1. (a) García-Vicuña, R. et. al.: Arthritis Rheum. 2004, 50 (12): 3866-3877. (b) Dawson, T. et. al.: Atherosclerosis 1999, 143 (1): 205-211. 2. Szalai, C. et al.: Atherosclerosis 2001, 158 (1): 233-239. 3. Maus, U.A. et al.: J. Immunol. 2003, 170 (6): 3273-3278. 4. (a) Deng, H. et al.: Nature 1996, 381 (6584): 661-666. (b) Liu, R. et al.: Cell 1996, 86 (3): 367-77. 5. (a) Yang, Y.-F. et al.: Eur. J. Immunol 2002, 32(8): 2124-2132. (b) Tokuyama, H. et al.: Intern Immunol 2005, 17(8): 1023-1034. (c) Schröppel, B. et al.: J. Am.Soc. Nephrol. 2004, 15 (7): 1853-1861. (d) Junker, A. et al.: Topics in Medicinal Chemistry (Springer) 2014; 1-55. 6. (a) Junker, A.; et al.: OBC 2014, 12 (1): 177-186. (b) Junker, A. et al.: J. Org. Chem. 2013, 78 (11): 5579-5586.

Time [min] 0.2 32-42 110-120

Figure 1: Whole body PET images of biodistribution studies of [18F]7b (10 MBq i.v.) after intravenous injection into C57BL/6 –WT mouse.

Financial support of the IRTG and this project by the DFG is gratefully acknowledged. We are also grateful for the financial support by the Collaborative Research Center 656 MoBil.

SL.06



Testing of potential inhibitors of human heparanase in a fluorescence activity assay Schoenfeld, A.; Vierfuß, S.; Lühn, S.; Alban, S.

Pharmaceutical Institute, Christian-Albrechts-University, Gutenbergstr. 76, 24118 Kiel, Germany

Heparanase, an endo-β-glucuronidase cleaving heparan sulfate (HS) chains at cell surfaces and in the extracellular matrix (ECM), is involved in angiogenesis, tumor progression and metastasis as well as in inflammation and kidney dysfunction. Therefore, heparanase is considered a promising therapeutic target and diagnostic marker. Recently, we have developed a simple, rapid, fully automatable fluorimetric activity assay using the synthetic sulfated pentasaccharide fondaparinux as substrate and bacterial heparinase II (HEP-II) instead of human heparanase (hHEP). The aim of this study was to evaluate this assay for inhibitor testing as well as to check whether the assay principle is applicable to measure the activity and inhibition, respectively, of the actual target enzyme hHEP.

Besides the known hHEP inhibitor suramin and the antiinflammatory and antimetastatic PS3, two series of β-1,3-glucan sulfates differing in their chain length and degree of sulfation, further semisynthetic sulfated glycans, and two sulfated polysaccharides isolated from algae were included to examine structure-activity relationships.

The inhibitory activity of sulfated glycans showed to be greatly dependent on both their degree of sulfation and their basic glycan structure, but independent of their molecular size. The β-1,3-glucan sulfates were superior to suramin as well as to the other glycans with similar degree of sulfations. The most active inhibitor was found to be the β-1,3-glucan sulfate PS3 (IC50 = 0.017 µM). By using hHEP instead of HEP-II comparable results were obtained. With an IC50 being about 160 times lower than that of suramin, PS3 exhibited again the strongest inhibitory effects. Inhibition of hHEP may therefore contribute to the potent antiinflammatory and antimetastatic activities of PS3 in vivo. In conclusion, the fluori-metric hHEP activity assay proved to be a simple, fully automatable tool for testing potential inhibitors. In case of HS mimetic inhibitors, the assay variant with HEP-II may provide a fast and inexpensive option for initial screening purposes.

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116

Monitoring Conformational Changes in PPARβ/δ by Cross-linking and Mass Spectrometry Schwarz, R.; Tänzler, D.; Kölbel, K.; Ihling, C.; Sinz, A.

Institute of Pharmacy, Department of Pharmaceutical Chemistry & Bioanalytics, Martin-Luther University Halle-Wittenberg, Germany

Chemical cross-linking, combined with an enzymatic digestion and mass spectrometric analysis of the reaction products, has evolved into an alternative strategy to identify protein-protein and protein-ligand interactions [1]. Peroxisome prolifer-ator-activated receptors (PPARs) belong to the subfamily of nuclear receptors that are involved in metabolic processes. One subtype, PPARβ/δ, is thought to be involved in the development of several chronic diseases and presents an important drug target [2]. Here, we present the monitoring of conformational changes in PPARβ/δ upon ligand binding by cross-linking studies combined with mass spectrometry. Expression and purification of the ligand binding domain of PPARβ/δ (amino acids 166-441) was optimized for obtaining high protein yields. Cross-linking reactions were performed using the amine-reactive homobifunctional cross-linker bis(sulfosuccinimidyl)glutarate (BS²G). After in-gel digestion, peptides were analyzed by high-resolution mass spectrom-etry (Orbitrap Fusion Tribrid, Thermo Fisher Scientific). All cross-links were evaluated with the in-house software Stav-roX [3]. The distance constraints imposed by the cross-links served to monitor conformational changes in PPARβ/δ upon binding of the agonists GW1516 (Fig.1) and GW0742 (Fig.2). In the absence of ligands a higher number of cross-links were identified, indicating a high flexibility of the ligand binding domain in the unbound state. After ligand binding, cross-links between lysines K322 and K422/K423 to the N-terminus suggested a large conformational change in PPARβ/δ. Apparently, amino acids 185-210 are easily accessible to the cross-linker as in the absence of ligands as well after ligand binding most of the cross-links were found in that region. In addition, photo-affinity labeling studies with the unnatural photo-amino acid para-benzoyl-L-phenylalanine (Bpa) are currently performed. Bpa was incorporated at specific positions into PPARβ/δ in the high flexible Ω-loop or the activation function helix 2 to investigate conformational changes upon ligand binding. Large hydrophobic amino acids, such as phenylalanine or tryptophan, are preferrably exchanged for Bpa. To inspect the conformation behavior of the activation function helix 2 the C-terminal tyrosine (Y441) was exchanged for Bpa.

Fig. 1: PPARβ/δ agonist GW1516

Fig. 2: PPARβ/δ agonist GW0742 References:

1. Sinz, A.: Mass Spectrom. Rev. 2006, 25(4): 663-682. 2. Berger, J. et al.: Annu. Rev. Med. 2002, 53: 409–435. 3. Götze, M. et al.: J. Am. Soc. Mass Spectrom. 2012, 23(1): 76-87.

SL.08


A dynamic pH junction method for monitoring the catalytic activity of cerebroside sulfotransferase Li, W.1; Zech, I.2; Gieselmann, V.2; Müller, C.E.1

1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Institut für Biochemie und Molekularbiologie, University of Bonn, Nussallee 11, 53115 Bonn, Germany

Cerebroside sulfotransferase (CST) is a promising new therapeutic target for metachromatic leukodystrophy (MLD), a rare and severe genetic disease. CST catalyzes the transfer of a sulfate group from the coenzyme 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to cerebroside as a substrate yielding cerebroside sulphate and adeno-sine-3′,5′-diphosphate (PAP).1 So far only a few weakly potent competitive inhibitors of the cosubstrate PAPS have been published, whereas no CST inhibitors competitive for the substrate cerebroside have been described.2 In the present study we developed a capillary electrophoresis- (CE-) based assay for monitoring the catalytic activity of CST. By using dynamic pH junction stacking, a low nanomolar limit of detection (LOD = 66.6 nM) was achieved for the reaction product PAP. Our CE method was sensitive, robust and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Several reference compounds were tested including cerebrosides, psychosine and Congo Red2,3 in order to validate the method. The newly developed CE method will be used to develop novel CST inhibitors, which are urgently needed for the treatment of MLD, a devastating disease that in the absence of therapy leads to early death of the young patients.

References:

1. Eckhardt, M.: Mol. Neurobiol. 2008, 37: 93–103. 2. Zaruba, M.; Hilt, D.; Tennekoon, G.: Biochem. Biophys. Res. Commun. 1985, 129: 522–529. 3. Honke, K. et al.: J. Biol. Chem. 1997, 272: 4864–4868.

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118

Confocal Raman microscopic (CRM) methodology for the analysis of the penetration of pharmaceutical actives into the skin Lunter, D.J.

Pharmaceutical Technology, Eberhard Karls University of Tuebingen, Auf der Morgenstelle 8, Tuebingen, 72076, Germany

CRM is increasingly used for the detection of xenobiotics within the skin. Conventionally, the skin is scanned from the stratum corneum downwards (depth scan/virtual cross section). In this context an alternative methodology is presented. It employs CRM on ex vivo cross sections of porcine skin. To investigate the usefulness of the methodology procaine was chosen as a model drug. It exhibits poor skin penetration as well as good detectability by CRM. These two features make it an ideal model substance to study penetration enhancement by CRM. Semisolid preparations that contained procaine HCl and the penetration enhancers propylene glycol or polyoxyethylene-23-lauryl ether (POE-lauryl ether) were used as model formulations. POE-lauryl ether was chosen as Shin et al. showed that it enhanced the penetration of local anesthetics [1]. They drew this conclusion on the basis of permeation experiments and the calculation of the enhance-ment ratio as the ratio of the flux with enhancer and the flux without enhancer. Propylene glycol was chosen as it is known to enhance the penetration/permeation of a variety of drugs and is frequently used in dermal preparations.

Excised postauricular porcine skin was incubated in Franz diffusion cells with semisolid formulations that contained either enhancer. An enhancer free formulation was used as control. After the incubation, the skin was cleaned, frozen and saggital cuts were made with a cryo-microtome (HM 560 Cryo-Star; Thermo Fisher, D-Langenselbold). The cuts were examined with a confocal Raman microscope (alpha 500, WiTec, D-Ulm) equipped with a 532 nm laser and a 10x (NA: 0.25) objective. Areas of 20x70 µm were mapped with a spatial resolution of 1.3 µm and a spectral resolution of 1 cm-1. Colour coded images that visualize the distribution of procaine within the skin were calculated. For a comparison of the relative procaine amount that was delivered to the skin by the penetration enhancers, the normalized peak areas of procaine in the scans were calculated and compared to the value that was obtained without enhancer. For compari-son, a permeation experiment was performed and the enhancement ratios were calculated.

The CRM investigation revealed that propylene glycol did not enhance the penetration of procaine whereas POE-lauryl ether had a distinct enhancing effect on the amount of procaine in the epidermis. Furthermore, it could be shown that procaine was predominantly located in the lipid rich domains in the skin. A behaviour that can be explained by the high partition coefficient of procaine (kp=100). In contrast, the permeation experiment and the enhancement ratios derived therefrom did not reveal any enhancement by propylene glycol or POE-23-lauryl ether.

It can therefore be concluded that CRM can give additional information in the investigation of dermal penetration. Infor-mation that is not accessible by the conventional method of calculating the enhancement ratio on the basis of permea-tion data. Furthermore, the proposed CRM-methodology can visualize the distribution of the drug within the skin and enhance the depth from which Raman spectra can be collected.

Acknowledgements: Institute of Experimental Medicine, University of Tuebingen, Schenk, M.

Reference:

1. Shin, C.-S. et al.: Int. J. Pharm. 2004, 287: 73-78.

SL.10


Determination of the Dissolution Behaviour of Celecoxib-Eudragit E 100-Nanoparticles using Cross-Flow Filtration Schichtel, J.1,2, Prinz, E.-M.1, Tuereli, A.E.1, Langguth, P.2 1 MJR PharmJet GmbH, Saarland University Medical Center, 66424 Homburg, Germany 2 Pharmaceutical Technology and Biopharmaceutics, Johannes Gutenberg-University, 55099 Mainz, Germany

This study deals with the development of a dissolution test method for nanoparticulate dosage forms. Thereto, nanopar-ticles (NPs) consisting of celecoxib as model drug and Eudragit E 100© were prepared using Microjet Reactor©. This technology (assembly depicted in Figure 1) uses two opposed high velocity linear jets. One jet conveys the solvent with API and polymer, the other jet the non-solvent. A third jet carrying inert gas facilitates fast depletion of the organic solvent. The produced particles have a Z-Average ranging from 100 to 250 nm, polydispersity index below 0.2 and entrapment efficiency of 70 to 80 % (scanning electron microscopy image of NPs shown in Figure 2). Dissolution tests were conducted using cross-flow filtration, a new approach in pharmaceutical dissolution testing. Current compendial methods implement the usage of dead-end filters to separate undissolved particles of the dosage form from dissolved drug molecules. The application of dead-end filters bears the risk of filter clogging and is extensive since new filters have to be used for every dissolution test. Prior to dissolution testing, the applicability of cross-flow modules with respect to their ability to retain NPs was successfully tested. Thereto, comparative dynamic light scattering and UV-spectroscopic measurements of nanosuspension and filtrate were performed at neutral pH where dissolution of Eudragit E 100© does not occur. These experiments revealed that the used filter modules are impermeable for the tested nanoparticles. For analysis of celecoxib an UV-Vis-spectroscopic method was developed which facilitates the quantification of celecoxib besides Eudragit E 100©. As dissolution medium hydrochloric acid of pH 1.2 was used since the stomach is the chosen site of release in vivo. Furthermore, Eudragit E© is only soluble at pH 5 or less [2]. Additionally, cetrimide, a quaternary ammonium salt, was added in a concentration of 0.3 % (m/v) to the medium. In this way, sink conditions for celecoxib dissolution are given [3]. To compare the effect of different filters dissolution tests were performed using three different filters: two cross-flow filtration modules with two different filter pore sizes as well as syringe filter holders to represent dead-end filtration. The suitability of the syringe filter holders to retain undissolved NPs was successfully tested as described above. The obtained results reveal that dead-end filtration well correlates with cross-flow filtration at larger pore size. In summary, it can be stated that cross-flow filtration can be a well appropriate alternative to classical dead-end filtration in pharmaceutical dissolution testing.

Figure 1: Schematic of MJR (mode of operation) Figure 2: SEM image of celecoxib-Eudragit E 100 NPs

References:

1. Baumstümmler, B.; Penth, B.; Penth, F.; Türeli, A.E.: US Patent (US20130012551 A1) 2013. 2. Rowe, R.C.; Sheskey, P.J.; Owen S.C.: Handbook of Pharmaceutical Excipients (American Pharmaceutical Association and Pharmaceutical Press) 2006. 3. Albers et al.: Eur. J. Pharm. Biopharm. 2009, 71(2): 387–394.

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120

Sensitivity of concentration-effect versus dose-effect analysis to detect small magnitudes of QTc prolongation in preclinical cardiovascular safety setting Gotta, V.1; van Ammel, K.2; Cools, F.2; Gallacher, G.J.2; Visser, S.A.G.3; Morissette, P.4; Sannajust, F.4; Danhof, M.1; van der Graaf, P.H.1

1 Systems Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden, The Netherlands 2 Global Safety Pharmacology, Janssen Research & Development, Janssen Pharmaceutica NV, Belgium 3 Quantitative Pharmacology and Pharmacometrics / Merck Research Laboratories, Merck & Co., Inc., Upper Gwynedd, PA, USA 4 SALAR-Safety and Exploratory Pharmacology / Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA

The heart-rate corrected QT interval (QTc) on the electrocardiogram is a simple biomarker for potential proarrhythmic risk. A QTc prolongation (∆QTc) of 2-8 ms in the dog correlates with a significant 10 ms prolongation in humans[1,2]. This simulation study aimed to investigate the sensitivity of concentration-effect (pharmacokinetic-pharmacodynamic, PKPD) analysis to detect small magnitudes of QTc-prolongation in a typical preclinical cardiovascular (CV) safety study in the conscious telemetered dog (crossover study in 4-8 animals receiving by oral route a vehicle and three dose levels, followed each by a wash-out phase). Results were compared with conventional dose-effect analysis (analysis of covari-ance, ANCOVA)[3,4]. A PKPD model predicting individual QTc was first developed from vehicle arms of 28 typical CV safety studies and one positive control study (D,L-sotalol administered orally). The model quantified between-animal, inter-occasion and within-animal variability and described QTc as a function of circadian variation and drug concentration (direct effect). This “true” model was used to repeatedly (n=500) simulate studies with typical drug-induced ∆QTc of 1 to 12 ms at high-dose peak concentrations. Simulated studies were re-analyzed by both PKPD and ANCOVA. Sensitivity (=true positive rate/power) was calculated as the percentage of studies in which a significant (α=0.05) drug effect was found (PKPD: likelihood-ratio test for linear concentration-effect slope; ANCOVA: comparison of dose versus vehicle and linear trend test for dose). One simulation scenario did not include a concentration-effect relationship and served to investigate false-positive rates. PKPD analysis/ANCOVA had a sensitivity of 80% (horizontal dashed line) to detect effects of 7/12 ms (n=4 animals), 5/10 ms (n=6) and 4.5/8 ms (n=8), respectively (Figure below). False-positive rates (estimates at true typical ∆QTc=0 ms) were higher using ANCOVA (39%) compared to PKPD analysis (1%). Results suggest superior sensitivity and specificity of PKPD approaches to quantify small QTc effects in preclinical safety testing. Their use may increase the confidence in observed effects and potentially allow reducing the number of animals while maintaining required study sensitivity.

Acknowledgements: This project was supported by the Dutch Top Institute Pharma (TIPharma) PK-PD platform 2.0.

References: 1. Chain, A. et al.: Br. J. Clin. Pharmacol. 2013, 76(5): 708-724. 2. Parkinson, J. et al.: J. Pharmacol. Toxicol. Methods. 2013, 68(3): 357-366. 3. Aylott, M. et al.: Pharm Stat. 2011, 10(3): 236-249.

4. Sivarajah, A. et al.: J. Pharmacol. Toxicol. Methods. 2010, 62(1): 12–19.

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POSTERS

122

ANTIINFLAMMATORY DRUGS (AD01-AD19)

Ceramide-1-Phosphate: a new player in modulating DC in allergic inflammation? Baudiß, K.; Ayata, K.; Vieira, R.P.; Idzko, M.

Department of Pneumology, University Hospital Freiburg, Breisacherstr. 66, 79106 Freiburg, Germany

Introduction: Sphingolipids are playing an essential role in normal cell und tissue homeostasis as well as in the development and progress of various diseases and disorders. The central molecule in the sphingolipid metabolism is ceramide, which can be converted into Shingosine-1-Phosphate (S1P) and Ceramide-1-Phosphate (C1P). While the role of S1P in the pathogenesis of airway inflammation has been extensively studied, little is known about C1P [1]. Aim: The aim of the current study was to elucidate wheater C1P can inhibit cardinal features of acute experimental asthma and might be a new modulator for DC in allergic inflammation. Methods and Results:. Balb/c mice were sensitized with ovalbumin-alum (OVA-alum) and challenged with OVA-aerosols for 3 days. After OVA challenge mice were sacrificed and bronchoalveolar fluid (BALF) was collected. The distribution of different cells were analysed by flow cytometer. The cytokine content in BAL fluid was measured by Elisa. C1P treated mice showed less inflammatory cells in BALF and lung tissue sections accompanied by decreased Th2 cytokine production in the mediastinal lymphnodes compared to vehicle treated mice. Furthermore C1P pulsed dendritic cells showed a limited priming capacity of Th2 immunity in a DC driven model of allergic airway inflammation in vivo. Mechanistically C1P inhibited the OVA-induced NF-kB activation in vitro and in vivo. Paramet-ric test were applied for statistical analysis Conclusion: In summary, C1P reduces the development of allergen-induced asthma in a mouse model by attenuating NF-kB activation and influencing dendritic cells. Our results suggest that C1P might be a therapeutic target for treatment of asthma.

References: 1. Gangoiti, P. et al.: Translational Oncogenomics 2008, 3: 81-98.

Effects of TH2 Cytokines on Filaggrin Deficient Skin Constructs Hönzke, S.; Schäfer-Korting, M.; Küchler, S.

Institute for Pharmaceutical Sciences, Pharmacology & Toxicology, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

Atopic dermatitis (AD) is a chronic inflammatory skin disease which is characterized by an impaired skin barrier function. Loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing risk factor for the manifestation of AD [1]. However, the exact pathogenesis is still ambiguous. In order to unravel the impact of filaggrin deficiency on the skin homeostasis, we established a FLG knock down skin model which exhibits a disturbed epidermal maturation and differentiation, altered skin lipid composition and organization as well as altered dermal drug absorption [2-3]. Aside from specific histological features, AD is characterized by an overshooting immune response. Particularly high levels of T helper cells (TH2) derived inflammatory cytokines contributes to the pathogenesis of AD [4]. Nevertheless, the effects of TH2 cytokines such as IL-4 and IL-13 on the skin barrier function and especially interdependencies with the FLG deficiency are not yet fully understood. Therefore, we systemically stimulated the FLG deficient skin model with TNF alpha, IL-4 and IL-13 for four days in order to induce inflammatory conditions in vitro. We detected significantly increased levels of the pro-inflammatory cytokines IL-6 and IL-8, whereas levels were even higher in the FLG deficient skin models compared to the normal models (503.4 ± 55.09 ng/ml vs 335.3 ± 11.86 ng/ml). Interestingly, even untreated FLG- models released slightly higher amounts of IL-8 and IL-6. Histological evaluations revealed major structural changes such as the induction of spongiosis, parakeratosis and an increase of epidermal thickness which was again most pronounced in the FLG-

models. Immunostaining revealed additional down regulation of filaggrin expression in FLG- models. By combining two characteristics of atopic skin, we were able to demonstrate that FLG- models have a higher susceptibility to inflammatory stimuli. Further investigations will show if the skin models are suitable test systems to assess anti-inflammatory effects in vitro and how Th2-derived cytokines further influence the skin homeostasis. Acknowledgement: Financial support by the Collaborative Research Center 1112 for the project C02 is gratefully acknowledged. References: 1. Palmer, C.N. et al.: Nat Genet. 2006, 38(4): 441-446. 2. Kuchler, S. et al.: Altern Lab Anim. 2011, 39(5): 471-480. 3. Vavrova, K. et al.: J Invest Dermatol. 2014, 134(3): 746-753. 4. Oyoshi, M.K. et al.: Adv Immunol. 2009, 102: 135-226.

Treatment with Peroxisome Proliferator-Activated Receptor Agonist Docosahexaenoic Acid Normalizes Filaggrin Expression in a Filaggrin Knock Down Skin Model Wallmeyer, L.1; Lehnen, D.1; Sochorová, M.2; Školová, B.2; Schäfer-Korting, M.1; Vávrová, K.2; Küchler, S.1 1 Freie Universität Berlin, Institute of Pharmacy, Pharmacology and Toxicology, Königin-Luise-Straße 2+4, 14195 Berlin, Germany 2 Charles University Prague, Faculty of Pharmacy, Heyrovského 1203, 50005 Hradéc Králové, Czech Republic

Loss-of-function mutations in the gene encoding for filaggrin (FLG) are the major predisposing factor for atopic dermatitis (AD). As for today, therapeutic options for FLG associated skin diseases only alleviate the symptoms such as dry and itchy skin or reduce the inflammation. No therapy exists preventing the development of these symptoms or restoring the disturbed skin barrier function. Peroxisome proliferator-activated receptor (PPAR) agonists are not only important therapeutics for the treatment of lipid disorders and diabetes but also exhibit beneficial effects in patients suffering from inflammatory skin diseases like AD. PPAR agonists are known to increase the expression of FLG in skin and positively influence the skin barrier homeostasis, skin barrier recovery and stratum corneum (SC) integrity [1]. The underlying mechanism, however, is still ambiguous. In order to study the effects caused by a lack of FLG in vitro, we established a FLG knock down skin model [2, 3]. Here, we evaluated the effects of the PPAR agonist docosahexaenoic acid (DHA) in a FLG deficient (FLG-) skin model in terms of FLG expression, skin lipid organization and composition and skin permeability. We detected an about 15.72-fold upregulation of FLG in DHA treated normal skin models (FLG+). FLG expression increased significantly about 2.69-fold in FLG- models upon DHA treatment even exceeding the FLG expression of DHA untreated FLG+ samples. These results were confirmed on the protein level and histological examination revealed a thickening of the SC upon DHA treatment (FLG-/DHA- 8 ± 1.2 µm vs. FLG-/DHA+ 12.8 ± 1.5 µm). In terms of skin lipid composition, a treatment with DHA normalized the pathologically increased free fatty acid (FFA) levels: FFA amounts were reduced from 22.0 ± 2.7 μg/mg to 15.3 ± 1.0 μg/mg in FLG- models following DHA treatment (FLG+ 13.3 ± 1.5 μg/mg). Furthermore, the skin lipid organiza-tion was significantly improved in FLG- constructs as determined by ATR-FTIR. Interestingly, skin absorption studies did not show an improvement of the skin barrier after DHA treatment. The beneficial effects on the skin barrier homeostasis are undoubted but further studies are necessary to completely understand the effects of PPAR agonists on the skin barrier function.

Acknowledgements: Financial Support by the Foundation SET (Stiftung zur Förderung der Erforschung von Ersatz- und Ergänzungs-methoden zur Einschränkung von Tierversu-chen) is gratefully acknowledged. This work was supported by the Czech Science Foundation (project No. 207/11/0365) and Charles University (SVV 265 001). References: 1. Michalik, L.; Wahli, W.: Biochimica et biophysica acta. 2007, 1771: 991-998. 2. Küchler, S. et al.: Altern Lab Anim. 2011, 39: 471-480. 3. Vávrová, K. et al.: J Invest Dermatol. 2014, 134: 746-753.

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Induction of glucocorticoid-induced leucine zipper (GILZ) contributes to anti-inflammatory effects of the natural product curcumin in macrophages Hoppstädter, J.; Hachenthal, N.; Sauer, K.; Kiemer, A.K.*; Diesel, B.

Pharmaceutical Biology, Campus, Bldg. C 2 2, Saarland University, 66123 Saarbrücken, Germany * To whom correspondence should be addressed: [email protected]

Background: Inflammation is characterized by the production of inflammatory mediators as well as a decrease in anti-inflammatory regulators. Glucocorti-coids are well-established anti-inflammatory compounds, but exert distinct side effects. The induction of the glucocorticoid-induced leucine zipper (GILZ, TSC22D3) protein plays a pivotal role in the therapeutic actions of glucocorti-

coids, e.g. by inhibiting the inflammatory transcription factor NF-B, but is not involved in glucocorticoids’ adverse actions [1]. We therefore sought for GILZ-inducing compounds, which do not act via the glucocorticoid receptor. Results: Macrophages obtained from GILZ knockout mice displayed an

inflammatory phenotype associated with increased NF-B and ERK activity, confirming an induction of GILZ as a promising therapeutic strategy against

inflammatory diseases. We observed that the natural product NF-B inhibitor curcumin induces GILZ protein in a dose- and time-dependent fashion in murine and human macrophages. Curcumin exerts its anti-inflammatory actions via induction of GILZ: GILZ knockdown by specific siRNA antagonized curcumin’s inhibitory action on lipopolysaccharide (LPS)-induced iNOS and

NF-B dependent luciferase activity. Consistently, curcumin failed to inhibit

TNF- production and ERK activation in GILZ knockout macrophages. Activation of the glucocorticoid receptor does not contribute to increased GILZ protein. We also observed neither increased GILZ mRNA nor protein stability. We rather suggest a translational involvement of the RNA-binding protein HuR (Elavl1) with HuR being translocated after curcumin treatment. Conclusion: We provide evidence for a steroid-independent GILZ-mediated anti-inflammatory action of curcumin, probably via posttranscriptional regulation of GILZ. Acknowledgments: This work was supported, in part, by the Deutsche Forschungsge-meinschaft (DFG, KI 702). We thank Indou Kepbane, Susanne Schütz and Oliver Wild for technical support.

Reference: 1. Beaulieu, E.; Morand, E.F.: Nat. Rev. Rheumatol.2011, 7(6): 340-348.

Omega-3 Fatty Acids (Omegaven) protect from Mitochondrial Dysfunction in a MCAO mouse model of stroke Berressem, D.; Koch, K.A.; Franke, N.; Klein, J.; Eckert, G.P.

Goethe Universität Frankfurt am Main, Germany

Recent investigations demonstrated efficacy of docosahexaenoic acid (DHA) to reduce stroke size and severity in the transient middle cerebral arterial occlusion (MCAO) model in rats when applied intravenously after reperfusion. In this study we investigated the beneficial effect of OMEGAVEN (Fresenius Kabi, Germany) a medical lipid emulsion for parenteral nutrition that contains the long-chain omega-3 fatty acids eicosapentaenic acid (EPA) and DHA in a model of transient stroke. Mice underwent transient MCAO and OMEGAVEN was administered intravenously (5 ml/kg b.w.) after stroke (90 min) at reperfusion that represents an early moment for potential intervention. The degree of damage, mitochondrial function and neuroinflammation were investigated. Treatment with OMEGAVEN significantly decreased the stroke area by 21% and lowered the severity of stroke by 50%. OMEGAVEN significantly improved mitochondrial membrane potential (MMP) and ATP levels in the ischemic brain hemisphere. These findings are accompanied by an enhanced mitochondrial function, e.g. improved respireation of the complexes responsible for oxidative phosphorylation in mitochondria isolated from the ischemic brain hemisphere. The inflammation markers COX-2 and iNOS significantly decreased after treatment with OMEGAVEN. This pilot study demonstrated that OMEGAVEN could represent a promising, approved lipid emulsion for the early therapeutic intervention in ischemic stroke.

Termination of inflammatory processes in the endothelium by inhibition of BMP2K Bischoff, I.1; Dai, B.2; Strödke, B.3; Bracher, F.3; Fürst, R.1 1 Institute of Pharmaceutical Biology, Goethe-University Frankfurt, Germany 2 Pharmaceutical Biology, Center for Drug Research, University of Munich, Germany 3 Department of Pharmacy - Center for Drug Research, University of Munich, Germany

During chronic inflammation in diseases such as Crohn’s disease or psoriasis, angiogenesis accompanied by leukocyte infiltration is an ongoing process. The termination of these processes, which normally occurs during the physiological situation of an acute inflammation, is not or only inadequately initiated under chronic conditions. Thus, the application of substances that induce the termination of inflammation seems a promising approach. We hypothesized the bone morphogenetic protein-2 (BMP-2)-inducible kinase (BMP2K/BIKE) might be a novel target of such substances. Surprisingly, the role of BMP2K in the endothelium has not been investigated up to now. During this study, C81, a small molecule, was used to inhibit BMP2K in order to determine potential anti-angiogenic and anti-inflammatory effects of the substance. In addition, C81 as well as BMP2K gene silencing were used to elucidate the role of BMP2K in these processes. Initial experiments demonstrated that C81 concentrations up to 3 µM did not exhibit any cytotoxic effect on a human microvascular endothelial cell line (HMEC-1) or on primary human umbilical vein endothelial cells (HUVEC), The performance of a migration assay revealed that increasing concentrations of C81 reduced the migratory capacity of HMEC-1. The treatment with C81 reduced the TNFα-triggered expression of the cell adhesion molecules ICAM-1, VCAM-1 and E-selectin on the endothelial cell surface with rising concentra-tions of C81 (flow cytometry). Similar results were detected in BMP2K-silenced HUVECs (siRNA), as ICAM-1 and VCAM-1 exhibited to be markedly reduced in BMP2K-silenced cells. Based on these findings, a cell adhesion assay using the monocytic leukemia cell line THP-1 and HUVECs was performed. Fluorescence-labelled THP-1 cells showed a significantly decreased adhesion to TNFα- or LPS-activated HUVECs upon C81 treatment, as determined by fluorescence measurements of cell lysates. Analysis of BMP-2-treated HUVECs using quantitative real-time PCR revealed that BMP2K seems not to be regulated by BMP-2 on the gene expression level. Our results indicate that BMP2K might be involved in inflammatory processes of the endothelium, Furthermore, these results suggest C81 as potential anti-inflammatory compound in vitro and, moreover, as a promising tool to interrupt BMP2K-mediated signalling events. Further studies are needed to elucidate the precise role of BMP2K in inflammatory and angiogenic processes and to clarify the underlying mechanisms.

Generic Cell-Permeable Probes of Eukaryotic and Bacterial Sialyl Transferases Inhibit Cellular Sialylation: New Targets in Inflam-mation and Cancer? Preidl, J.J.1; Gnanapragassam, V.S.2; Horstkorte, R.2; Rademann, J.1

1 Institut für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany, 2 Institut für Physiologische Chemie, Martin-Luther-Universität, Hollystr. 1, 06114 Halle, Germany.

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion between cells, with bacteria, and virus particles. Synthesis of neuraminic acid-containing glycosides is accomplished by intracellular sialyl transferases. Hypersialylation of cells is found in inflammation and enables immune cells to intrude into infected tissue. Moreover, strongly hypersialydated cells are capable to leave their primary tissue environment, to migrate, and to form metastases in remote tissues. Hypersialylation is, therefore, strongly indicative for a bad prognosis of neoplasia and inhibition of this event could be an alternative therapeutic strategy against cancer. We have developed and applied the first nanomolar fluorescent inhibitors of sialyl transferases. The obtained carbohydrate-nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed

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that the anionic mimetics intruded rapidly and efficiently into cells in vesicles translocating to cellular organelles surrounding the nucleus of CHO-cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation. Finally, the established binding assay enabled high-throughput screening of a chemical library of drug-like molecules and small-molecule inhibitors of sialyl transferases were identified.

Acknowledgements: Support is gratefully acknowledged from the DFG (FOR 806, SFB 765, TRR 67) and the Berlin School of Integrative Oncology. Reference: 1. Preidl, J.J. et al.: Angew. Chem. 2014, 126: 5808-5813; Angew. Chem. 2014, 53: 5700-5705.

For abstract see short lecture SL.07.

Cellular Assay Methods for Detection of Compounds Enhancing the Generation of Lipoxins Lehmann, C.1; Homann, J.2; Ferreirόs, N.2; Parnham, M.J.1; Geisslinger, G.1,2; Stark, H.3; Steinhilber, D.4; Kahnt, A.S.4

1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Theodor Stern Kai 7, 60596 Frankfurt/Main, Germany 2 Institute of Clinical Pharmacology, Goethe University Hospital, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany 3 Heinrich Heine University, Institute of Pharmaceutical and Medicinal Chemistry, Universitätsstraße 1, 40225 Düsseldorf, Germany 4 Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany

Uncontrolled inflammation is a characteristic of chronic diseases such as rheumatoid arthritis, diabetes and atherosclerosis. Recent findings indicate that the resolution of inflammation is an active process controlled through endogenous mediators and mechanisms that switch off acute inflammation by suppression of pro-inflammatory gene expression and cell trafficking and induce inflammatory cell apoptosis and phagocytosis [1]. Lipoxins (LX) are a unique class of arachidonic acid (AA) derived lipid mediators displaying pro-resolving activities during the resolution phase of acute inflammatory reactions. In exchange between distinct cell types such as neutrophils and endothelial cells, neutrophils and platelets or even in single cells, AA undergoes a double oxygenation by the sequential action of two different lipoxygenases (LO) (either 15- / 5-LO or 5- / 12-LO) to form LX [2, 3, 4]. Besides LO triggered LX synthesis, a series of epimeric 15-LX has been found. This group of ‘alterna-tive’ LX is aspirin triggered [2]. Here, acetylation by aspirin of cyclooxygenase-2 (COX-2) abolishes prostaglandin H2 (PGH2) synthesis while retaining the oxygenase activity of the enzyme, leading to the production of 15(R)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15(R)-HETE) instead. Epimeric 15-HETE is then further processed by 5-LO to give rise to 15-epi-LXs which share many anti-inflammatory properties with the regular LX [2,5]. Diverse cell-based, in-vitro co-culture systems have been described in literature [2, 3, 4]. Some of them we were able to establish. First of all, we spiked freshly isolated 5-LO positive, peripheral blood mononuclear leukocytes (PMNL) with 10 µM 15(S)-HETE, 15(R)-HETE and 17R-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17(R)-HDoHE) as a proof of principle. We were able to detect different epi- and native lipoxins as well as

17(R)-Resolvin D1 (17(R)-RvD1) via chiral LC-MS/MS analysis. In addition, we have detected LX biosynthesis so far in a variety of in-vitro systems. Co-culture experiments with PMNL/platelets, apoptotic PMNL/different macrophage phenotypes (M1/M2) and Human Umbilical Vein Endothelial Cells (HU-VEC)/PMNL with/without aspirin provided various native and 15-epi-LXs. Incubations of macrophages alone failed to generate detectable LX via LC-MS/MS. Potential lipoxin enhancing drug candidates are being tested in the different cell-based systems.

References: 1. Willoughby, D.A. and Gilroy, D.W.: Nature Reviews Immunology, 2002, 2: 787-795. 2. Serhan, C.N. et al.: Molecular Medicine, 1996, 2: 583-596. 3. Serhan, C.N. et al.: The Journal of Pharmacology and Experimental Therapeutics, 1998, 287: 779-790. 4. Serhan, C.N. and Sheppard, K.N.: Journal of Clinical Investigation, 1990, 85: 772-780. 5. Romano, M.: The Scientific World Journal, 2010, 10: 1048-1064.

A smart cell-based screening system for inhibitors of leukotriene biosynthesis Garscha, U.; Gerstmeier, J.; Werz, O.

Chair of Pharmaceutical/ Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller University Jena, Philosophenweg 14, 07743 Jena, Germany

Leukotrienes (LT) are potent lipid mediators released during chronic inflamma-tion, cancer, allergy, and cardiovascular diseases [1]. During the initial step of the LT biosynthesis, 5-Lipoxygenase (5-LO) transforms liberated arachidonic acid (AA) to 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and subse-quently to leukotriene A4 (LTA4). The latter is further converted by LTA4 hydrolase to the pro-inflammatory mediator LTB4, or conjugated with reduced glutathione by leukotriene C4 synthase (LTC4-S) to LTC4 [2]. Consequentially, for many years, inhibition of 5-LO has played a pivotal strategy to reduce LT biosynthesis as mean for pharmacological intervention with related diseases. However, in intact cells 5-LO requires the nuclear membrane-bound 5-lipoxygenase-activated protein (FLAP) that supplies 5-LO with AA and facilitates the conversion of AA to 5-LO products [3]. Therefore, FLAP represents an additional interesting target during inflammatory processes. Unfortunately, so far a suitable screening system for new FLAP inhibitors is still missing. Here we present a smart cell-based model to evaluate putative 5-LO and FLAP inhibitors. HEK293 cells were stably transfected with 5-LO alone or together with FLAP. Upon stimulation by Ca-ionophore and 3 µM AA, co-expression of FLAP increased product formation by 5-LO due to enhancing LTA4-formation.This FLAP-mediated effect could be significantly reduced by the known FLAP inhibitor MK886. On the other hand, MK886 was not able to inhibit the LT biosynthesis in cells that expressed only 5-LO. Zileuton, a well-characterized direct 5-LO inhibitor, reduced 5-LO product formation with equal potency, independently of FLAP expression. Together, our test system allows evaluating the inhibitory potency of LT synthesis inhibitors and, for the first time, enables to discriminate between putative 5-LOX and/ or FLAP inhibitors. Furthermore, site-directed mutagene-sis approaches will help to elucidate new potential sides that are targeted in cellular LT biosynthesis. References: 1. Peters-Golden, M. et al.: Leukotrienes. N. Engl. J. Med. 2007, 357(18): 1841-1854. 2. Panossian, A. et al.: FEBS Lett. 1982, 150(2): 511-513. 3. Dixon, R.A. et al.: Nature. 1990, 343(6255): 282-284.

Characterization and cellular localization of protein isoforms of human 5-lipoxygenase Ball, A.-K.1; Saul, M.J.1,2; Hofmann, B.1; Steinhilber, D.1; Häfner, A.-K.1

1 Institute of Pharmaceutical Chemistry, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany 2 Institute of Biology, Schnittspahnstraße 10, 64287 Darmstadt, Germany

Human 5-lipoxygenase (5-LO) is the key enzyme in leukotriene (LT) biosyn-thesis which play an important role in many diseases like asthma bronchiale, atherosclerosis and in many types of cancer. The 5-LO catalyzes two reaction

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steps, first oxygenation of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HpETE). In a second step, 5-HpETE is converted to the instable epoxide leukotriene A4 (LTA4) that can be further metabolized by the LTA4 hydrolase to LTB4 or by the LTC4 synthase to the cysteine containing leukotrienes LTC4, D4 and E4, causing chemotaxis, vasoconstriction and tumor growth [1-3]. 5-LO is expressed in many cell types of the human immune system like polymorphonuclear leukocytes, mono-cytes/macrophages and B-cells [4-6]. Recently, we were able to identify novel in-frame mRNA splice variants in B-cells and T-cells named delta 4 and delta p12. Co-transfection of delta 4 or delta p12 with 5-LO wild type (WT) in HEK293 cells shows an influence of the activity in contrast to transfection of WT alone. In crude cell lysates the effect was less pronounced. Moreover, we made the observation that 5-LO is able to form dimers [7]. Thus, we hypothesize that 5-LO and its isoforms can form heterodimers, regulating its activity. By investigating the cellular localization of WT and isoforms, we could determine that the 5-LO isoforms are only present in the nuclear fraction whereas WT 5-LO can be found in both, nuclear and non-nuclear fractions.

References: 1. Samuelsson, B. et al.: Science, 1987, 237: 1171-1176. 2. Funk, C.D.: Science, 2001, 294: 1871-1875. 3. Shimizu, T. et al.: PNAS, 1986, 83: 4175-4179. 4. Steinhilber, D.: Pharm. Acta Helv., 1994, 69: 3-14. 5. Spanbroek, R. et al.: PNAS, 1998, 95: 663-668. 6. Janssen-Timmen, U. et al.: PNAS, 1995, 92: 6966-6970. 7. Häfner, A.-K. et al.:Biol Chem, 2011, 392: 1097-1111.

Synthesis and pharmalogical characterization of N-phenylbenzenesulfonamides as dual 5-lipoxygenase and micro-somal prostaglandin E2 synthase-1 inhibitors Cheung, S.-Y.1; Hanke, T.1; Fischer, K.2; Listing, M.2; Werz, O.2; Schubert-Zsilavecz, M.1

1 Goethe University of Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Friedrich-Schiller University of Jena, Philosophenweg 14, 07743 Jena, Germany

Prostaglandins (PGs) and leukotrienes (LTs) are powerful bioactive lipid mediators that have a large number of biological actions in the human body [1,2]. The common precursor of PGs and LTs is arachidonic acid (AA). The 5-lipoxygenase (5-LO) and the microsomal prostaglandin E2 synthase-1 (mPGES-1) are both enzymes which are involved in the arachidonic acid cascade. The 5-LO is the initial enzyme which catalyzes AA to the correspond-ing LTs; whereas the mPGES-1 is responsible for the conversion of PGH2 into PGE2 which is one of the most prominent mediator of inflammation, pain and fever. A novel pharmacological approach for anti-inflammatory therapy is the dual inhibition of 5-LO and mPGES-1. In contrast to the traditional NSAIDs the dual inhibition of PGs and LTs might be superior over single interference with PGs in terms of anti-inflammatory effectiveness as well as regarding reduced side effects [3]. In this study we wanted to explore the structure-activity relationship of N-phenylbenzensulfonamide derivatives as dual 5-LO/mPGES-1 inhibitors. Lead structure of this series was 4-(N-octyl-4-methylbenzenesulfonamido)benzoic acid (compound 1, see Fig. below), which was originally identified by a virtual screening approach by Waltenberger, B. et al. [4]. For this compound a facile three-step synthesis was developed and structural optimization should be carried out in three directions while maintaining the central N-phenylbenzenesulfonamide scaffold (see Fig. below).

References: 1. Funk, C.D.: Science 2001, 294(5548): 1871−1875. 2. Samuelsson, B., Morgenstern, R., Jakobsson, P.J.: Pharmacol Rev 2007, 59(3): 207–224. 3. Koeberle, A., Werz, O.: Curr. Med. Chem. 2009, 16(32): 4274–4296. 4. Waltenberger, B. et al.: J. Med. Chem. 2011, 54(9): 3163–3174.

Zafirlukast – a Dual Modulator of Soluble Epoxide Hydrolase and Peroxisome Proliferator-Activated Receptor γ Diehl, O.; Schader, T.; Wittmann, S.K.; Steri, R.; Schubert-Zsilavecz, M.; Maier, T.J.; Steinhilber, D.; Proschak, E.; Kahnt, A.S.

Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany

Cardiovascular diseases are the major causes of death and disability in diabetic patients due to micro- and macroangiopathic complications. The gold standard in the treatment of type II diabetes are up to now thiazolidinediones (TZDs) which are potent activators of PPARγ with robust insulin-sensitizing activities. However, their clinical use is limited due to excessive weight gain, fluid retention and increased osteoporosis risk in treated patients. Meta-analyses of clinical trials have implicated rosiglitazone in increasing the risk of congestive heart failure, myocardial infarction, cardiovascular disease and all-cause mortality leading to tightly restricted access in the United States and a recommendation for market withdrawal in Europe. Troglitazone was withdrawn from the market due to hepatotoxicity and pioglitazone seems to trigger bladder cancer. Another drawback is the poor effect of TZDs on the occur-rence of macrovascular events, although the equilibration of blood glucose levels reduces microvascular complications. Therefore, there is an unmet medical need for safer PPARγ modulating drugs and the combination of PPARγ agonism with vasoprotective and anti-inflammatory properties in a compound might have beneficial effects in the treatment of type II diabetes. In previous studies we found that zafirlukast, a CysLT1 receptor antagonist frequently used as add-on therapy in asthma, displays anti-inflammatory properties in the low micromolar range due to inhibition of the microsomal prostaglandin E2 synthase. Further studies revealed that the compound also displays PPARγ agonism (EC50 3.2 µM, max. 126%) in the same concentration range. This was confirmed by reporter gene assays as well as by influence of zafirlukast on mouse adipocyte differentiation. Here, zafirlukast (3 – 10 µM) triggered triglyceride accumulation as well as target gene upregulation in a concentration dependent manner. In addition, we found the compound to inhibit soluble epoxide hydrolase (sEH) (IC50 1.9 µM), an enzyme which is involved in atherosclerosis formation by depletion of endothelium-derived hyperpolarizing factors (epoxyeicosatrienoic acids). Therefore, we postulate that the combination of PPARγ agonism with sEH and mPGES-1 inhibition represents a potent approach for the treatment of metabolic disease accompa-nied with type II diabetes mellitus. In vivo studies will answer this question in the future.

Acknowledgements: The work has been supported by the Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation – Pharma (TRIP).

Multi-dimensional optimization of N,4-diaryl-1,3-thiazol-2-amines as potent 5-lipoxygenase inhibitors Woltersdorf, S.1; Kretschmer, S.B.M.1; Rödl, C.B.1; Vogt, D.1; Steinhilber, D.1; Stark, H.1,2; Hofmann, B.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany; E-Mail: [email protected] 2 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany; E-Mail: [email protected]

Leukotrienes (LTs) are important lipid mediators derived from polyunsaturated fatty acids which play an important role as regulators in immunity and the inflammation process. These mediators have fundamental functions on pathophysiological processes in a complex network of interactions. They play a key role in the pathogenesis of inflammation as well as in acute and various chronic diseases, e.g. asthma, allergic rhinitis, cardiovascular disease and

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certain types of cancer [1]. The LT biosynthesis is initiated by the 5-lipoxygenase enzyme (5-LO). It catalyzes the conversion of free arachidonic acid to LTA4 which is subsequently converted into further LT subtypes. [2] Up to date, there is only one approved direct 5-LO inhibitor in clinical use: Zileuton. It acts by chelating the catalytic iron in the active site of the enzyme. However, this drug exhibits a non-optimal pharmacodynamic and pharmacoki-netic profile. Therefore, novel potent 5-LO inhibitors are of great interest for an anti-inflammatory therapy. [3] Starting from SKI-II, a known 5-LO inhibitor [4], we prepared a series of N,4-diaryl-1,3-thiazol-2-amine based compounds with a heterogeneous substitution pattern and investigated their influence on inhibition of human 5-LO activity. The chemical structure of the thiazole-2-amine scaffold was further optimized at positions R1, R2 and R3 with regard to the cytotoxicity profile, maintaining high 5-LO inhibitory activity and selectivity.

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From this series a comprehensive structure-activity relationship analysis was performed. With this, we could deduce the role of the chemical substitutes R1, R2 and R3 on 5-LO inhibitory potency, selectivity and cytotoxicity. Among all tested compounds, in vitro screening revealed that ST-1853 is the most potent derivative of this series. It blocks the 5-LO activity with an IC50 value of 0.05

(0.039-0.066) M and demonstrates no signs of cytotoxicity.

Acknowledgements: This work was supported by Else Kröner-Fresenius-Stiftung, TRIP, LOEWE, OSF and Fonds der Chemischen Industrie. H.S. and B.H. share senior authorship.

References: 1. Gualde, N. et al.: Trends. Mol. Med. 2008, 14(10): 461-469. 2. Dennis, E.A. et al.: J. Lipid. Res. 2009, 50(6): 1015-1038. 3. Steinhilber, D., Hofmann, B.: Basic Clin. Pharmacol. Toxicol. 2014: 114(1): 70–77. 4. Suh, J. et al.: Chem. Biol. Drug. Des. 2012, 80(1): 89-98.

Regulation of 5-lipoxygenase promoter activity by transcription factors AP2, GATA-1, PU.1, Ets-1/2, and WT1 Fettel, J.; Wöbke, T.K.; Steinhilber, D.; Sorg, B.L.

Institut of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany

Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes, which are mediators of proinflammatory and immune modulatory responses [1]. 5-LO is involved in the pathogenesis of atherosclerosis, asthma and several types of cancer. In order to fully understand the pathomechanisms underlying these conditions, which partly include 5-LO gene dysregulation, a comprehensive understanding of 5-LO gene regulation is necessary. Further-more, novel possibilities for therapeutic intervention might arise from the complete mechanistic picture of 5-LO gene regulation. At this, the identification of transcription factors involved in the transcriptional regulation of the 5-LO gene is of particular interest. The proximal 5-LO gene promoter is highly GC-rich and comprises nine GC-boxes (GGGCGG) [2] which are targeted by the transcription factors Sp1 and Egr-1 [3]. Besides these well characterized motifs the 5-LO promoter harbors several other putative transcription factor binding sites although their distinct functional significances are unknown. Among those are three putative binding sites for the activation protein 2 (AP2) [2]. Moreover, in silico analysis postulated putative binding sites for GATA-1, PU.1, and Ets-1/2 [4]. Additional-ly, the proximal 5-LO promoter contains a consensus motif for the transcription factor Wilms' tumor 1 (WT1), which overlaps with the sequence recognized by Egr-1 [5]. To investigate the direct effect of these transcription factors on 5-LO promoter activity, reporter gene studies in HeLa cells were carried out with successive promoter deletion constructs of the 5-LO gene. 5-LO promoter activity was increased by the transcription factors AP2, GATA-1, PU.1, Ets-1/2, and WT1. The effect of Ets-1 was proven by the use of a dominant-negative reporter construct. Additionally, the effects accomplished by AP2 and WT1 were

characterized in detail regarding their binding sites in the 5-LO promoter sequence. Three AP2 binding sites were found to be functional in the 5-LO promoter. Interestingly, different effects were found for four different WT1 isoforms, which most likely act via the GC-boxes in the proximal 5-LO promoter. 5-LO promoter regulation by WT1 was strictly dependent on the presence of the amino acid stretch lysine-threonine-serine (KTS) in the WT1 protein. Taken together, the results revealed novel aspects of 5-LO promoter activity regulation. They may prove valuable for the understanding of 5-LO gene (dys)regulation in pathogenesis and are part of the mechanistic picture available for therapeutic intervention within the 5-LO pathway.

References: 1. Haeggström, J.Z., Funk, C.D.: Chem. Rev., 2011, 111: 5866-98. 2. Hoshiko, S., Rådmark, O., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, 1990, 87: 9073-9077. 3. Silverman, E.S. et al.: Am. J. Respir. Cell. Mo.l Biol., 1998, 19: 316-323. 4. Klan, N.: Dissertation: Functional analysis of the human 5-LO promoter (Goethe University, Frankfurt/Main) 2003. 5. Madden, S.L. et al.: Science, 1991, 253: 1550-1553.

Multi-parameter optimimization of 1,3-thiazole-2-amine deriva-tives with potent 5-lipoxygenase inhibitory activity Kretschmer, S.B.M.1; Rödl, C.B.1; Vogt, D.1; Woltersdorf, S.1; Stark, H.1,2; Steinhilber, D.1; Hofmann, B.1 1 Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany; E-Mail: [email protected] 2 Heinrich Heine University, Institute of Pharmaceutical and Medicinal Chemistry, Universitätsstraße 1, 40225 Düsseldorf, Germany; E-Mail: [email protected]

5-Lipoxygenase (5-LO) mediated biosynthesis of leukotrienes (LTs) from arachidonic acid plays a pivotal role in immunity and inflammation. LTs are involved in the pathogenesis of asthma and allergic rhinitis, but may also play a role in certain types of cancer and cardiovascular diseases [1]. Currently only one 5-LO inhibitor (Zileuton, trade name Zyflo®) is approved by the FDA. However its usage is limited due to its unfavourable pharmacokinetic profile with a short half-life and safety issues concerning liver toxicity [2]. Therefore, there is an urge for novel 5-LO inhibitors with favourable pharma-codynamic and pharmacokinetic profiles. Substituted 1,3-thiazole-2-amines represent a new class of inhibitors of 5-LO [3]. Yet only little is known about the molecular mechanisms of 5-LO inhibition and other key factors in drug development. In order to develop a successful, safe and efficacious drug candidate a small set of 1,3-thiazole-2-amines with distinct substitution patterns was synthesized and tested in various cell-based and cell-free assays. Starting from N-4-hydroxyphenyl-4-(4-chloro-phenyl)-1,3-thiazole-2-amine (ST1083) within this study certain derivatives were tested to investigate the manner of inhibition as well as the dependency on different stimuli or conditions, cytotoxicity and specificity with regard to the impact on other key enzymes involved in eicosanoid metabolism.

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As a result from these studies a highly promising new lead ST-1853 is profiled, displaying high potency in various 5-LO dependent in vitro assays. Together with the non-cytotoxic profile as well as the selectivity this new lead establishes the basis for further characterization of the molecular mode of action and optimization for a novel anti-inflammatory drug approach.

Acknowledgements: This work was supported by Else Kröner-Fresenius-Stiftung, TRIP, LOEWE, OSF and Fonds der Chemischen Industrie.

References: 1. Peters-Golden, M. and Henderson, W.R.: N. Engl. J. Med. 2007, 357(18): 1841-1854. 2. Steinhilber, D., Hofmann, B.: Basic Clin. Pharmacol. Toxicol. 2014, 114(1): 70–77. 3. Suh, J., et al.: Chem. Biol. Drug. Des. 2012, 80(1): 89-98.

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Development and evaluation of ST-1829 based on 5-benzylidene-2-phenylthiazolones as promising agent for anti-leukotriene therapy Lill, A.P.1; Rödl, C.B.1; Steinhilber, D.1; Stark, H.1,2; Hofmann, B. 1 1 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany; E-Mail: [email protected] 2 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany; E-Mail: [email protected]

Arachidonic acid released from cytoplasmic membrane upon external stimuli is the source of one important group of inflammatory mediators: the leukotrienes (LTs). The key enzyme of their biosynthesis is the iron-containing, heme-free 5-lipoxygenase (5-LO). LTs play a pivotal role in inflammation, allergic disorders, asthma, cardiovascular diseases and cancer. Up to now, only one LT biosynthesis interfering drug is marketed with limited success: the the iron chelating 5-LO inhibitor Zileuton (Zyflo®). The need to discover new active ligands for anti-leukotriene therapy is still urgent [1,2]. This study presents the synthesis and development of a potent and direct 5-LO inhibitor based on the molecular pharmacologically characterized 5-benzylidene-2-phenylthiazolone C06 whose further pharmacological investiga-tion was precluded due to its low solubility [3,4]. Through optimization of C06, extensive evaluation of the structure-activity relationships including profound assessment of the thiazolone core and consideration of the solubility we developed the 5-benzyl-2-phenyl-4-hydroxythiazoles as new and effective 5-LO inhibitors. ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol, showed an improved 5-LO inhibitory activity in a cell-based (IC50 value 0.14 µM) and a cell-free assay (IC50 value 0.03 µM) as well as a greatly enhanced solubility. Furthermore, it keeps its promising inhibitory potency, even in the presence of blood serum, excluding excessive binding to serum proteins. Together with a non-cytotoxic profile, the thiazolone-based parent compound could successful-ly be optimized which thereby marks a major step towards an effective anti-inflammatory therapy.

Acknowledgements: This work was supported by Fonds der Chemischen Industrie, Deutsche Forschungsgemeinschaft DFG (SFB 1039), by the EU COST Actions BM0806, BM1007, CM1103, and CM1207 as well as the Hesse LOEWE Schwerpunkte Fh-TMP, OSF and NEFF, the Else-Kröner-Stiftung, TRIP and the Deutsches Konsortium für Translationale Krebsforschung, DKTK (HS). A.P.L. and C.B.R contributed equally to this work. H.S. and B.H. share senior authorship.

References: 1. Peters-Golden, M., Henderson, W.: N. Engl. J. Med. 2007, 357(18): 1841-1854. 2. Werz, O., Steinhilber, D.: Pharmacol. Ther. 2006, 112(3): 701-718. 3. Hofmann, B. et al.: J. Med. Chem. 2011, 54(6): 1943-1947. 4. Hofmann, B. et al.: Br. J. Pharmacol. 2012, 165(7): 2304-2313.



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ANTICANCER DRUGS AND EPIGENETICS (ACE01-ACE33)

A fluorescence polarisation based phospho-Tyr Myt1 kinase activity assay Platzer, C.1; Rohe, A.1; Schutkowski, M.2; Sippl, W.1; Schmidt, M.1

1 Institute of Pharmacy, Department of Medicinal Chemistry, Martin-Luther-University-Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany 2 Institute of Biochemistry and Biotechnology, Department of Enzymology, Martin-Luther-University-Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany

The human Myt1 kinase is a negative regulator of Cdk1/Cyclin B complex, hence, important for the G2/M transition in the cell cycle especially in cancer cells. It may act as a drug target in anti-cancer therapy [1], but an activity assay for assessing potential inhibitors is lacking so far. Because the natural human Myt1 substrate Cdk1 is unsuitable for assay development [2] 2304 short chain peptides were investigated with Myt1 on a microarraychip. 11 peptides were recognized by Myt1 and EFS_HUMAN_302 was identified to be the best Myt1 substrate. With this substrate an activity assay could be developed. Here the steps of development of a fluorescence polarisation based phospho-Tyr assay is presented. Therefore the kinase reaction of Myt1 full length was optimized by adaption of reaction time, temperature, buffer system and other reaction conditions. So the pH optimum of Myt1 was identified at 7.5 in HEPES buffer. The assay relies on the immunodetection of the phosphorylated substrate using a phospho-tyrosin antibody and the fluorescence labeld pentapeptide (6-FAM-) KI(pY)VV. This assay extends the spectrum of methods to investigate the Myt1 kinase, which consist of two binding assays so far. So it allows the exactly characterisation of Myt1 inhibitors.

References: 1. Chow, J.P., Poon R.Y.C.: Oncogene 2013, 32(10): 4778-4788. 2. Rohe, A., et al: Bioorg. Med Chem. Lett. 2012, 22(2): 1219-1223.

Evaluation of the receptor tyrosine kinase RON as therapeutic target in pediatric sarcoma Schleithoff, C.1,2, Lechtape, B.1, Tillmanns, A.1, Schaefer, C.1, Hempel, G.2, Dirksen, U.1, Potratz, J.1 1 Universitätsklinikum Münster, Albert-Schweitzer-Campus A1, 48149, Germany 2 Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstaße 48, 48149, Germany

The receptor tyrosine kinase (RTK) RON (recepteur d‘origine nantais) is a cell-surface receptor, involved in the regulation of cellular migration and with potential relevance to cancer cell metastasis. Previous data suggested a cross-talk of RON with the insulin-like growth factor receptor (IGF1R) in Ewing sarcoma. Therapeutic inhibition of IGF1R has shown clinical efficacy, but resistance is being observed, possibly due to alternative RTK activations. This led us to investigate the role of RON in pediatric sarcoma, alone and in cross-talk with IGF1R. RON is expressed (mRNA and protein) and constitutively activated, i.e. phosphorylated, as were downstream signals ERK and AKT in Ewing sarcoma and rhabdomyosarcoma cell lines. In tumor samples RON mRNA expression was significantly higher in 6 Ewing sarcoma patients with metastases than in 15 patients with localized disease. shRNA knockdown of RON reduced cellular migration in wound healing and Transwell assays. An N-terminal RON blocking antibody was tested, but did not show any effect on cell proliferation or migration, neither alone nor in combination with an IGF1R blocking antibody. These data encouraged us to investigate RON variants such as short form (sf) RON that lacks the N-terminal antibody-binding domain but sustains tyrosine kinase activity. A small molecule inhibitor binding and blocking this tyrosine kinase domain showed some activity. A second sfRON isoform was detected in patient samples. Additional RON variants are currently being investigated. RON is expressed and activated in Ewing sarcomas with a role in sarcoma cell migration. RON variants were detected in cell lines and tumor samples with potential impact on therapeutic targeting strategies.

Acknowledgements: The project is supported by the Deutsche Krebshilfe.

Bicyclic acetals as potential inhibitors of Golgi alpha-mannosidase II Borek, C.1; Irsheid, L.1; Weickert, A.2; Seibel, J.2; Engels, B.2; Stauber, R.3; Schirmeister, T.1

1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY

2 Institute of Physical and Theoretical Chemistry, University of Würzburg, GERMANY 3 Ear, Nose, Throatclinic and Policlinic, University of Mainz, GERMANY

Golgi α- mannosidase II (GMII) plays a crucial role in the N-glycosylation pathway. In various tumor cell lines, the distribution of the N-linked sugars on the cell surface is modified and correlates with the progression of tumor metastasis [1]. GMII is therefore a molecular target for anticancer agents and its inhibition has shown tumor repression [2]. GMII, a member of the family 38 glycoside hydrolases, cleaves two mannose units (α-(1,3) and α-(1,6)) of the intermediate GlcNAcMan5(GlcNAc)2.The active site of the enzyme consists of two aspartate residues and a zinc cation. GMII acts as a retaining glycosidase and cleaves the sugars in a two-step-SN2-mechanism in which a covalent glycosyl-enzyme complex is formed. The mechanism preserves the configura-tion of the anomeric C-atom [3,4]. Several natural product-based or synthetic inhibitors have been investigated. However, the clinical use of the known potent inhibitor swainsonine is restricted due to the side effects resulting from inhibition of lysosomal α-mannosidases [5]. The aim of our work is the computational design and syntheses of selective, covalent-reversible GMII-inhibitors. QM/MM calculations and molecular docking have shown that bicyclic acetals are favorable candidates, in terms of both, high affinity to the target enzyme and reaction kinetics. Based on L-gulose, we synthesize 1-6 bridged derivatives which are promising lead structures.

References: 1. Fujita, T. et al.: Org. Lett. 2004, 6 (5): 827-830. 2. Van den Elsen, Y.M.H., Kuntz, D.A., Rose, D.R.: The EMBO Journal 2001, 20 (12): 3008-3017. 3. Petersen, L. et al.: J. Am. Chem.Soc. 2010, 132: 8291-8300. 4. Zhong, W. et al.: J. Am. Chem.Soc. 2008, 130: 8975-8983. 5. Cheng,T-J.R. et al: Chem. Asian J. 2013, 8: 2600-2604.

The impact of latent heparanase on integrin-mediated adhesion and migration of melanoma cells in metastatic spread – A novel target for therapeutic interference? Hoß, S.G.1; Gerber, U.1; Schlesinger, M.1; Naggi, A.M.2; Ilan, N.3; Vlodavsky, I.3; Bendas, G.1 1 University Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn, Germany 2 G. Ronzoni Institute for Chemical and Biochemical Research, Via G. Colombo 81, 20133 Milan, Italy 3 Rappaport Faculty of Medicine, Technion, P.O.B. 9649, 31096 Haifa, Israel

Background: Heparanase is an endoglycosidase that cleaves glycosidic chains of cell surface heparan sulfate proteoglycans (HSPG), which is important in cancer progression, metastasis development and inflammatory diseases. The latent, non-enzymatically active precursor form of heparanase mediates signaling functions e.g. activation of Akt, Src or Rac via cell surface HSPGs, which can result in stronger adhesion and migration, but the molecular mechanisms remain elusive. Integrins appear as promising candidates for upregulated functionality by heparanase, since integrin signaling overlaps in part with HSPG pathways especially with those of syndecan-4 (SDC-4). In light of our recent findings on the dominant contribution of VLA-4 to melanoma metastasis and interference by heparin [1], latent heparanase might be a potential target for heparin in this tumor entity. Aim/objectives: This study aims to investigate i) whether and how latent heparanase affects VLA-4 functionality on melanoma cell lines, and ii) whether heparin can interfere with this activity. Methods: A MV3 cell clone with reduced SDC-4 expression was generated and binding of VLA-4 to VCAM-1 was determined by flow cytometry in presence/absence of latent heparanase (2 µg/mL). Integrin-mediated cell migration was investigated using a modified scratch assay. For a better understanding of invasive aspects of metastasis a transmigration assay using

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a modified Boyden chamber was performed. Interference of heparanase actitvity by different heparins was investigated and mainly confirmed by surface acoustic wave (SAW) biosensor technology, different chemical entities were used to interfere with steps of intracellular signaling in a selective manner. Results: Latent heparanase affects VLA-4 activity in binding the cellular ligands and in melanoma cell migration/transmigration via a SDC-4 dependent pathway. An insight into the underlying mechanisms will be provided. Heparin can interfere with the heparanase activity. To differentiate heparin effects directly related to VLA-4 from those affecting heparanase we employed different heparin derivatives that either bind solely latent heparanase, or bind both heparanase and VLA-4 directly using SAW technology. Comparing MV3 wt and SDC-4 knock-down cells we could show that blockade of heparanase is a vital part in heparin activity for reducing VLA-4 function and thus a potential novel target in the antimetastatic approaches using low molecular weight heparin. Conclusions: We identified latent heparanase as a novel target of heparin in tumor progression of melanoma cells and thereupon support the motivation to use heparins as anti-metastatic drugs in clinics. Nonetheless, the clarification of intracellular underlying mechanisms and involved pathways need further investigation.

Reference: 1. Schlesinger, M. et al.: Thromb. Res. 2014, 133: 855-862.

Tryptophan-Induced Quenching for the Detection of Allosteric Akt Inhibitors – Development of an Inter-Domain Interaction Sensing System Weisner, J.1; Fang, Z.1; Rauh, D.1 1 Technical University Dortmund, Department of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, 44227 Dortmund, Germany

The survival kinase Akt (Protein Kinase B/PKB) plays a pivotal role in many cellular signal transduction pathways that are responsible for regulation of e.g. cell proliferation, cell growth and apoptosis[1]. Dysregulation of this mul-tidomain enzyme is directly associated with neoplastic transformation, malignant progression as well as increased resistance to chemo- and radiotherapy in a variety of solid tumors[2]. Novel allosteric ligands, first identified and characterized in 2005, were shown to exclusively bind to the protein in the presence of the regulatory PH domain[3]. These small molecules target a unique pocket that is formed at the interface of the kinase and the PH domain in the inactive closed conformation which allows for a specific modulation of kinase localization and activity and thus offering great potential in medical therapy[4]. In order to allow for the selective detection of such allosteric inhibitors in high-throughput screenings an inter-domain interaction sensing system was developed based on Tryptophan-Induced Quenching (TrIQ)[5] and the previously established FLiK and iFliK technologies[6,7]. Therefore, artificial tryptophans were individually introduced to the kinase domain being located in close proximity to a fluorescent probe coupled to the PH domain in the inactive state. The newly generated constructs were assessed via steady-state fluorescence measurements resulting in the identification of a suitable combination of fluorophore and artificial tryptophan. This novel TrIQ-FLiK assay (Tryptophan-Induced Quenching of Fluorescent Labels in Kinases) readily distinguishes between ATP-competitive and allosteric inter-domain Akt inhibitors.

References: 1. Manning, B.D., Cantley, L.C.: Cell 2007, 129(7): 1261-1274. 2. Rudner, J. et al.: Radiat. Oncol. 2010, 5(108). 3. Barnett, S.F. et al.: Biochem. J. 2005, 385(2): 399-408. 4. Wu, W.I. et al.: PLoS One 2010, 5(9), e12913. 5. Mansoor, S.E., Dewitt, M.A., Farrens, D.L.: Biochemistry 2010, 49(45): 9722-9731.

6. Simard, J.R. et al.: J. Am. Chem. Soc. 2009, 131(37): 13286-13296. 7. Fang. Z. et al.: ACS Chem. Biol. 2014, in press.

Lysophospholipid induced antimetastatic effects - Insights into the underlying mechanisms Ross, T.1; Jakubzig, B.1; Schlesinger, M.1; Raynor, A.2; Jantscheff, P.2; Gorzelanny, C.3; Massing, U.2; Bendas, G.1 1 University of Bonn, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn, Germany 2 Tumor Biology Center, Freiburg, Clinical Research, Breisacher Str. 117, 79106 Freiburg, Germany 3 Experimental Dermatology, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany

Based on the findings that empty liposomes, applied to tumor bearing mice, possess antimetastatic effects [1] lysophosphatidylcholine (LysoPC) was regarded as the active agent resulting from the liposomal phospholipid degradation within the tumor tissue. Thereupon we demonstrated that a LysoPC pretreatment of human as well as mice melanoma cells strongly attenuated their metastatic spread in a lung invasion model in mice [2]. In vitro studies revealed attenuated integrin functions in cell adhesion as well as in cell migration as the functional consequence of LysoPC for affecting metastasis. Since LysoPC did not possess apoptotic or cytotoxic effects, the underlying molecular mechanisms of reduced integrin functionality remained open and are the aim of this study. Using gas chromatography it becomes apparent that upon exposure to LysoPC, melanoma cells massively incorporate LysoPC associated fatty acids into the cell membrane, affecting the lipid composition of the membrane dramatically, which goes along with morphological changes of the membrane observed by electron microscopy. We could confirm that the shift in lipid composition alters membrane properties, in dependence on the LysoPC species employed (C18:0 /C18:1), examined by fluorescence based methods as well as atomic force microscopy (AFM). Using trimethylammoniumdiphenylhexatriene as fluorescence anisotropy probe, we detected a significant rigidification of the membrane of MV3 melanoma cells by saturated LysoPC (C18:0). These finding could be confirmed by a fluorescence recovery after photo bleaching (FRAP) technique. The increased membrane rigidity induced by the saturated LysoPC corre-sponds with a diminished receptor-mediated migration capacity of the cells, which is induced by an affected focal adhesion complex via the signaling function of the proteoglycan syndecan 4 and different protein kinase C isoforms. Consequently we suppose that the reduced integrin functionality is a result of membrane rigidification and affected lateral raft formation with strong consequences for underlying signaling pathways. Our data shed a new light on liposomal drug carrier approaches in cancer therapy with respect to novel, yet not considered activities of phospholipid degradation products.

References: 1. Graeser, R. et al.: Pancreas 2009, 38(3): 330-337. 2. Jantscheff, P. et al.: Mol Cancer Ther 2011, 10(1): 186-197.

Structural analysis of novel covalent inhibitors of the epidermal growth factor receptor Becker, C.; Engel, J.; Rauh, D.

Technische Universität Dortmund, Fakultät für Chemie und Chemische Biologie, Otto-Hahn-Str. 6, 44227 Dortmund email to: [email protected]

The epidermal growth factor receptor (EGFR, ERBB1) is one of the most prominent members of the ERBB family of receptor tyrosine kinases and plays an important role in the regulation of cell proliferation and cell survival. A dysregulation of the receptor leads to the emergence of different cancer types [1,2]. In 10 % of all cases, non-small cell lung cancer (NSCLC) is caused by a single point mutation L858R within the kinase domain of EGFR [3]. NSCLC patients harbouring EGFR L858R respond well to the small molecule kinase inhibitors erlotinib and gefitinib, at least until these patients acquire a second-ary point mutation at the gatekeeper amino acid (T790M), which causes a drug

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resistance and relapse of the disease [4,5]. Recent developments proved covalent inhibitors to have a great potential to overcome drug resistant mutations in NSCLC [6,7]. Here we report our efforts to elucidate the structure and binding mode of novel covalent, mutant selective EGFR inhibitors. We were able to crystallize these compounds in complex with the drug-resistant gatekeeper mutant T790M and clearly confirm their covalent binding mode by protein X-ray crystallography [8].

Figure: Crystal structure of EGFR T790M in complex with a covalent inhibitor (only electron density is shown). References: 1. Arteaga, C.L., et al.: Cancer Cell 2014, 25(3): 282-303. 2. Zandi, R., et al.: Cell Signaling 2007, 19(10): 2013-2023. 3. Sharma, S.V., et al.: Nat Rev Cancer 2007, 7(3): 169-181. 4. Wong, K.K.: Lung Cancer 2008, 60 Suppl. 2: S10-18. 5. Heuckmann J.M., et al.: J Clin Oncol 2012, 30(27): 3417-3420. 6. Walter, A.O., et al.: Cancer Discov 2013, 3(12): 1404-1415. 7. Zhou, W., et al.: Nature 2009, 462(7276): 1070-1074. 8. Stamos, J., et al.: J. Biol. Chem. 2002, 277: 46265-46272.

RNA-based therapeutic strategies for targeting the oncogenic Pim1 kinase Lange-Grünweller, K.1; Weißer, A.1; Weirauch, U.2; Aigner, A.2; Grünweller, A.1; Hartmann, R.K.1

1 Institut für Pharmazeutische Chemie, Pharmazie, 35037 Philipps-Universität Marburg, Germany 2 Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Klinische Pharmakologie, 04107 Universität Leipzig, Germany

The serine/threonine kinase Pim1 is overexpressed in several aggressive solid tumors and lymphomas with a bad prognosis for patients. Pim1 activates cell proliferation and inhibits apoptosis, and it seems that the Pim kinase family (Pim1-3) have no essential function in healthy cells and adult tissues. We have recently shown that Pim1 is a target of miRNA regulation by miR-33a and miR-15b [1-3] and that Pim1 together with c-Myc can regulate the expression of the oncogenic miRNA-cluster miR-17-92 at the transcriptional level [4]. Therefore, inhibition of Pim1 seems to be an interesting antitumor strategy. Here, we used RNAi strategies to inhibit Pim1 in mouse xenograft tumor models of colon carcinoma and glioblastoma. Delivery of miR-33a mimics or Pim1-specific siRNAs into tumors was achieved by forming nanoplexes of RNA with branched low molecular weight polyethylenimine (PEI). This approach was also used to explore a new antisense strategy in vivo which is called U1-Interference (U1i) [4]. Mechanistically, the abundant U1 snRNP, a splicing subparticle, is recruited to the last exon of a pre-mRNA by a chemically modified oligonucleotide with dual specificity. This U1 adaptor is able to simultaneously bind the targeted pre-mRNA and the U1 snRNA. The recruit-ment of the U1 snRNP results in a blockage of polyadenylation, followed by

pre-mRNA degradation. We found that RNA-based Pim1 targeting reduced tumor growth significantly without changing liver enzyme activities or inducing unwanted immune responses. In a next step, we combined RNAi with statin treatment to downregulate Pim1. Statins are competitive HMG-CoA-Reductase inhibitors with pleiotropic antitumor effects and importantly, we found that statins can downregulate Pim1 at low micromolar concentrations. Here we show that statin treatment in combination with Pim1-specific siRNA or other Pim1 inhibitors improves statin-dependent effects on Pim1, thereby decreasing the statin concentrations for Pim1 inhibition to the sub-micromolar range. We will now further evaluate statin effects on Pim1 in mouse xenografts of colon carcinoma and glioblasto-ma. References: 1. Thomas, M. et al.: Oncogene 2012, 31(7): 918-928. 2. Ibrahim, A.F. et al.: Cancer Research 2011, 71(15): 5214-5224. 3. Weirauch, U. et al.: Neoplasia 2013, 15(7): 783-794. 4. Weirauch, U. et al.: Nucleic Acids Therapeutics 2013, 23(4): 264-272.

Novel strategies to overcome the cisplatin resistance of tumor cells – Liposomes and low molecular weight heparin as promis-ing modulators

Pfankuchen, D.1; Stölting, D.P.1; Royer, H.D.2; Bendas, G.1 1 Pharmaceutical Department, University of Bonn, D-53121 Bonn, Germany 2 Department of Human Genetics & Anthropology, University of Düsseldorf, D-40225 Düsseldorf, Germany

Cisplatin is a well-established cytostatic agent in the therapy of ovarian carcinoma. However, the therapeutic application and benefit of cisplatin is often restricted by the development of chemoresistance. While resistance mechanisms are multifactorial and not fully understood, we recently reported on strong differences in apoptotic pathways affected by free and liposomal cisplatin [1]. Furthermore, we also found a chemosensitizing effect of LMWH [2]. However, the molecular mechanisms of the latter findings remained open. The aim of this study was to obtain an insight into chemoresistance mecha-nisms by using liposomal cisplatin as well as heparin to assess similarities or differences in their mode of action as chemosensitizers. Cisplatin liposomes with a POPC/Chol/m-PEG-PE (6.5/3/0.5) lipid composition were prepared by hydration technique as described [1]. The LMWH tinzaparin was used up to therapeutic threshold concentrations (50 µg/mL). Cytotoxicity (MTT-assay), cellular platinum accumulation (fAAS), transporter expression (SDS-PAGE/WB) and expression of apoptosis proteins (antibody array) were investigated in A2780 ovarian cancer cells and the cisplatin resistant cell line A2780cis. Intracellular pathways after treatment with liposomal cisplatin were analyzed by gene array data (photometric, microarray scan) and subsequent ranked with the GeneGO software pathway tool (n-p, t-t, Mann-Whitney test for comparison, p < 0.05). Based on the transporter status of A2780cis cells it became evident that the superior cytotoxicity of cisplatin liposomes is not to explain by raising influx dependent cytotoxicity or by bypassing native uptake mechanisms. . These results refer to a more complex mode of action of liposomes. Gene array and protein expression data reveal that liposomal cisplatin affects apoptotic pathways differently from the free drug bringing up a different view on liposomes as modulators of apoptosis more than simple carriers for a drug payload. In contrast, LMWH affected the chemoresistance in A2780cis cells by alterations in transporter expression independent of the cytostatic drug(-formulation), but did not act via a blockade of cell adhesion molecule mediated drug resistance (CAM-DR), as the integrin status of resistant cells checked by flow cytometry revealed. Also for LMWH a different expression profile of apoptosis proteins was evident, suggesting alterations in apoptotic pathways to be the missing link between extracellular tinzaparin concentrations and its beneficial effect on cisplatin cytotoxicity in resistant cells. Future and ongoing studies will provide an insight into the mechanistical basis of these surprising findings.Altogether, our data underline chemoresistance as a matter of pathway rather than mere intracellular cisplatin concentrations. References: 1. Stölting, D.P. et al.: Anticancer Res. 2014, 34(1): 525-530. 2. Stölting, D.P. et al: Int. J. Clin. Pharmacol. Ther. 2013, 51(1): 70-73.

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Structure-based design and synthesis of covalent TBK1-Inhibitors Kaitsiotou, H.; Basu, D.; Rauh, D.*

Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Straße 6, 44227 Dortmund, Germany

Protein kinases regulate cellular processes such as proliferation, differentia-tion, apoptosis, or inflammatory and antiviral responses. The cytosolic Ser/Thr kinase TANK-binding kinase 1 (TBK1) is an important mediator of antiviral and inflammatory immune responses.[1] Besides its regulating role in the mediation of antiviral responses, TBK1 has been proposed as a potential target in cancer therapy.[2,3] However, its detailed biological function in tumor biology remains unclear. Therefore, deciphering the complex regulation and activation mechanism and associated signaling pathways of TBK1 was brought into focus and encouraged the conduction of numerous studies to identify and develop tool compounds as well as novel TBK1 inhibitors. The recently reported crystal structure of TBK1 in complex with the fairly potent, but non-selective, type I inhibitor BX795 has provided the structural basis for medicinal chemistry approaches.[4] Here we present the structure-based design approach to generate covalent modulators of TBK1 for deciphering its role in cancer biology. Covalent binding probe-molecules represent promising auxiliaries to investigate the function of TBK1 in regulating cellular signaling of the antiviral immune response, as well as its role in cancer development and progression. Based on the scaffold of BX795 we designed and synthesized analogues incorporating Michael acceptors for covalent biding which may enhance inhibitor selectivity and residence time and thereby may serve as molecular probes for further chemical biology approaches.

References: 1. Fitzgerald, K.A. et al.: Nat Immunol 2003, 4: 491-496. 2. Barbie, D.A. et al.: Nature 2009, 462: 108-112. 3. Wei, C. et al.: Proc Natl Acad Sci U S A 2014, 111: E601-610. 4. Ma, X. et al.: Proc Natl Acad Sci U S A 2012, 109: 9378-9383

Design and Synthesis of covalent Inhibitors to overcome drug resistance in EGFR Lategahn, J.; Flaßhoff, M.; Engel, J.; Becker, C.; Rauh, D. Technische Universität Dortmund, Department of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, D-44227 Dortmund, Germany eMail: [email protected]

The discovery of mutations in the epidermal growth factor receptor (EGFR) has marked a dramatic change in the treatment of non-small cell lung cancer. Patients with EGFR-mutant lung carcinoma receiving EGFR inhibitors have a median overall survival of more than 2 years, contrasting with the survival of unselected patients receiving chemotherapy.[1] Acquired resistance to these targeted drugs is in 50% of the cases mediated by a secondary point mutation in the kinase domain of EGFR, the gatekeeper position (T790M).[2,3] The particular size and physicochemical properties of the amino acid found at this position are critical determinants for kinase inhibitor affinity and selectivity. Gatekeeper mutations affect the thermodynamic and kinetic binding character-istics of all 4-amino-quinazoline-based inhibitors (originally developed to target wild-type EGFR).[4] Multi targeted EGFR/VEGFR inhibitor AEE788 was developed by Novartis, but failed to inhibit the T790M drug resistant mutant variant of EGFR.[5,6] Here we present our efforts to develop novel EGFR inhibitors based on the pyrrolopy-rimidine scaffold that overcome drug resistance by reduced spatial dimension in proximity to the gatekeeper side chain. Moreover covalent alkylation of a unique cysteine (C797) by accurate positioning of a reactive Michael acceptor system is achieved. Biochemical characterization substantiates our approach of targeting gatekeeper mutant EGFR by combining covalent binding and spatially flattened compounds.

References: 1. Heuckmann, J.M. et al.: Journal of clinical oncology 2012, 30: 3417-3420. 2. Pao, W., et al.: PLoS medicine 2005, 2: e73. 3. Kosaka, T. et al.: Clinical cancer research 2006, 12: 5764-5769. 4. Sos, M.L. et al.: Cancer research 2010, 70: 868-874. 5.Traxler, P. et al.: Cancer research 2004, 64: 4931-4941. 6. Yun, C.H. et al.: PNAS 2008, 105: 2070-2075.

V-ATPase regulates epithelial-mesenchymal transition in breast cancer cells Merk, H.1; Müller, R.2; Vollmar, A.M.1; Liebl, J.1 1 Department of Pharmacy, Pharmaceutical Biology, University of Munich, Buten-andtstrasse 5-13, 81377 Munich, Germany 2 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO 151150, 66041 Saarbrücken, Germany

Breast cancer represents one of the leading causes of cancer related death of females and its incidence is growing. Despite the initial effectiveness of chemotherapy, treatment resistance limits therapeutic success of breast cancer treatment. The relatively high rate of relapse and metastases of aggressive breast cancer is attributed to breast cancer stem cells (CSCs) which have self-renewing and high tumor-initiating potential, are resistant to standard therapy and cause establishment of metastases. CSC formation is closely linked to epithelial-mesenchymal transition (EMT) which confers mesenchymal properties on epithelial cells [1]. Therapeutic strategies that target breast CSCs may substantially improve breast cancer treatment and patient prognosis. Vacuolar H+-ATPases (V-ATPases) are ubiquitous multimeric ATP-dependent proton pumps that acidify intracellular compartments and translocate protons across the plasma membrane. The enzyme comprises the cytoplasmic V1 domain that carries our ATP hydrolysis and the membrane-bound V0 integral subunit that is responsible for proton transport from the cytoplasm to the endosomal/lysosomal lumen or the extracellular space. V-ATPase is essential for endocytotic processes, receptor internalization and recycling, and lysosomal degradation [2]. Of note, recent reports show that V-ATPase is responsible for the degradation and recycling of epithelial adhesion proteins e.g. E-cadherin and increased V-ATPase activity has been shown during EMT [3, 4]. We hypothesized that the proton pump V-ATPase is such a target affecting EMT and CSC formation. For our study we used the V-ATPase inhibitor Archazolid A. Archazolid A is a natural compound, first isolated from cultivated myxobacteria Archangium gephyra and is also available by chemical synthesis [5]. Like the known V-ATPase inhibitors concanamycin or bafilomycin A1, it binds to the V0 subunit c and inhibits V-ATPase activity. To investigate whether Archazolid A influences EMT, we used immortalized human mammary epithelial cells (HMLEs) with tamoxifen-inducible TWIST transcription factor overexpression. Activated TWIST is a direct suppressor of E-cadherin and activates EMT markers like N-cadherin or vimentin and therefore promotes EMT. Archazolid A treatment during EMT decreased migratory capability of HMLEs. Moreover, HMLEs which have already undergone EMT and thus show mesenchymal properties show decreased migration after Archazolid A treatment. To analyze a potential implication of V-ATPase in breast CSCs, mammosphere assays were performed. The mammosphere assay is based on the fact that only breast CSC can survive in suspension culture and mature tumor cells die by anoikis. In fact, Archazolid A decreased mammosphere formation of mesenchymal HMLEs that have undergone EMT. In summary, our results indicate that the V-ATPase inhibitor Archazolid A inhibits migration of breast CSC and point to a function of V-ATPase in EMT and breast CSC formation. Thus, V-ATPase inhibition by Archazolid A might be investigated as potential new strategy for the treatment of invasive and metastatic breast cancer. Supported by DFG FOR 1406

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References: 1. Sendurai, A. Mani et al.: Cell, 2008, 133(4):704-715. 2. Forgac, M.: Nat Rev Mol Cell Biol., 2007, 8(11):917-929. 3. Le, T.L.; Yap A.S.; Stow, J.L.: J Cell Biol, 1999, 146:219-232. 4. Cao, X. et al.: Am J Physiol Renal Physiol, 2012, 302:F1121-F1132. 5. Sasse, F. et al.: J Antibiot, 2003, 56(6):520-525.

Synthesis and characterization of new substrate-analogue matriptase inhibitors Maiwald, A.; Hammami, M.; Wagner, S.; Heine, A.; Klebe, G.; Steinmetzer, T.

Institute of Pharmaceutical Chemistry, Marbacher Weg 6, Philipps University, 35032 Marburg, Germany

Matriptase is a type II transmembrane protein that contains an extracellular trypsin-like serine protease domain at the C-terminus. In vitro experiments revealed that matriptase can activate a number of substrates involved in tumor progression and metastasis such as the proforms of hepatocyte growth factor/scatter factor (HGF), urokinase-type plasminogen activator (uPA), protease-activated receptor 2 (PAR-2), the cancer-related growth factor macrophage-stimulating protein 1 (MSP-1), or the metalloproteases MMP-3 and MMP-1. Furthermore, matriptase is often upregulated in cancer, e.g. in gastrointestinal tract, breast, ovary, prostate, cervix and lung tumors. There-fore, matriptase appears to be a promising target for cancer therapy. Moreo-ver, recent studies suggest matriptase as a major PAR-2 activator in osteoar-thritis leading to increased collagenase expression involved in cartilage degradation. Several groups have described substrate-analogue inhibitors containing a C-terminal 4-amidinobenzylamide in combination with an N-terminal sulfonyl residue as inhibitors for various trypsin-like serine proteases such as thrombin, factor Xa, factor VIIa, uPA, plasmin and plasma kallikrein. Therefore, we have screened analogues of this type available from previous studies as matriptase inhibitors. The most potent compound contains an N-terminal benzylsulfonyl group in P4 position and a D-homoPhe-Pro moiety as P3-P2 segment. However, this compound suffers from poor selectivity and is a stronger thrombin and factor Xa inhibitor. Based on this result several new analogues have been prepared by incorporation of substituted D-homophenylalanines in P3 position, whereby the highest inhibitory potency was found for derivatives containing D-homotyrosine. Further replacement of the P2 proline by alanine and elimination of the N-terminal benzylsulfonyl group changed the selectivity profile and provided a first substrate-analogue inhibitor (MI-470), which exhibits a stronger affinity for matriptase compared to thrombin and factor Xa.

OH

H2NNH

HN

O

O

NH

NH2

Ki Matriptase = 26 nM

Ki Thrombin = 302 nM

Ki Factor Xa = 570 nM

MI 470

Due to the insufficient amount of available matriptase we used trypsin for crystallization in complex with inhibitor MI-470. The refined structure of the trypsin/MI-470 complex was superimposed with the known crystal structure of matriptase. The obtained model reveals that the D-homoTyr side chains binds into a well defined binding pocket above Trp215 of matriptase, which is surrounded by Phe99 on the right and Gln175 on the left side.

V-ATPase inhibition affects iron metabolism: a novel therapeutic option for breast cancer Schneider, L.S.; von Schwarzenberg, K.; Vollmar, A.M.

Department of Pharmacy, Pharmaceutical Biology, University of Munich, Butenandtstraße 5-13, 81377 Munich, Germany

Within the last decade evidence is increasing that the vacuolar H+-ATPase (V-ATPase), a heteromultimeric proton pump, plays a vital role in the survival of tumor cells. However, the precise mode of action still awaits molecular explanation. To investigate V-ATPase mediated cytotoxicity we used the myxobacterial derived compound archazolid, a highly potent V-ATPase inhibitor. Here we show that V-ATPase inhibition clearly induces apoptosis in breast cancer cells in vitro. Remarkably, these findings were recapitulated in vivo using a 4T1 mammary mouse model which showed reduced tumor growth upon archazolid treatment. With regard to the molecular mechanisms of V-ATPase related cell death we found that archazolid stabilizes the HIF1α protein which is associated with the apoptosis induction in p53 wild type tumor cells. We unveil that the stabilization of HIF1α is due to iron depletion in the cytosol and associate this with disrupted transferrin/transferrin-R internalization upon V-ATPase inhibition. As a consequence, activity of the iron dependent enzyme ribonucleotide reductase is diminished leading to S-phase block and double strand breaks. Finally, we connect the HIF1α expression as well as the occurrence of double strand breaks with the stabilization of the tumor suppressor protein p53. This eventually connects V-ATPase inhibition to fundamental cellular processes such as DNA synthesis, DNA repair and apoptosis. Hence, our study reveals a novel mode of action for V-ATPase induced cell death in tumor cells and suggests V-ATPase inhibition as a promising and viable strategy for breast cancer therapy.

Supported by DFG FOR 1406

Acknowledgments: We thank Prof. Dr. Dirk Trauner (University of Munich) for synthesizing archazolid, Prof. Dr. Dirk Menche (University of Bonn) for isolating archazolid and Dr. Rebekka Kubisch and Prof. Dr. Ernst Wagner (University of Munich) for performing the in vivo studies.

CDK5 regulates angiogenesis via stabilizing HIF-1α in endothelial and liver tumor cells: a novel signaling mechanism with potential importance for HCC therapy

Herzog, J.1; Ehrlich, S.M.1; Liebl, J.1; Fröhlich, T.2; Mikulits, W.3; Vollmar, A.M.1; Zahler, S.1 1 Department of Pharmacy, Pharmaceutical Biology, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 2 Gene Center Munich, University of Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany 3 Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, Spitalgasse 23,1090 Vienna, Austria

The cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase which has recently been shown to regulate angiogenesis [1]. However, the underlying signaling mechanisms are still unclear. We hypothesized that CDK5 might be involved in hypoxic signaling. Hypoxia is often a key feature of tumor progres-sion, promoting the expression of angiogenic factors by hypoxia inducible factors (HIFs) [2]. The aim of this study was to characterize the link between CDK5 and HIF in endothelial cells (EC) and liver tumor cells (HUH7) in vitro and in vivo, since hepatocellular carcinoma (HCC) is one of the most vascular-ized solid tumors [3]. We could show that pharmacological inhibition of CDK5 with roscovitine as well as transient and stable knockdown lead to a significantly reduced protein level of HIF-1α in EC and HUH7 cells. Additionally, the transcription of HIF target genes involved in the regulation of angiogenesis such as VEGF A or VEGFR1 was decreased. Of note, immunhistochemistry of HIF-1α in murine liver cancer tissue sections confirmed a reduced protein level of the transcrip-tion factor by CDK5 inhibition. As treatment of CDK5 knockdown cells with a proteasomal inhibitor (MG132) results in an increase of HIF-1α, we assume that CDK5 is involved in the

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stabilization of the transcription factor. Our data showing that CDK5 co-immunoprecipitates with HIF-1α and CDK5 phosphorylates HIF-1α in vitro underline the hypothesis that both proteins directly interact. In mass spectrom-etry analysis, Serine 687 could be identified as a CDK5 phosphorylation site on HIF-1α putatively involved in the regulation of the HIF-1α level. Remarkably, mutation studies could confirm the importance of this phosphorylation site for HIF-1α stability. As in vivo proof of our concept, CD31 staining of tissue sections from xenograft and orthotopic liver cancer tumor models revealed a decreased number of microvessels for CDK5 knockdown tumors and tumors from mice treated with roscovitine or its derivatives. In summary our data indicate a direct phosphorylation of HIF-1α by CDK5 at Serine 687, resulting in a stabilization of the transcription factor thereby promoting angiogenesis. Since the expression of HIFs in HCC patients is associated with a poor prognosis [3], CDK5 might be a promising target for therapy. Acknowledgements: This study was supported by the German Research Council (DFG) grant ZA 186/7-1.

References: 1. Liebl, J. et al.: J. Biol. Chem. 2010, 285(46): 35932-35943. 2. Liao, D., Johnson, R.S: Cancer Metastasis Rev 2007, 26: 281-290. 3. Li, S. et al.: Hepat Mon. 2011, 11(10): 821-828.

Chemical-modified miRNA inhibitors as high-potential anticancer molecules Harloff, M.; Lange-Grünweller, K.; Hartmann, R.K.; Grünweller, A. 1 Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany

MicroRNAs (miRNAs) are small (21-23 nt) non-coding RNAs with important regulatory functions, which inhibit gene expression at the post-transcriptional level via RNA-Interference (RNAi). Approximately 30 % of the protein-coding genome is regulated by miRNAs. Thus miRNAs are involved in critical pathogenic processes in human diseases. Several studies have shown that small regulatory RNAs are deregulated in cancer cells, thus miRNA replace-ment or miRNA inhibition are interesting new anticancer strategies. In the case of up-regulated miRNAs, complementary antisense oligonucleotides (ASO), so called antimiRs, are used to block miRNAs sterically through Watson-Crick base-pairing. Given that RNA is generally not stable in biological fluids, a lot of different chemical modifications and delivery strategies have been developed to improve the properties of ASOs, e.g. 2’- O-methylations in combination with a phosphorothioate backbone are often used in antisense designs. Another well-known strategy is the incorporation of Locked Nucleic Acids (LNA) into ASOs, which leads to substantially increased nuclease stability. In our group especially short all-LNA antimiRs are used to block miRNAs. These miRNA inhibitors, which we termed LNA-antiseeds, target the highly conserved seed region of miRNAs, making this a valuable strategy to inhibit entire miRNA families. We already have shown that LNA (PO) 14-mer antiseeds against ongogenic miR-17-5p and miR-20a efficiently derepress the p21 tumor suppressor. Moreover, we showed functional delivery of LNA-antiseeds into cancer cells upon complexation with the branched cationic polymer polyethyl-enimine (PEI F25-LMW) [1]. With longer antimiRs, however, it is possible to block specifically single miRNAs. We are currently performing studies with different ASO designs against the oncogenic miRNAs miR-19a, miR-21 and miR-155 to test their inhibitory potential in a glioblastoma model. We have analyzed the effects of oncogenic miRNA inhibition on cell proliferation and on the derepression of some miRNA targets with the goal to identify the most efficient ASOs for further exploring their anticancer effects in vivo. Another goal is to optimize antisense strategies by developing miRNA inhibitors with novel chemical modifications. In this project, we like to improve the efficiency of miRNA inhibitors by coupling artificial chemical nucleases (guanidine-analogs). Moreover, specificity of ASO-dependent inhibition is another important issue. The coupling of photocaging groups to miRNAs or miRNA inhibitors to activate them with a light signal in a temporally and spatially controlled manner is a promising new approach that we try to establish for specific RNA targeting.

References: 1. Thomas, M. et al.: RNA Biol. 2012, 9(8): 1088–1098.

Pim-1 dependent transcriptional regulation of the oncogenic miR-17-92 Cluster Schulte, F.W.1; Seidler, S.1; Thomas, M.1; Lange-Grünweller, K.1; Weirauch, U.2; Aigner, A.2; Grünweller, A.1; Hartmann, R.K.1 1 Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany 2 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Leipzig, Germany

MicroRNAs (miRNAs) are small non-coding RNAs, important for the post-transcriptional regulation of gene expression. In general, miRNAs are derived from RNA polymerase II primary transcripts (pri-miRNA) that are further processed to ~70 nt precursors (pre-miRNA) and after nuclear export to mature miRNAs by the activity of two endonucleases. miRNAs are incorpo-rated into the miRNA-induced silencing complex (miRISC) and act as repressors of translation by imperfect base-pairing to their target sites in mRNAs. The majority of miRNAs is encoded in intronic regions, either individually or as miRNA clusters that are cotranscribed. Several miRNAs are involved in tumourigenesis, accounting for their designation as tumour-suppressing or as oncogenic miRNAs. Such miRNAs can downregulate targets involved in the regulation of apoptosis or cell cycle progression. Thereby they are interesting targets for cancer therapies.

Especially the human miRNA cluster miR-17-92, which encodes six miRNAs, is overexpressed in several solid tumours and some hematopoietic malignan-cies. Because of numerous targets of its individual miRNAs, the miR-17-92 locus exerts pleiotropic functions during development, proliferation, apoptosis and angiogenesis. To understand transcriptional regulation of oncogenic miRNAs is an important issue for cancer therapy; unfortunately, the most miRNA promoters have not been characterized or even identified yet. In the case of the miR-17-92 cluster, expression is in part controlled by a host gene promoter, which is regulated by the transcription factor E2F. Interestingly, the AT-rich region between the host gene promotor and the miRNA coding region reveals a host gene promoter independent transcriptional activity. Deletion analysis shows a strong reliance on the binding of c-Myc to a functional c-Myc binding site (E3 site), a conserved E-Box element about 1.5 kb upstream of the miRNA coding region. Furthermore, the proto-oncogenic kinase, Pim-1, its phosphorylation target HP1γ and c-Myc colocalize to this E3 region, as inferred from chromatin immunoprecipitation. Analysis of pri-miR-17-92 expression revealed that siRNA-mediated knockdown of E2F3, c-Myc or Pim-1 negatively affects cluster expression, with a synergistic effect caused by c-Myc/Pim-1 double knockdown [1]. We have recently shown that Pim-1 is also a target for miRNA regulation by miR-33a [2]. Interestingly, a miR-33a mimic reduces transcriptional activity of the A/T-rich sequence as well. In some preliminary experiments we observed an influence of statins (HMG-CoA-Reductase inhibitors) on the expression levels of single miRNAs like miR-33a. As a consequence of these results we want to analyze the effects of statins on miR-17-92 cluster expression levels by quantitative PCR (qRT-PCR) and further on global miRNA levels via high-throughput sequencing. With these approaches we hope to get a deeper insight into the observed anticancer effects of statins. References: 1. Thomas, M. et al.: Int. J. Mol. Sci. 2013, 14: 12273-12296. 2. Thomas et al.: Oncogene 2012, 31(7): 918-928.

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Bacterial Tumor Cell Targeting using the Autodisplay Technolo-gy Weckenbrock, W.V.; Blaßhofer, F.; Jose, J.

Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany

In the last decades several approaches have been made towards using bacterial infection in the treatment of cancer diseases. In the 1940´s it was already shown that attenuated strains of Clostridium tetani accumulated in cancer tissue lacking oxygen, thus predominantly affecting malignant cells and sparing healthy tissue [1]. Similar experiments using attenuated Salmonella strains have even reached phase I clinical trials [2]. Autodisplay is a well-established system for surface display of proteins on gram-negative bacteria [3]. The surface expression of functionally active antibody fragments directed against a tumor factor could allow to use genetically modified cells as bacterial drugs e.g. in gastroenterologic cancer therapy. The antibody fragment 425 directed against the Epidermal Growth Factor Receptor (EGFR) is a suitable candidate for targeting strategies, since it is known for its specificity and high affinity towards EGFR. In this study the surface display of a functional single chain variable fragment (scFv) of anti-EGFR antibody 425 on the surface of Escherichia coli was performed. The affinity of E. coli cells displaying the antibody fragment towards tumor cells overexpressing EGFR was demonstrated in FACS and fluores-cence microscopic assays. Specificity of binding to the EGF-receptor was proven in experiments using siRNA down-regulation of this receptor. Apart from usage for directed infection of malignant tissues and drug targeting applications, autodisplay of antibody fragments provides the possibility of a simple and rapid screening tool for new antibodies against pre-given antigens, for example via fluorescence activated cell sorting.

References: 1. Parker, R.C. et al.: Proc Soc Exp Biol Med 1947, 66: 461-467. 2. Toso, J.F. et al.: Journal of Clinical Oncology 2002, 20: 142-152. 3. Jose, J.: Appl Microbiol Biotechnol 2006, 69: 607-614.

Cisplatin resistance is associated with altered signalling in NSCLC cells

Sarin, N.1; Engel, F.2; Kalayda, G.V.1; Roberto, S.1; Frötschl, R.2; Cinatl jr., J.3; Rothweiler, F.3; Michaelis, M.4; Jaehde, U.1 1 Institute of Pharmacy, Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany 3 Institute of Medical Virology, Goethe University Hospital Frankfurt,Frankfurt/Main, Germany 4 Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, UK

Background: Platinum complexes are still widely used in the treatment of several cancer entities, among them non-small cell lung cancer (NSCLC). The treatment outcome is often limited by the development of resistance, with the exact mechanisms still posing many questions. There are hints that altered signalling plays a role in the development of cisplatin resistance. Aim: This projects aims at revealing signalling differences between cisplatin-sensitive and cisplatin-resistant NSCLC cells. Methods: Cisplatin-sensitive A549 and resistant A549Pt NSCLC cells were characterized for cisplatin cytotoxicity (MTT assay) and drug uptake. Addition-ally, the activation of the ERK signalling was analyzed on the proteome level. Gene expression was measured with a whole genome array. Cells were treated with 10 µM cisplatin for 24 h after 4 h of serum starvation. Results: Cisplatin cytotoxicity is markedly reduced in resistant cells (pEC50 4.141 ± 0.013; mean ± SD, n=3) as compared to the sensitive cell line (pEC50 = 4.332 ± 0.048; n=4). Intracellular platinum accumulation in resistant cells was reduced to 0.363 ± 0.116 ng platinum/µg protein (n=8), compared to sensitive cells exhibiting 0.939 ± 0.151 ng platinum/µg protein (n=9). Although basal ERK activation and expression were higher in cisplatin-resistant cells,

cisplatin treatment led to ERK activation in both cell lines. Gene expression analysis exhibits similarities as well as differences of activated signalling pathways between sensitive and resistant cell lines and between treatment conditions. Conclusions: Although cisplatin-resistant NSCLC cells exhibit a reduced cellular uptake and a higher basal ERK expression, ERK activation was not different from the sensitive cells. The preliminary pathway analysis suggests that sensitive cells treated with cisplatin regulate the same pathways which are altered in resistant cells.

Effect of GRP78 knockdown on cisplatin cytotoxicity in ovarian cancer cells Kullmann, M.1; Kotz, S.2; Hellwig, M.1; Metzger, S.2; Kalayda, G.V.1; Jaehde, U.1 1 Institute of Pharmacy, Department of Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn 2 Cologne Biocenter, University of Cologne, Zülpicher Straße 47b, 50674 Cologne

Cisplatin is an effective treatment for different cancer entities. Besides toxic side effects, acquired resistance occurs frequently and compromises therapy outcome. An increased intracellular formation of biologically inactive adducts with proteins or peptides seems to be a relevant factor contributing to resistance. This project aimed at identifying intracellular binding partners of CFDA-cisplatin (CFDA-Pt), a fluorescent cisplatin analogue. [1] The effect of these proteins on cisplatin cytotoxicity was examined by siRNA-mediated knockdown. The ovarian cancer cell line A2780 and its cisplatin-resistant subline A2780cis were exposed to 25 µM CFDA-Pt for 2 h. Fractionated cell lysates were separated by 2D gel electrophoresis. Protein spots were digested and analysed by high-resolution mass-spectrometry (ESI-MS/MS). Amongst others, we investigated glucose-regulated protein 78 (GRP78) as binding partner of CFDA-Pt. We performed densitometric Western Blot analysis of GRP78 following cisplatin treatment. SiRNA directed against GRP78 was transfected into A2780 and A2780cis cells with the K2® transfection system. Cisplatin cytotoxicity was compared in non-transfected and transfected cells using an MTT-based assay. Following treatment with CFDA-Pt and fractionation of cell lysates several intracellular protein-adducts were identified, including adducts with GRP78. Knockdown of GRP78 was achieved with a relative protein expression of 59 ± 9 % and 37 ± 8 % in A2780 and A2780cis cells, respectively, each compared to untreated controls. No significant change of cisplatin cytotoxicity after GRP78 knockdown was observed (pEC50A2780 = 5.356 ± 0.07 vs. pEC50A2780+GRP78kd = 5.369 ± 0.03 and pEC50A2780cis = 4.745 ± 0.06 vs. pEC50A2780cis+GRP78kd = 4.625 ± 0.05). In conclusion, we established a method for identification of intracellular CFDA-Pt-protein adducts. There appears no significant influence of GRP78 knock-down on cisplatin cytotoxicity. Further Pt binding partners will be assessed to identify proteins contributing to cisplatin resistance.

This project is supported by the Deutsche Forschungsgemeinschaft (JA 817/4-1).

Reference: 1. Molenaar, C. et al.: J. Biol. Inorg. Chem. 2000, 5: 655–665.

Tumor-specific Ligands for Targeted Delivery of Polymer-based Nucleic Acid Complexes Kietz, A.1; Vornicescu, D.2; Keusgen, M.2; Aigner, A.1; Höbel, S.1 1 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, Faculty of Medicine, University of Leipzig 2 Institute for Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg

The specific knockdown of disease-related genes by induction of RNA-Interference (RNAi) or micro-RNA replacement are highly attractive interven-tion strategies for the treatment of cancer. One of the major hurdles to be overcome in gene therapy approaches is the efficient delivery of therapeutical-ly active nucleic acids. Upon systemic administration in vivo, targeted delivery

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systems are an essential prerequisite to enhance therapeutic effects and to reduce unwanted actions. Within the group of non-viral delivery systems, several compounds based on cationic polymers and lipids are under intense investigation. Among those compounds, polyethylenimine (PEI) takes a prominent position due to the so-called “proton-sponge-effect”. The molecular structure of PEI and its ability to form complexes with nucleic acids (polyplexes) provide the basis for the generation of targeted nanoparticles by modification with cell-specific ligands (see Figure 1).

Figure 1. Mode of action of Tumor-specific polyplexes.

Figure 2. SPR-Meaasurements of Tumor-Specific Polyplexes.

Here, we present a tumor-specific delivery system based on covalent coupling of the antibody cetuximab to our low molecular weight PEI F25-LMW [1]. Exhibiting high biocompatibility and biological activity this PEI proved to be an efficient platform for the delivery of DNA and small RNAs in vitro and in vivo [2,3]. Ligand-modification of PEI was performed via a PEG-spacer to reduce non-specific interactions, which is of high interest especially for systemic administration of the polyplexes. This coupling procedure yielded a target-specific gene carrier as demonstrated by surface plasmon resonance (SPR) measurements [4]. Using this method, we were able to real-time investigate the specific binding of cetuximab-modified PEG-PEI as well as cetuximab-modified PEI/siRNA complexes to immobilized epidermal growth factor receptor (EGFR) without labelling (see Figure 2). Furthermore, ligand-mediated uptake of the polyplexes by EGFR-overexpressing cells was shown by flow cytometry experiments and by carrier-mediated transfection of a reporter gene. In addition, we report on the generation of a novel tumor-specific protein (TSP) as targeting ligand. Employing a bacterial expression system, a recombinant peptide is produced that binds to cell surface structures highly abundant on various tumor cells. Although quantitative amounts of TSP are expressed, only a very small portion of the protein is part of the soluble fraction. Due to an optimized purification procedure relying on denaturing conditions and subsequent column-based refolding of the protein, sufficient amounts are obtained. As demonstrated by flow cytometry measurements, TSP is biologi-cally active and ready to use for coupling to PEI. Taken together, we established coupling of the antibody cetuximab to PEI via a PEG-spacer resulting in a targeted drug delivery system that is a very promising platform for therapeutic knockdown strategies in vivo. Moreover, we present the development of a tumor-specific protein for the generation of novel tumor-targeted polyplexes. Acknowledgments: Deutsche Krebshilfe (A.A.), DFG Forschergruppe Nanohale (A.A.), Junior Research Grant from the Medical Faculty, University of Leipzig (S.H.)

References: 1. Werth, S. et al.: J. Control. Release. 2006, 112(2): 257-270. 2. Höbel, S. et al.: Eur. J. Pharm. Biopharm. 2008, 70(1): 29-41. 3. Höbel, S. et al.: J. Gene. Med. 2010, 12(3): 287-300. 4. Höbel, S., Vornicescu, D. et al.: Anal. Chem. 2013, Nov 20.

Halogenated Gold(I) NHC Complexes and their Antiproliferative Effects on Tumor Cells and Bacteria Schmidt, C.1; Sergeev, G.2; Franke, R.2; Brönstrup, M.2; Ott, I.1 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braun-schweig, Beethovenstr. 55, 38106 Braunschweig, Germany; 2 Department of Chemical Biology, Helmholtz Centre for Infection Research GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany

Thioredoxin reductase (TrxR) is an example for a relevant enzyme, which protects cells against oxidative stress and apoptosis. It is upregulated in carcinoma cells and represents a possible target in cancer chemotherapy. Especially gold(I) containing compounds inhibit TrxR in vitro, due to the high affinity of gold to selenocysteine moieties. A well-known drug is auranofin,

which is established in the current therapy of rheumatoid arthritis. It shows antiproliferative effects and triggers a remarkableTrxR inhibition. N-heterocyclic carbene (NHC) metal complexes have already shown antiprolif-erative effects on tumor cell lines as well as bacteria and have proven their potential as anticancer and antibacterial agents. [1-6] New halogenated mono- and bis-NHC-gold(I)-complexes of the imidazole-, benzimidazole- or phenylimidazole-type were synthesized, purified and characterized (see figure 1). To study the effects on tumor cell growth, they were tested against tumorigenic HT-29 colon carcinoma cells, MCF-7 breast carcinoma cells, MDA-MB-231 breast carcinoma cells and non-tumorigenic RC-124 human kidney cells. All complexes showed cell growth inhibition with IC50 values in the low micromolar range. In particular the activities of the bis-NHC-complexes were comparable to auranofin. The potency of TrxR inhibition of the gold(I) derivatives was determined with IC50 values in the submicromolar range. Cellular uptake studies were per-formed using high resolution continuum source atomic absorption spectrosco-py to quantify the intracellular concentration. Assays for antibacterial activities also proved the antiproliferative activities of the new halogenated gold(I) NHC complexes on different gram positive as well as gram negative bacterial strains. Agar diffusion tests were performed and the minimum inhibitory concentrations (MIC) were calculated. For some bacterial strains the gold(I) complexes showed lower MIC values than the established antibiotics ciprofloxacin and vancomycin, which were used as references. In conclusion, halogenated mono- and bis-NHC-gold(I)-complexes trigger strong antiproliferative effects in tumor cells as well as bacteria. Their mode of action involves the inhibition of thioredoxin reductase and related enzymes.

References: 1. Hickey, J.L. et al.; J Am Chem Soc. 2008, 130: (38):12570-12571. 2. Oehninger, L. et al.; Dalton Trans. 2013, 42, (10):3269-3284. 3. Liu, W. et al.; Chem. Soc. Rev. 2013, 42: 755-773. 4. Hackenberg, F. et al.; Organometallics 2013, 32: 5551−5560. 5. Öznur, D. et al.; MonatshChem. 2013, 144: 313–319. 6. Fernández, G.A. et al.; J. Inorg. Biochem. 2014, 135: 54–57.

Synthesis and biological studies of new alkynylgold(I)(NHC)-complexes as anticancer agents Prochnicka, A.; Ott, I. Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braun-schweig, Beethovenstraße 55, D-38106 Braunschweig,Germany

Current research activities deal with a great variety of metal-complexes, containing gold, ruthenium or platinum. Cisplatin and Carboplatin, for example, are used as anticancer agents and Auranofin is established in the therapy of rheumatoid arthritis. Organometallic gold(I) complexes are a new potent group of drugs, that show activity against tumor-relevant enzymes like thioredoxin reductase (TrxR) and have good antiproliferative activity in-vitro. [1] They are part of the attempt to develop new anticancer agents, which particularly show fewer side effects, a good tumor cell selectivity and no drug resistance. N-heterocyclic carbenes (NHC), phosphanes or alkynyls as ligands of gold(I) offer a high potential for the design of new anticancer agents. [2] Cellular uptake studies indicated, that such gold(I) complexes can accumulate in cancer cells. Many of these gold(I) complexes also showed selective activity against TrxR, which is one of the overexpressed enzymes in cancer cells. [3] A decrease in respiration suggested mitochondria as a further possible target. The group of alkynyl-gold(I)(phosphane)-complexes triggered anti-angiogenic effects, as shown in former work with zebrafish embryos. [4] The choice of stably coordinated ligands is critical in the design of new gold metallodrugs. Based on previous results we chose alkynyl and NHC ligands for coordination to gold(I) (see figure 1). The resulting alkynyl-gold(I)(NHC)-complexes offer a system with high stability under physiological conditions. The structural versatility of the organic ligands allows a broad variety of structural modifications and the preparation of compound libraries.

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In this pilot study alkynylgold(I)(NHC)-complexes, containing different NHC ligands derived from imidazole and benzimidazole and 1-ethynyl-4-methoxybenzene, were synthesized, purified and analyzed by nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy and elemental analysis. Cytotoxic and antiproliferative effects were evaluated in three tumor cell lines (MDA-MB-231 breast carcinoma, MCF-7 breast carcinoma and HT-29 colon carcinoma) as well as in healthy human kidney cell line (RC-124). To get more information about the biological properties of the new compounds, the activity against TrxR was measured and the cellular uptake was quantified using high resolution continuum source atomic absorption spectroscopy.

Fig.1 Design of alkynylgold(I)(NHC)-complexes

References: 1. Ott, I.: Coord. Chem. Rev. 2009, 253: 1670-1681. 2. Oehninger, L., Rubbiani, R., Ott I.: Dalton Trans. 2013, 42: 3269-3284. 3. Rubbiani, R. et al.: Med. Chem. Commun. 2013, 4: 942-948. 4. Meyer, A. et al.: Angew. Chem. 2012, 51: 8895-8899.

Comparison of three pharmacokinetic/toxicity models describing neutropenia caused by topotecan in cancer patients Henrich, A.1,2; Parra-Guillen, Z.1; Kloft, C.1 1 Dept. Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany 2 and Graduate Research Training program PharMetrX

Objectives: One of the most important dose-limiting toxicities in common anticancer therapies is myelosuppression leading e.g. to neutropenia. The pharmacokinetic/pharmacodynamic (PK/PD) model proposed by Friberg et al [1] describes the time-course of this life-threatening adverse event by assuming a linear, non-saturable effect of the drug concentration on the proliferation rate constant (kprol) of progenitor cells in the bone marrow. Another, probably more physiological approach is the maximum effect model (Emax), which would facilitate the use of parameters obtained in vitro such as the EC50 value of myelotoxicity assays. Thus, the objective of this work was to develop Emax models to link PK and PD of topotecan, an anticancer agent. These models shall be compared with the linear model concerning the ability to predict neutropenia. Methods: Concentration-time data for neutrophils were included from 71 patients pooled from three different trials receiving topotecan monotherapy [2]. Individual topotecan concentration-time profiles were simulated and a linear (model 1) and two different Emax models (model 2 and 3) for neutropenia were developed using NONMEM 7.2. Model 2 was a saturable Emax model with Emax = 1 and an additional elimination rate constant from the compartment with proliferating cells (kel), and model 3 was an Emax model with an estimated maximum effect. Both Emax models were compared with a linear model 1. Model evaluation was done using precision of parameter estimates, goodness of fit plots and visual predictive checks utilising PsN 3.5.3 and Xpose4 4.4.0. Results and Discussion: Adequate description of the time course of the neutrophil data, including nadir and time to recovery was possible with all three PK/PD models. As expected, different values for kprol were estimated for model 2 in comparison to model 1 and 3, given that this parameter in those models is a mixture of proliferation and elimination. The three PK/PD models were able to provide precise estimates for system-related parameters. Both Emax models, especially model 3 did not lead to precise drug-related parameter estimates (EC50 and Emax), probably caused by the cytotoxic drug effect (Edrug), which might be still in the linear part of the PK/PD relationship.

Conclusion: Model 2, including a saturable drug concentration-effect relationship on kprol with an additional elimination rate constant of proliferating cells, was able to successfully characterise the data. Given the more physio-logical approach, this model might be able to use in vitro measurements of cytotoxicity assays to predict the outcome of new drugs prior to treatment. Nevertheless, further experiments are needed in order to assess the validity of this model for different scenarios. References: 1. Friberg, L.E. et al.: J. Clin. Oncol. 2002, 20(24): 4713–4721. 2. Léger, F. et al.: Clin. Pharmacol. Ther. 2004, 76(6): 567-578.

Heterogeneous antibody based activity assay for lysine specific demethylase 1 (LSD1) on a histone peptide substrate Schulz-Fincke, J.1,2; Schmitt, M.1; Ladwein, K.I.1; Carlino, L.3; Willmann, D.4; Metzger, E.4; Schilcher, P.5; Imhof, A.5; Schüle, R.4; Sippl, W.3 ; Jung, M.1 1 University of Freiburg, Institute of Pharmaceutical Sciences 2 German Cancer Consortium, (DKTK); German Cancer Research Center, (DKFZ) 3 Martin-Luther-University of Halle-Wittenberg, Institute of Pharmacy 4 University of Freiburg Medical Center, Department of Urology/Women´s Hospital and Center for Clinical Research

5 Ludwig Maximilians University of Munich, Adolf-Butenandt Institute and Munich Center of Integrated protein science (CIPS)

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. Currently, work on Medicinal Chemistry and testing of new inhibitors is ongoing.

We thank DKFZ and DKTK for funding of research of synthesis and assay development of new LSD1 inhibitors.

References: 1. Spannhoff, A. et al.: ChemMedChem 2009, 4: 1568. 2. Arrowsmith, C.H. et al.: Nat. Rev. Drug Discov. 2012, 11: 384. 3. Willmann, D. et al.: Int. J. Cancer 2012, 131: 2704–2709. 4. Schmitt, M.L. et al.: J. Biomol. Screen. 2014, March [Epub ahead of print]

Carbamate inhibitors of NAD+-dependent histone deacetylases (Sirtuins) Swyter, S.1; Beese, K.2; Link, A.2; Jung, M.1

1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany 2 Institute of Pharmaceutical Sciences, Ernst-Moritz-Arndt-University Greifswald, 17489 Greifswald, Germany

Introduction: There are seven different human Sirtuins (Sirt 1-7), which are all NAD+-dependent enzymes [1]. Originally, Sirtuins have been classified as class III Histone deacetylases (HDACs). But it was shown that other non-histone targets like p53 or α-tubulin exist [2], as well as other enzymatic activities like deacylation of fatty acids [3]. Sirtuins are potential drug targets in age dependent diseases e.g. cancer or diabetes. The inhibition of Sirtuin-mediated p53-deacetylation is an example for a possible approach in the treatment of cancer [2]. Starting from lactone containing inhibitors like splitomicin [4], we studied other analogues with carbamate or thiocarbamate structures. Here we present screening results on Sirtuin 1-3.

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Assay screening: The screening is performed by a trypsin-coupled homogeneous assay, using the low-moleculare substrate ZMAL [5]. All compounds were pretested on auto-fluorescence, quenching effects and trypsin inhibition. Results: We were able to find some selective inhibitors with an IC50 in the low µM range, e.g. Benzoxazinones:

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Acknowledgement: We thank the DFG for funding (RTG 1976) References: 1. Trapp, J., Jung, M.: Curr. Drug Targets 2006, 7(11), 1553-1560. 2. Hoffmann, G. et al.: J Biol Chem. 2014, 289(8), 5208-5216. 3. Feldman, J.L., Baeza, J., Denu JM.: J Biol Chem. 2013, 288(43), 31350-31356. 4. Neugebauer, R.C. et al: J. Med. Chem. 2008, 51(5), 1203-1213. 5. Heltweg, B., Trapp, J., Jung, M.: Methods. 2005, 36(4), 332-337.

Investigation of genome-wide alterations of epigenetic signa-tures in the presence of the leukaemia-initiating MLL-AF4 and AF4-MLL complexes Löscher, D.; Kühn, A.; Kowarz, E.; Marschalek, R.

Institute of Pharmaceutical Biology, Biocentre, Goethe-University, Max-von- Laue-Str. 9, 60438 Frankfurt/Main, Germany It has been convincingly demonstrated that aberrant epigenetic modifications are a driving force in many cancers. Epigenetics is based on DNA methylation, and moreover, on covalent post-translational modifications of histone core proteins. Based on these findings, changes in the pattern of DNA or histone modifications are e.g. associated with genetic instability and are correlated with the rapid development of pre-cancerous cells. In some human diseases, such as MLL- rearranged leukaemia, these epigenetic changes seem to be sufficient for disease onset, without the requirement of further mutations1. Chromosomal translocations that involve the MLL (Mixed lineage leukaemia) gene result in the production of novel MLL fusion proteins which initiate critical steps of malignant transformation and lead either to the development of acute lyphoblastic leukemia (ALL) or acute myeloid leukemias (AML)2. The t(4;11) chromosomal translocation is one of the most frequent MLL translocations known today and is a major cause of infant acute lymphoplastic leukemia (ALL). Infant ALL is an aggressive disease with very poor outcome. t(4;11)(q21;q23) chromosome translocations fuse MLL in-frame with the AF4 gene and produce both the MLL-AF4 and AF4-MLL fusion proteins. In mammals the MLL protein is an example of a developmentally important protein that controls the epigenetic maintenance of gene activation. MLL possesses a C-terminal SET-domain that methylates histone H3 on Lysine 4 at

the start sites of transcriptionally engaged genes3. H3K4 methylation is associated with transcriptional activation. These H3K4-modified regions are largely constrained to the transcription start site regions of genes that are transcriptionally active. The fusion partner AF4 is part of a nuclear machinery that activates RNA-Polymerase II transcriptional elongation by P-TEFb and mediates coordinated chromatin remodeling4 but exerts also chromatin modifying activity (H3K79, H3K36, etc). First results with affinity-purified AF4-MLL fusion protein complex showed that the purified complex exhibits properties of both MLL and AF4 wildtype proteins. It exhibits H3K4 and H3K79 histone methyltransferase activity and is able to activate P-TEFb kinase which leads to an strong activation of the RNA-Pol II and thus influences the elongation process5. In contrast the MLL-AF4 fusion protein lacks the C-terminal SET-domain, but is still capable of enhancing gene expression by recruitment of the endogenous AF4 complex, thereby causing extended H3K79 signatures6. Therefore we want to further investigate the mechanism that causes the disease by studying genome-wide alterations of epigenetic signatures in the presence of MLL-AF4, AF4-MLL or the combination of both reciprocal fusion proteins. This work is funded by the DFG grant Ma 1876/10-1 to RM. References: 1. Scholz, B. and Marschalek R.: Br. J. Haematol. 2012, 158(3): 307-322. 2. Hess, J. L.: Trends Mol Med. 2004, 10(10): 500-507. 3. Dou, Y. et al.: Nat. Struct. Mol. Biol. 2006, 13(8): 713-719. 4. Bitoun, E., Oliver P.L., Davies K.E.: Hum. Mol. Genet. 2007, 16(1): 92-106. 5. Benedikt A. et al.: Leukemia 2011, 25(1): 135-144. 6. Krivtsov A. V. et al.: Cancer Cell 2008, 14(5): 355-368.

Investigating the interplay between the Iroquois homeoprotein family and the MLL-AF4 complex

Kühn, A.; Löscher, D. ; Kowarz, E.; Marschalek, R.

Pharmazeutische Biologie, Max-von-Laue Straße 9, Biozentrum, Frankfurt Campus Riedberg

The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is the most frequent target gene of chromosomal translocations and rearrangements in childhood leukemia and therapy related leukemia. Until now 80 different translocation partners of the MLL-gene have been cloned, all of which causing either acute myeloid (AML) or acute lymphoblastic (ALL) leukemia [1]. In our working group we focus on the t(4;11) leukemia, where the MLL gene of chromosome 11 is fused to the AF4 gene of chromosome 4 leading to the 2 chimeric proteins MLL-AF4 and AF4-MLL, respectively. Within all ALL-patients, the t(4;11)-mediated leukemia is the most common form of MLL-rearrangements. Especially pediatric leukemic patients bear this form of translocation and mostly having a poor outcome with an overall survival of only about 25-45%, due to a high risk of relapse despite the treatment with high risk protocols. Until 2009 it was generally accepted that MLL-fusion genes were recognized as transcriptional deregulators of HOXA genes claiming that all MLL fusion proteins work by a similar concept: they increase and maintain high level transcription of MEIS1 and HOXA gene family members [2]. In contrast to these findings Trentin et al. could show that only about one half of the examined t(4;11)-patient cohort exhibits the expected overexpression of HOXA-gene [3]. The other part showed a decreased HOXA level. An idiosyn-cratic upregulated gene in this “HOXA-low” patient group was the transcription factor Iroquois 1 (IRX1). Further work confirmed the presence and importance of IRX1 in t(4;11) patients [4,5]. IRX1 is a member of the homeoprotein family and binds to the promotor regions of target genes in a sequence specific manner [6]. Therefore it is classified as a transcription factor being important for pattern formation, especially for lung, brain and heart development [7] [8]. Furthermore it acts as a tumor suppressor gene in gastric cancers [9]. Our focus stands now on the investigation of the interplay between the Iroquois family members and MLL and its derivatives. At first we performed microarray analysis to explore which genes were up- or down-regulated in the presence of ectopically expressed IRX1. During our work we were able to demonstrate that IRX1 itself causes the down-regulation of HOXA-genes in a dominant fashion, explaining the observed downregulation of HOXA genes in the “HOXA-low” patient group. Furthermore we were able to demonstrate that Iroquois proteins (IRX1 and IRX2) seem to counteract the molecular actions deriving from the MLL-AF4 protein.

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This work is funded by the DFG grant MA1876/10-1 to RM.

References: 1. Meyer, C. et al.: Leukemia. 2013, 27(11):2165-2176. 2. Ayton, P.M. and Cleary, M.L.: Genes Dev. 2003, 17(18): 2298-2307. 3. Trentin, L. et al.: Eur. J. Haematol. 2009, 83(5): 406-419. 4. Stam, R.W. et al.: Blood. 2010, 115(14): 2835-2844. 5. Kang, H. et al.: Blood. 2012, 119(8): 1872-1881. 6. Bilioni, A. et al.: Proc. Natl. Acad. Sci. U.S.A. 2005, 102(41): 14671-14676. 7. Doi, T. et al.: J. Pediatr. Surg. 2011, 46(1): 62-66. 8. Gómez-Skarmeta, J.L. and Modolell, J.: Curr. Opin. Genet. Dev. 2002, 12(4): 403-408. 9. Guo, X. et al.: Oncogene. 2010, 29(27): 3908-3920.

Inhibition of class I HDACs abrogates the dominant effect of MLL-AF4 at the 5-lipoxygenase promoter by activation of MLL Ahmad, K.1; Katryniok, C.1; Scholz, B.2; Merkens, J.2; Marschalek, R.2; Steinhilber, D.1 1 Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany 2 Goethe University Frankfurt, Institute of Pharmaceutical Biology, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany

5-Lipoxygenase (5-LO), which is encoded by the ALOX5 gene and mainly expressed in leukocytes, is an enzyme that catalyzes the first two steps in the biosynthesis of leukotrienes derived from arachidonic acid. Physiologically, leukotrienes are a part of the innate immune system. In the pathophysiological context, leukotrienes are associated with inflammatory, allergic and cardiovas-cular diseases as well as certain types of cancer [1]. Thus, there is a reasona-ble interest to understand the regulation of ALOX5 gene expression. Previously, it was shown that the ALOX5 promoter is activated by the pan-histone deacetylase (HDAC) inhibitor TSA leading to enhanced transcript initiation [2-3]. However, the molecular mechanism behind the induction of 5-LO transcript initiation by HDAC inhibitors remained unknown. By chromatin immunoprecipitation, we observed that induction of 5-LO mRNA expression by HDAC inhibition correlates with histone H3 lysine 4 trimethylation (H3K4me3). The SET domain of the MLL (mixed lineage leukemia) protein catalyzes the formation of H3K4me3 [4,5]. In order to study the role of MLL on 5-LO promoter activity, an ALOX5 promoter construct was cotransfected with expression constructs for MLL or the leukemogenic fusion proteins MLL-AF4 (der11) and AF4-MLL (der4) [6,7]. The constitutively active MLL derivative MLL-AF4 stimulated 5-LO promoter activity by more than 50-fold whereas wildtype MLL was inactive. Addition of class I HDAC inhibitors (which lead to MLL activation) induced 5-LO promoter activity in an MLL-dependent fashion. Interestingly, in the presence of constitutively active MLL-AF4, addition of HDAC inhibitors attenuated its activity in an MLL-dependent manner. Thus, these results reveal that HDAC class I inhibitors can attenuate the oncogenic effects of MLL-AF4 by activation of wildtype MLL, suggesting that these compounds might be of considerable therapeutic interest for the treatment of leukemias with constitutively active oncogenic MLL fusion proteins. In subsequent experiments, we found that class I HDAC inhibitors are indeed very effective inhibitors of cell growth of a leukemia cell line that carry the respective t(4;11) translocation leading to these MLL fusion proteins.

Acknowledgments:

We are grateful to Else Kröner-Fresenius-Stiftung (Dr. Hans-Kröner-Graduiertenkolleg) and CEF for financial support.

References: 1. Rådmark, O. et al.: Trends Biochem. Sci. 2007, 32(7): 332-341. 2. Klan, N. et al.: Biol. Chem. 2003, 384(5): 777-785. 3. Schnur, N. et al.: Biochim. Biophys. Acta. 2007, 1771(10): 1271-1282. 4. Milne, T.A. et al.: Mol. Cell. 2002, 10(5): 1107-1117. 5. Wang, P. et al.: Mol. Cell. Biol. 2009, 29(22): 6074-6085. 6. Marschalek, R.: FEBS J. 2010, 277(8): 1822-1831. 7. Meyer, C. et al.: Leukemia 2013, 27(11): 2165-2176.

Modulation of Dicer-mediated microRNA processing by 5-lipoxygenase - roles in inflammation & cancer

Scholl, F.1; Basavarajappa, D.1; Weigert, A.2; Suess, B.3; Steinhilber, D.4; Rådmark, O.1 1 Department of Medical Biochemistry and Biophysics (Kemi II), Karolinska Institutet, Scheeles väg 2, 17177 Stockholm, Sweden 2 Institute of Biochemistry I, Goethe University Frankfurt, Theodor-Stern-Kai 7, building 74, 60590 Frankfurt, Germany 3 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt, Germany 4 Biology Department, TU Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany

Leukotrienes (LTs) are inflammatory mediators also known to be involved in cancer. They are synthesized by 5-Lipoxygenase (5-LO)[1]. In vitro studies revealed that 5-LO can interact with Dicer, an enzyme responsible for the microRNA (miRNA) maturation [2]. MicroRNAs are transcribed as primary miRNAs (pri-miRNA) and further processed by Drosha to intermediates (pre-miRNA). These intermediates then undergo a final cleavage to mature miRNAs by Dicer [3]. The aim of this study is to determine if 5-LO has any impact on biosynthesis of miRNAs in the monocytic cell line Mono Mac 6. Possibly, interaction of 5-LO with Dicer in the cell could modulate Dicer activity. We first performed a microarray to compare the miRNA expression in 5-LO knockdown cells (Δ 5-LO) and cells treated with a control shRNA[4]. The strongest effect appeared for hsa-miR-99b-5p and hsa-miR-125a-5p. Interestingly, these miRNAs are organized in a common pri-miRNA and enclose a third miRNA, hsa-let-7e-5p. However, an approximately 2-fold decrease of miR-125a-5p and miR-99b-5p in Δ 5-LO could be confirmed whereas let-7e-5p did not show a significant change. Since the pri-miRNA level are unaffected by Δ 5-LO, the data suggest that the regulation occurs at the level of miRNA processing. Further on, we investigated whether the downregulation of the mature miRNAs could be mimicked by blocking leukotriene biosynthesis. But neither the inhibition of 5-LO led to a decrease in miRNA production nor led the additional treatment with leukotrienes to an upregulation. This showed that the 5-LO protein and not its products is necessary for maturation of specific miRNAs. MiR-125a and miR-99b are already known to be involved in inflammatory processes [5, 6] . We could show that interleukin 6 is increased in the absence of miR-125a and miR-99b and especially tumornecrosis-factor-α (TNF-α) is significantly elevated in cells lacking both miRNAs. Interestingly Woo et al. showed that TNF- α has an impact on 5-LO activity [7]. To summarize, our findings suggest that 5-LO can directly interact with Dicer to fine-tune inflammatory responses. This occurs by a negative feedback mechanism with miRNAs and TNF- α as linker.

We thank the Else Kröner-Fresenius-Stiftung (Dr. Hans Kröner Graduierten-kolleg) for financial support.

References: 1. Radmark, O. and Samuelsson, B.: J. Intern. Med. 2010, 268(1): 5-14. 2. Dincbas-Renqvist, V. et al.: Biochim. Biophys. Acta 2009, 1789(2): 99-108. 3. Ha, M. and Kim, V.N.: Nat. Rev. Mol. Cell Biol. 2014. 4. Basavarajappa, D. et al.: Proc. Natl. Acad. Sci. USA 2014. 5. Banerjee, S. et al.: J. Biol. Chem. 2013, 288(49): 35428-35436. 6. Singh, Y. et al.: J. Biol. Chem. 2013, 288(7): 5056-5061. 7. Woo, C.H. et al.: J. Biol. Chem. 2000, 275(41): 32357-32362.

Compound C causes resistance to tubulin inhibition independent of AMPK – possible role of c-Myc ubiquitination? Scherzberg, M.-C.1, Steinhilber, D.1, Ulrich-Rückert, S.1 1 Institute of Pharmaceutical Chemistry Goethe-University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt a.M., Germany

Tubulin inhibitors belong to the most frequently used anti-cancer drugs. In recent years it became evident, that beyond affecting the mitotic spindle apparatus, also anti-mitotic activities influencing crucial cellular functions seem

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to underlie the established efficacy [1]. C-Myc is a transcription factor, which expression levels closely correlate with the proliferation of mammalian cells [2]. Therefore its degradation is tightly controlled by the ubiquitin-proteasome system [3]. Compound C is a widely used kinase inhibitor to investigate biological responses mediated by AMPK although recent studies propose that Compound C exerts effects which cannot be attributed to AMPK inhibition [4,5]. The inhibition of tubulin polymerisation or –depolymerisation with various tubulin inhibitors significantly reduces cell growth of HT-29 cells (***p<0.001) determined by crystal violet staining. Interestingly, combination with the AMPK inhibitor Compound C significantly counteracts this effect (***p<0.001) without actually affecting the AMPK signalling pathway, which was confirmed by RNAi experiments. FACS analysis revealed that Compound C considerably attenuates tubulin inhibitor induced G2/M arrest and increases the number of cells in G0/G1 phase (e.g. 17% for vinblastine, *p<0.05). Western Blot experiments showed multiple bands for c-Myc after tubulin inhibition for 8-16h followed by complete disappearance after 24h, which suggests an intensive posttranslational modification of the c-Myc protein leading to subsequent degradation. Combination with Compound C partly prevents this occurrence of higher molecular bands for c-Myc. To evaluate if these multiple bands for c-Myc might be due to ubiquitination and proteasomal degradation cells were treated with proteasome inhibitor MG132 and blotted for c-Myc protein. Interestingly, the same pattern of multiple bands also observed with tubulin inhibitors was evident. Furthermore, similar to Com-pound C, also MG132 was able to significantly reverse tubulin inhibitor induced growth arrest (***p<0,001). We should take into consideration that Compound C induced resistance to microtubule interfering agents in human colon cancer cells might be due to an altered process of c-Myc ubiquitination and degradation, but the exact mechanism remains to be elucidated.

References: 1. Fürst, R., Vollmar, A.: Pharmazie 2013, 68(7): 478-83. 2. Schuhmacher, M., Eick, D.: Transcription 2013, 4(4): ahead of print. 3. Farrell, A., Sears, R.: Cold Spring Harb Perspect Med 2014, 4(3). 4. Vucivecic, L. et al.: Autophagy 2011, 7(1): 40-50. 5. Kim, Y. et al.: Atherosclerosis 2011, 219(1): 57-64.

Ionizing radiation-induced glioblastoma cell migration in vivo Butz, L.1,2; Stegen, B.2; Zips, D.2; Buschauer, A.3; Huber, S.M.2; Ruth, P.1

1 Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen 2 Department of Radiation Oncology, University of Tübingen, Hoppe-Seyler-Str. 3, 72076 Tübingen 3 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, 93040 Regensburg

Invasion of the brain by glioblastoma cells reportedly requires Ca2+-activated IK and BK K+ channels in the plasma membrane. These channels are involved in both, the generation of Ca2+ signals that program cell migration/invasion and cell volume changes that motorize migration. Moreover, ionizing radiation (IR, 2 Gy) has been shown to stimulate glioblastoma cell migration in vitro by increasing K+ channel activity. Importantly, pharmacological targeting of these channels abolishes IR-induced migration in vitro. The present project aims to monitor in an orthotopic mouse model the migration of glioblastoma cells during fractionated radiation and to define the role of K+ channels herein. Human U87MG Kat glioblastoma cells (30,000) which express the Katushka fluorescence protein and which have high copy numbers of functional IK and BK K+ channels were injected stereotactically into the right striatum of immunocompromised NSG (NOD scid gamma) mice and grown for 7 days to solid glioblastomas with a volume of about 1 mm3.On days 7 until 11 after tumor challenge, mice received daily fractions of 0 (control group) or 2 Gy X-ray to a total dose of 10 Gy (irradiated group) delivered to an 5 x 3 mm2 area of the tumor bearing right hemisphere by the use of a 6 MV linear accelerator. The mouse torso and the mouse head outside the target-volume were shielded by the multi leaf collimator and an 8 cm thick on-body lead block, respectively. Film dosimetry using a mouse head phantom suggested a steep dose gradient at the field margin with a residual dose of below 2% at the shielded brain area. On day 21, mice were sacrificed, dissected brains were fixed for 24 h with 2% paraformaldehyde in PBS, postincubated for 24 h in 30% sucrose in PBS,

frozen, sliced into 20 µm thick sections and the Katushka fluorescence of the glioblastoma cells was analyzed by fluorescence microscopy. As a result, tumor challenge resulted in reproducible formation of U87MG Kat tumors. In addition, mice tolerated the 5 fractions of partial head irradiation very well. Importantly, fractionated migration increased number of emigrated cells (437 ± 51 vs. 253 ± 30, n= 5-6 mice, p < 0.02) suggesting radiation-induced hypermigration in vivo. This hypermigration was paralleled by a radiation-induced increase in tumor-associated SDF-1 protein abundance as demonstrated by immunofluorescence microscopy. Moreover, preliminary data suggest that the BK K+ channel inhibitor paxilline (8 mg/kg BW i.p.) applied 6 h prior to and 6 h after each radiation fraction abolishes the radiation-induced hypermigration while having no effect on tumor spreading of un-irradiated glioblastomas. In conclusion, the orthotopic U87MG Kat mouse model seems to be well suited to study hypermigration of glioblastoma cells during fractionated radiation in vivo and to disclose potential effects of fractionated radiation and K+ channel targeting. Acknowledgements: This work has been supported by the Wilhelm-Sander-Stiftung (Grant: 2011.0831.1)

Characterization of 5-lipoxygenase overexpression in tumor cell lines Dos Santos Capelo, R.; Brüggerhoff, A.; George, S.; Steinhilber, D.; Kahnt, A.S.

Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany.

Accumulating evidence from laboratory and epidemiological studies suggests that aberrant 5-lipoxygenase (5-LO) activity can promote carcinogenesis. In contrast to healthy tissues the enzyme is frequently over expressed in solid malignancies of the prostate, colon and lung, to name a few. There is a correlation between 5-LO overexpression and cancer cell formation, prolifera-tion and metastasis. Nevertheless, the enzyme’s direct role in the formation of solid tumours remains elusive so far. Profound understanding of the enzyme’s role in carcinogenesis would help to predict if combination of leukotriene inhibitors with chemotherapy regimens constitutes a promising approach to successfully treat 5-LO overexpressing tumors. We screened different tumour cell lines for 5-LO expression. Out of these we chose 2 over expressing cell lines (Capan-2, HT-29) and checked them for the presence of other enzymes of the leukotriene machinery (5-LO-activating

protein (FLAP) and cytosolic phospholipase A2(cPLA2)) by western blot

analysis. FLAP and cPLA2were expressed in both cell lines, pointing to functional leukotriene generation in these cell lines. Next, intact cells were treated with different stimuli to trigger leukotriene formation. Interestingly, no enzyme activity was found. In contrast, broken cell preparations showed robust 5-LO activity leading to the conclusion that the enzyme was functional but not activated in these cells due to an unknown inhibitory mechanism. 5-LO activity is tightly regulated by different factors such as glutathione peroxidases controlling the intracellular redox tone and activating and inactivating phos-phorylations. We could show that the enzyme is phosporylated on Ser523, a PKA-dependent modification known to inhibit enzymatic activity in both cell lines. Nevertheless, preincubation of HT-29 cells with different PKA inhibitors prior to stimulation did not reconstitute enzyme activity. Also incubation with diamide, to impair GPx activity and enhance oxidative stress had no impact on leukotriene formation. Therefore, further studies are needed to elucidate the role of 5-lipoxygenase in tumor progression and survival.

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Detection of Extracellular Glyosylphosphatidylinositol-Anchored Proteins Within Phospholipid Complexes Using a Biosensor for the Prediction and Stratification of Type 2 Diabetes Müller, G.; Tschöp, M.

Helmholtz Center Munich, Institute for Diabetes and Obesity, Am Parkring 13, 85478 Garching-Hochbrück, Germany

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) have been shown to exhibit high susceptibility for release in ECGAPP from the surface of mamma-lian cells in vitro and in vivo in response to cellular and metabolic stress, such as high glucose, fatty acids and reactive oxygen species [1,2], that is prevalent during the pathogenesis of type 2 diabetes (T2D). The underlying non-classical secretory mechanism is commonly thought to rely on the anchorage of GPI-APs in the outer leaflet of the plasma membrane phospholipid bilayer by the glycosylphosphatidylinositol moiety, exclusively, that is covalently attached to the carboxyl-terminus of the polypeptide chain. However, the presence of ECGAPP in the plasma of T2D patients, that are faced with elevated blood glucose and free fatty acids levels, has not been studied so far. This is presumably due to conceptual restrictions (i.e. reductionistic and holistic thinking) and technological challenges. To overcome these hurdles, a novel type of chip-based biosensor will be used for the specific detection and biophysical characterization of ECGAPP. Its principle relies on the generation of horizontal surface acoustic waves (SAW) of defined frequency and amplitude within the gold surface of a microfluidic four-channel chip. Any interaction of (macro)molecules with the gold surface will result in correspond-ing changes in the shape of the SAW, altering both their frequency and amplitude. These alterations reflect mass loading (i.e. binding of ECGAPP) to and biophysical properties (i.e. size and shape depending on the ECGAPP protein composition as well as viscoelasticity and rigidity depending on the ECGAPP phospholipid composition, cholesterol content and open/empty-closed/filled configuration) at the chip surface. The major advantages of the SAW vs. the commonly used surface plasmon resonance biosensor rely on the possibility of measurement of large (lipid-containing) macromolecules even in the presence of serum [3] as well as on the potential high sensitivity towards alterations in the composition (proteins, phospholipids) and structure (micelles, nanodiscs, vesicles, particles) of the ECGAPP. Albeit SAW biosensing per se does not enable the delineation of the type of ECGAPP contained in a given sample, the SAW signatures will be characteristic for the overall contents of all ECGAPP, either as summation signals or as 1D-/2D-signatures of high informative value.

References: 1. Müller, G. et al.: Br. J. Pharmacol. 2009, 158: 749-770. 2. Müller, G. et al.: Cell. Signalling 2009, 21: 324-338. 3. Gronewold, T.: Anal. Chim. Acta 2007, 603: 119-128.

One substrate – seven products with different prenylation positions in one-step reactions by using fungal prenyltransfer-ases Fan, A.; Li, S.-M.*

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany

Prenylated indole alkaloids are widely distributed in nature and show diverse biological and pharmacological activities, usually distinct from their non-prenylated precursors. Prenyltransferases catalyze the transfer reactions of prenyl moieties onto indole nucleus and contribute largely to the structural diversity of these compounds. Based on their sequences and biochemical properties, prenyltransferases can be divided into different subgroups.[1;2] One of the most investigated subgroup is the dimethylallyl tryptophan synthase (DMATS) superfamily. So far, more than 30 such prenyltransferases have been identified and characterised biochemically[3-5]. The majority of the DMATS superfamily can be classified into tryptophan and tryptophan-containing cyclic dipeptide prenyltransferases according to their substrates. In this study, we demonstrate the acceptance of cyclo-L-homotryptophan-D-valine, an unnatural cyclic dipeptide, by three tryptophan prenyltransferases and five cyclic dipeptide prenyltransferases. Seven products with one prenyl moiety at each position of the indole nucleus and one diprenylated derivative were isolated from enzyme assays of cyclo-L-homotryptophan-D-valine with dimethylallyl diphosphate as prenyl donor. Our results presented here expand potential usage of these enzymes in the production of prenylated derivatives.

Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft (to S.-M. Li). A. Fan is a recipient of a fellowship from China Scholarship Council. References: 1. Li, S.-M.: Appl.Microbiol.Biotechnol. 2009, 84; 631-639. 2. Heide, L.: Curr.Opin.Chem.Biol. 2009, 13: 171-179. 3. Winkelblech, J.; Li, S.-M.: Chembiochem. 2014, 15:1030-1039. 4. Yu, X.; Li, S.-M.: Methods Enzymol. 2012, 516: 259-278. 5. Miyamoto, K. et al.:, Bioorg.Med.Chem. 2014, 22: 2517-2528.

Zebrafish as model organism for pharmacological research on soluble guanylyl cyclase Dittmar, F.1; Bähre, H.1,2; Kaever, V.1,2; Seyfried, S.3; Seifert, R.1 1 Institute of Pharmacology, Hannover Medical School, 30625 Hannover, Germany 2 Research Core Unit Metabolomics, Hannover Medical School, 30625 Hannover, Germany 3 Max-Delbrück-Centrum für molekulare Medizin (MDC), 13125 Berlin, Germany

The zebrafish Danio rerio has become an important model organism for a wide range of scientific research questions regarding vertebrates [1]. Current studies are mainly focused on development, genetics and disease [1]. The beneficial features of the zebrafish include its small size, rapid development,

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short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1]. Furthermore, application of drugs is simple as zebrafish absorb compounds from their surrounding media [2]. The aim of our study was to examine the impact of various activators and stimulators on the soluble guanylyl cyclase (sGC) in Danio rerio. Therefore, embryos were treated with these compounds from one day post fertilization (dpf) to five dpf. First of all, we determined if cyclic nucleotides occur naturally in several developmental stages and organs of this vertebrate. The well-established purine nucleotides cAMP and cGMP as well as the little-known pyrimidine nucleotides cCMP and cUMP were detected in embryos, larvae and some organs of adult zebrafish via high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Treatment of embryos with the NO-synergistic sGC stimulator 3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (Bay 41-2272) increased cGMP concentrations. Current studies comprise similar treatment with the NO-independent sGC activator cinaciguat (Bay 58-2667). Future research will focus on sGC expression pattern in different tissues and the entire organism as well as treatment of redox-mutants with the abovementioned sGC drugs. Analysis of the NO-sGC system in Danio rerio will provide valuable information on drugs for cardiovascular diseases.

References: 1. Lawrence, C.: Aquaculture 2007, 269: 1–20. 2. Langheinrich, U.: BioEssays 2003, 25: 904–912.

Surface modifications of polyethylene sinter bodies for serologi-cal diagnosis of borreliosis Alasel, M.; Dassinger, N.; Vornicescu, D.; Keusgen, M.

Institute of Pharmaceutical Chemistry, Marbacher Weg 6-10, D-35032, Marburg, Germany

Abstract: Allyl alcohol was utilized as a monomer to introduce hydroxyl groups on the surface of 3D-polyethylene sinter bodies. Using UV photografting technique, allyl alcohol could be polymerized on the surface of the polyeth-ylene providing active hydroxyl groups that can be linked via (3-aminopropyl)triethoxy silane (APTES) to polysaccharides like mannan. In the next step, a fusion protein consisting of the lectin binding domain ConA and a Borrelia surface antigene has been immobilized by self-organization. Keywords: UV photografting, monomer, allyl alcohol, diagnostic immunoas-say. Introduction For the immobilization of biomolecules on solid polymer surfaces, a primary functionalization of these materials is necessary. Generally functionalization of polyethylene takes place using wet chemistry, dry chemistry like plasma activation or photochemistry. One of the easiest methods in photochemistry is by ultraviolet light (UV) in order to activate chemical bounds. This technique can be also applied to polyethylene polymers, which later were utilized in serological diagnosis of Lyme Borreliosis disease [1]. Results and Discussion Functionalization of the 3D-polyethylene surface has been successfully performed utilizing the radical reaction of allyl alcohol with the polymer after their exposure to UV light in the presence of an initiator (benzophenone). Surface modification has been confirmed by Fourier transform infrared spectroscopy (FTIR) (Figure1) and surface electron microscopy (SEM) measurements. After this primary surface modifications, hydroxyl groups were linked to self assembled mono layers of (3-aminopropyl)triethoxy silane (APTES) to provide the surface with amine groups. For amine coupling reaction, mannan was first activated using N,N-disuccinimidyl carbonate (DSC) followed by kovalent immobilization via amide bounds. Using a fusion protein with one lectin (Concanavalin A; ConA) part and another antigen (lyme antigen) part, serological diagnosis of Lyme Borreliosis was possible. Test could be successfully performed with different Borrelia + and – sera.

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Conclusions Functionalization of the 3D-polyethylene sinter bodies has been successfully achieved using photografting technique. The obtained hydroxyl groups could be utilized for further immobilizations. The final coating by the polysaccharide mannan allows fixation of genetically designed fusion proteins with a ConA moiety by self-organization. The latter step is a rather smooth procedure, which does not influence the biological functionality of the fusion protein in a negative manner.

Acknowledgements: I would like to thank Yousef Jameel scholarship fund for funding me during my research.

References: 1. Bandopadhay, D. et al.: Journal of Applied Polymer Science 2004, 92: 3046–3051.

Crystal Structure of Blood Coagulation Factor XIII: Template for the Design of a Novel Anticoagulant Stieler, M.1; Weber, J.2; Hils, M.2; Kolb, P.1; Heine, A.1; Büchold, C.2; Paster-nack, R.2; Klebe, G.1 1 Institut of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35037 Marburg, Germany 2 Zedira GmbH, Roesslerstrasse 83, 64293 Darmstadt, Germany

Blood coagulation factor XIII (FXIII) is the last enzyme of the blood coagulation cascade and represents one of the most promising target for the development of safer alternatives to presently administered anticoagulants such as heparins, vitamin K antagonists or direct acting thrombin and factor Xa inhibitors. All the latter drugs affect the level of thrombin, which activates not only fibrin assembly for coagulation but also stimulates platelets, a prerequisite for primary clot formation. Thus, undesirable and even life-threatening bleeding episodes can result. While other enzymes of the cascade are serine proteases, FXIII is a plasma transglutaminase catalyzing isopeptide bond formation. It acts downstream of thrombin, effectively determining mechanical stability, half-life and lysis rate of clots. FXIII has been discussed as ideal target to interfere with coagulation, but no inhibiting drug candidates are available to explore its pharmacological potential. This is mainly due to the fact that a protein structure representing the relevant active state is unknown. Here we report the first high-resolution crystal structure (1.98 Å) of FXIII in the active state complexed with an irreversible inhibitor. Only crystal structures of inactive, homodimeric FXIII have been reported so far where the active site is completely buried and any access to the catalytic center is obscured. Such structures are unsuitable for drug design. In the present structure, three calcium ions recruit polar functional groups of the protein and establish local metal ion coordination sites which induce rearrangements of two domains along with local adaptations of the catalytic domain to expose the enzyme in its active state. The observed transformations establish the substrate and co-substrate binding sites for isopeptide bond formation and suggest involvement of a catalytic triad and a diad in the enzyme mechanism which is considered valid for the entire transglutaminase family.

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Figure 1: FTIR spectra of unmodified (―) and modified ( )

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We acknowledge the beamline support of Bessy II in Berlin for practical help and the HZB for travel grants. Furthermore we acknowledge the BMBF for financial support. References: 1. Stieler, M. et al: Angewandte Chemie (Int. Edition) 2013, 52(45): 11930-11934. 2. Shebuski, R. J. et al: Blood 1990, 75(7): 1455-1459.

α-Aminoxy Peptoids: A Unique Peptoid Backbone with a Prefer-ence for Cis Amide Bonds Syntschewsk, V.1; Ciglia, E.1; de Sousa Amadeu, N.2; Vasylyeva, V.2; Janiak, C.2; Kurz, T.1; Gohlke, H.1; Hansen, F.K.1 1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany 2 Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany

α-Peptoids (Fig. 1, A) are oligomers of N-substituted glycine and feature several advantages over peptides as potential bioactive compounds, among them proteolytic stability and increased cell permeability. [1,2] Peptoid libraries have been utilized as protein binding agents and as inhibitors of protein-protein interactions, [3] although primary screening hits identified from peptoid libraries have usually not displayed high activity or potency. [4]

Figure 1: A) Selected peptoid backbones, B) X-ray structure of an α-aminoxy tripeptoid, C) helical arrangement of a hexamer derived from MD simulations.

Due to the absence of internal hydrogen bonding, peptoids are less conforma-tionally restrained than peptides. This may adversely affect their binding affinity to proteins. Thus, it is important to study the preferred conformation of the peptoid backbone. Peptides possess mostly trans-amide bonds, apart from proline, whereas a high degree of cis-amide bonds is observed in α-peptoids. [5] Based on the fact that cis- and trans-amide conformations are almost isoenergetic for N-alkyl α-peptoid monomers, studies have shown that the nature of the side chain can significantly modulate the ratio of cis-/trans-amide bond conformers. [6] Hence stable, helical secondary structures in α- -peptoids can be achieved by an optimally controlled cis-/trans isomerism. This tactical incorporation of specific cis- or trans-directing side chains enables the control of the folding properties of α- and β-peptoids. However, this comes at the expense of side chain diversity. A preferable peptoid backbone should therefore not only predispose a peptoid to adopt a specific conformation but also allow the introduction of different substituents. Thus, it is worthwhile to address this limitation and to design peptoid derivatives with conformationally constrained backbones. In 2002, Shin and Park reported the preparation of α-aminoxy peptoid pentamers. However, the folding properties were not investigated. [7] Learning from the backbone-controlled secondary structures of α-aminoxy peptides, we reasoned that α-aminoxy peptoids can form stable secondary structures due to an increased energetic difference between cis and trans conformers. We

therefore studied the conformational preference of α-aminoxy peptoids in established peptoid model systems. A comprehensive analysis of model peptoids by 1D and 2D NMR spectroscopy, X-ray analysis (Fig. 1, B), and computational conformational analysis (Fig. 1, C) revealed that α-aminoxy peptoids prefer a cis-configuration even in the presence of side chains that usually are giving inhomogeneous mixtures of cis- and trans-configured amides. Thus, α-aminoxy peptoids represent an interesting novel pep-tidomimetic backbone architecture featuring a distinct folding pattern. References:

1. Miller, S.M. et al.: Bioorg. Med. Chem. Lett. 1994, 4(22): 2657–2662.

2. Kwon, Y.-U.; Kodadek, T.: J. Am. Chem. Soc. 2007, 129(6): 1508–1509. 3. Reddy, M.M.; Bachhawat-Sikder, K.: Kodadek T. Chem. Biol. 2004, 11(8): 1127–1137. 4. Suwal, S.; Kodadek, T.: Org. Biomol. Chem. 2013, 11(13): 2088–2092.

5. Hodges, J.A.; Raines, R.T.: Org. Lett. 2006, 8(21): 4695-4697. 6. Yoo, B.; Kirshenbaum, K.: Curr. Opin. Chem. Biol. 2008, 12(6): 714–721. 7. Shin, I.; Park, K.: Org. Lett. 2002, 4(6): 869–872.

Thrombin Revisited: Using a well established Protein to gain new insights on Protein-Ligand Interactions Collins, C.; Weimer, D.; Biela, A.; Heine, A.; Klebe, G.

Institute for Pharmaceutical Chemistry, Phillips-University Marburg, Marbacher Weg 6, 35032, Marburg, Germany

Thrombin is an important drug target in the prevention of blood clotting making it a well studied protein. Due to the large prevalence of Cardiovascular Disease throughout the world population, research in this field is immense. Anticoagulants such as thrombin inhibitors play a key role in preventing heart attacks and strokes. One of the newest being Dabigatran, an oral thrombin inhibitor. Researchers are able to discover and characterize these new drugs with the help of methods such as protein crystallography, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR). Using crystallog-raphy, structures of the protein with the inhibitor can be determined and the exact position of binding can be elucidated. ITC is a method which uses the change in Enthalpy ΔH (the heat of an interaction) and the Gibbs Free Energy ΔG to determine the thermodynamic profile. With this thermodynamic data the binding modes of compounds can be compared which aids in the process of drug development and allows a basis to rank the various compounds. This method can measure the data without the requirement of labelling, immobiliza-tion or any other chemical modification.[1] To further study the interaction, SPR can be utilized to study the binding kinetics. In order to improve the process of drug development it is important to fully understand the interactions between proteins and the molecules they bind with. It is now understood that so many factors weigh in on the binding process. The binding affinity is not only dependent on the non-covalent interactions of the protein and its partner but also on the interaction of both components with their solvent.[2] This makes the prediction of an interaction much more complex than it was first imagined. Water molecules, for example, play a crucial role in this process. They intercede the binding partners, enhancing the binding by forming excess hydrogen bonds and expanding the binding surface.[3] We are just now starting to learn about the circumstances in which these water molecules can positively affect the thermodynamics of binding. By turning to Thrombin, a familiar protein, we are able to use the established techniques to concentrate on these small but vital interactions which occur in the binding processes of all proteins to their ligands. Only if the sum of these interactions is understood will we someday be able to make predictions more efficiently. This understanding also allows for the utilization of this knowledge to develop new leads. These could then precisely trigger certain positive effects such as the inclusion of water to improve the thermodynamics of an interaction. For the study a series of ligands was synthesized to attempt to alter the binding of waters in the binding pocket making it possible to evaluate the importance of these waters in the protein ligand binding process. The inhibitors consist of a tripeptide-like D-Phe-Pro-XXX scaffold which varies only in the P1 side chain (the moiety which binds in the S1 pocket of Thrombin). [3] Previous studies on inhibitors of this kind have shown that, depending on the type of P1 substituent, a well-defined water molecule can be observed in the active site. By coordinating with four neighbouring atoms, it can be assumed that this water mediates hydrogen bonds necessary for the binding process. In the next series inhibitors were especially designed to try to change the interaction of this tetra-coordinated water. [3] By studying the structures and thermodynamic properties of these ligands bound to the protein, taking the

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involved waters into consideration, we are able to broaden our understanding on the intricate requirements necessary for optimal interactions.

References: 1. Ladbury, J. et al: Nature Reviews Drug Discovery 2010, 9: 23-27. 2. Baum, B. et al: JMB 2010, 397: 1042-1054. 3. Biela, A. et al: JMB 2012, 418(5): 350–366.

Determination of the DNA sequence of the alliinase of Allium stipitatum REGEL Mielke, M.G.; Keusgen, M.

Philipps-Universität Marburg, Marbacher Weg 6, 35037 Marburg, Germany

The genus Allium consists of more than 800 species worldwide and therefore belongs to one of the largest genera in the plant kingdom [1,2]. This genus is divided into 15 subgenera with Melanocrommyum being the second largest [1]. A characteristic attribute of Allium species is the occurrence of a C-S-lyase, also called alliinase, and cysteine sulphoxides. This enzyme cleaves these cysteine sulphoxides after cell disruption leading to the formation of bioactive aroma compounds [3]. In Europe, common species like Allium sativum (garlic) and Allium cepa (onion) are well characterised in respect to their cysteine sulphoxides, but also to their alliinases [4,5]. Allium stipitatum, however, plays an important role in Central Asian cuisine, but research on its alliinase has hardly been done, yet. Furthermore, A. stipitatum contains a pyridinyl cysteine N-oxide that is also subjected to alliinase-reaction (see fig.) [6]. The resulting reaction products show high antimycobacterial activity, inducing an increasing interest in alliinase-reaction chemistry of that species [7]. One of the predominant proteins in a protein extract of A. sativum is the alliinase with a monomeric molecular weight of about 50 kDa. A protein extract of A. stipitatum however, only shows a faint protein spot in the alliinase region (see fig.), but alliinase-reaction can be easily detected in such a protein extract. Other protein spots with molecular weights estimated at 21 and 28 kDa are prevalent. To get information on the alliinase of A. stipitatum, already known alliinase sequences were aligned and highly conserved regions were

used to design specific primers for alliinase DNA. Thus it was possible to obtain an interior part of the gene of interest. The gene was subsequently extended versus 5’ and 3’ ends using the method of restriction enzyme site-directed amplification [8] leading to the whole DNA sequence. Amplification of the gene from cDNA, derived of mRNA, not only verified the gene expression in bulbs of A. stipitatum, but also provided valuable information about length and position of introns. The alliinase is consisting of five exons and therefore four introns. Despite the difference of the relative amounts of alliinase in protein extracts, alignment of the amino acid sequences showed 83 % of conformity with a difference in length of four amino acids.

N+

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di(2-pyridyl) disulphide N,N'-dioxide

Acknowledgments: Leibnitz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Dr. Reinhard M. Fritsch References: 1. Fritsch, R.M. et al.: Phyton (Horn, Austria) 2010, 49(2): 145-220.

2. Friesen, N. et al.: Aliso 2006, 22: 372-395. 3. Stoll, A. and Seebeck, E.: Helvet Chim Acta. 1949, 32(1): 197-205. 4. Nock, L.P. And Mazelis, M.: Plant Physiol. 1987, 85(4):1079-1083. 5. Kuettner, E.B. et al.: J Biol Chem. 2002, 277(48): 46402-46407. 6. Kubec, R. et al.: J Agric and Food Chem. 2011, 59(10): 5763-5770. 7. O’Donnell, G. et al.: J Nat Prod. 2009, 72(3): 360-365. 8. Gonzáles-Ballester, D. et al.: Anal Biochem. 2005, 340(2): 330-335.

α-Aminoxy Peptides as α-Helix Mimetics: Solid-Phase Synthesis and Conformational Investigation Diedrich, D.1; Ciglia, E.1; Rüther, A.2; Kurz, T.1, Lüdeke, S.2; Gohlke, H.1; Hansen, F.K.1 1 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany 2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany

Over the last few years much effort has been devoted to the investigation of peptidomimetics consisting of unnatural amino acids to adopt defined secondary structures such as helices or turns.[1,2] Recently, α-aminoxy peptides, analogs of β-peptides in which the β-carbon is replaced by an oxygen atom, were found to be promising candidates for foldamers.[2-4] It was shown that even short α-aminoxy peptides can form stable folded structures composed of strong intramolecular hydrogen bonds between the C=Oi and the N-Hi+2 proton, so called N-O turns.[3] For example, 1.88 helices were observed for D-α-aminoxy peptides containing only three residues and the backbone of these peptides is more rigid than that of natural peptides.[3] Notably, the aminoxy amide bond is resistant to enzymatic degradation. Altogether, these properties make α-aminoxy peptides promising peptidomimetic foldamers. As part of our research towards the development of novel α-helix mimetics for the modulation of protein-protein interactions (PPIs),[5,6] we became interested in α-aminoxy peptides as peptidomimetics. We reasoned that the mimicry of three residues located on one α-helical face requires peptides constructed from at least six homochiral α-aminoxy acids. Unfortunately, the synthesis of longer α-aminoxy peptide sequences under solution phase conditions is relatively cumbersome. Herein, we present an improved synthetic access to α-aminoxy oligopeptides based on a straightforward solid-phase supported approach using dimeric building blocks to synthesize a mini library of oligomers. The conformational properties of selected oligomers were studied by CD spectroscopy and MD simulations in order to get a more profound understanding of the folding properties of α-aminoxy peptides.

References: 1. Martinek, T.A., Fülüp, F.: Chem. Soc. Rev. 2012, 41(2): 687–702. 2. Li, X., Yang, D.: Chem. Commun. 2006, 32: 3367–3379. 3. Li, X., Wu, Y.-D., Yang, D.: Acc. Chem. Res. 2008, 41(10): 1428–1438. 4. Draghici, B., Hansen, F.K. et al.: RSC Adv. 2011, 1(4): 602–606. 5. Spanier, L., Ciglia, E. et al.: J. Org. Chem. 2014, 79(4): 1582–1593. 6. Ciglia, E., Vergin, J. et al.: PLoS ONE 2014, 9(4): e96031.

Heterologous expression of the thiopeptide antibiotic GE2270 from Planobispora rosea in Streptomyces coelicolor requires deletion of ribosomal genes from the cluster Flinspach, K.1; Kapitzke, C.1; Tocchetti, A.2; Sosio, M.2; Apel, A.K.1* 1 Pharmaceutical Biology, Eberhard Karls University, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Email: [email protected] 2 KtedoGen Srl, Via G. Fantoli 16/15, 20138 Milan, Italy

The thiopeptide antibiotics comprise about one hundred natural compounds, including micrococcin, thiostrepton, thiomuracin or berninamycin. They are biosynthesized from ribosomally formed preproteins which undergo extensive posttranslational modifications (1). They are active especially against Gram-

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positive bacteria including MRSA and against malaria parasites, and they have antiproliferative activity against human cancer cells (2). This makes them promising drug candidates. However, their low water solubility and their poor pharmacokinetics have so far prevented their clinical use. Structural modifica-tion by genetic engineering may provide a strategy to make these compounds available for medical use. The rare actinomycete Planobispora rosea produces the thiopeptide antibiotic GE2270 which is the precursor of NAI-ACNE, an antibiotic against Propioni-bacterium acnes that has completed phase 1 clinical trials. In order to facilitate genetic experiments for the generation of new GE2270 derivatives, the GE2270 biosynthetic gene cluster together with an adjacent set of 22 genes coding for ribosomal proteins was cloned into a SuperCos3-based cosmid. Repeated attempts to introduce this cosmid into the heterologous expression host Streptomyces coelicolor M1146 remained unsuccessful. However, exconjugants could finally be obtained after the ribosomal genes were deleted from the cosmid, and GE2270 was thereupon formed in the heterologous host. Production of this antibiotic was further increased when the constitutive ermE* promoter was introduced into the cluster upstream of the GE2270 resistance gene. Expression of ribosomal genes from Planobispora rosea may have a toxic effect on Streptomyces coelicolor, possibly due to the rather large phylogenetic distance between of P. rosea and S. coelicolor. In contrast, conjugation of the entire cosmid into Nonomuraea sp. ATCC39727, which is more closely related to P. rosea, was possible without removal of the ribosomal genes.

References: 1. Bagley, M.C. et al.: Chem. Rev. 2005, 105: 685-714. 2. Young, T.S. and Walsh C.T.: Proc. Natl. Acad. Sci. U S A 2011, 108: 13053-13058.

Discovery and Structural Optimization of Peptides Inhibiting E. coli RNA Polymerase Kamal, A.; Haupenthal, J.; Hüsecken, K.; Negri, M.; Fruth, M.; Hartmann, R.W.; Empting, M.

Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug Design and Optimization and Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbrücken, Germany

The recent rise in antibiotic resistance by bacterial pathogens has rendered several antibiotics ineffective, threatening our ability to cure infectious diseases. Therefore, the search for new antibiotics with novel targets and modes of action is of great importance.(1,2) As such, peptides targeting RNA polymerase (RNAP) has been designed interfering with RNA polymerase function via inhibiting protein-protein-interaction between the β' subunit of RNAP and the σ70 subunit interaction and thus Holo-enzyme formation, resulting in inhibition of transcription initiation. The interface between core RNAP and σ70 represents a promising binding site. Nevertheless, detailed studies investigating its druggability are rare.(3,4) Techniques such as, ELISA, an abortive transcription assay, molecular dynamics simulations, Circular dichroism (CD) spectroscopy, rational amino acid replacement study and side-chain-to-side-chain macrocyclization revealed several peptides with the ability to impede transcription initiation via binding to the coiled-coil region in β′ and that its flexible N-terminus inhibits the enzyme by interaction with the β′ lid-rudder-system (LRS). This work revisits the β′ coiled-coil as a hot spot for the protein−protein interaction inhibition and expands it by introduction of the LRS as target site.(5,6)

References: 1. Chopra, I. et al.: Curr. Opin. Invest., Drugs 2007, 8: 600−607. 2. Hüsecken, K. et al.: ACS Chemical Biology, 2013, 8(4): 758-766 3. Sharp, M et al.: Genes Dev., 1999, 13: 3015−3026. 4. Lesley, S. et al.: Biochemistry, 1989, 28: 7728−7734. 5. Greenfield, N.J., Nature Protocols, 2007, 1: 2876 - 2890 6. Bergendahl, V. et al.: Appl. Environ. Microbiol., 2003, 69: 1492−1498.

Expression, purification and crystallisation of MKK7 Wolle, P.; Mayer-Wrangowski, S.; Rauh, D.

Technische Universität Dortmund, Otto-Hahn-Str. 6, 44227 Dortmund, Germany

The mitogen-activated protein kinase kinase 7 (MKK7) is a dual-specific protein kinase and a member of the c-Jun N-terminal protein kinase (JNK) signalling pathway, which is involved in the regulation of numerous physiologi-cal processes during cellular development and in response to stress [1]. MKK7 is phosphorylated and thereby activated by MAP3K and phosphorylates JNK together with MKK4 on specific Tyr and Thr residues in the activation loop [2]. For a better understanding of the cellular function of MKK7 in disease states, we focus on the development of potent and selective inhibitors. As a starting point, we set out to solve the crystal structure of MKK7 in complex with classic ATP competitive inhibitors. For this, we cloned various constructs of MKK7, which differ in mutations in the activation loop and modifications at the N-terminus. All constructs were expressed in E. coli and purified by affinity chromatography, ion exchange chromatography and size exclusion chroma-tography. Suitable crystallisation conditions were selected by screening all constructs against 386 different conditions at 4 °C and 20 °C. With these conditions, we could obtain the first complex structure of MKK7. This complex gave key insights into the pharmacological perturbation of MKK7 and now serves as starting point for further compound development.

References: 1. Asaoka, Y.; Nishina, H.: J. Biochem. 2010, 148(4): 393-401. 2. Haeusgen, W.; Herdegen, T.; Waetzig,V.: European Journal of Cell Biology, 2011 90: 536-544.

Interactions of differently phospholylated ERK1 with various metal ions studied by affinity capillary electrophoresis Mozafari, M.1; Nachbar, M.1; Redweik, S.1; Alhazmi, H.A.1; Albishri, H.M.2; El-Hady, D.A.2,3; El Deeb, S.1,4; Wätzig, H.1 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Bee-thovenstrasse 55, 38106 Brunswick, Germany. 2 Chemistry Department, Faculty of Science, King Abdulaziz University, 80203 Jeddah, Saudi Arabia. 3 Chemistry Department, Faculty of Science, Assiut University, 71516-Assiut, Egypt 4 Department of Pharmaceutical Chemistry, Al-Azhar University-Gaza, Gaza, Palestine

Extracellular signal-regulated kinase (ERK) is a key regulatory enzyme in the widely used signalling cascade of phosphorylation-dephosphorylation cycles and plays a pivotal role in many aspects of biological processes such as proliferation, differentiation and cell cycle progression. The ERK family belongs to the mitogen-activated kinases (MAPKs) and in accordance with their modified groups belongs to the serine/threonine kinases [1]. Secondary structure transitions such as protein phosphorylations are integral modulators of signal transduction. Many cellular processes are regulated by phosphoryla-tion reaction inducing conformational changes and activates of proteins [2]. Due to the complexity of molecular interactions, simplified peptide models have emerged as a useful tool for investigating the molecular interaction phenome-non. Phosphorylated residues can induce structural changes through metal binding [3]. We investigated a peptide from the activation loop of the signal protein ERK1, consisting of 19 amino acids using affinity capillary electrophoresis (ACE). Affinity capillary electrophoresis has become a powerful method for separation of peptides and proteins, so does for investigation of interactions between various ligands and macromolecules. ACE identifies changes in the electro-phoretic mobility of proteins and peptides due to changes in mass and charge through binding or interaction with the ligand [4]. The determination of the mobility changes was carried out by using mobility ratios of EOF-marker and the peptide to avoid the migration time shifts which are not related to interactions. The difference of the mobility ratio of the peptide with the ligand (Ri) and without the ligand (Rf) was normalised to Rf (ΔR/Rf) [4]. The interaction of the unphosphorylated form of the above-mentioned peptide with various metal ions e.g. Ba2+, Ca2+, Mg2+, Mn2+, Cu2+, Ni2+ was investigated. Furthermore the results of the interactions of mono- and diphosphorylated form with the same metal ions were compared.

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Among all the investigated metal ions, we found strong interactions of Ni2+ and Cu2+ with the mono- and diphosphorylated peptide which changed the overall charge of the peptide positively and negatively respectively. Possibly Ba2+-ion shows significant interactions as well. These different changes are probable due to the conformation changes of peptide after binding to metal ions and the further interactions of binding metal ions with the ions of surrounding solution. Only a weak interaction with other metal ions could be found.

References: 1. Kolch, W. et al.: Biochemical Society 2000, 351: 289-305. 2. Nam, H-S. et al.: Journal of Chromatography A 2002, 976: 79-85. 3. Broncel, M. et al.: Organic & Biomolecular Chemistry 2010, 8(10): 2575-2579. 4. Redweik, S. et al.: Electrophoresis 2012, 33(22):3316-3322.

Borreliosis Immunoassay using Reflectometric Interference Spectroscopy (RIfS) Dassinger, N.; Vornicescu, D.; Keusgen, M.

Philipps Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6-10, 35037 Marburg

An improved assay for the detection of Borrelia infections in animals and humans based on Reflectometric Interference Spectroscopy (RIfS) has been developed. A recombinant fusion protein was used as recognition element on the sensor surface. One moiety of the protein consists of the Borrelia antigene VlsE (Variable major protein sequence Expressed) sequence and the other is the lectine Concanavalin A (ConA). It was possible to immobilize the fusion protein on sugar coated biochip surfaces via the ConA-part. With this assay, a rapid detection of anti-Borrelia antibodies in serum will be possible. Introduction In previous investigations it has been shown that Reflectrometric Interference Spectroscopy (RIfS) is suitable for the detection of bacteria [1]. Based on these investigations, an assay for the detection of Borrelia in humans and animals has been designed. If Borrelia enters the human body, antibodies against the VlsE (Variable major protein sequence Expressed) antigene occur within days. [2]. These antibodies are a valid marker for borreliosis. The VlsE protein consists of invariable domains at the amino and the carboxyl termini and a variable domain in the centre. The variable domain contains 6 variable regions named VRI to VRIV as well as 6 invariable regions named IR1 to IR6 [3]. For the here presented experiments, the IR 6 epitope of the Borrelia garinii clone P7-1 was chosen. In order to bind the VlsE protein to the biochip surface loaded with mannan, the genetic sequence of VlsE was fused to the gene of the binding domain of the lectin Concanavalin A (ConA). Results and Discussion The C6-ConA protein was successfully expressed in Escherichia coli. After inclusion body purification and refolding of the protein, the functionality was tested on a mannan coated RIfS-chip surface. C6-ConA binds to the mannan surface via the ConA part and the VlsE epitope is able to bind the borreliosis specific antibodies in a positive serum sample (see fig. 2).

Figure 2: RIfS sensorgramm. Mannan coated chip with injection of blocking solution after 300 sec. Application of C6-ConA (50µg/ml) after 1822 sec and injection of positive serum sample after 4822 sec. Conclusions The fusion protein C6-ConA is a very promising protein for detection of borreliosis antibodies in serum samples. In combination with RIfS, a rapid detection of a Borrelia infection seems to be possible.

Acknowledgements: This work was supported by „Arbeitsgemeinschaft Industrieller Forschungsvereinigungen“ (AiF) References: 1. Merkl, S. et al.: Phys. Status Solidi A. 2014, DOI: 10.1002/pssa.201330436 2. Chandra, A. et al.: Clinical Immunology 2011, 141(1) : 103-110. 3. Liang, F.T. et al.: J. Clin Microbiol 2000, 38 (11) : 4160-4166.

New Biochemical Features of Indole Prenyltransferases from Streptomyces Winkelblech, J.1,2 1 Philipps-Universität Marburg, Institut für Pharmazeutische Biologie und Biotechnologie, Deutschhausstrasse 17A, 35037 Marburg (Germany), E-mail: [email protected]] ² Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg Hans-Meerwein-Strasse, 35032 Marburg (Germany)]

Prenyltransferases contribute largely to the structural diversity of natural products, which are important resources for drug discovery and development. Attachment of prenyl moieties derived from prenyl diphosphate, usually dimethylallyl diphosphate (DMAPP), to various aliphatic or aromatic acceptors often increases the biological and pharmacological activity of the resulted compounds. Indole prenyltransferases from the dimethylallyltryptophan synthase (DMATS) superfamily represent one of the most investigated enzymes for the prenyl transfer reactions. These enzymes show high flexibility towards their aromatic substrates and high regioselectivity of the prenylation position on the indole ring. In contrast, most of them have a restricted substrate specificity towards prenyl donors and utilize solely DMAPP as substrate (1). Two indole prenyltransferase genes SAML0654 and Strvi8510 were identified in Streptomyces ambofaciens and Streptomyces violaceusniger, respectively. Their deduced proteins with a sequence identity of 63 % on the amino acid level to each other were overproduce in E. coli and used for enzyme assays. HPLC analysis of the incubation mixtures revealed that L- tryptophan and derivatives including D-tryptophan, 4-, 5-, 6- and 7-methyl-DL-tryptophan were well accepted by both enzymes in the presence of DMAPP. Structure elucidation of the isolated enzyme products and determination of kinetic parameters demonstrated that L-tryptophan was accepted as the best substrate and both enzymes function as 6-dimethylallyltryptophan synthases (6-DMATS). Detailed biochemical characterization of 6-DMATSSa from S. ambofaciens and 6-DMATSSv from S. violaceusniger revealed a number of new features for indole prenyltransferases. For example, these enzymes represent the first examples of tryptophan prenyltransferases, which accepts both DMAPP and geranyl diphosphate (GPP) as prenyl donors and catalyzed the same regiospecific prenylation. Furthermore, the prenylation of some hydroxynaphtalenes and the unusual prenylation position on their unsubstitut-ed rings also represent a new feature for bacterial indole prenyltransferases (2).

Acknowledgements: We thank the Deutsche Forschungsgemeinschaft and LOEWE program of the State of

Hessen (SynMikro) for financial supports.

References: 1. Yu, X. and Li, S.-M.: Methods Enzym 2012, 516: 259-278. 2. Winkelblech, J. and Li, S.-M.: Chembiochem 2014, 15: 1030-1039.

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DMAT synthases catalyze the formation of alkylated indoles by using unnatural allyl and aryl diphosphates Liebhold, M.1; Xie, X.2; Li, S.-M.1 1 Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17a, 35037 Marburg, Germany 2 Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany

Prenylated indole alkaloids such as ergot alkaloids exhibit diverse pharmaco-logical and biological effects [1,2], distinct from respective unprenylated precursors [3]. The transfer reaction of the prenyl residues onto the aromatic nucleus is catalyzed by prenyltransferases in nature [4]. The members of the dimethylallyl tryptophan synthase (DMATS) superfamily belong to the recently most investigated enzymes from this group and show high flexibility towards aromatic substrates. They attach the prenyl moiety not only on simple indoles or cyclic dipeptides, but also on flavonoids and hydroxynaphthalenes [5]. On the other hand, these enzymes mostly accept DMAPP as prenyl diphosphate [5,6,7,8].

The presented studies (Figure) show the acceptance of unnatural β-unsaturated allyl diphosphates such as monomethylallyl and 2-pentenyl diphosphate by L-tryptophan and tryptophan-containing cyclic dipeptide prenyltransferases. In the first case (Figure A), the natural prenylation position was partly or completely shifted, which depends on the available DMAPP analogue. As shown in Figure B, in contrast to the natural reaction of the used enzymes, a mixture of C2- and C3-reversely alkylated diastereomeres was detected. Furthermore, the flexibility of several DMATS prenyltransferases towards more space-demanding alkyl donors was demonstrated by using benzyl diphosphate. Since FgaPT2 yielded the highest amount of products, diverse L-tryptophan derivatives were subsequently incubated in the presence of this enzyme. Structure elucidation proved the formation of C5-benzylated indole derivatives (Figure C).

References: 1. Williams, R.M.; Stocking, E.M.; Sanz-Cervera, J.F.: Topics Curr. Chem. 2000, 209: 97-

173. 2. Li, Y.-X. et al.: Mar. Drugs 2013, 11(12): 5063-5086. 3. Botta, B. et al.: Curr. Med. Chem. 2005, 12(6): 717-739. 4. Li, S.-M.: Nat. Prod. Rep. 2010, 27(1): 57-78. 5. Yu, X.; Li, S.-M.: Methods Enzymol. 2012, 516: 259-278. 6. Chooi, Y.H. et al.: J. Am. Chem. Soc. 2012, 134(22): 9428-9437. 7. Pockrandt, D. et al.: Appl. Microbiol. Biotechnol. 2014, 98(11): 4987-4994. 8. Tarcz, S.; Xie, X.; Li, S.-M.: RCS Adv. 2014, 4(35): 17986-17992.

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A target-mediated drug disposition model to characterize the pharmacokinetics of bosentan after intravenous administration in healthy subjects Volz, A.-K.1; Krause, A.2; Markert, C.3; Haefeli, W.E.3; Dingemanse, J.2; Lehr, T.1 1 Saarland University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany 2 Actelion Pharmaceuticals Ltd, Department of Clinical Pharmacology, Gewerbestrasse 16, Allschwil, 4123, Switzerland 3 Heidelberg University, Department of Clinical Pharmacology and Pharmacoepidemiolo-gy, Im Neuenheimer Feld 410, Heidelberg, 69120, Germany Background and Objectives The endothelin receptor antagonist bosentan (TracleerTM) is one of the most frequently used drugs in the therapy of pulmonary arterial hypertension. Its pharmacokinetic (PK) behaviour is influenced by interaction with different transporters and metabolic enzymes [1]. The aim of this analysis was the development of a population PK model to characterize the PK of bosentan after single intravenous application of different doses in healthy male subjects. Methods Model development was performed using intravenous data from a single ascending dose study (10, 50, 250, 500, and 750 mg) of bosentan [2]. Several structural models were evaluated using NONMEM 7.2. Model selection was performed based on statistical and graphical procedures. A covariate analysis was applied using a stepwise forward inclusion (p=0.05) and backward elimination (p=0.001) procedure. Results Overall, 706 plasma concentration–time data from 54 healthy subjects were available. The PK of bosentan were best described by a 2-compartment target-mediated drug disposition (TMDD) model with internalization of the bosentan-target-complex and a turnover model for the target production- and degrada-tion rate (Fig. 1). Interindividual variability for clearance, volumes of distribution

and kint was moderate ( 38 %CV). None of the covariates investigated influenced the PK of bosentan systematically. A sine function on the production rate of the target (ksyn) mimicked a circadian rhythm and improved the model significantly (p<0.001). Conclusion A TMDD model was successfully developed describing the plasma concentra-tion-time profiles of bosentan. The model revealed that binding of bosentan to a target, presumably endothelin receptors, influences the PK significantly. In addition, a new hypothesis on the circadian fluctuation of the free target was generated. The model presented is a first step towards unravelling the PK characteristics of bosentan and will serve as a valuable tool for future model development following oral and multiple-dose administration.

Fig.1. TMDD model of bosentan References: 1. Dingemanse, J. and van Giersbergen, P.L.M.:Clinical Pharmacokinetics 2004, 43 (15): 1089–1115. 2. Weber, C. et al. :Clinical Pharmacoogy & Therapeutics 1996, 60: 124–137.

Prevention of type 2 diabetes by participation in the pharmacy-based lifestyle intervention program GLICEMIA: a multicenter, randomized, controlled trial Schmiedel, K.1; Schlager, H.1; Friedland, K.2

1 Scientific Institute for Prevention in Health Care (WIPIG), Maria-Theresia-Str. 28, 81675 Munich, Germany 2 Friedrich-Alexander-University, Department of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, Cauerstr. 4, 91058 Erlangen, Germany

Lifestyle intervention can be effective in decreasing risk for type 2 diabetes. A recent meta-analysis has shown that diabetes prevention programs in outpatient settings can be effective [1]. However, conveniently accessible opportunities to prevent type 2 diabetes are still not introduced in Germany. The purpose of this study was to assess the efficacy of a prevention program carried out in community pharmacies in reducing the risk for diabetes. Community pharmacies in Bavaria, South Germany, were randomly assigned to the intervention (n = 21) or to the control group (n = 21). Eligible participants had an increased risk for diabetes (FINDRISC score ≥ 7) and were at least 35 years old. All participants received written information about diabetes prevention. In the intervention group (n = 565) the GLICEMIA program combined three appointments with individual counselling and five educational group sessions. The control group (n = 575) obtained only information about their health status in three assessments. Primary outcome was the change of the risk for diabetes indicated by the FINDRISC score after 12 months. In the intention-to-treat population (n = 1,092), 68.6 % of the participants were female and the median age was 57.5 years (IQR: 49.2 - 65.5). Highly prevalent risk factors were overweight and sedentary lifestyle (81.9 % and 72.7 %, respectively). The primary endpoint, a reduction of the FINDRISC score, was achieved by a significantly higher proportion in the intervention group than in the control group (39.1 % versus 21.0 %, p < 0.001). Statistically significant differences between the groups were also demonstrated in the secondary endpoints weight reduction, physical activity and physical quality of life. The goal of a minimum weight reduction by 5 % was achieved by 21.6 % in the intervention group and 8.3 % in the control group (p < 0.001). Moreover, the blood pressure decreased and the mental quality of life increased in both groups. Pharmacists can help patients to reduce their risk for diabetes. The prevention program GLICEMIA can be successfully conducted in community pharmacies and is effective decreasing the risk for type 2 diabetes in the high-risk population in Germany. Acknowledgments: This study received funding through the Dr. August and Dr. Anni-Lesmüller Foundation, the Bavarian State association of Corporate Health Insurers (BKK), the funding initiative for prevention (Förderinitiative Prävention e. V.) and the Bavarian health promotion initiative “Gesund.Leben.Bayern.” by the Bavarian State Ministry of Public Health and Care Services.

Reference: 1. Dunkley, A.J. et al.: Diabetes Care. 2014, 37(4): 922−933.

Predicting the impact of oral anticoagulants on the human coagulation pathway using a comprehensive systems pharma-cology model Dings, C.; Schäftlein, A.; Lehr, T.

Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany

Background and Objectives: The introduction of the oral anticoagulation drugs (OAC) warfarin, dabigatran, rivaroxaban, edoxaban and apixaban changed the landscape of the treatment of thrombotic diseases substantially [1]. The pharmacokinetics (PK) and pharmacodynamics (PD) of the OAC are well known. Nevertheless, a mathematical coagulation model in humans, which summarizes the influence of different OAC on clotting times such as e.g. activated partial prothrombin time (aPTT) or prothrombin time (PT), is not available yet. The aim of this work was to extend a previously published human coagulation model [2] by the effects of the OAC warfarin, dabigatran, rivaroxaban,

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edoxaban and apixaban on the coagulation pathway and further by the inclusion of the Ecarin clotting time as an important clinical coagulation marker. Materials and Methods: A recently published coagulation pathway model [2] was rebuilt in Matlab version R2013a. Published population PK models of warfarin [3], dabigatran [4], rivaroxaban [5] and edoxaban [6] as well as a self-developed PK model of apixaban based on digitized concentration-time profiles [7] were included. The link between the PK and the PD of the drugs was recognized using an Emax inhibition rate model on vitamin K, thrombin or factor Xa. The effects of the OAC on the coagulation system were visualized using Matlab and SAS version 9.3 and compared to the observed clotting times after administration of the respective investigated drug [4-7]. Model development of the apixaban PK model was performed using NONMEM 7.2. Results: A linear one compartment model with first order absorption for warfarin and rivaroxaban and a linear two compartment model for apixaban, edoxaban and dabigatran were found to be adequate. The human coagulation cascade was described by 47 ordinary differential equations (ODE) and 158 parameters. 13 ODE and 51 parameters connected the PK of the OAC to the coagulation network. Overall, the model successfully predicted observed concentration-time profiles of the OAC as well as the impact of the drugs on clotting times like aPTT or PT. In addition, the Ecarin clotting time (ECT) was implemented in the model and showed an adequate predictive performance. Conclusion: The developed OAC-coagulation model is a powerful tool for a wide range of applications. For example, the model enables a comparison of different OAC and support the clinical important prediction of switching algorithm between the OAC. Besides, timing of antidote dosing to reduce major bleedings (e.g. Vitamin K for warfarin) or pharmacodynamic interactions between the OAC on every coagulation factor of interest can be investigated. References: 1. Kitslaar, D.B. et al.: Curr Treat Options Cardiovasc Med. 2014, 16(8): 326. 2. Wajima, T. et al.: Clincial pharmacology & Therapeutics 2009, 86(3): 291. 3. Lane, S. et al.: Br J Clin Pharmacol. 2012, 73(1): 66. 4. Liesenfeld, K.H.: et al. J Thromb Haemost 2011, 9: 2168. 5. Girgis, I.G. et al.: The Journal of Clinical Pharmacology 2014. 6. Song, S.H. et al.: The Journal of Clinical Pharmacology 2014. 7. Frost, C. et al.: Br J Clin Pharmacol. 2013, 75(2): 476.

Pharmaceuticals in an Afghan hospital: boon or bane? Mielke, M.G.*; Neumann, S.*; Keusgen, M.

*equally contributing authors Philipps-Universität Marburg, Institute for Pharm. Chemistry, Marbacher Weg 6-10, D-35032 Marburg, Germany

Falsifications of pharmaceuticals are an increasing problem worldwide as was recently demonstrated by operation Pangea VII, where 111 countries participated in seizing about 9.4 million counterfeit medicines [1]. Although the European market seems to be quite safe, if not ordering medicines by internet other countries in Africa, Asia and South America have more problems regarding drug falsifications [2]. Afghanistan can be considered as developing country and the health care system is still to be built up. The hospital in Mazar-e Sharif is being helped to establish an up-to-date treatment of the population in the region by the GIZ [3]. Nevertheless, pharmaceuticals in this hospital seem to be incredibly cheap (0.20 € up to 2.63 €), so falsifications with no active agent or a concentration, that is too low, could be assumed. To examine if this is the case, the active compounds of some medicines of the hospital in Mazar-e Sharif were analysed (see fig.). These pharmaceuticals were produced in different countries nearby Afghanistan. Until now, some pain killers of the NSAID category and several antibiotics were analysed based on the European Pharmacopoeia and USP. Extraction methods were validated by examining the content of some pharmaceuticals produced for the German market. All Afghan pharmaceuticals investigated so far fulfilled the Pharmaco-poeias’ requirements regarding identity and content.

Acknowledgements: Civil Hospital Mazar-e Sharif, Dr. Matthias Körner

References: 1. Interpol: http://www.interpol.int/Crime-areas/Pharmaceutical-crime/Operations

(25.06.2014) 2. WHO: http://www.who.int/mediacentre/factsheets/fs275/en/ (27.06.2014) 3. Deutsche Gesellschaft für Internationale Zusammenarbeit:

http://www.giz.de/de/weltweit/14695.html (25.06.2014)

HPLC method development and validation for the determination of anidulafungin in microdialysis samples of healthy volunteers and intensive care patients Weiser, C.1; Kauzor, D.1; Brosig, H.1; Kees, F.2; Zeitlinger, M.3; Kloft, C.1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr.31, 12169 Berlin, Germany 2 Dept. of Pharmacology, University of Regensburg, Universitätsstr. 31, 93053 Regens-burg, Germany 3 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

Objectives: Anidulafungin (ANI), a new echinocandin, has been approved for the treatment of invasive Candida infections. Up to now, knowledge about pharmacokinetics for ANI is very rare particular in special patient populations and at the target site. For the analysis of several upcoming pharmacokinetic microdialysis studies e.g. in healthy volunteers and intensive care patients, which will be performed at the University Hospital in Vienna, a suitable HPLC assay for the determination of ANI had to be developed and validated. A low quantification limit of the assay was required in order to reliably quantify also low tissue fluid concentrations of ANI. In addition, unspecific adsorption of the drug shall be avoided by appropriate sample preparation. Methods: A Thermo Scientific Dionex Ultimate 3000 HPLC with a DAD 3000 detector was chosen for the quantification of ANI. Different ways of sample preparation and adsorption prevention measures were investigated using additives (methanol, ethanol and human serum albumin (HSA)) to prevent adsorption and different volumes of methanol for subsequent protein precipitation. The mobile phase, based on [1], and consisting of methanol and ammonium dihydrogen phosphate, was investigated regarding the composition and flow rate. Method development was also important for the column (Accucore Phenyl-Hexyl column (50 x 4.6 mm, 2.6 µm) vs. Merck LiChrospher 100 RP-18 column (125 x 4 mm, 5 µm), both with a guard column (RP 18, 5 µm)) and column temperature (25 - 40°C). After the assay development, validation was performed in accordance to the EMA guideline for bioanalytical method validation [2]. Results and Conclusions: The successful HPLC assay development comprised a LiChrospher RP18 column (125 x 4 mm, 5 µm), a C18 guard column, isocratic mode with methanol and ammonium dihydrogen phosphate 85:15 (v/v) at a flow rate of 1.0 mL/min, autosampler and column temperature at 10° C and 40° C, respectively, detection at 310 nm, injection volume of 20 µL and fulfilled all predefined prerequisites. The total run time was 5 minutes with a retention time for ANI of 2.5 minutes. Adsorption of ANI was prevented by addition of HAS (0.5%) and subsequent protein precipitation using methanol. Validation was successfully performed according to the criteria of the EMA guideline for bioanalytical method validation for the concentration range of 0.1 – 20 µg/mL.

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The developed and validated assay allows quantification of ANI from in vitro microdialysate samples as crucial precondition allow in vivo μD investigations in healthy volunteers and patients. As next step the in vitro µD experiments will be performed to decide settings for the clinical studies. References: 1. Sutherland, C.A. et al.: J Chromatogr Sci 2011, 49 (5): 397-400. 2. European Medicines Agency (EMA): Guideline for bioanalytical method validation 2012

Quality assurance for pharmaceutical laboratories in developing countries Maul, K.J.1; Diergardt, T.2; Feldmann, D.3; Wätzig, H.1 1 Technische Universität Braunschweig – Institute of Medicinal and Pharmaceutical Chemistry, Beethovenstraße 55, 38106, Germany 2 Physikalisch-Technische Bundesanstalt – Braunschweig, Bundesallee 100, 38116, Germany 3 Bausch+Lomb, Brunsbütteler Damm 165-173, 13581, Germany

Quality control is a major issue in the development, production and testing of medicine. This should be practiced in all laboratories which deal with this highly valuable good. Quality infrastructure in the East African Community (EAC) is discussed if the requirements of the pharmaceutical sector are met. At present the pharmaceutical industry can only produce for their own domestic market. They are not allowed to sell their products anywhere else due to the lack of quality standards assurance. In this project initiated by the Physikalisch-Technische Bundesanstalt called “Establishment of a regional Quality Infrastructure for the pharmaceutical sector in the EAC“ proficiency tests, a tool for quality assurance, were conducted. The first in October 2013 was about analyzing Amoxicillin 500 mg tablets. Among the 15 participants were laboratories from five different countries. According to the USP monograph assay and dissolution were performed. The requirements of the USP are 90-120 % for the assay and at least 75 % for the dissolution. The interlaboratory standard deviation for the assay is 12.7 mg/tablet (RSD%=2.62) and for the dissolution 4.87 % (RSD%=5.28). As assigned value the mean of all laboratories was used and the z-score for each laboratory was calculated (Figure 1). The result for the assay is very satisfying the overall average of 97.1 % is the exact same result the manufacture of the tablets states in its release certificate.

Figure 1: Z-score charts: The z-scores of each participant are shown for assay and dissolution. A z-score between -2 and 2 (white area) is very good. Z-scores between -3 to -2 and 2 to 3 (black area) accepta-ble.

The Erlangen Teacher Practitioner Project: Advances in Clinical Pharmacy Teaching in Germany Dircks, M.G.1,2; Dörje, F.2; Friedland, K.1 1 Molecular and Clinical Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg, Cauerstraße 4, 91058 Erlangen, Germany 2 Pharmacy Department, Erlangen University Hospital, Palmsanlage 3, 91054 Erlangen, Germany

Following the implementation of “Clinical Pharmacy” into the pharmacy course curriculum (AAppO) in 2001, the German Pharmaceutical Society (DPhG) published recommendations for its structure in a paper of consent [1] including the involvement of a teacher practitioner to ensure a patient-centered education. To systematically evaluate the benefits of bed-side teaching of fourth year pharmacy students in a German university hospital setting a randomized teaching and learning study was carried out. A course was created consisting of 2x1,5h of class-room teaching and 2x4h of practical teaching on a psychiatric ward in small student groups. Learning aims concentrated on patient and interprofessional communication techniques and included: taking medication histories, identification and handling of medication-based problems and pharmaceutical counseling of psychiatric patients. The 42 students of the control group only participated in the theoretical part while the 42 students of the intervention group took part in the complete course. The effects were assessed by an objective structured clinical examination (OSCE) [2] consisting of five practical and five theoretical tasks testing for clinically applied knowledge and communication skills. In addition, a questionnaire was conducted asking for students’ opinions about course structure, relevance of teaching content and overall satisfaction. The intervention group achieved a significantly better overall result in the OSCE assessment (46.4±9.5 vs. 28.2±9.0 of 90 points; p<0.001) with most positive effect in assessed communication skills (27.4±5.4 vs. 16.3±6.0 of 40 points; p<0.001). The performance in the theoretical tasks was poor (12.1±4.1 vs. 8.1±3.2 of 30 points; p<0.001) indicating that knowledge-based applied clinical pharmacy skills still need to be improved. This could be achieved by more practical training in clinical pharmacy. The findings are supported by the questionnaire: 93% of the students rated the course as practice-orientated, 90% felt better prepared for patient contact and 92% gave a positive answer when asked for overall satisfaction. Many students suggested an extension of the course in the free text field of the questionnaire. In conclusion, the results of this quantitative teaching study demonstrate significant learning benefits by the teacher practitioner course and the high usefulness of bed-side teaching in pharmacy student education. Hence, it should be implemented as a mandatory course in the pharmacy course curriculum in Germany.

Acknowledgments: This work was supported by Bayerische Akademie für Klinische Pharmazie/Dr. August und Dr. Anni Lesmüller-Stiftung, Munich, Germany. The authors are grateful for the expert advice of Dr. Annette Freidank, Pharmacy Department, Fulda Hospital, Germany and PD Dr. Wolfgang Frobenius, MME, Department of Gynacology and Obstetrics, Erlangen University Hospital, Germany.

References: 1. Konsensuspapier der AG zur Ausbildung im Fach Klinische Pharmazie: 1. Teil: Rahmenbedingungen und Organisation. http://www.klinische-pharmazie.org/. Assessed June 10, 2014. 2. Smee, S.: BMJ 2003, 326(7391): 703-706.

In vitro microdialysis characterising vancomycin as precondition for an upcoming trial in neonates Kauzor, D.1; Schröpf, S.²; Fürtig, M.1; Ruhe, D.1; Kloft, C.1 1 Dept. Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany 2 Dept. of Pediatrics, Dr. von Haunersches Kinderspital, Lindwurmstraße 4, 80337 Munich, Germany

Background: Infections with gram-positive bacteria are commonly treated with vancomycin. This wide use leads to increasing rates of vancomycin-resistant bacteria which are favoured by suboptimal dosing. To avoid non-therapeutic vancomycin concentrations, the pharmacokinetics (PK) has to be well characterised as has been done for adults, but not in neonates. Extrapolation of PK parameters from adults to other patient populations may increase the risk of adverse effects (overdosing) or therapy failure (underdosing). Especially in neonates PK studies are difficult to perform with multiple blood sampling due to the limited availability of blood volume. An alternative is the microdialysis technique which benefits from sampling without removing body fluids: a catheter with a semipermeable membrane is inserted in the particular interstitial fluid (ISF) allowing measure-ments of drug concentrations continuously and directly at the target site. In an upcoming trial, microdialysis will be performed in the ISF of subcutaneous tissue of neonates treated with vancomycin (VAN). Prior to clinical trials, an in vitro microdialysis characterisation of the particular drug is crucial in order to

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set optimal settings for a consistent relative recovery (RR) in vivo; being the ‘catheter calibration factor’ as the ratio between measured concentrations in microdialysate samples and ISF in neonates. Methods: In vitro characterisation of vancomycin was performed with a standardised in vitro microdialysis system [1] and CMA 63 microdialysis catheters (membrane length 10 mm, molecular weight cut-off 20 kDa) which are approved for neonates and will be used in the clinical trial. Three catheters were used simultaneously for the characterisation of VAN to investigate intercatheter variability. Drug concentration was set to 50 µg/mL of vancomy-cin for all investigations; the surrounding medium was heated to 37° C to mimic body temperature. As perfusate, Ringer’s solution (RS) was used, and the surrounding medium consisted of phosphate buffered saline (PBS) to mimic ISF with a constant pH value. RR was determined for different flow rates (2.0, 1.0 and 0.5 µL/min) and pH values (6.6, 7.0, 7.4 and 8.0) of the surround-ing medium in both, delivery (VAN diluted in RS in the perfusate) and recovery (VAN diluted in PBS in the surrounding medium) experiments, to assess equality of RR in both diffusion directions. Five replicates were sampled in each experiment. An HPLC assay for the quantification of VAN from microdialysate samples was developed and validated according the EMA guideline [2]. Results: RR was found to be dependent of flow rate and pH value. RR increased with lower flow rates and was highest at a pH value of 7.0 in delivery as well in recovery experiments. Highest RRs were obtained with a flow rate of 0.5 µL/min (pH: 7.0). Lowest RRs were observed at a pH value of 8.0 and a flow rate of 2 µL/min. RRs calculated from delivery were on average approxi-mately 20% lower than RR from recovery experiments. Conclusion: The results demonstrate a strong dependency of RR from flow rate, as well as from pH value of the surrounding medium. Based on the RR findings, a flow rate of 1 µL/min is to be recommended for the clinical trial due to a compro-mise in high RR, acceptable µD sampling intervals and VAN concentration changes over time. As RR seems to be dependent on the pH value of the surrounding medium, monitoring of pH values of the microdialysate samples as surrogates for the pH values in interstitial fluid shall be performed in the trial. Equality of RR in both directions was not observed in vitro and has to be further investigated. As next step, the RR dependency of drug concentration shall be examined to further establish conditions in the upcoming trial which shall contirubute to improve vancomycin therapy and might support the implementation of therapeutic drug monitoring.

We thank Prof. Markus Zeitlinger, UKH Wien for the support concerning microdialysis issues.

References: 1. Kirbs et al.: EJPS 2014. 2. EMA guideline on bioanalytical method validation 2012.

Status quo and future directions for the implementation of a Medication Management Service – An online survey in German Pharmacy Stores Britz, H.*; Keßler, V.*; Schneider, I.*; Müller, C.*; Schäftlein, A.; Lehr, T. 1 Saarland University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany * Equally contributing first-authors

Background and Objectives The implementation of medication management as a standard service for German pharmacies is under discussions [1]. Despite the increasing interest, there might be open issues for German pharmacist around medication management, like a common definition of the process or reimbursement. Aim of this project was to develop and conduct an online survey for German pharmacists on the medication management service in German pharmacy stores to depict the Status Quo of knowledge about the medication manage-ment and the expectations about the potential future service in a huge population of officinal pharmacists. Methods An anonymized online survey for German pharmacies on medication man-agement was developed [2]. Besides questions about demographic data and information on the respective pharmacy store, pharmacists were asked questions on status quo and future directions of medication management. The

survey is accessible online [2] and will be promoted in August and September 2014. The survey will be continued until at least 200 responses are available. Results from the survey were summarized electronically; statistics and graphics were generated using SAS 9.3. Results So far, 12 pharmacists responded to the online survey. Even so the number is small so far, a few interim results are presented in the following, final results will be presented at the meeting. Overall, 70% of the pharmacists perform already medication management in their pharmacy store. However, there is a huge diversity in what is actually done and what is believed to be part of the medication management service. Only 30% of the pharmacists are aware of the definitions about medication management provided by the German pharmaceutical society (DPhG) [3]. After providing the definition to the pharmacists, 80% would consider the definition of an “extended” medication management including information about and through the patient as implementable. Pharmacists believe that total costs for medication management should be shared as follows: 91% should be paid by health care providers and 9% by patients. Pharmacists expected that they would spend on average 50 minutes for a first face-to-face meeting, 60 minutes for the analysis of the medication and 25 minutes for a follow up meeting. On average, a reimbursement of €50 per hour was considered as acceptable for a medication management service by pharmacists. Conclusion Medication management is recognized by German pharmacists and consid-ered as a potential new standard service in pharmacy stores. The results of this survey may help to the guide the future implementation of a regular medication management service in pharmacy stores. References: 1. http://www.abda.de/leitbild.html 2. www.medikationsmanagement.info 3. http://www.dphg.de/news-folder/detailansicht/implementierung-des-medikationsmanagements-als-neue-pharmazeutische-dienstleistung/f196c965646ee2fac82c21c165e1f939/

Acceptance of a Medication Management Service in German Pharmacy Stores – A Saarland Patient Survey Müller, C.*; Schneider, I.*; Keßler, V.*; Britz, H.*; Schäftlein, A.; Lehr, T.

Saarland University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany * Equally contributing first-authors

Background and Objectives Medication management is considered as an important tool to improve the safety and efficacy of patient’s therapy. The implementation of medication management as a standard service for German pharmacies is intensively discussed these days [1]. However, little is known about the interest and perception of patients on the potential future service. Aim of this project was to develop and conduct a survey in patients about the acceptance of medication management service in German pharmacy stores. Methods A patient dedicated anonymized survey on medication management was developed. Besides age and sex, various questions were asked about patient’s medication (number, self-reported knowledge, resource, contact person). Next, medication management was introduced to the randomly selected patients by a standardized paragraph which was recited by the interviewer. Afterwards, questions about medication management (interest, willingness to pay, sharing of costs) were asked. The survey was conducted in the pedestrian zone in Saarbrücken by four undergraduate pharmacy students in April 2014. Results from the survey were summarized manually; statistics and graphics were generated using SAS 9.3. Results Overall, 212 patients (60% females) participated in the survey. Most patients (48.9%) were between 51 and 70 years old. 74% of the patients had a regular intake of medications; the average number of medications per patient was 3. The self-reported knowledge of the patients about their medications was high (average 82%). The majority of the patients had a fix pharmacy store (67%) where they receive their medication from and which the contact for questions about the medication.

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In total, 70% of the patients are willing to use a medication management service. Of this proportion, 71% were willing to pay on average €18 out of their pocket for this service. Patients believe that total costs for medication management should be shared as follows: 67% paid by health care providers, 22% by patients, 9% by pharmacists and the remaining 2% by doctors. Conclusion Medication management was perceived very well by the patients as a potential new standard service in pharmacy stores. In addition to the acceptance of the new service provided, patients were also willing to pay for medication management. Even so the study presented may have a recall bias of the patients and potentially a selection bias of the patients selected by the interviewer, the results are promising. The outcomes of this survey may help to the guide the future implementation of a regular medication management service in pharmacy stores.

Reference: 1. http://www.dphg.de/news-folder/detailansicht/implementierung-des-medikationsmanagements-als-neue-pharmazeutische-dienstleistung/f196c965646ee2fac82c21c165e1f939/

Utilisation of electronically generated medication plans in German community pharmacies: status quo Botermann, L.1,2, Griese, N.2, Krueger, K.2, Eickhoff, C.2, Schulz, M.2, Kloft, C.1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany 2 Dept. of Medicine, ABDA – Federal Union of German Associations of Pharmacists, Jaegerstr. 49/50, 10117 Berlin, Germany

Objectives: A current and complete medication plan is of central importance for medication safety. Therefore the German Ministry of Health (BMG) integrated a standardised medication plan (MP) as a safety measures in their “Action Plan to promote medication safety in Germany”. A specification1 defines the requirements for the standardised MP in form and content. The aim of this study was to evaluate the status quo of the technical capabilities and the utilisation of electronically generated printable MPs for patients in German community pharmacies. Methods: An online survey was developed and sent to 3,380 community pharmacists. The questionnaire contained items on the digital storage of patient data, the technical capabilities to generate and print a MP for patients and its utilisation in pharmacy practice. Pharmacists were also asked to mail an example of a medication plan. These sent documents were then analysed according to specific criteria: The first criterion was whether it was actually to be considered as a plan for patients and not for health care professionals. In the next step it was analysed to which extent the document followed the specification for a standardised MP by the BMG. Results: The response rate was 10% (n=326). 80% (n=261) of the respondents saved patient-based medical data digitally. For 35% (n=91) of these pharma-cists this also included dosages for selected patients. 44% (n=144) of all respondents indicated that their pharmacy software offered the technical capabilities to print a medication plan, whereas 36% (n=117) were uncertain about this. 15% (n=42) of the questioned pharmacists stated to print MPs or selected ambulant patients. Only two of the sent medication plans were for patients and based on the BMG specification. Conclusions: At present not all German community pharmacies have the technical capabilities to generate a MP with their pharmacy software systems. Furthermore, other medication lists are mistaken to be a MP for patients. For a successful nationwide implementation of the standardised MP it is necessary to improve (i) the technical capabilities of pharmacy software systems and (ii) the knowledge of community pharmacist.

References: 1. Spezifikation für einen patientenbezogenen Medikationsplan, Version 2.0 - für Modellvorhaben (2013). (Accessed July 10, 2014, at http://www.akdae.de/AMTS/Medikationsplan/index.html)

Systemic review of treatment options for functional gastrointestinal diseases: STW 5 is equivalent to MCP in the treatment of functional dyspepsia and has a superior safety profile Madisch, A.1; Vinson, B.R.* 2; Kelber, O.2; Kraft, K.3; Storr, M.4 1 Gastroenterology, Medical Clinic I, KRH Clinic Siloah , Hannover, 2 Clinical Research / Medical Information, Scientific Department, Steigerwald Arzneimittel-werk GmbH, Darmstadt, 3 Chair of Naturopathy, Center for Internal Medicine, University Medicine, Rostock, 4 Neurogastroenterology, Medical Clinic and Policlinic II, Clinic of Munich University Munich-Großhadern, Munich, Germany

INTRODUCTION: The use of metoclopramide (MCP) has recently been restricted due to the risk of extrapyramidal side effects, based on provisions of the European Regulatory Agency EMA and national regulatory authorities. Thus, MCP is no longer available for the treatment of chronic conditions such as dyspepsia and gastro-oesophageal reflux diseases. This necessitates revisiting of the clinical data for alternative treatments with equivalent clinical efficacy and a more benign safety profile, which were less visible in the past. AIMS & METHODS: As herbal medicinal products (HMPs) are widely used for functional gastrointestinal diseases including functional dyspepsia (FD) and irritable bowel syndrome (IBS) and with a favorable safety profile, a systematic review was conducted, in compliance with the PRISMA statement, to identify HMPs with a therapeutic efficacy comparable to MCP, using a data base search complemented by cross referencing and hand searching for assuring completeness. RESULTS: Six comparison studies using HMPs and MCP were identified, four of them with Ginger in the treatment of emesis and therefore out of scope, while two were conducted in FD and were therefore included into the evalua-tion. In these studies, STW 5, an herbal combination medicine was used. One study is an RCT in 94 patients [1], showing despite its small size a clear equivalence of both treatments, with a trend towards a faster onset of action and a lower number of UEs in the STW 5 group. The second, a retrospective epidemiologic analysis study was conducted in 960 patients treated with MCP or STW 5 [2] and confirmed these data in routine clinical practice, with a significantly higher proportion of symptom-free patients and a lower number of days off work in the STW 5 group. A literature search assessing the safety of STW 5 identified studies in more than 50.000 patients with FD and IBS [3], with a high degree of safety also in children [4, 5] and without any severe side effects and without an interaction potential with other medicines. CONCLUSION: For the treatment of functional gastrointestinal diseases such as FD, STW 5 was identified as a treatment with clinical efficacy equivalent to MCP but a superior safety profile. This allows an excellent long term treatment for patients with FD in the post MCP age.

References: 1. Nicolay, K. et al.: Gastro-Entero-Hepatologie 1984, 4: 24-28. 2. Raedsch, R. et al.: Z Gastroenterol 2007, 45: 1041-1048. 3. Ottillinger, et al.: Wien Med Wochenschr 2013, 163: 65-72. 4. Kelber, O.: Z Phytotherapie 2010, 31: 40-47. 5. Radke, M.: Gastroenterologe 2011, 6: 486-495.

1 + 1 ≠ 2? Gene expression profiling as a novel approach for developing fixed drug combination products Kelber, O.1; Abdel-Aziz, H.1; Okpanyi, S.N.1; Ulrich-Merzenich, G.2 1 Scientific Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany; 2 Center for Internal Medicine, Medical Clinics III, University Clinics Bonn, Bonn, Germany

Introduction: Combinations of drugs are common in many indications. But despite their established positive effects with regard to compliance, the number of available fixed combination products is comparatively small. Aim: The regulatory rules of FDA and EMA for the justification of fixed combination products are based up to now on the theses of Crout from 1974 [1]. Methods: Therefore an overview of the present state of scientific approaches in this field is given. Results: The equivalence or superiority of combinations, in comparison to the combination partners, is shown in studies with several arms, as the Vishnu-

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or the Diener study [2, 3]. In contrast, gene expression profiling allows a description of the pharmacological profile of the combination in comparison to the combination partners. According to this, the profile of the combination is different to that of the partners and the risk profile can be more favourable compared to that of the partners [4]. Discussion: A combination is, according to the equation 1 + 1 ≠ 2, a completely new active substance with an own pharmacological profile [4]. Instead of the combination partners [1], therefore the standard therapy in the indication should be used in comparison studies, as in other new active substances.

Acknowledgments: This contribution is dedicated to Prof. Dr. Dr. h.c. mult. Hildebert Wagner, who has described in a multitude of scientific contributions the multi-target effect of multi-drug-combinations. To Dr. Christiane Otto, Dr. Uwe Gessner, Dr. Carolin Stäbler and Dr. Christoph Theurer, Bayer Vital, Leverkusen, Prof. Dr. Thomas Effert, Universität Mainz and Prof. Dr. Alexander Panossian, Swedish Herbal Institute, Vallberga, best thanks for scientific discussions.

References: 1. Crout, J.: J Clin Pharmacol 1974; 14:249. 2. Eckles, R. and Voelker M.: Clin Pharm Drug Dev 2014; 3:118-125. 3. Diener, H.C. et al.: Cephalgia 2005; 25:776-787. 4. Ulrich-Merzenich et al.: Phytomedicine 2012; 19:322-329.

Investigations on the acceptance of new direct oral anticoagu-lants in long-term treatment of thrombo-embolic prophylaxis in patients with atrial fibrillation Hergenröther, A.1; Thuermann, P.A.2 ; Weiss, C.3; Harenberg, J.1 1 Instiute of Clinical Pharmacology, Medical Faculty Mannheim, Heidelberg of University, Germany 2 Institute of Clinical Pharmacology, University of Witten/ Herdecke, Germany 3 Department of Medicial Statistics, Medical Faculty Mannheim, Heidelberg of University, Germany

Background and aims: Oral anticoagulants are world-wide frequently prescribed for prevention and treatment of thrombotic and embolic diseases [1]. However, severe and life-threatening side effects, numerous drug interactions as well as regular blood coagulation monitoring limit their use. Direct oral anticoagulants (DOAC) such as dabigatran, rivaroxaban, apixaban and edoxaban became recently available as therapeutic alternatives. DOAC can be administered without regular coagulation monitoring with a similar therapeutic efficacy and a positive risk/benefit profile compared to VKA. Disadvantages include higher expenses than their conventional competitors [2]. In the present study we aimed to analyse whether patient’s personal characteristics may contribute to the decision-making process concerning the prescription and application of the DOAC as opposed to the conventional VKA. The superior aim of this study is to optimise treatment and therapeutic success by integrating a patient’s personal characteristics and preferences into the prescribing process. Materials and methods: Data were acquired over a period of 15 months. Patients treated (for a minimum of 2 months) with either VKA or DOAC were recruited from registered physicians and public pharmacies and written informed consent was obtained according to ethical approval of the study. The anticoagulation therapy survey was developed based on the results of a previous focus group interview [3]. In addition to a standardised questionnaire to assess personality traits (Freiburger Persönlichkeitsinventar, FPI-R) [4], a new questionnaire was developed and validated. After a pilot study (44 participants) the full-scale survey on anticoagulation therapy employing a shortened version of the questionnaire was completed. Biographic data and medical history, concomitant diseases and comedication were documented in an EXCEL datasheet (EXCEL, Microsoft Office, 2007). Statistical analyses using the chi-squared test (SAS version 9.2) were performed investigating the association between personal characteristics and the patient’s disposition for a change in anticoagulation therapy. Results: In total 117 anticoagulation patients aged between 24 and 92 years with 39.3 % female participants were included. 70.9 % of them specified atrial fibrillation as indication for anticoagulant therapy. At the time of the interview 84 participants were on VKA, whereas 33 patients took one of the DOAC. 23 of these patients had been previously treated with VKA. Although patients on VKA were quite satisfied with their treatment, 39 (47 %) of them were willing to

accept a change to DOAC. Patients willing to accept a change in therapy were slightly younger than those who refused. Gender differences were not observed. 74.4 % of the currently VKA-treated patients reported stable INR values. 52.1% of patients who had already switched to DOAC indicated complications in maintaining adequate INR-values prior to the change. The following factors were statistically significantly associated with the acceptance to change therapy: lack of a need for coagulation monitoring, doctor’s recommendation, personal interest in alternative drugs, prospect of an improvement in health-related quality of life, as well as the personal character-istics extrovert/introvert. Conclusion: About 50 % of the participants of our survey were willing to change anticoagulant therapy from VKA to DOAC. High variation in INR-values, opinion of the practitioner and several personality traits motivate patients to accept a change. Further investigations are required to prove the validity of these results.

References: 1. Moser, M., Bode, C.: Kardiologe 2012, 6:148–156. 2. Harenberg, J., Weiss, C.: Haemostaseologie 2013, 33:62-70. 3. Zolfaghari, S. et al.: Semin Thromb Hemost 2014, 40:121-128. 4. Fahrenberg, J., Hampel, R. and Selg, H. (2010): Freiburger Persönlichkeitsinventar, 8.

Auflage. Hogrefe Verlag Göttingen

Caco-2 cells – a suitable in vitro model to predict drug-drug interactions caused by induction of drug transporters? Brück, S.; Strohmeier, J.; Busch, D.;Siegmund, W.; Oswald, S.

Clinical Pharmacology, University of Greifswald, Center of Drug Absorption and Transport (C_DAT), Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany

Background: Intestinal absorption of many drugs is influenced by transport proteins. Induction of these proteins by concomitant treatment with inducers such as rifampicin or St. John’s wort (SJW) was shown to cause unwanted drug-drug interactions (DDIs). Although it would be desirable to predict these interactions using well established cellular transporter models, in vitro methods for evaluation of transporter induction are not yet established. Therefore, we investigated whether Caco-2 cells may be a suitable experimental model to predict the aforementioned DDIs. Methods: For induction studies, Caco-2 cells were incubated for 48 hours with pharmacologically relevant but non-toxic concentrations of carbamazepine (500 µM), efavirenz (10 µM), the SJW constituents hypericin (0.5 µM) and hyperforin (1 µM), and rifampicin (100 µM), respectively. Afterwards, mRNA expression and protein abundance were determined using TaqMan® low density arrays and LC-MS/MS-based targeted proteomics. Functional studies with respect to ABCB1 were performed using Transwell® assays with [3H]-digoxin and [3H]-talinolol as substrates. In parallel, mRNA expression and protein content of clinically relevant intestinal transporters in native Caco-2 cells and jejunal tissue specimens were compared. Results: After incubation with prototypical inducers, effects on mRNA expression and protein content could not be observed in Caco-2 cells. Consequently, the efflux ratios (B to A/A to B) for ABCB1 probe drugs in Caco2 monolayers were not significantly affected by the tested inducers. In contrast to this, ABCB1 function was nearly abolished by PSC833. With the exception of ABCB1, the gene expression and protein content of ABCC2, ABCC3, PEPT1, OATP2B1 and others were markedly different in Caco-2 cells compared to that from human jejunum. Conclusion: Established in vivo inducers of drug transport do not influence expression and function of drug transporting proteins in Caco-2 cells. Therefore, Caco-2 cells are no suitable in vitro model to predict transporter induction in man.

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DRUG DESIGN/MEDICINAL CHEMISTRY/ANALYTICS (MC01-MC62)

Synthesis, Enantiomeric Separation and Biological Evaluation of Fosmidomycin Thia Isosters as 1-deoxy-D-xylulose 5-phosphate reductoisomerase Inhibitors Lienau, C.1; Kunfermann, A.2; Illarionov, B.3; Held, J.4; Gräwert, T.3; Behrendt, C.T.1; Werner, P.3; Hähn, S.1; Eisenreich, W.2; Riederer, U.3; Mordmüller, B.4; Bacher, A.3; Fischer, M.3; Groll, M.2; Kurz, T. 1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany 2 Center for Integrated Protein Science Munich, Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany 3 Hamburg School of Food Science, Universität Hamburg, Grindelallee 117, 20146 Hamburg, Germany 4 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany

Multidrug-resistant pathogens endanger human health worldwide. Anti-infective drugs addressing novel targets are therefore urgently needed. Being absent in mammalians, the non-mevalonate pathway of isoprenoid biosynthe-sis (MEP pathway) is a promising target for the development of new antibiotics [1,2]. In several pathogenic bacteria and apicomplexan protozoa - including Plasmodium spp. - the MEP pathway is the only source of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr, IspC), a key enzyme within the MEP pathway, can be inhibited by the natural product forsmidomycin. Reverse α-aryl substituted carba- and oxa-analogs of fosmidomycin are highly active Dxr inhibitors [3,4,5]. We report on the synthesis, enantiomeric separation and biological evaluation of reverse α-aryl substituted β-thia isosters of fosmidomy-cin, which show potent antiplasmodial in vitro activity [6].

Fig. 1 S-Enantiomer of fosmidomycin thia isoster 5a in complex with PfIspC

References: 1. Jomaa, H. et al.: Science 1999, 285: 1573-1576. 2. Rohmer, M. et al.: Curr. Opin. Investig. Drugs 2004, 5: 154-162. 3. Behrendt, C.T. et al.: J. Med. Chem. 2011, 54: 6796-6802. 4. Behrendt, C.T. et al.: ChemMedChem 2010, 5(10):1673-1676. 5. Brücher, K. et al.: J. Med. Chem. 2012, 55: 6566-6575. 6. Kunfermann, A., Lienau, C. et al.: J. Med. Chem. 2013, 56: 8151-8162.

Enantiomeric purity of Magnesium bis(L-hydrogenaspartate) dehydrate Wahl, O.; Holzgrabe, U.

University of Würzburg, Institute for Pharmacy and Food Chemistry, 97074 Würzburg, Germany

Magnesium supplementation by means of organic magnesium salts is a very popular practice [1] e.g. with magnesium bis(L-hydrogenaspartate) dihydrate. This substance is monographed in the European Pharmacopoeia. Here, we examined the enantiomeric purity using a chiral capillary zone electrophoresis applying (2-hydroxypropyl)-β-cyclodextrin as a chiral selector coupled to laser induced fluorescence detection after CBQCA derivatization [2] and a HPLC-fluorescence method with chiral derivatization using o-phthaldialdehyde and N-acetyl-L-cysteine [3] as an orthogonal method. Two API batch samples and three drug products of the salt were analyzed by means of both methods. The concentration of the D-enantiomer of aspartic acid ranged from 0.03 to 0.12 per cent.

The substance is prepared from different magnesium salts and L-aspartic acid via neutralization and subsequent precipitation [4]. A closer look to the synthesis revealed the elevated D-aspartic acid content to be caused by the dissolution of L-aspartic acid at acidic pH values following the reaction pathway shown below [5].

Acknowledgements: Thanks are due to the Federal Institute of Drugs and Medical Devices (Bonn, Germany) for financial support and the European Directorate for the Quality of Medicines & HealthCare for the sample and reference substance supply.

References: 1. Garrison, S.R. et al.: Magnesium for skeletal muscle cramps, Cochrane Db. Syst. Rev., 2012. 2. Novatchev, N., Holzgrabe, U.: J. Pharm. Biomed. Anal., 2002, 28(3-4): 475-486. 3. Aswad, D.W.: Anal. Biochem., 1984, 137(2): 405-409. 4. Shaolin Huang, H.J., Feng Liu, Fei Wang, Yong Zhang: Method for preparing magnesium aspartate, CN 101239925 (A), Beijing Jingwei Xinkang Pharma, China, 2008. 5. Erbe, T., Bruckner, H.: Eur. Food Res. Technol., 2000, 211(1): 6-12.

Analytical technologies for the detection of counterfeit medi-cines: Are simple field assays still suitable for the routine quality control of pharmaceuticals in developing countries? Höllein, L.; Holzgrabe, U.

University of Würzburg, Institute of Pharmacy and Food Chemistry, Am Hubland, 97074 Würzburg

The increasing prevalence of poor-quality pharmaceuticals circulating in low- and middle-income countries, especially in the field of antibiotic and antimalar-ial medicines, is alarming because side effects, resistances or complete therapy failures may occur after the ingestion of such products [1]. Regulatory authorities in the developing world are scarcely able to control the situation within the national markets. The lack of human resources, limited technical equipment or narrow financial means at the respective testing institutions are only few reasons why counterfeit and substandard medicines can be easily distributed in these countries. Laboratories in resource-restraint environments are not always capable of adhering to compendial methods which are described in the major pharmaco-poeias, e.g. the European Pharmacopoeia. The application of sophisticated High-Performance Liquid Chromatography (HPLC) found in almost every monograph is still considered the gold standard, but demands for delicate consumables (chemicals, solvents, columns), expensive apparatus or skilled analysts for operation and maintenance [2]. These are critical bottlenecks during laboratory work in developing countries making a routine application almost impossible. The provision of adapted technologies for the analysis of essential medicines has been anticipated since many years and one of the major achievements in this area was the introduction of the Minilab® through Merck and the Global Pharma Health Fund in the late 1990s using colour reactions for identification

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and thin-layer chromatography (TLC) for a semiquantitative determination of the content for meanwhile 70 essential active pharmaceutical ingredients (APIs) [3]. Beside the simplicity of its protocols it also holds certain drawbacks being a failure rate of approximately 60 % during the quantitative assays [4]. Therefore we suggest the application of simplified liquid chromatographic methods instead which we developed for common antimalarial agents [5]. Only very simple chemicals and columns (e.g. no ion-pairing reagents) are necessary and they are able to quantitatively determine the respective APIs, resolve distinct impurities and can detect replacements or mix-ups. Our methods can be run on basic HPLC instruments at simple laboratories (e.g. no gradient pumps are required) and can be used for intermediate testing between the Minilab screenings and confirmatory assays using established HPLC protocols. Combining these technologies to three analytical ‘levels’ could be a promising solution to reliably detect poor-quality medicines circulating in the distribution chains.

References: 1. Tremblay, M.: Curr. Drug Saf. 2013, 8: 43-55. 2. Deconinck, E. et al.: J. Chromatogr. Sci., 2013, 51: 791-806. 3. Jahnke, R.W.O. et al.: Drug Inf. J. 2001, 35: 941-945. 4. World Health Organization: Survey of the Quality of Selected Antimalaria Medicines Circulating in Six Countries of Sub-Saharan Africa. 2011. 5. Hoellein, L. and Holzgrabe, U.: J. Pharm. Biomed. Anal, 2014, article in press.

Development of MIP-Inhibitors Seufert, F.1; Juli, C.1; Hein, M.1; Norville, I.H.2; Jenner, D.2; Sarkar-Tyson, M.2; Harmer, N.J.3; Weiwad, M.4; Schweimer, K.5; Rösch, P.5; Stacy, R.6; Myler, P.6; Begley, D.7; Fox, D.7; Lorimer, D.7; Sotriffer, C.A.1; Holzgrabe, U.1 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

2 Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK

3 Biocatalysis Centre, University of Exeter, Devon, EX4 4QD, UK

4 Institute of Biochemistry and Biotechnology, MLU Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germany

5 Department of Biopolymers, University of Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany

6 Seattle Structural Genomics Center for Infectious Disease, Seattle, WA

7 Emerald BioStructures, Bainbridge Island, WA

Macrophage infectivity potentiator proteins (MIP proteins) are virulence factors that facilitate the infection of cells with different pathogenic bacteria like Legionella pneumophila (Lp) or Burkholderia pseudomallei (Bp). Both pathogens manifest in different diseases, Lp is the causative agent of Legionnaires‘ disease, whereas Bp induces melioidosis. MIPs of both gram-negative bacteria show peptidyl prolyl cis/trans isomerase (PPIase) activity. These surface proteins belonging to the family of the FKBP binding protein form a stable complex with the immunosuppressive drugs FK506 or rapamycin which inhibit the enzymatic function of the mentioned MIPs (1, 2). Due to the immunosuppressive effects Legionnaires‘ disease and melioidosis can’t be treated with these drugs; thus novel inhibitors have to be developed. With the aid of crystal structures and nuclear magnetic resonance analyses of Bp and Lp MIP a key chemical scaffold was identified. Biophysical characterisation of this scaffold showed that binding site specificity in solution is possible. This series purveyed a low-micromolar affinity for Bp and Lp MIP without displaying toxicity in human macrophages or having immunosuppressive effects. In an in vitro assay, the compounds substantially reduce the Bp MIP activity, leading to a decreased macrophage infectivity and intracellular growth of Bp (3). This, the skeleton (1) can be regarded as a lead structure. The derived compound library was tested towards several MIP proteins. SARs were established.

Acknowledgments: Financial support DFG (SFB 630)

References: 1. Norville I.H. et al.: Infection and Immunity 2011, 79(11): 4299–4307. 2. Juli, C. et al.: J. Med Chem. 2011, 54: 277–283. 3. Begley D. et al.: Antimicrob Agents Ch. 2014, 58(3): 1458-1467.

Nonyl-linked bis-tacrine as a promising candidate for inhibition of Trypanothione reductase Schmidt, I.1; Schurigt, U.2; Krauth-Siegel, R.L.3; Jiménez-Ruiz, A.4; Holzgrabe, U.1 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 2 Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany 3 Center of Biochemistry, University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany 4 Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Carretera Madrid-Barcelona km 33,600, E-28871 Alcalá de Henares, Madrid,Spain

Parasites belonging to the order of Trypanosomatida are the causative agents for a variety of important infectious tropical diseases like African sleeping sickness, Chagas` disease and Leishmaniasis. Unfortunately, therapy is limited to the low number of currently available drugs and, due to spreading drug resistance, to their effectiveness. Therefore the development of new and cheap agents is urgently needed. A promising target for anti-trypanosomal drug development is the trypanothione reductase (TR) system required for DNA synthesis and defense against oxidative stress. Since it is unique for the parasites and absent in mammalian hosts, no selectivity problems arise. The search for inhibitors of TR is difficult because of its large binding pocket and, besides, the TR activity would need to be reduced by >85 % to prevent parasite proliferation [1]. The antiprotozoal activities of dimeric tacrine derivatives against Trypanosoma brucei and Leishmania major in a full cell assay were in high nanomolar range of concentration. In search of the mode of action, we found out that the TR of Trypanosoma cruzi, Trypanosoma brucei and Leishmania infantum is inhibited by our compounds. So further kinetic experiments were conducted and resulted in a competitive mode of inhibition. To give an example, Compound 1 possesses a very low Ki-value (< 1 µM) for Trypanosoma cruzi and Trypano-soma brucei TR and doesn´t inhibit human glutathione reductase, which is a cognate enzyme to TR in humans.

Compound 1

Acknowledgments We acknowledge the Deutsche Forschungsgemeinschaft (SFB 630) for support.

References: 1 Krauth-Siegel R.L., Leroux A.E.: Antioxid Redox Signal 2012, 17(4):583-607.

Development and validation of an HPLC method for determina-tion of vancomycin in human plasma and its comparison with PETINIA (Siemens) Usman, M.; Hempel, G.

Institute of Pharmaceutical and Medicinal Chemistry - Clinical Pharmacy, Westfälische Wilhelms Universität Münster, Corrensstr. 48, 48149 Münster, Germany

Vancomycin (VAN), a glycopeptide antibiotic is used against infections caused by gram positive bacteria particularly methicillin-resistant staphylococcus aureus (MRSA) infections [1-4]. Under dosing of VAN leads to drug resistance in bacteria and over dosing is associated with toxicity [5] therefore therapeutic drug monitoring (TDM) is recommended for optimizing VAN therapy [6]. For TDM plasma/serum-level determination of VAN is inevitable. In this investigation, a selective and sensitive method of high performance liquid chromatography (HPLC) was developed and validated for the quantifica-tion of vancomycin (VAN) in human plasma. The separation was carried out by

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using a mobile phase NH4H2PO4(50 mM)-acetonitrile (88:12, v/v) (pH 2.2) at isocratic flow rate 0.360 mL/min on a nucleodur C18 column (125 mm × 4.6 mm, particle size 5 µm) with UV detection at 205 nm. Sample preparation was done by deproteination of plasma with 70 % perchloric acid and a liquid/liquid extraction to remove lipophilic compounds. Validation was performed according to the European Medicines Agency (EMA) guideline. The method showed linearity over the range of 0.25-60 mg/L with a coefficient of determi-nation r2 ≥ 0.998 and a lower limit of quantification of 0.25 mg/L. No interfer-ence was observed in blank plasma samples at the retention time of VAN (Fig. 1). The percentage recovery and coefficient of variation (C.V %) values for accuracy and precision were within the acceptable limits with a mean percentage recovery (n = 5) of VAN between 91.46 % and 115.03 %. The latter value is for LLOQ where the acceptable limit is ±20 %. For precision, the C.V is ≤ 13.48 % except for LLOQ (17.82 %) where acceptable limit is 20 %. Stability was proved at room temperature for 24 hours, after repeated freeze-thaw cycles and storage at -20ºC for three months. A good correlation was observed (r2 = 0.898) by comparing with the results of particle enhanced turbidimetric inhibition immunoassay (PETINIA, Siemens) technique in 216 serum samples (Fig. 2).

Figure 1: Chromatograms of blank plasma (a) and plasma spiked with vancomycin 60 mg/L (b)

Figure 2: Correlation of VAN concentrations in 216 serum samples analyzed by PETINIA (Siemens) and HPLC.

Acknowledgments: We acknowledge the help of Dr. Manfred Fobker, Universitätsklinikum Münster (UKM) for this project.

References: 1. Van Bambeke, F.: Curr. Opin. Pharmacol. 2004, 4(5): 471-478. 2. Lundstrom, T.S.; Sobel, J.D.: Infect. Dis. Clin. North. Am. 2004, 18(3): 651-668, x. 3. Wilhelm, M.P.; Estes, L.: Mayo Clin. Proc. 1999, 74(9): 928-935. 4. Kullar, R. et al.: Clin. Infect. Dis. 2011, 52(8): 975-981. 5. Ingram, P.R. et al.: J. Antimicrob. Chemother. 2008, 62(1): 168-171. 6. Martin, J.H. et al.: Clin. Biochem. Rev. 2010, 31(1): 21-24.

Design and synthesis of covalent-reversible Inhibitors to over-came the drug-resistant gatekeeper Mutation in EGFR. Smith, S.; Basu, D.; Engel, J..; Rauh, D.

Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, D-44227, Dortmund, Germany

Acquired drug resistance is a major bottleneck in targeted cancer therapies. Cancer patients that suffer from non-small cell lung cancer (NSCLC)[1] caused by a point mutation (L858R) in EGFR (epidermal growth factor receptor), exhibit a dramatic relapse which is caused by secondary drug resistance mutations. The first-line treatment with erlotinib and gefitinib[2] proofs ineffective because of the newly formed gatekeeper mutation (T790M)[3], which increases the off-rate if the conventional reversible inhibitors[4]. Therefore new drug scaffolds with a minimal off-rate and a maximal drug-target residence time had to be developed.[5] Covalent inhibitors are equipped with Michael-acceptors that target a unique cysteine (C797) located at the lip of the

ATP binding site of EGFR. Some of these inhibitors including WZ-4002[6] and CO1686[7] have shown great potential in overcoming drug resistance in EGFR-T790M and are currently in further clinical development. Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRI) represent another class of interesting inhibitors that can be optimized for extended drug-target residence times. For CRI’s it was shown that the fast addition of thiols to electron-deficient olefins leads to a covalent bond that can break reversibly under proteolytic conditions (Fig. 1).[8] Although CRI’s are only tool compounds at this point, they might very well develop into innovative therapeutics to overcome some of the problems associated with conventional covalent inhibitors, such as toxicity or off-target activities.

Figure 1 Schematic representation of the mechanism of covalent reversible inhibition.

Here, we aim towards a better understanding of the kinetic mechanisms of CRI’s and to investigate their potential in targeting EGFR-T790M in cellular and in vivo systems. In a proof of concept study, we set out to design, synthesize and test compounds that can address C797 in drug resistant EGFR-T790M with an electron-deficient olefin.

References: 1. Wong, K.K.: Lung cancer 2008, 60: Suppl 2, S10. 2. Zandi, R. et al.: Cellular signalling 2007, 19: 2013. 3. Heuckmann, J.M. et al.: Journal of clinical oncology 2012, 30: 3417. 4. Kubo, K. et al.: Journal of medicinal chemistry 2005, 48: 1359. 5. Rabiller, M. et al.: Archiv der Pharmazie 2010, 343: 193. 6. Zhou, W. et al.: Nature 2009, 462: 1070. 7. Walter, A.O. et al.: Cancer discovery 2013, 3: 1404. 8. Serafimova, I.M. et al.: Nature chemical biology 2012, 8: 471.

Structure-Based Design, Synthesis and Biochemical Evaluation of Ligands Addressing a Lipophilic Binding Site in the MAP-Kinase p38α Bührmann, M.; Wiedemann, B.M.; Hardick, J.; Ecke, M.; Rauh, D.

Fakultät für Chemie und Chemische Biologie, Technische Universität, D-44227 Dortmund, Germany

Protein kinases catalyze the phosphotransfer from ATP to protein substrates and are involved in a number of signaling pathways. Hence, dysfunction of the fine-tuned regulation of kinase activity may result in diseases such as inflammation and cancer. Targeting kinase function with small organic molecules represents promising therapeutic strategies. However, due to the conserved nature of the kinase active site, ATP competitive inhibitors are often plagued by limited selectivity [1]. Allosteric ligands bind to distal binding sites and often stabilize catalytically inactive conformations. Such ligands are valuable starting points towards the development of more selective kinase inhibitors [2,3]. The mitogen-activated protein kinase (MAPK) p38α is a key player in inflammation and contains a unique hydrophobic pocket at its C-terminus about 30 Å away from the ATP-pocket. This allosteric site has so far no known biological function [4]. In a chemical biology approach, we aim to generate potent binders that may serve as molecular probes and help to dissect the functional role of this pocket in p38α. Recently, we have developed a fluorescence-based high throughput screening (HTS) direct binding assay for the allosteric pocket of p38α and identified scaffolds which bind exclusively to this site [5]. Using protein X-ray crystallography, we validated the screening results (Figure 1). We now present the structure-based design, organic synthesis and biochemical evaluation of follow-up compounds.

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Figure 1. Crystal structure of p38α in complex with 1 bound to the lipid binding site at the C-terminus (boxed)[5].

References: 1. Rabiller, M. et al.: Arch. Pharm. 2010, 343: 193–206. 2. Fang, Z.; Grütter, C.; Rauh, D.: ACS Chem. Biol. 2013, 8(1): 58–70. 3. Schneider, R. et al.: J. Am. Chem. Soc. 2012, 134(22): 9138–9141. 4. Diskin, R.; Engelberg, D.; Livnah, O.: J. Mol. Biol. 2008, 375(1): 70–79. 5. Getlik, M. et al.: PLoS ONE 2012, 7(7): e39713.

Synthesis of small molecules restoring the function of TRPML1 mutant isoforms causing mucolipidosis type IV Keller, M.1; Cheng-Chang, C.2; Kortum, F.1; Hawarden, A.1; Prothiwa, M.1; Hüwel, D.1; Schiffmann, R.3; Urban, N.4; Schaefer, M.4; Wahl-Schott, C.2; Biel, M.2; Grimm, C.2; Bracher, F.1 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany 2 Department für Pharmazie – Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany 3 Institute of Metabolic Disease, Baylor Research Institute, Dallas, TX, USA 4 Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Germany

Mucolipidosis type IV (ML IV) is a very rare autosomal recessive lysosomal storage disorder (LSD) characterized by severe neurological problems, progressive neurodegeneration, and ophthalmologic abnormalities [1]. Mutations in the TRPML1 gene are causative for ML IV. Some of these mutations lead to a loss or partial loss of function but do not cause severe mislocalization of the protein [2]. Starting from SF-22 [3], a TRPML1 activator discovered recently in a random screening, we generated several series of chemically modified SF-22 analogues with the aim to further improve the efficacy and potency profile of SF-22. Therefore we performed systematic modifications in any structural motif of the lead structure. These compounds were tested for their potential to restore TRPML1 mutant channel function. Using the whole-lysosome planar patch-clamp technique, we found out that some of the synthetic small molecules are acting as agonists and strongly increase the activity of the TRPML1 receptor. Trafficking defects as well as accumulation of heavy metals (zinc) in lysosomes of ML IV patient-derived cell lines could be rescued by the treatment with these small molecules. These findings demonstrate that small molecules can be used to restore TRPML1 channel function and to rescue disease associated symptoms in patient cells expressing certain TRPML1 point mutations.

References: 1. Schiffmann, R. et al.: Proc Natl Acad Sci USA 1998, 95: 1207-1212.

2. Altarescu, G. et al: Neurology 2002, 59: 306-313. 3. Grimm, C. et al: Chem. Biol. 2010, 17: 135-148.

Synthesis and pharmacological evaluation of Dithiocarbamates as novel anthelmintic inhibitors against Schistosomiasis Mäder, P.1; Blohm, A.2; Grevelding, C.G.2; Schlitzer, M.1 1 Institut für Pharmazeutische Chemie, Philipps-University Marburg, Marbacher Weg 6-10, 35032 Marburg 2 Institut für Parasitologie, Justus-Liebig-Universität Gießen, Schubertstraße 81, 35392 Gießen

Schistosomiasis, also known as bilharzia, is a chronic parasitic disease infecting more than 250 million people, and at least 200.000 deaths are associated with the disease.[1] After Malaria, it is the second most important parasitic disease worldwide occurring in over 70 countries in tropical and subtropical regions. [2] The schistosome species are all transmitted through the contact with water containing free-living larval forms of the parasite (cercariae) originating from intermediate host snails. [2] Due the lack of a vaccine, the therapy is currently limited to a single drug, praziquantel, which is the only drug effective against all schistosome species, and it has been used for more than 40 years. Every year, millions of people are treated with praziquantel, so that upcoming fear of drug resistance encourages the search for novel anthelmintic drugs against schistosomiasis. [2] [3] Dithiocarbamates as anthelmintic compounds were identified from a screening with disulfiram. Disulfiram was used for the treatment of chronic alcoholism by inhibiting the acetaldehyde dehydrogenase that converts acetaldehyde to acetic acid. [4] It has been observed that disulfiram also disintegrates the surface structure of schistosomes, the tegument, leading to the death of the parasites. We synthesized 22 dithiocarbamates instead of dithiocarbamate disulfides. The basic structure is shown in figure 1.

Figure 1: basic structure. All compounds with a methyl-, amide-, carboxylic acid or methylester as substituent for R3 are inactive, but the compounds with a lipophilic substituent for R3 such as benzyl or cyclic acetal showed a much better activity than disulfiram. The significant effects of dithiocarbamate compounds on adult schistosomes in vitro provides convincing evidence that these compounds have promise with respect to a new direction for chemotherapy of human schistosomiasis

References: 1. Maha-Hamadien, A. et al.: PLoS Medicine. 2007, 4(1): 130-138. 2. Thétiot-Laurent, S. et al.: Angew. Chem. Int. Ed. 2013, 52: 7936-7956. 3. Sayed, A. et al.: Nature Medicine. 2008, 14(4): 407-412. 4. Wickström, M. et al.: Biochem. Pharm. 2007, 73: 25-33.

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Fatty acid composition analysis in polysorbate 80 with high performance liquid chromatography coupled to charged aerosol detection Ilko, D.1; Braun, A.1; Germershaus, O.2; Meinel, L.1; Holzgrabe, U.1 1 Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany 2 Institute for Pharma Technology, University of Applied Sciences Northwestern Switzerland, Gruendenstrasse 40, 4132 Muttenz, Switzerland

Polysorbate 80 (PS 80), a sorbitan oleic acid ester copolymerized with about 20 mole of ethylene oxide per mole sorbitan, is a frequently-used non-ionic surfactant and stabilizing agent in protein formulations. Its fatty acid (FA) composition is characterized by gas chromatography after derivatization in the European (EP) [1] and United States Pharmacopoeia (USP) [2]. We developed an alternative method using HPLC in combination with a Corona® charged aerosol detector (CAD). After saponification with potassium hydroxide the FA fraction was collected with liquid-liquid extraction using methyl-tert-butyl ether. Method validation in terms of specificity, repeatability, limits of quantification, linearity, range, accuracy and robustness was performed. In addition, the method was expanded to the evaluation of the free FAs. Having determined the entire FA composition, the acid value according to EP and USP can be calculated. The characterization of four different PS 80 batches revealed considerable variability regarding their FA composition. Two FAs currently not mentioned in pharmacopoeias were found additionally. Petroselinic acid, a double-bond positional isomer to oleic acid was present in every batch. In addition, 11-hydroxy-9-octadecenoic acid, an oxidation product of oleic acid was found by means of HPLC-MS/MS.

References: 1. Polysorbate 80 Monograph 01/2011:0428 in European Pharmacopoeia, 8th Edition, European Directorate for the Quality of Medicines & HealthCare (EDQM), 2014, Strasbourg, France. 2. Polysorbate 80 Monograph in United States Pharmacopoeia, USP 37 NF 32, The United States Pharmacopeial Convention, 2014, Rockville, MD, USA.

Synthesis of simplified amide analogues of the antimycobacterial cyclodepsipeptide pyridomycin Klemm, M.; Imming, P.

Martin-Luther-Universitaet Halle-Wittenberg, Institut fuer Pharmazie, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany

Maeda et al. isolated pyridomycin from cultures of Streptomyces pyridomyceti-cus in 1953 [1]. Pyridomycin is a twelve-membered cyclic depsipeptide containing five stereocentres, substituted with a semicyclic (Z)-s-butylidene group, 3-pyridylmethyl side chain and an exocyclic hydroxypicolinoyl ring [2]. It shows activity against Mycobacterium tuberculosis at 0.6 –1.2 µg/ml by inhibiting NADH-dependent enoyl acyl carrier protein reductase InhA directly [3] and has the unique feature to block both substrate and co-factor binding sides of InhA [4]. As little is known about pyridomycin SAR, analogues are promising candidates for further investigation. In our group, we synthesized simplified analogues of pyridomycin. We established a sequence with formation of the amide bond first to reduce the risk of inter- or intramolecular transacylations followed by the formation of the two ester bonds. [5] The semicyclic (Z)-s-butylidene group was replaced by different aliphatic side chains as it was recognized to be unessential for biological activity [6]. The ring closure poses the crucial step of the whole synthetic procedure because of possible di/polymerization, resulting in difficult purification. We decided to synthesize cyclic depsipeptides in which both or either of the ester bonds is replaced by amide bonds combining the advantages of less likelihood for transacylations with better yields for the ring closure step implementing macrolactamization instead of macrolactonization [5, 6]. We present synthetic approaches to a pyridomycin-analogous backbone replacing one of the ester bonds by an amide bond. The cyclic depsipeptides are synthesized via a building block strategy along the amide and ester bonds implying the need for orthogonal protecting groups. As the exocyclic side chain

will need to bear additional functional groups, requiring protective groups, exocyclic amidation is the last step of the synthesis. We extend this by discussing a stereoselective synthesis for 2,3-diaminobutyric acid by a Mitsunobu reaction starting from allo-threonine needed as starting material for 12-membered cyclic peptides, replacing both ester bonds by amide bonds. Pyridomycin

References: 1. Maeda, K. et al.: J Antibiot (Tokyo) 1953, 6(3): 140. 2. Koyama, G. et al.: Tetrahedron Lett. 1967, 37: 3587–3590. 3. Hartkoorn, R.C., et al.: EMBO Mol Med 2012, 4: 1–11. 4. Hartkoorn, R.C., et al.: Nature Chemical Biology 2014, 10: 96–98. 5. Laqua, K. et al.: paper in preparation. 6. Horlacher et al.: ACS Med. Chem. Lett. 2013, 4(2): 264–268.

Molecular details of the reaction of nitro, nitroso, hydroxylamino and amino benzothiazinones with Mycobacterium tuberculosis DprE1 Richter, A.1; Rudolph, I.1; Chung, C.2; Singh, O.2; Argyrou, A.2; Ballell, L.3; Voigt, K.4; Möllmann, U.4; Imming, P.1

1 Institut fuer Pharmazie, Martin-Luther-Universitaet, Wolfgang-Langenbeck-Str. 4, Halle (Saale), 06120, Germany 2 GSK Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom 3 Diseases of the Developing World, GSK, Severo Ochoa 2, Tres Cantos, Madrid, 28760, Spain 4 Leibniz Institute for Natural Product Research and Infection Biology Hans-Knöll- Institute, Beutenbergstr. 11a, Jena, 07745, Germany

Benzothiazinones (BTZs) display very strong antimycobacterial activity in vitro [1]. Some derivatives of this compound class, for example BTZ043 [2] and PBTZ169 [3], are very active against Mycobacterium tuberculosis in vitro with MICs < 1nM. The outstanding in vitro activity is not yet fully translated to in vivo effectivity, with relatively high doses of 50-200 mg/kg daily required for considerable reduction of CFUs in mice [1,4]. BTZs inhibit the mycobacterial enzyme decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE1), interfering with the construction of a functioning cell wall. Important for the activity is a nitro group in the molecule, which is reduced by FADH2/DprE1 to a nitroso BTZ that binds covalently to the thiol group of Cys-387 near the catalytic centre [5]. In Fig. 1 we fully depict the complex process of this two-step mechanism-based inhibition:

Figure 1. Steps of DprE1 inhibition by BTZs

BTZs with nitro, nitroso, hydroxylamino and amino groups were directly incubated with DprE1 of Mycobacterium tuberculosis. The nitroso BTZs were synthesized and purified for the first time [6]. The covalent binding to the target was corroborated by mass spectral data, consistent with previous findings [7, 8]. BTZ-DprE1 X-ray structures with

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different BTZs were solved and indicated a very similar binding mode of all BTZs investigated, notwithstanding their different in vitro or in vivo activities. For a deeper insight into the molecular activity of BTZs, long-term enzyme kinetic data were collected and evaluated. This information improves the understanding of the complex BTZ-DprE1 interaction better, trying to find an answer to the question what are the key determinants for highly active DprE1 inhibitors. References: 1 Makarov, V. et al.: Science 2009, 324: 801 – 804. 2 Möllmann, U.; Makarov, V.; Cole, S.T.: WO2009010163A1 2009. 3 Makarov, V.; Cole, S.T.: WO2012/066518A1 2012. 4 Makarov, V. et al.: EMBO Mol. Med. 2014, 6 (3): 372-383. 5 Trefzer, C. et al.: J. Am. Chem. Soc. 2012, 134 (2): 912 – 915. 6 Davey, M.H. et al.: J. Org. Chem. 1999, 64 (13): 4976-4979. 7 Trefzer, C. et al.: J. Am. Chem. Soc. 2010, 132 (39): 13663 – 13665. 8 Neres, J. et al.: Sci. Transl. Med. 2012, 4 (150): 150ra-121.

Synthesis of peptide and depsipetide analogues of Hirsutellide A using a solid phase peptide synthesis approach for antimyco-bacterial activity testing Asfaw, H.; Imming, P.

Martin-Luther-Universitaet Halle-Wittenberg, Institut fuer Pharmazie, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany

Hirsutellide A is an 18-membered symmetrical cyclic depsipeptide that was isolated from a cell extract of the entomopathogenic fungus Hirsutella kobayasii BCC 1660 and reported to exhibit antimycobacterial activity with a minimum inhibitory concentration (MIC) of 6–12 µg/mL towards M. tuberculosis H37Ra [1]. The structure of Hirsutellide A was elucidated by analyses of spectroscopic data and shown to contain L-allo-isoleucine, (R)-2-hydroxy-3-phenylpropanoic acid and sarcosine. Though a synthetic strategy for one of stereoisomers of Hirstullide A was reported using a standard solution phase approach [2], it was found to be time consuming and purification of the intermediate products was troublesome. Hence, to synthesize a variety of derivatives for SAR study, a more rapid and efficient synthetic route is needed. We report a new solid phase peptide synthetic approach for preparation of some peptide and depsipeptide analogues of Hirsutellide A to define its SAR with a hope of generating potent antituberculotic agents. The synthesis involved a stepwise solid phase synthesis of the linear hexapeptide and hexadepsipeptide precursors based on an Fmoc protecting group strategy on chlorotrityl resin followed by macrocyclization in solution phase under conditions of highly dilution. All amide bond formations on the solid support were done by using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) with diisopro-pylethylamine (DIPEA) as the coupling agent. The ester bond of the linear hexadepsipetide was formed using N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) mediated coupling. While ring closure for the peptide analogue was achieved via macrolactamization using HATU/DIPEA, the cyclic depsipeptide analogue was formed via macrolactoni-zation using 2-methyl-6-nitrobenzoic anhydride (Shiina’s reagent) [3].

References: 1. Vongvanich, N. et al.: J. Nat. Prod. 2002, 65: 1346-1348. 2. Xu, Y. et al.: Tetrahedron Lett. 2005, 46: 437- 4379. 3. Shiina, I. et al.; Tetrahedron Lett. 2002, 43, 7535-7539.

Detection and separation of microparticles using surface modified polymer particles Shopova, T.1; Holländer, A.2; Keusgen, M.1

1 Institute for Pharmaceutical Chemistry, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Germany

2 Fraunhofer Institut for Applied Polymer Research, Gieselbergstraße 69, D-14476 Potsdam, Germany]

Because of a global increase of infections by microorganisms and viruses, there is a continuous demand for rapid detection methods. In the here presented approach, the surface of polyethylene microparticles was modified in a manner that bacteria and fungi will be bound reversibly. The polyethylene particles were filled in a chromatographic tube and retention was investigated for artificial microparticles as surrogates for cells, for bacteria, and fungi. A light scattering detector was used for analysis. The goal of this investigation is the rapid separation and detection of microorganisms in a chromatography-like manner.

Introduction: Bacterial and viral infections have increased rapidly during last years, especially in developing countries. There are many reasons for this as incorrect application of antibiotics, the increasing globalization, contamination of large amounts of foods, etc. Conventional methods for the detection of bacteria are culture techniques (cultivation on selective agar plates and examining the cultures by metabolic markers) as also various immunoassays. The major drawback is that these methods are labour-intensive and it takes several days to obtain results and confirmation (between 2 and 3 days for immunoassays and up to 7-10 days for culture methods). There are also some biosensoric approaches for rapid detection of bacteria [1]. But nevertheless, there is an increasing demand for inexpensive, rapid and sensitive diagnostic methods for the analysis of microorganisms. The here presented approach is based on polymer surfaces with various chemical compositions [2]. Polyethylene seems to be a suitable material [3].

Results and Discussion: Preliminary investigations showed that surface modified polyethylene beads can be used for separation of functionalized microparticles. As shown in Figure 1, amino-functionalized polyethylene beads were filled in a column and microparticles [carboxy-functionalized polymethylmethacrylate (PMMA-COOH)] could be eluted under various pH conditions. PMMA-COOH particles were firstly dispersed in water and than applied to the column. During the elution with water, no particles could be detected (Fig 1, violet). Only when using sodium hydroxide solution (pH 11) as an eluent, the detector showed a significant signal (Fig 1 green). PMMA-COOH particles without H2O washing led to rapid elution (Fig 1, red). Additionally, experiments with the bacteria Escherichia coli and the yeast Saccharomyces cerevisiae were carried out also showing different reversible retentions of these cells. The ongoing research is focussed on improved polyethylene beads as well as forming monolithic filling materials for the column.

Figure 1: Elution of 1 µm PMMA-COOH particles from amino–polyethylene beads under various conditions.

Conclusions: Surface functionalized polyethylene beads seem to be a promising material for the separation of microparticles, especially microorganisms.

Acknowledgments: The Authors kindly want to thank Mr. Van Elsäcker for his support.

References: 1. Mazumdar, S. et al.: Biosensors and Bioelectronics 2007, 22: 2040-2046. 2. Kumar, A. et al.: Nature protocols 2010, 5: 1737-1747. 3. Holländer, A.: Patent WO002006077020A2 2006.

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Inhibitors of “New Permeability Pathways” Steiner, I.S.1; Baumeister, S.2; Lingelbach, K.2; Schlitzer, M.1 1 Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg 2 Philipps-Universität Marburg, Entwicklungsbiologie und Parasitologie, Karl-von-Frisch-Straße 8, 35043 Marburg

Upon Malaria-infection intensive remodeling of the host red blood cell takes place. Novel pathways, commonly known as “New Permeability Pathways” (NPP), are established to take up solutes. These novel structures are not fully identified yet, but at least some may have several characteristics of anion channels.[1] Examples of known NPP inhibitors are furosemide and NPPB.[2] We have prepared several new compounds which inhibit solute uptake of cultured malaria parasites. As an additional design feature these compounds are fluorescent. These fluorescent compounds are intended to be used as tools for further identification and characterization of proteins which constituted the NPP:

Inhibitor-Design NPPB On long term some of these structures may ultimately be used as basis for a development of drug candidates. References: 1. Baumeister, S. et al.: Protoplasma 2010, 240: 3-12. 2. Kirk, K.; Horner, H.A.: Biochem. J. 1995, 311: 761-768.

Synthesis, characterization and screening of Dengue Virus S2B-NS3 protease inhibitors Wu, H.1; Holloway, S.1; von Hammerstein, F.1; Berger, T.1; Weidner, T.1, Kiefer, W.1, Bodem, J.2; Bock, S.2; Snitko, M.2; Kanitz, M.3 Steuber, H.3; Diederich, W.3; Schirmeister, T.1 1 Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099 Mainz, Germany 2 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D-97078 Würzburg, Germany 3 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6-10, D-35032 Marburg, Germany

Dengue fever is a mosquito-borne tropical disease caused by the dengue virus (DENV). It is transmitted to humans by the Aedes aegypti and Aedes albopictus mosquitoes. [1] This viral infection is becoming continually a global threat, as there are nearly 3.6 billion people living in the areas, tropical and subtropical regions of the world (predominantly in Southeast Asia, Africa and the Americas), where the infection is common. [2,3] The DENV infection can result in classic dengue fever, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). [4] Worldwide there are over 50 million infections reported annually and the infection causes over 20,000 deaths each year. [5] DENV is a single positive-stranded RNA virus of the family Flaviviridae with four distinct serotypes. [6,7] The dengue virus genome encodes a serine protease with a classical catalytic triad (His51, Asp75 and Ser135) [8,9] which is responsible for the post-translational proteolytic processing of the polypro-tein precursor and essential for the viral replication [10,11], making it an important and attractive therapeutic target. [12]

Our projects include synthesis, characterization and testing of DENV2/3 NS2B-NS3pro inhibitors based on the structure of variously substituted diaryl (thio)ethers. The possible binding modes are analyzed by docking studies. The synthesized compounds are screened both in vitro in fluorometric enzyme assays using different fluorogenic AMC-derived substrates and in cell culture. Additionally, Microscale Thermophoresis (MST) is used to investigate the binding of selected diaryl (thio)ethers to DENV NS2B-NS3pro.

Substitution layout of the diaryl (thio)ethers: X = NO2, CF3, NH2, H, COOH, aromatic or aliphatic residues Y = S, C, O Z = variously substituted aromatic, heterocyclic or aliphatic residues

[University of Mainz for financial support] References: 1. Yildiz, M. et al.: ACS Chem. Biol. 2013, 8: 2744-2752. 2. Murray, N.E.A. et al.: Clinical Epidemiology 2013, 5: 299-309. 3. Guzman, M.G. et al.: Nat. Rev. Microbiol. 2010, 8: S7-S16. 4. Martina, B.E. et al.: Clin. Microbiol. Rev. 2009, 22: 564-581. 5. Wilder-Smith, A. et al.: Arch. Med. Res. 2002, 33(4): 330-342. 6. Li, H. et al.: J. Virol. 1998, 73(4): 3108-3116. 7. Chambers, T.J. et al.: Annu. Rev. Microbiol. 1990, 44: 649-688. 8. Bazan, J.F.; Fletterick, R. J.: Virilogy 1989, 171: 637-639. 9. Melino, S., Paci, M.: FEBS Journal 2007, 274: 2986-3002. 10. Falgout, B. et al.: J. Virol. 1991, 65: 2467-2475. 11. Zhang, L.; Mohan, P.M.; Padmanabhan, R.: J. Virol. 1992, 66: 7549-7554. 12. Zheng, Y. et al.: Bioorg. Med. Chem. Lett. 2006, 16: 36-39.

The Apicoplast: An Organelle of particular Interest in antimalarial Drug Development Schäfer, E.-M.1; Ortmann, R.1; Degenhardt, I.; Boomgaren, M.; Dahse, H.-M., Hillebrecht, A.; Seeber, F.2; Baumeister, S.3; Lingelbach, K.3; Klebe, G.1; Schlitzer, M.1 1 Institut für pharmazeutische Chemie, Fachbereich Pharmazie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Deutschland. 2 Robert-Koch-Institut, FG 16: Erreger von Pilz- und Parasiteninfektionen und Mykobakte-riosen. D-13353 Berlin, Deutschland 3 Institut für Entwicklungsbiologie und Parasitologie, Fachbereich Biologie, Philipps-Universität Marburg, Karl-von-Frisch-Str.8, D-35043 Marburg, Deutschland.

Among infectious diseases Malaria still is one of the biggest problems for public health especially in developing countries.[1] Due to rising resistance the clock’s been ticking on the development on highly active antimalarial drugs. The apicoplast represents a crucial target with its various singular and essential pathways and functions. Fosmidomycine is a drug in clinical development that inhibits DOXP-reductase, an enzyme of the non-mevalonate pathway of isoprenoid synthesis that is located in the plasmodial apicoplast and thus is active against erythrocytic phases of the parasite. We were able to increase the in vitro activity of FR900098.[2]

An important house keeping function of the apicoplast is the modification of proteins. At the end of protein biosynthesis a formyl residue needs to be split off the N-terminal of the protein for its activation. This is done by the enzyme peptiddeformylase. Actinonine is a known natural product that not only inhibits this enzyme but also the proliferation of malaria parasites and thus is not applicable. We were able to find a synthetic ten fold more active inhibitor of the peptiddeformylase.

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A promising target in the apicoplast is the redox-system ferredoxin/ferredoxin-NADPH-reductase. This system is essential for the synthesis of iron-sulphur clusters and its distribution to enzymes of various essential pathways like the lipoic-acid synthesis or the non-mevalonat isoprenoid synthesis. Therefore an inhibition of this system meant an inhibition of these essential pathways and lead to the parasites death. Some active compounds have been found and represent a basis for further development.[3]

References: 1. World Malaria Report 2013, World Health Organisation, Genf, Schweiz. 2. Bormann, S. et al.: JID, 2004, 189: 901-908. 3. Seeber, F.; Aliverti, A.; Zanetti, G.: Curr. Pharm. Des. 2005, 11: 3159-3172.

Synthesis and characterization of liposome-bound thrombin inhibitors Endreas, W.1; Brüßler, J.2; Bakowsky, U.2; Steinmetzer, T.1 1 Institute of Pharmaceutical Chemistry, Philipps University, 35032 Marburg, Germany 2 Institut of Pharmaceutical Technology and Biopharmacy, Philipps University, 35032 Marburg, Germany

Thrombin is a serine protease and the final enzyme of the blood coagulation cascade. Meanwhile, various direct thrombin inhibitors such as oral dabigatran etexilate and parenteral r-hirudin, bivalirudin or argatroban have been approved and can be used as anticoagulants for various prophylactic and acute applications. Moreover, thrombin is easily available and there are several methods to determine its activity in vitro. Therefore, it is an excellent tool for studying receptor/ligand interactions. Many peptidic protease inhibitors suffer from poor bioavailability, metabolic instability or short half-life due to rapid elimination. In case of parenteral inhibitors it is possible to overcome some of these problems by modification with larger polymers, such as polyethylenglycols. Here we describe a different strategy by developing potent and selective thrombin inhibitors suitable for presenting them on the surface of liposomes. For this purpose our previously described thrombin inhibitors containing L-amino acids in P3 position such as Lys or Asp were used. X-ray structure analysis of the complex with thrombin revealed that the side chain of their P3 residue is directed into the solvent and might be used for further modification without losing potency [1]. Therefore, we have prepared several new thrombin inhibitors, whereby the P3 side chain was modified with short and defined amino- or carboxyl-functionalized ethylene glycol linkers. These derivatives can be either used for direct anticoagulant modification of artificial surfaces and are suitable for further coupling with fatty acids and incorporation into liposomes. Several of the palmitoylated derivatives inhibit thrombin in the subnanomolar range, e.g., an inhibition constant of 0.65 nM was determined for analogue MI-902. This inhibitor was used for the preparation of liposomes. Analysis of the supernatant after liposome preparation by HPLC, MS and thrombin activity tests revealed that the inhibitor was completely incorporated. These liposomes possess potent thrombin inhibitory activity in enzyme kinetic studies and strong anticoagulant activity in plasma clotting assays. Replacement of the thrombin inhibitor by biotin provided a related analog MI-908. This compound together with the thrombin inhibitor MI-902 was used for the development of bifunction-alized liposomes, as shown below. The additional biotinylation can be used for the further characterization of the liposomes. Our studies show that it is possible to develop liposomes suitable for the protease inhibition. This concept should be applicable for other enzyme or protease inhibitors as well.

NHHN

SNH

O

O

H HO

OON

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SO2

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HNO

NH

NH2O

HN O

OO

NH

O

palmitoyl chain

inhibitor segment

linker

O

linker

MI-902 MI-908

Reference: 1. Steinmetzer, T. et al. : ChemMedChem. 2012, 7: 1965-1973.

Regulation of cNMP concentrations in RFL-6 fibroblasts by activators and stimulators of soluble guanylate cyclase Böttcher, M.M.1; Kaever, V.1, 2, Stasch, J.-P.3 Seifert, R.1

1 Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 2 Research Core Unit Metabolomics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 3 Cardiology Research, Bayer HealthCare AG, Apratherweg 18 a, 42096 Wuppertal, Germany

Soluble guanylate cyclase (sGC) plays an important role in the physiology and pathophysiology of numerous cardiovascular diseases [1]. sGC is activated by binding of nitric oxide (NO) to the heme group of the enzyme. Recently, two classes of compounds have been discovered that amplify the function of sGC, the so-called sGC stimulators and sGC activators. The sGC stimulators, such as BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine) directly stimulate sGC, both independently of NO and in synergy with NO. In contrast, sGC activators, such as BAY 58-2667 (4-[[4-carboxybutyl-[2-[2-[[4-(2-phenylethyl)phenyl]methoxy]phenyl]etyl]amino]methyl]benzoic acid) preferen-tially activate sGC, when it is in an oxidized or heme-free state [2]. Purified sGC does not only form cGMP, but also synthesizes other cyclic nucleotides such as cAMP, cCMP and cUMP [3]. Therefore, the objective of this project is to examine whether sGC stimulators and activators induce production of other cyclic nucleotides in intact cells. As model system we use RFL-6 lung fibroblasts that express sGC endogenously [4]. RFL-6 fibroblasts were treated with BAY 41-2272 and BAY 58-2667. Their effects on cNMP concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry [5]. BAY 41-2272 increased cGMP in a time-dependent manner. Moreover, at later time points, BAY 41-2272 also increased cAMP. Currently, we examine the effects of a combination of the sGC stimulator and NO as well as with a phosphodiesterase (PDE)5 inhibitor on cNMP concentrations. In future studies, longer time course experiments will be conducted in order to detect a potential increase in cCMP or cUMP concentration. The time course studies are particularly important in view of the fact that in sGC-transfected HEK293 cells, NO increased cCMP and cUMP only after 60- 120 minutes [5].

Fig. 1: Effect of BAY 41-2272 [10 µM] on cNMP concentrations in RFL-6 cells. (Two-way ANOVA with Bonferroni post-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001)

References: 1. Stasch, J.P. et al.: Circulation 2011, 123: 2263-2273.

cAM

P

cCM

P

cGM

P

cUM

P

0

10

20

30

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50

Basal

5 min

10 min

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**

*

***

***

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[ p

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g p

rote

in]

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2. Follmann et al.: Angew Chem Int Ed Engl. 2013, 52: 9442-9462. 3. Beste, K.Y. et al.: Biochemistry 2012, 51: 194-204. 4. Förstermann, U. et al.: Proc. Natl. Acad. Sci. USA 1991, 88: 1788–1792. 5. Bähre, H. et al.: Biochem. Biophys. Res. Commun. 2014, 443: 1195-1199.

In silico library design for BID inhibitors Wegscheid-Gerlach, C.1; Barho, M.T.1; Kraus, A.L.1; Oppermann, S.2; Culmsee, C.2; Schlitzer, M.1 1 Institute for Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Germany 2 Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-Universität Marburg, Karl-von-Frisch-Str. 1, D-35032 Marburg, Germany

Bid has been discovered as a potential target for progressive neuronal death in age-related neurological diseases. By inhibition of the BH3-only protein BID, a prevention of mitochondrial damage and a delayed neuronal death after oxygen-glucose-deprivation can be achieved. Recently, two new structural classes of lead structures have been identified [1, 2], which prevent tBID-induced toxicity.

To obtain more insights in the structure-activity relationships of these compounds a virtual library design approach was chosen. Building blocks were handpicked by application of basic medicinal chemistry rules to introduce structural variations. Furthermore, a structure-based approach called KNOBLE [3] was used to select building blocks with direct information of the binding pocket of BID. Bound fragments are extracted using structural information derived from similar subpockets which can be identified within the Cavbase [4], a database developed from the database RELIBASE. Subsequently, a substructure search taking feasible synthetic strategies into account was performed giving rise to a subset of putative building blocks with suitable functional groups for the later synthesis. Afterwards, an enumeration of all compounds is done. A fast filtering is achieved by evaluation of the entirely assembled molecules‘2D and 3D descriptors. Finally, all compounds with desired properties are evaluated by a docking run using FlexX as implemented in LeadIT [5].

References: 1. Oppermann, S. et al. J Pharmacol Exp Ther. 2014, 350(2):273-289. 2. Barho, M. et al. ChemMedChem 2014, accepted. 3. Gerlach, C. et al. Angew Chem Int Ed Engl. 2007, 46(47):9105-9109. 4. S. Schmitt, D. Kuhn, G. Klebe, J. Mol. Biol. 2002, 323: 387. 5. a) Rarey, M., et al. J. Mol. Biol. 1996, 261(3):470–489. b) BioSolveIT, St. Augustin, Vers. 2.1.7

Validation of an analytical method for simultaneous quantifica-tion of Midazolam and 1’-Hydroxymidazolam in human plasma, using QTOF liquid chromatography/mass spectrometry. Radke, C.1; Fabian, J.2; Hempel, G.1 1 Department of Pharmaceutical and Medicinal Chemistry – Clinical Pharmacy, University of Münster, Corrensstrasse 48, 48149 Münster, Germany 2 Department of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstrasse 48, 48149 Münster, Germany

Introduction: Midazolam (MDZ) is a widely used sedative in intensive care units, especially for mechanically ventilated critically ill patients (e.g. septic patients). It is often administered as a continuous infusion and therefore increasing concentrations of MDZ and its main metabolite 1’-Hydroxymidazolam (1’-OHM) can be expected. The aim of our investigation was to develop and validate a quadru-pole time-of-flight (QTOF) based mass spectrometry method for the simultane-ous quantification of both, MDZ and 1’-OHM, in human plasma, suitable to assess increasing concentrations in septic intensive care patients. Method: Blank plasma samples were spiked with the analyte and the internal standard (IS) Clobazam and were afterwards extracted by a liquid-liquid extraction (LLE) procedure. A Dionex Ultimate 3000 LC system (ThermoScientific, Waltham,

MA, USA) was used for separation of the samples which was performed on a Luna C18(2) (2 x 100 mm, particle size 3 µm, Phenomenex, Torrance, CA, USA) using a gradient method at a flow rate of 0,3 ml/min and an injection volume of 3 µl. For sample acquisition and quantification, a micrOTOF-Q II (Bruker Daltonik, Bremen, Germany) mass spectrometer, operating at a mass range from m/z 290 to m/z 360, was used. Validation was assessed according to the EMA guideline on bioanalytical method validation. Results: The method showed valid results which meet the specifications of the EMA guideline. Using a 1/x weighted quadratic regression, the calibration curve showed an excellent fit over a concentration range of 3.5 – 1500 ng/ml for both, MDZ and its main metabolite 1’-OHM. Within-run accuracy for MDZ ranged between 97.7 – 99.7% with a precision of 2.0 – 6.2%, whereas the results for 1’-OHM ranged between 99.5 – 107.2% and 3.9 – 6.4%. Between-run accuracy for MDZ and 1’-OHM ranged over 98.7 – 100.7% and 96.5 – 101.7%, with a corresponding precision of 6.2 – 9.2% and 6.9 – 10.7%, respectively. Absolute recoveries for MDZ, 1’-OHM and Clobazam were 78.5±4.2%, 76.0±5.3% and 68.0±2.1% (mean ± SD), respectively. Selectivity was examined using six individual sources of blank plasma. Furthermore three possibly co-administered drugs (Bisoprolol, Piperacil-lin/Tazobactam, Dobutamin) as well as the constitutional isomer 4-Hydroxymidazolam were examined for interference. No interfering chroma-tographic and mass spectrometric peaks could be detected at the respective retention times neither in blank plasma samples nor in the drug-spiked samples. In addition, no matrix effects could be detected for the three substances. A dilution factor of five and ten was tested and accuracy and precision were within the specified limit of ± 15%. Stability of stock solutions and sample probes were not studied, because former investigations determined a long-term stability of up to ten months at -20°C, a short-term stability of up to three days at room temperature and no affection of stability due to three freeze-thaw cycles [1]. Conclusion: Although triple quadrupole (QqQ) mass spectrometer are nowadays state of the art for drug quantification, this investigation is the first method to determine the concentration of MDZ and its main metabolite 1’-OHM in human plasma, using a QTOF mass spectrometer. The wide concentration range of the calibration curve offers the possibility to monitor increasing plasma concentra-tions, due to constant drug infusion, which might be useful for pharmacokinetic studies in special subpopulations, e.g. critically ill patients.

Acknowledgments: This investigation was supported by Bayer Technology Services GmbH (Leverkusen, Germany).

Reference: 1. Shimizu, M. et al.: J Chromatogr B Analyt Technol Biomed Life Sci. 2007, 847(2): 275 – 281.

Active Site Inhibitors Disturbing the Dimerization of tRNA-Guanine Transglycosylase (TGT) Ehrmann, F.R.1; Barandun, L.J.2; Debaene, F.3; Betz, M.1; Heine, A.1; Sanglier-Cianférani, S.3; Diederich, F.2; Klebe, G.1 1 Institut of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35037 Marburg, Germany 2 Laboratory of Organic Chemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland 3 Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC-DSA, Université de Strasbourg, CNRS UMR7178; 25 rue Becquerel, 67087 Strasbourg, France

tRNA-Guanine Transglycosylase (TGT) is a putative target enzyme to fight pathogenicity of Shigella flexneri, the causative agent of Shigellosis. This bacterial dysentery occurs predominantly in developing countries and is responsible for several million cases of death per year. The bacterium is usually transmitted via fecal-oral ingestion through contami-nated food and water or through person-to-person contact. Shigellosis is highly infectious, as only 10–100 bacteria are sufficient to cause the disease in an adult person. After oral uptake, the bacteria invade the intestinal mucosa, which causes the typical symptoms such as bloody diarrhea, abdominal cramps, and dehydration. The primary goal in the treatment is the replacement of fluid and salt loss caused by diarrhea. In severe cases, antibiotics (such as Ampicilin, Cotrimox-

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azole, Nalidixic acid, and Ciproflaxin) are administered, but they are often not accessible in developing countries. Recent studies show a dramatic growth of resistant strains, making the develop-ment of new drugs an urgent need. TGT catalyses only as a functional homodi-mer a base exchange reaction in the wobble position of tRNAHis, Tyr,

Asp, Asn. Here, guanine is replaced by a modified base which leads to an effective translation of several virulence factors of Shigella. Thus, inhibition of TGT reduces the patho-genicity of Shigella significantly.

Antimicrobial resistance pattern for the different

Shigella species

Break apart of the TGT homodimer

We have developed a small series of lin-benzoguanine type inhibitors substituted with different modified ribose sidechains. All inhibitors exhibit binding affinities in nanomolar range and confirm the assumption as promising parental compounds which fit perfectly into the active site. Interestingly, a superposition of crystal structures with spiking ligands, designed to destabilize and break the interface formation, show that one ribose ligand imposes similar effects onto a flexible loop (β1α1-loop), which shields the interface from water access. Native mass spectrometry (nanoESI-MS) confirmed that this ligand has a similar effect onto dimer stability as the spiking ligands and represents an active site inhibitor with a dual mode-of-action.

We acknowledge the beamline support of Bessy II in Berlin for practical help and the HZB for travel grants

References: 1. Immekus, F. et al: ACS Chem Biol 2013, 8(6): 1163-1178. 2. Barandun, L.J.: Dissertation 2013, ETH Zurich, Zurich (Switzerland). 3. von Seidlein, L. et al: PLoS Med 2006, 3(9): e353.

Fast Estimation for Cellular Metal Ion Uptake Using Affinity Capillary Electrophoresis (ACE) Alhazmi, H.A.1; Nachbar, M.1; Mozafari, M.1; El Deeb, S.1,2; Albishri, H.M.3; El-Hady, D.A.3,4; Wätzig, H.1 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Bee-thovenstrasse 55, 38106 Brunswick, Germany. 2 Department of Pharmaceutical Chemistry, Al-Azhar University-Gaza, Gaza, Palestine 3 Chemistry Department, Faculty of Science, King Abdulaziz University, 80203 Jeddah, Saudi Arabia. 4 Chemistry Department, Faculty of Science, Assiut University, 71516-Assiut, Egypt

Developing organometallic complexes is still growing for the treatment of different disorders such as cancer and infection. In fact, these complexes are pro-drugs, and have the role to transfer metal ions to target binding sites [1]. Therefore, investigations of metal ions behaviors and their interactions with the plasma proteins such as human serum albumin and transferrin could give a preliminary information about the cellular metal ion uptake prior to the development of a new pro-drug. Hence, techniques to reliably investigate these interactions are urgently needed. Recently, Affinity Capillary Electropho-resis (ACE) has been provided as an important tool for this purpose [2,3]. In a recent study, the performance of this technique has been improved by applying an appropriate rinsing protocol. Using 0.1 M EDTA in the rinsing protocol became mandatory to desorb the metal ion from the capillary wall and prevent the influence of EOF changes on the precision and accuracy of results. ACE can now be performed in approximately 10 min including rinsing procedures. An excellent precision, corresponding to RSD% of < 1.0% was achieved. The

influence of the noble metal ions (Pd2+, Ir3+, Ru3+, Rh3+, Pt4+, Os3+, Au3+, Au+, Ag+, Cu2+) on plasma proteins were investigated by ACE, giving deep insight into the strength of the interactions between these species and plasma proteins. Accordingly, the cellular uptake of these species could be estimated. Furthermore, the interaction of the recently developed Rh1+ N-heterocyclic carbene complex (prospective anticancer) with plasma proteins were investigated [4]. The results showed that the cellular uptake of Rh1+ could be decreased in the presence of BSA while could be increased in the presence of HSA or transferrin. Hence, ACE technique could become first choice for in-vitro studies of the cellular metal ion uptake and protein metal ion interactions.

Acknowledgment: We gratefully acknowledge the financial support by the funding of King AbdulAziz University, Jeddah, Saudi Arabia, provided by Vice President Prof. Dr. A. O. AlYoubi. We also thank Jazan University and Saudi Arabian cultural office in Berlin for supporting our work by a grant to H. AlHazmi. Furthermore, we thank Polymicro Technologies for providing the capillaries.

References: 1. Romero-Canelón, I., Sadler, P.J.: Inorg. Chem. 2013, 52: 12276 - 12291. 2. AlHazmi, H. A. et al.: submitted to Electrophoresis. 3. El Deeb, S. et al..: Trends Anal. Chem. 2013, 48: 112-131. 4. Oehninger, L. et al.: Chem. Eur. J. 2013, 19: 17871-17880

Synthesis, Biological Evaluation and Binding Modes of Reverse Fosmidomycin Analogs against the Antimalarial Drug Target IspC Konzuch, S.1; Umeda, T.2; Held, J.3; Brücher, K.1; Lienau, C.1; Behrendt, C.T.1; Gräwert, T.4; Bacher, A.4; Illarionov, B.4; Fischer, M.4; Mordmüller, B.3,5; Tanaka, N.2; Kurz, T.1

1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany 2 School of Pharmacy, Showa University, Tokyo 142-8555, Japan 3 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany 4 Hamburg School of Food Science, Universität Hamburg, Grindelallee 117, 20146 Hamburg, Germany 5 Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon

Inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase of Plasmodium falciparum (PfIspC) of the non-mavalonate isoprenoid pathway (MEP pathway) is a promising strategy for the development of novel antiplasmodial drugs.[1,2] The enzyme is inhibited by the antibiotic fosmidomycin, which has been used successfully to treat malaria patients in clinical studies.[1,2] Reverse α-aryl substituted carba-, oxa- and thia- analogs of fosmidomycin are well-known as highly potent inhibitors against PfIspC.[3,4,5] In this work a series of new reverse fosmidomycin derivatives was synthesized and biologically evaluated in order to provide new insights into their structure activity relationships. The most potent α-aryl substituted analog 2c inhibits PfIspC as well as Plasmodium falciparum growth and exceeds the inhibitory potential of the natural product fosmidomycin by more than one order of magnitude. The binding mode of three derivatives in complex with PfIspC, NADPH and Mg2+ was clarified by X-ray analysis.[6]

Figure 1: Interactions of the S-enantiomer of inhibitor 2c in the active site of PfIspC.

References: 1. Jomaa, H. et al.: Science 1999, 285: 1573-1576. 2. Rohmer, M. et al.: Curr. Opin. Investig. Drugs 2004, 5: 154-162. 3. Behrendt, C.T. et al.: J. Med. Chem. 2011, 54: 6796-6802. 4. Brücher, K. et al.: J. Med. Chem. 2012, 55: 6566-6575.

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5. Kunfermann, A. et al.: J. Med. Chem. 2013, 56: 8151-8162. 6. Umeda, T. et al.: Sci. Rep., 2011, 1: 1-8.

Triarylmethyl radicals: Synthesis, EPR characterization and pharmaceutical applications Elewa, M.; Frank, J.; Mäder, K.; Drescher, S.; Imming, P. Institut fuer Pharmazie, Martin-Luther-Universitaet, Wolfgang-Langenbeck-Str. 4, D-06120, Halle (Saale), Germany

Triarylmethyl (trityl) radicals are paramagnetic spin probes which are used in electron paramagnetic resonance (EPR) spectroscopy and imaging. Tris(tetrachloro, tetrathia, and tetraoxatriaryl)methyl radicals show intense, single and narrow EPR lines at concentrations in the µM range. Furthermore, they show good water solubility when present as tricarboxylates, and a good stability in biological systems. Trityl radicals (Figure 1) were synthesized and characterized. The influence of pH, viscosity, oxygen concentration and solvent parameters on the EPR signal of tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl radical (PTMTC) are shown in Figures 2, 3 and 4. PTMTC contains three carboxylic acid groups leading to pH sensitivity. To investigate the effect of viscosity on line width, different glycerol-water mixtures were tested (Figure 2). Between 10 and 40 % (m/V) glycerol content, a slight decrease in the EPR signal line width was detected. This could be due to the decrease of oxygen solubility with increasing percentage of glycerol [1]. The increase above 60 % reflects the effect of increased viscosity by increasing glycerol content. Apparently, viscosity by itself up to a value of about 40 % of glycerol has no effect on the EPR signal line width. This fact is important for in-vivo applications as blood has a viscosity of 3–4 (mPa.s) at 37 °C [2], which is only reached at 50 % of glycerol in water at 40 °C [3]. EPR signal line width increase is directly proportional to oxygen concentration [4, 5]. A 1 mM solution of PTMTC in phosphate buffer (pH 7.4) shows a linear relationship between line width and oxygen concentration (Figure 3). The small slope of the line is due to the fact that oxygen concentration hardly increases with increasing oxygen partial pressure, a physicochemical fact that should be kept in mind for any oxygen determination in water. A 1 mM solution of the radical in methanol-water mixtures (10 % – 100 % methanol) were tested (Figure 4). On increasing methanol concentration, the EPR signal line width increased because oxygen solubility increases consider-ably with increasing methanol concentration [1], leading to line broadening [4]. In conclusion, PTMTC is an interesting paramagnetic spin probe with favourable EPR spectroscopy and imaging characteristics (single EPR signal with narrow line width). Line width is affected by different parameters (viscosi-ty, oxygen concentration and pH [not shown]). Each of these parameters can be measured accurately when others are kept constant. In aqueous solutions, viscosity and oxygen partial pressure do not have a pronounced effect on line width.

Acknowledgments: We thank the Egyptian Ministry of Higher Education and Scientific Research for financial support to M.E.

References: 1. Kutsche, I. et al.: J. Chem. Eng. Data. 1984, 29(3): 286–287. 2. Joubert-Huebner, E. et al.: Perfusion 2000, 15: 69–76. 3. Physical properties of glycerine and its solutions (Glycerine Producers Association) 1963. 4. Driesschaert, B. et al.: Bioorg. Med. Chem. Lett. 2008, 18: 4291–4293. 5. Kuppusamy, P. Zweier, J.L.: NMR Biomed. 2004, 17: 226–239.

Molecular Docking Studies to Explain SARs of Tertiary Amine Substituted Acetylcholinesterase Inhibitors Wehle, S.1; Darras, F.H.2; Sotriffer, C.A.1; Decker, M.1,2 1 Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany 2 Institut für Pharmazie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany

Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative brain disorder and the most common form of dementia with 24 million people affected worldwide [1]. No truly disease-modifying therapies are known so far, and cognitive deficits that characterize AD can only be addressed by sympto-matic pharmacotherapy. Currently, the most common clinical treatment for AD is the use of acetylcholinesterase (AChE) inhibitors which increase acetylcho-line levels in the brain and symptomatically improve cognition and memory. Quinazolinones are moderate micromolar AChE inhibitors both at the electric eel and human isoforms of the enzyme [2]. Quinazolines (i.e. reduced form of quinazolinones) connected with tertiary amines of variable size via a three-carbon-linker have been identified as highly potent dual-acting hAChE inhibitors and hH3 antagonists [3]. Analogous substitution of quinazolinone scaffolds leads to a loss in hH3 affinity, but to a remarkable increase in AChE inhibitory affinity up to the nanomolar range [4]. A correlation of basicity with IC50 values is observed. Computational docking methods were applied to investigate the putative binding mode of this group of inhibitors in the human AChE gorge and, concomitantly, an inhibitor library was synthesized for these studies and for further optimization.

The 36 compounds of the compound library were docked to the binding site of the human AChE crystal structure (PDB-ID: 4EY7) using GoldSuitev5.2 and v5.1 by incorporation of seven structural water molecules in the docking process [4, 5]. These water molecules were found to be essential for inhibitor-protein stabilization by at least one hydrogen bond of the quinazolinone carbonyl to a water molecule. An “inverted binding mode” was found in 88% of all top-5 poses. These poses show the basic amine placed at the active center near the choline-attracting residue Trp86 (human AChE numbering), potentially stabilized by a cation-π interaction, whereas the quinazolinone is placed at the entrance of the binding gorge showing aromatic interactions with Trp286 and hydrogen bonds to the water molecules. The “classical” orientation with the scaffold placed near the catalytic active site (CAS) was found in just 12% of the top-5 poses. Therefore, the high affinity and the surprisingly high AChE selectivity over butyrylcholinesterase is only partly due to the heterocyclic part of the molecules; SARs are mainly dependent on the tertiary amine structure. It seems essential for the optimization of novel heterocyclic AChE inhibitors to investigate the putative binding mode by SARs in connection with molecular docking studies.

References: 1. Ballard, C. et al.: Lancet 2011, 377(9770): 1019-1031. 2. Decker, M. et al.: Bioorg. Med. Chem. 2006, 14(6): 1966-1977. 3. Darras, F.H. et al.: ACS Chem. Neurosci. 2014, 5(3): 225-242. 4. Darras, F.H., Wehle, S. et al.: Bioorg. Med. Chem. 2014, in press. 5. GOLDSUITE 5.1, GOLDSUITE 5.2, CCDC Software, www.ccdc.cam.ac.uk

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Synthesis of L- S-(2-pyrrolyl)-cysteine sulphoxide Feizabad, M.S.; Keusgen, M.

Institute of Pharmaceutical Chemistry, Philipps-Universität Marburg D-35037, Germany

The genus Allium L. has a large diversity, and more than 800 species worldwide are described until now. Nearly all of them occur in the semiarid regions of Europe, North America, North Africa and Asia. The most common Allium species are garlic, (Allium sativum) and Onion (A. cepa) [1, 2]. In Middle Asia, Allium species of the subgenus Melanocrommyum have a large range of usage. In several cases, the amount of cysteine sulphoxides and their metabolites are rather high, so that the plants are used as spicy vegetables or are even not edible [3]. A recently discovered, new cysteine sulphoxide containing a pyrrolyl residue seems to typical for many bulbs of species belonging to the subgenus Melanocrommyum. Typical examples are A. giganteum and A. rosenorum, found in Central Asia and West Asia, especially in Iran, Tajikistan, Uzbekistan and Afghanistan. This new cysteine sulphoxide has been identified as L-S-(2-pyrolyl)-cysteine sulphoxide [5]. The members of the above mentioned subgenus have antibiotic antifungal, antidiabetic, or wound healing effectivity that shows the medical importance of synthesizing this cysteine sulphoxide found in them. The aim of this study was to synthesis this pyrrole-containing cysteine sulphoxide.

5. Chemical structure of L-S-(2-pyrrolyl)-cysteine sulfoxide Synthesis has been done in three steps. At first, N-benzyl-pyrrole and 3-chloro-L-alanine hydrochloride were used as starting blocks, with the addition of thiourea, iodine and potassium iodide, in extreme alkaline medium (sodium hydroxide and hydrazine), soved in aqueos ethanol (50%-50%). The intended product was 3-(1-benzyl-1H-pyrrol-2-yulsulphanyl) propionic amino acid [4]. In the next step, this product was oxidized using hydrogen peroxide in acetic acid as solvent, in order to obtain S-(1H-benzyl-2-pyrrole)-cysteine sulphoxide. In the last step, the benzyl group should be reductively removed with palladium/C 20% and hydrogen in methanol as solvent.. With the exception of the last step, all reaction pruducts could be confirmed by TLC, HPLC-MS and NMR. For most important products, preparative HPLC was performed.

Acknowledgments: We are grateful to Matthias Brauschke for technical support.

Refrences: 1. Yoshida, H. et.al.: Biosci., Biotechnol. Biochem. 1999, 63: 588-590. 2. Kumari, K.; Augusti, K.T.: Planta Medica.1995, 61: 72-74 3. Keusgen, M. et.al.: J. Ethnobiol. Ethnomed. 2006, 2: 18. 4. Rudyakova, E.V. et.al.: Russian Journal of Organic Chemistry. 2008, 44(10): 1539-1543

New tripeptide inhibitors of the West Nile virus NS2B-NS3 protease Kouretova, J.1, Hammamy, M.Z.1, Haase, C.2, Hilgenfeld, R.2, Steinmetzer, T.1 1 Institute of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032 Marburg, Germany 2 Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

West Nile virus (WNV) is a mosquito-borne flavivirus, which was first identified in the West Nile part of Uganda and has spread later to Asia, America and Europe. The majority of the infected humans shows no symptoms but may develop a mild flu-like illness. A small number of infected people, mainly children and the elderly, develop fatal meningitis or encephalitis leading to a mortality rate of around 10 %. Despite increasing demand, there is no specific treatment of WNV infections available, so far. A potential target for the treatment of WNV infections could be the viral NS2B-NS3 protease, which is essential for cleaving the WNV-polyprotein into various mature viral proteins. The NS3 protein contains a serine protease domain, which cleaves their substrates preferentially at the C-terminus of two basic amino acids.

Based on the preferred P3-P1 amino acids containing an N-terminal phenyla-cetyl protection [1] we have prepared a series of peptide derivatives as WNV NS2B-NS3 protease inhibitors by modifying the prime site segment. Surpris-ingly, the strongest inhibitory potency was found for simple protected tripep-tides containing a C-terminal arginylamide moiety, all elongated derivatives exhibit reduced activity. Incubation experiments and subsequent HPLC analysis revealed that these compounds are nearly stable against cleavage by the WNV-protease, only a minor amount of the cleavage product was detected after 4 hours. Therefore, these compounds seem to be very poor substrates of the WNV NS2B-NS3 protease, which can be considered as competitive inhibitors. The best Ki-values < 0.15 µM were obtained for peptides with the general structure phenylacetyl-Lys-Lys-Arg-NH2 containing an N-terminal guanidinomethyl substitution at the P4-residue.

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Ki = 0.12 µM for C4 modification

References: 1. Hammamy, M.Z. et al.: ChemMedChem 2013, 8: 231-241.

Improving binding free energy estimates for InhA inhibitors by a hybrid scoring function/molecular dynamics approach Narkhede, Y.; Sotriffer, C.A.

Institute for Pharmacy and Food Chemistry, Am Hubland, D– 97074, Wuerzburg, Germany

Due to the high infection rates and the recent emergence of extremely drug resistant forms, infection by Mycobacterium tuberculosis still represents a significant challenge for global health [1]. The NADH-dependent enoyl-ACP-reductase InhA of the type II mycobacterial fatty acid biosynthesis pathway is a well-validated target for inhibiting mycobacterial growth [2]. Numerous inhibitors of InhA have already been synthesized and tested for in-vitro activity. Despite the progress in this area, very few drug candidates have been reported or are in development [3]. Of all inhibitors reported in literature, the diaryl ethers, pyrrolidine carboxamides and arylamides are the most prominent chemical classes for further development [3]. The present work illustrates the use of hybrid scoring approaches consisting of classical scoring functions and molecular dynamics based methods to improve the prediction of binding free energies of pyrrolidine carboxamides as InhA inhibitors. The pyrrolidine carboxamides with a narrow pIC50 range coupled with diverse binding modes presented a challenge for molecular docking as well as the ensuing binding free energy calculations using the Linear Interac-tion Energy approach (LIE) [4]. Starting from representative crystal structures, the binding modes for 44 compounds were predicted using molecular docking. The Glide XP [5] and SFC scoring functions [6] together with the LIE method were used in multiple logistic regression models to classify the compounds into low-, medium- and high-affinity groups, respectively. Taking into consideration the experimental uncertainty of binding activity data, this approach appears more reasonable and practically useful than classical regression methods and pure correlation-based metrics.

References: 1. WHO Global TB report, 2013, 1-3. 2. Molle, V. et al: Mol. Microbiol., 2010, 78: 1591–1605. 3. Pan, P. et al.: Curr. Top. Med. Chem. 2012, 12(7):672-693. 4. Gutiérrez-de-Terán, H. et al.: Computational Drug Discovery and Design, (Springer New York) 2012, 890: 305-323. 5. Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012 6. Sotriffer, C.A. et al.: Proteins, 2008, 73: 395–419.

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Catechol-based substrates of Chalcone Synthase as a scaffold for novel inhibitors of PqsD Allegretta, G.; Weidel, E.; Empting, M.; Hartmann, R.W.

Helmholtz Institute for Pharmaceutical Research Saarland – Department of Drug Design and Optimization, Saarland University, Campus C 2.3, 66123 Saarbrücken, Germany

A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair the communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS [1] and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the active site, the catalytic residues and the electrostatic surface potential at the entrance of the substrate tunnel. [2,3,4] Hence, we evaluated selected substrates of the vegetable enzyme as potential inhibitors of the bacterial protein. This similarity-guided approach leads to the identification of a new class of PqsD inhibitors having a catechol structure as an essential feature for activity, a saturated linker with two or more carbons and an ester moiety bearing bulky substitu-ents. The developed compounds showed PqsD inhibition with IC50 values in the single-digit micromolar range. The binding mode of these compounds was investigated by SPR experiments revealing that their interaction with the protein is not influenced by the presence of the anthranilic acid bound to active site cysteine. Importantly, some compounds reduced the signal molecule production in cellulo.

Acknowledgments: “Helmholtz Institute for Pharmaceutical Research Saarland – Department of Drug Design and Optimization”, Maurer, C., Kirsch, B. References: 1. Dulcey, C.E. et al.: Chem. & Biol. 2013, 20(12): 1481–1491. 2. Bera, A.K. et al.: Biochem. 2009, 48(36): 8644–8655. 3. Ferrer, J.–L. et al.: Nat. Struct. Biol. 1999, 6: 755–784. 4. Dao, T.T.H.; Linthorst, H.J.M.; Verpoorte, R.: Phytochem. Rev. 2011, 10(3): 397–412.

Johannes Franz Wilhelm Valentin (1884–1959) - a pioneering pharmacist in the field of chromatography Michler, V.1; Friedrich, C.1

Institut für Geschichte der Pharmazie, Universität Marburg, Roter Graben 10, 35037 Marburg, Germany

The chromatographic technique was invented by an an italian-born russian biologist named Mikhail Semjonovich Tswett (1872–1919).[1] He separated plant pigments in 1903 by allowing them to percolate down columns of calcium carbonate or inulin. This new procedure attracted little attention until it was rediscovered by a group of german chemists at the University of Heidelberg in 1931.[2] After that chromatography entered a period of intensive development and is nowadays the most versatile technique in analytical chemistry. However, thus far little is known how this technique entered the pharmaceuti-cal analytics.

Based on our research, Johannes Franz Wilhelm Valentin (1884–1959), a pharmacist from the University of Königsberg, was the first german scientist who used the chromatography to analyze pharmaceutical products.[3] The initial publication of Valentin was released in Mai 1935.[4] He made the technique of chromatography available for analyzing pharmaceutical prepara-tions. The first pharmaceutical substances he investigated were Peru Balsam

and Tinctura Digitalis.[4] He demonstrated how easy, fast and more feasible these methods are compared to the existing monographs in the German Pharmacopoeia 6 in 1926. This poster will summarize the life and work of Hannes Valentin, a pioneering pharmacist from the University of Königsberg and later on from the University of Greifswald.

References: 1. Michler, V.: CVET, Michail Semënovič. In: Personendatenbank zum Vorhaben "Wissenschaftsbeziehungen im 19. Jahrhundert zwischen Deutschland und Russland auf den Gebieten Chemie, Pharmazie und Medizin" bei der Sächsischen Akademie der Wissenschaften zu Leipzig. Online-Ausgabe: http://drw.saw-leipzig.de/10283.html (23. Mai 2014) 2. Kuhn, R.; Winterstein, A.; Lederer, E.: Hoppe Seyler‘s Zeitschrift für physiologische Chemie, 1931, 197: 141–161. 3. Friedrich, C.; Seidlein, H.- J.: Pharmazie, 1984, 39 : 262–269. 4. Valentin, J.: Pharmazeutische Zeitung, 1935, 80 : 469-471.

Targeting drug resistance in EGFR with covalent inhibitors – a structure-based design approach Engel, J.1; Getlik, M.1; Richters, A.1; Heuckmann, J.M.2; Grütter, C.1; Thomas, R.K.2; Rauh, D.1

1 Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, D-44227, Dortmund, Germany 2 Universität zu Köln, Abteilung Translationale Genomik, Weyertal 115b, D-50931 Cologne, Germany

Aberrant activity of the epidermal growth factor receptor kinase (EGFR) plays a pivotal role in the development and growth of tumor cells and is associated with the onset and progression of non small cell lung cancer (NSCLC).[1,2] Patients harboring activating mutations in the catalytic domain of EGFR show a significant clinical response to reversible Type-I inhibitors gefitinib and erlotinib.[3] However, patients responding to these drugs develop secondary drug resistance mutations and suffer from a dramatic relapse. In 50% of these cases, Thr790 at the gatekeeper position of the kinase domain, was mutated to a sterically more demanding methionine.[4] In order to overcome this resistance, covalent inhibitors represent the most promising strategy by covalently targeting the unique Cys797 in the active site of EGFR.[5,6,7] Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686 which remarkably takes advantage of increased residence time to EGFR by alkylating Cys797 simultaneously preventing toxic effects. [6] Here, we present the structure-based approach to design novel and covalent EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against about 1500 compounds. Using protein X-ray crystallography we determined the binding mode of this inhibitor in the related tyrosine kinase cSrc. The complex crystal structure did not only provide valuable insights into the mode of action but also highlighted strategies for chemical optimization that led to a series of derivatives which displayed strong inhibitory activities against EGFR and its mutant variants.

References: 1. Zandi, R. et al.: Cell Signaling 2007, 19(10): 2013-2023. 2. Heuckmann, J.M. et al.: J. Clin. Oncol. 2012, 30(27): 3417-3420. 3. Pao, W. et al.: Proc. Natl. Acad. Sci. USA 2004, 101(36): 13306-13311. 4. Kobayashi, S. et al.:N. Engl. J. Med. 2005, 352(8): 786-792. 5. Zhou, W. et al.: Nature 2009, 462(7276): 1070-1074. 6. Walter, A.O. et al.: Cancer Discov. 2013, 3(12): 1404-1015. 7. Sos, M.L. et al.: Cancer Res. 2010, 70(3): 868-874.

Buffer conditions regulate the binding pose of dualsteric ligands at the muscarinic M2 receptor Krebs, F.; Chirinda, B.; Mohr, K.

Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany

G-protein-coupled receptors, the largest superfamily of membrane bound receptors, control and regulate several processes in the human body and

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therefore serve as targets for a variety of drugs. Muscarinic acetylcholine receptors, with their subtypes M1-M5, belong to this superfamily of receptors. Throughout the five subtypes, the orthosteric binding site is highly conserved [1]. Consequently, synthetic drugs that target this binding site generally lack subtype selectivity. In contrast, the allosteric vestibule is not as highly conserved among the subtypes and therefore could function as a possible binding epitope for allosteric modulators yielding certain subtype selectivity. Recently the muscarinic M2 receptor was crystallized in its active state bound simultaneously with both an orthosteric agonist and a specifically designed allosteric modulator [2]. Dualsteric ligands are small molecules, which consist of covalently linked moieties that address the orthosteric and the allosteric binding site, respective-ly. As a consequence, the binding pose of such a bitopic ligand depends on the individual affinities of these moieties for their respective binding site [3]. A previous study showed that the binding affinity of positively charged allosteric ligands is highly sensitive to the cation concentration of the incubation buffer [4]. Here the aim of the study was to investigate the influence of different salt compositions in the buffer solution on the binding pose. The affinities of the bitopic ligands and their orthosteric and allosteric fragments were investigated in either equilibrium binding or dissociation kinetics experiments with the radioactive tracer [³H]NMS. All experiments were conducted with membrane homogenates from CHO cells stably expressing the human M2 receptor. A change of buffer conditions from HEPES-buffer (10 mM HEPES, 10 mM MgCl2, 100 mM NaCl, pH = 7.4) to Na,K,Pi-buffer (1 mM KH2PO4, 4 mM Na2HPO4, pH = 7.4) led to a prominent increase in affinity. In contrast, the affinity for [³H]NMS remained unchanged. The increased affinity for the allosteric fragment in Na,K,Pi-buffer conditions caused the bitopic compound to bind in a higher fraction in the purely allosteric pose, which was reflected by a promoted [³H]NMS binding to the receptor. In contrast, under HEPES-buffer conditions the affinity of the orthosteric fragment dominated the binding pose of the dualsteric compound, as reflected by displacement of [³H]NMS from the orthosteric pocket. These findings show that modification of buffer conditions is a new means to gain insight into the binding pose of orthosteric/allosteric class A GPCR ligands.

Test compounds were kindly provided by Prof. Dr. U. Holzgrabe, Würzburg, Germany, and Prof. M. De Amici, Milan, Italy and co-workers. F.K. is a member of the research training group GRK 1873 - funded by the German Research Foundation (DFG). B.C. is a member of the research training group BIOTECH PHARMA. References: 1. Wess, J. et al.: Crit Rev Neurobiol 1996, 10: 69-99. 2. Kruse, A. et al.: Nature 2013, 504: 101-106. 3. Bock, A. et al.: Nat Chem Biol. 2014, 10: 18-20. 4. Schröter, A. et al.: Naunyn Schmiedebergs Arch Pharmacol. 2000, 362: 512-519.

Aldose Reductase as a target to prevent diabetic complications: Investigations of the structural and thermodynamic background of the opening of the specificity pocket Rechlin,C.1; Scheer,F.2; Toth, P.2; Heine, A.1; Diederich, W.2; Klebe, G.1

1 Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35037 Marburg, Germany 2 Zentrum für Tumor- und Immunbiologie, Philipps-Universität Marburg, Hans- Meerwein- Str. 3 , 35032 Marburg, Germany

Diabetes is a global burden of which 347 million people suffered in 2008 alone. Estimations indicate that this number will increase so that by 2030 4.4 % of all age groups will suffer from this disease. [1] Diabetes does not only have immediate symptoms but also leads to some long-term complications. Blindness, end-stage renal disease and different neuropathies are microangi-opathies which count to these complications. [2] The human aldose reductase (ALR2) is involved in the first reaction step in the so-called polyol pathway which is one of the major mechanisms for the development of diabetic complications. Glucose is reduced to sorbitol with the help of the cofactor NADPH. Higher oxidative stress is the result of the reduced NADPH level. Based on this knowledge inhibitors of the human aldose reductase (ARIs) have been evaluated in several clinical studies concerning the effectiveness and safety.[3] They seem to have an effect

especially on motor nerve function in patients with mild to moderate diabetic sensorimotor polyneuropathy. [4] Despite these promising findings the ALR2 is a challenging target for drug design, as it exhibits an highly conserved anion binding pocket and the very flexible specificity pocket forming the binding site. The specificity pocket only opens if the inhibitors provide favorable interactions. In the past this pocket has been addressed to obtain selective inhibitors for ALR2. Taking into account that ALR2 belongs to a large protein family, selectivity is a requirement for successful drug design. [5] We would like to understand how the opening of the specificity pocket works in detail. The structure of the crystallization buffer ingredient citrate complexed to the binding site of ALR2 (pdb: 2j8t) has been determined. As citrate is only partially occupied this structure provides some insights into the putative apo-structure and suggests the specificity pocket is closed. This indicates that the enzyme with an opened pocket does not correspond to the energetically most favorable situation. An inhibitor that protrudes into this area of the binding site has to open this pocket first. By how much does the inhibitor pay for the opening and is the contribution compensated by suitable interactions in the newly formed pocket? We are investigating this process using derivatives of the so called “IDD-ligands”, a series of 2-benzylcarbamoyl-phenoxy-acetic acids. Members of this class could be found which leave the pocket closed while the most potent ones open the pocket. Our aim is to determine the smallest sidechain which is able to open the pocket. X-ray crystallography and Isothermal titration calorimetry (ITC) were used to follow the opening of the pocket from the structural and the energetic point of view. References: 1. Danaei et al.: LANCET. 2011, 378: 31-40. 2. Brownlee: NATURE. 2001, 414: 813-820. 3. Hu et al.: PLOS ONE. 2014, 9: 2. 4. Bril et al.: DIABETES CARE. 2009, 32;7: 1256-1260. 5. Sotriffer et al.: PROTEINS. 2004, 56: 52-66.

Chiral separation of amino acids by CE and HPLC using online derivatization with ortho-phthalaldehyde and a chiral mercap-tane. Kühnreich, R.; Holzgrabe, U. Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

The aim of this study was to develop a method for the separation of chiral racemic amino acids, which is fast and allows for automatization. Ortho-phthalaldehyde (OPA) is a commonly used derivatization reagent for amino acids to improve sensitivity and retention on reversed phase HPLC columns. By using a chiral mercaptan as coreagent for the derivatization, enantiomeric compounds can be converted to diastereomers, allowing a separation without the use of an expensive chiral column. Amino acids were derivatized with ortho-phthalaldehyde, using N-acetyl-L-cysteine (NAC) or N-isobutyryl-L-cysteine (NIBLC) as mercaptan reagent in a 20 mM borate buffer (pH 10.0) to form diastereomeric isoindole derivatives, which can be separated by HPLC using reversed phase columns or capillary electrophoresis (CE) using selectors like cyclodextrines (CD). Besides enantioseparation, CDs can be used for the separation of closely related compounds, which have similar electrophoretic mobilities. For the online derivatization on CE, a modified method by Kaale et al.1 was used. The derivatized amino acids were separated in a 50 mM borate buffer (pH 9.2) and different neutral CDs (di-o-methyl-β-cyclodextrine, tri-o-methyl-β-cyclodextrine, hydroxypropyl-β-cyclodextrine, β-cyclodextrine, γ-cyclodextrine, hydroxypropyl-γ-cyclodextrine) and charged CDs (carboxymethyl-β-cyclodextrine, sulphated β-cyclodextrine) were tested. The derivatization on HPLC was performed with a custom injection program of the autosampler, where the derivatization takes place in the needle directly before injection. For the separation, a Kinetex PFP column (150x4.6 mm; 2.6 µm) and a Phenylhexyl column (150x4.6 mm; 3.0 µm) was used. The mobile phase consists of various buffers with pH in the range from 2.5 to 5.5 and acetonitrile. For both, HPLC and CE, separation conditions for the separation for 17 amino acids were found. The resolution was found to be at least 1.5. Acknowledgments: We like to thank BfArM for their financial support.

References: 1. Kaale, E. et al.: Electrophoresis 2001, 22(13): 2746–2754.

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Decoration of living cell surfaces by copper catalyzed azide-alkyne cycloaddition Gutmann, M.1; Wurzel, J.1; Memmel, E.2; Seibel, J.2; Meinel, L.1; Lühmann, T.1

1 Institute for Pharmacy and Food Chemistry, University of Würzburg, Germany ² Institute for Organic Chemistry,University of Würzburg,Germany

The cell surface is responsible for the interaction among cells and plays a key role in anchorage of the cell in macromolecular systems such as the extracellu-lar matrix (ECM). This study aims at the modification of cell surfaces by deploying the bioorthogonal copper catalyzed azide-alkyne cycloaddition (CuAAC) and by studying the associated copper toxicity in the cellular system. We aimed to address two essential points, (i) the modification of the cell surface after CuAAC in respect to cell viability and (ii) the characterization of the cell surface with fluorescent dyes and a model protein plk-eGFP after click chemistry over time. The cell surface of adherent NIH3T3 fibroblasts and suspended HEK 293 F cells was modified to introduce azide-functionalities by a glycoengineering step using tetraacylated-N-azidoacetylglucosamine as previously described [1]. To proof the presentation of the azide-modified glycoproteins on the cell surface, we applied the azide-alkyne click reaction (50 µM CuSO4 / 250 µM THPTA / 2,5 mM sodium ascorbate for 5 minutes at room temperature), followed by surface staining with the fluorescent dyes sulfo-Cy5-alkine or acetylene-fluor 488, respectively [2, 3]. Moreover, an enhanced green fluorescent protein (plk-eGFP) modified with propargyl-L-lysine in position 5 was recombinantly expressed in BL21-DE3 cells and purified and used in a similar manner for cell surface modification. Site-directed immobilisation was investigated by confocal laser scanning microscopy (CLSM) at different time points. Copper cell toxicity after CuAAC was monitored by qRT-PCR analysis of apoptotic genes and FACS measurements thereafter [4]. High viability of the cells after the glycoengineering step and optimized CuAAC was shown. Here, we investigated both the apoptotic downstreaming process by use of qRT-PCR and the metabolic and membranous status using a fluoresceindiacetate (FDA) and propidiumiodide (PI) staining monitoredby FACS analysis. An overall good survivability ~80-100% was observed up to 20 minutes under click conditions regarding the expression of the examined apoptotic genes and the ratio of FDA and PI staining analyzed by FACS. In a subsequent set of experiments, the cell surface was decorated by means of metabolic glycoengineering and further modified by CuAAC between azido-functionalized NIH3T3 fibroblasts and HEK 293 F cell surfaces and alkyne groups carrying fluorescent dyes. Strong fluorescence was observed on the cell membrane with some background scattering within the cytosol directly after the click reaction by CLSM. Later time points (12 hours) demonstrated reduced membrane fluorescence. To introduce a more relevant biomacromol-ecule onto the cell surface, we chose as a model protein, plk-eGFP, for site-directed immobilisation. In line with the optimized protocols, we confirmed the site-directed immobilisation of plk-eGFP onto the cell surface. After click reaction between azido-functionalized NIH3T3 fibroblasts and plk-eGFP, specific fluorescence of eGFP was observed on the cell membrane with fast reduction (1 hour) in fluorescence over time.

References: 1. Homann, A. et al.: Beilstein journal of organic chemistry 2010, 6: 24. 2. Memmel, E. et al.: Chemical communications (Cambridge, England) 2013, 49 (66): 7301-7303. 3. Uttamapinat, C. et al.: Nature protocols 2013, 8 (8): 1620–1634. 4. Hong, V. et al.: Bioconjugate Chemistry 2010, 21: 1912-1916.

Site-directed modification of myostatin-inhibitors for muscle regeneration Braun, A.1; Gutmann, M.1; Li, L.1; Ebert, R.2; Jakob, F.2; Lühmann, T.1; Meinel, L.1

1 Institute for Pharmacy and Food Chemistry, University Würzburg, Germany 2 Orthopedic Center for Musculoskeletal Research, Würzburg, Germany

Introduction

Myostatin is a potent negative regulator of myogenesis and inhibits myoblast differentiation in compromised skeletal muscles. As myostatin is upregulated during aging and in muscle-wasting diseases including sarcopenia, we targeted this protein by 20 – 32 mer peptide antagonists in order to provide an effective therapy. By introducing a propargyl-derivative of glycine into the molecule, thus rendering it amenable for click chemistry, we featured the therapeutic for functionalized surface decoration.

Materials and Methods

The myostatin inhibitors were manufactured by solid phase peptide synthesis following the protocol of [1] with a propargyl-modified glycine analogue introduced into the sequence for the click chemistry option. Following purification using reversed phase chromatography on an ÄKTA purifier system, the synthesized peptides were analyzed by RP-HPLC and MALDI-MS for the verification of successful synthesis and characterization of stability upon freeze drying. Potency was profiled by a luciferase-based reporter gene assay in HEK 293 cells stably expressing the SBE-luciferase reporter gene [2] and bioactivity of the alkyne-functionalized peptides collated with the unmodified sequences. A C2C12 myoblast differentiation assay was deployed for the evaluation of the inhibitory effect on myostatin as determined with fluorescence microscopy and Western Blot. The accessibility of the alkyne group for click chemistry was demonstrated by performing click reaction with the fluorescent dye Cy3 azide.

Results and Discussion

Successful synthesis and purification of the functionalized peptides was demonstrated by mass spectrometry and HPLC. The luciferase assay confirmed the inhibiting activity on myostatin signalling and comparison of the potency of functionalized and original peptides resulted in similar outcome. The restoration of C2C12 differentiation ability after incubation of myostatin treated cells with the peptide inhibitor indicated bioactivity as demonstrated by the formation of multinucleated myotubes and positive MyHC expression of differentiating myoblasts. Performance of the click reaction with Cy3-azide [3] proved the functionality of the ‘clickable’ group. The successful coupling of the myostatin antagonists to azido-silk fibroin coated polystyrene particles demonstrated the ability for surface decoration. In conclusion, a potent ‘clickable’ myostatin inhibitor was developed for advanced controlled release approaches. This system is further profiled for boosting muscle function and regeneration in ongoing studies.

Acknowledgements: The financial support from the Bavarian Research Foundation (FORMOsA grant) is gratefully acknowledged.

References: 1. US 2004/0181033 A1 2. Cash, J.N. et al.: J. Biomol. Screen. 2013, 18: 837-844. 3. Lutz, J.F. et al.: Adv. Drug Deliv. Rev. 2008, 60: 958–970.

Part of this research has been presented at the CRS Local Chapter meeting in Kiel (27./28. February 2014)

Screening, synthesis and characterization of novel ligands for Farnesoid X Receptor (FXR) Flesch, D.; Achenbach, J.; Gabler, M.; Merk, D.; Steri, R.; Proschak, E.; Schubert-Zsilavecz, M.

Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt

The Farnesoid X Receptor (FXR) is not only a key regulator of bile acid metabolism, furthermore this nuclear receptor acts in various pathways of the fatty acid- and carbohydrate metabolism as well as liver protection and -regeneration [1,2]. Therefore it is an attractive target to treat several metabolic disorders. A Phase III study with Obeticholic acid (OCA, 6α-ethyl-chenodeoxycholic acid, 6-EDCA, INT-747) in primary biliary cirrhosis and phase II studies in non-alcoholic hepatosteatosis and alcoholic hepatitis are underway to verify the value of an FXR agonist in disease models of hepatic injury [3]. To generate starting points for novel synthetic FXR modulators we initiated an in silico search with a combined ligand- and structure-based virtual screening.

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The most promising structures to be identified were an anthranilic acid derivative and an imidazo[1,2-a]pyridine, which showed moderate activation of the receptor [4]. Subsequently these scaffolds were chemically modified to generate structure-activity-relationships (SAR) revealing an insight into pharmacophore properties of the specific residues. All structures were characterized biologically in a luciferase-based, full-length FXR transactivation assay [5].

In detail the imidazo[1,2-a]pyridine scaffold was chemically altered considering the thiophene- and methylenedioxyphenyl partial structures as well as the substitution pattern of the imidazo[1,2-a]pyridine core. The published "mini-SAR" was enlarged to systematically and gradually evaluate modifications to the structure to activity and maximal activation towards FXR. Therefore several derivatives were synthesized using the groebke-blackburn three-component synthesis of imidazo[1,2-a]pyridines using the respective aminopyridines, aldehydes and isocyanides. The results of biological evaluation promote ligands that activate FXR in a nanomolar activity range and that can be optimized regarding physicochemical properties for further in vitro and in vivo characterization.

The work has been supported by the Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation - Pharma (TRIP)

References: 1. Wang Y.D. et al.: Cell Res. 2008, 18(11):1087-1095. 2. Fan M. et al.: Biochim Biophys Acta. 2014 May 27. pii: S1874-9399(14)00135-7. [Epub ahead of print] 3. https://clinicaltrials.gov/ct2/results?term=obeticholic+acid&Search=Search 4. Achenbach J. et al.: Med. Chem. Commun.,2013, 4: 920-924 5. Merk D. et al.: Bioorg Med Chem. 2014, 22(8): 2447-2460.

Design, synthesis, SAR exploration and optimization of novel bacterial RNA polymerase inhibitors targeting the switch region Elgaher, W.A.M.; Sahner, J.H.; Groh, M.; Haupenthal, J.; Hartmann, R.W.

Helmholtz Institute for Pharmaceutical Research Saarland, Department of Drug Design and Optimization, Campus C2.3, Saarland University, 66123 Saarbrücken, Germany

The evolution of antibiotic resistant bacteria represents a true threat to public health that demands the development of new antibiotics with alternative mode of action. [1] The “switch region” of RNA polymerase (RNAP), targeted by the α-pyrone antibiotics e.g. myxopyronins, was proved to be a promising target for antibacterial drug discovery. [2] Based on a hit candidate discovered by virtual screening, a small library of 5-aryl-3-ureidothiophene-2-carboxylic acids (class I) was synthesized resulting in compounds with increased RNAP inhibition. Hansch analysis revealed π (lipophilicity constant) and σ (Hammett constant) of the substituents at the 5-aryl moiety to be crucial for activity. [3] In a further step, six new classes were synthesized via two analog design approaches: First, design of regioisomers to explore the optimum configuration of the aryl-ureidothiophene-carboxylic acids (class II‒IV). Second, bioisosteric replacement of the thiophene core by different heterocyclic rings to improve the physicochemical properties (class V‒VII). Structure activity relationship (SAR) studies, supported by molecular modeling, revealed the structural requirements necessary for interaction with the binding site. The new compounds displayed good antibacterial activities against Gram positive bacteria and Gram negative E. coli TolC, accompanied by a reduced re-sistance rate compared to the known antibiotic rifampicin together with a low mammalian cytotoxicity. [4]

References: 1. Coates, A.R.M., Halls, G., Hu, Y.: Br. J. Pharmacol. 2011, 163: 184−194. 2. Srivastava, A. et al.: Curr. Opin. Microbiol. 2011, 14: 532−543. 3. Sahner, J.H. et al.: Eur. J. Med. Chem. 2013, 65: 223−231.

4. Elgaher, W.A.M. et al.: RSC Adv. 2014, 4: 2177−2194.

Mobility Shift Affinity Capillary Electrophoresis: A Fast and Suitable Method for Early Stage Protein Metal Ion Interaction Screenings Nachbar, M.1; Mozafari, M.1; Alhazmi, H.A.1; Albishri, H.M.2; El-Hady, D.A.2,3; El Deeb, S.1,4; Redweik, S.1; Wätzig, H.1 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Bee-thovenstrasse 55, 38106 Brunswick, Germany. 2 Chemistry Department, Faculty of Science, King Abdulaziz University, 80203 Jeddah, Saudi Arabia. 3 Chemistry Department, Faculty of Science, Assiut University, 71516-Assiut, Egypt 4 Department of Pharmaceutical Chemistry, Al-Azhar University-Gaza, Gaza, Palestine

The importance of protein drugs for inflammatory and antitumor therapy is growing steadily. Hence, for reaching an optimal therapeutic success the right conformation and therefore the overall charge is of major interest. Protein conformations are not only highly affected by the pH, temperature and the ionic strength of the surrounding solution; they are also influenced by the containing ions. These ions can change the conformation by binding to specific motifs at the protein e.g. the EF hand motif or by unspecific bonds like electrostatic attraction between a cation and negatively charged amino acids. Changes of a protein's charge can easily be examined using affinity capillary electrophoresis (ACE). This work reveals how protein drugs can be affected by metal ions using a proline-rich digest obtained from galectin-3 (CBPep) as a model for these interactions. The mode of operation for ACE is based on the shift of electrophoretic mobility after ions bind to a protein. Thus, the time until the analyte is detected is changed according to the change of the overall charge of the protein. So if the protein is getting more negative the time is increased or if the protein is getting more positive the time is decreased. The influence of various ions was determined by using mobility ratios of the EOF marker and the protein to prevent effects of migration time shifts which are not related to interactions. The difference of the mobility ratio of the protein with the ligand (Ri) and without the ligand (Rf) was normalised to Rf (ΔR/Rf)1. The result demonstrates the change in the overall charge of the protein-ion-complex and also the strength of the interaction. During the experiments various metal ions e.g. Mn2+, Cu2+ and Ba2+ as well as complexes of metal ions which have a low solubility at physiological pH (e.g. Fe3+) were tested to determine their ability to interact with CBPep. The analysis of CBPep did not show the strong, sequence-specific interactions with calcium ions that were reported for mass spectrometry binding experi-ments in the vacuum state2. Only very weak interactions with other metal ions e.g. (Mg2+, Mn2+, Ba2+ , Sr2+, SeO3

2-, Fe3+, Ni2+, Cu2+, Zn2+, Au3+) were found. These findings suggest that the binding mode in aqueous solutions is different, which is also encouraged by preliminary molecular modelling results for the CBPep-Ca2+-complex in vacuum state.

References: 1. Redweik S., Xu Y., Wätzig H.:ELECTROPHORESIS 2012, 33(22):3316-3322 2. Lehmann WD. et al.: RAPID COMMUN MASS SP 2006, 20(16):2404-2410

C2- and O-Linked Melatonin Dimers as Bivalent Ligands Target-ing Dimeric Melatonin Receptors Zlotos, D.P.1; Sadek, M.S.1; Tadros, S.A.A.1; Gerbier, R.2,3,4; Jockers, R.2,3,4 1 The German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City, 11835 Cairo, Egypt 2 Inserm, U1016, Institut Cochin, Paris, France 3 CNRS UMR 8104, Paris, France 4 Univ. Paris Descartes, Sorbonne Paris Cite, Paris, France

Melatonin MT1 and MT2 receptors are among the first G-protein coupled receptors whose homo- and hetero-dimerization have been demonstrated using bioluminescence resonance energy transfer (BRET) [1,2]. Recently, we have reported the synthesis and pharmacological evaluation of a series of dimeric melatonin analogues obtained by connecting two melatonin molecules through N1 with spacers of 15-24 atoms [3]. The BRET studies provided

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evidence for the binding of these compounds to MT1 and MT2 homo- and heterodimeric receptors. Here, we describe the synthesis and pharmacological evaluation of two novel series of dimeric melatonin analogues obtained by linking two melatonin pharmacophores through C2 and O with spacers of 18-24 atoms. The findings are important for the design of novel bivalent melaton-ergic ligands selectively targeting MT1/MT1-homodimers, MT2/MT2-homodimers, and MT1/MT2-heterodimers.

NH

NHAcONH

O

ONH

AcHN

NH

NHAcMeO

NH

O

NH

AcHN

OMe

NH

O

NH

(CH2)n

O

NH

NH

NHAcMeO

NH

ONH

AcHN

OMe

NH

O

NH

(CH2)12

O

NH

O

O

NH

O

(CH2)m

n = 6,8,10,12

m = 4,6,8,10,12

Acknowledgments: Prof. Dr. Ulrike Holzgrabe, Würzburg University, Deutscher Akademi-scher Austauschdienst (DAAD)

References: 1. Ayoub, M.A. et al.: J. Biol. Chem. 2002, 277: 21522. 2. Zlotos, D.P. et al.: J. Med. Chem. 2014, 57: 3161. 3. Journé, A-S. et al.: Med. Chem. Commun. 2014, 5: 792.

Phenylazocarboxamides as structural analogues for cinnamoyl amides in D3 receptor ligands Bartuschat, A.L.1; Fehler, S.K.1; Hübner, H.1; Prante, O.2; Gmeiner, P.1; Heinrich, M.R.1 1 Department of Chemistry and Pharmacy, Medicinal Chemistry, FAU Erlangen-Nürnberg, Schuhstr. 19, 91052 Erlangen, Germany 2 Department of Nuclear Medicine, Molecular Imaging and Radiochemistry, FAU Erlangen-Nürnberg, Schwabachanlage 6, 91052 Erlangen, Germany

Cinnamoyl amide derivatives such as 1 represent a potent class of dopamine D3 receptor ligands, as shown by Saur et al..[1] By replacing the C-C double bond with the structurally comparable azo unit we were able to find a new class of dopamine D3 receptor ligands 2. These ligands are readily accessible from phenylazocarboxylic esters which are valuable building blocks for combinatorial synthesis.[2,3] Due to the electron-withdrawing properties of the azocarbonyl moiety, the aromatic core of phenylazocarboxylic esters is highly activated towards nucleophilic substitution. Besides the introduction of aromatic amines, aliphatic amines and phenols, substitutions with [18F]fluoride can be conducted at short reaction times, under mild conditions and with high yields, which in turn allows a very efficient access to [18F]-labeled D3 ligands such as 3.[4] Moreover, new results on the effects resulting from structural variations of the lead compound 2 will be presented.

References: 1. Saur, O. et al.: Arch. Pharm. Chem. Life Sci. 2007, 340(4): 178−184. 2. Höfling, S.B. et al.: Angew. Chem. 2010, 122(50): 9963−9966. 3. Jasch, H. et al.: J. Org. Chem. 2012, 77(3):1520−1532. 4. Fehler, S.K. et al.: Chem. Eur. J. 2014, 20(2): 370−375.

Performance Qualification in Surface Plasmon Resonance Analysis Steinicke, F.; Oltmann-Norden, I.; Wätzig, H.

TU Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany

Surface Plasmon Resonance (SPR) is a dominant tool for characterization of biomolecular interaction. This technique facilitates label-free binding analysis studies of biomolecules such as affinity, kinetic, thermodynamics and specifity analysis in real-time. Therefore SPR is an important application in drug discovery and proteomics.

In this study a concept was investigated for performance qualification, long-term precision and accuracy for kinetic measurements of biomolecular interactions using SPR on a Biacore X100 instrument. So far there is no research done which offers reliable data for the parameters of our interest. The method used was an already established single cycle kinetic assay by GE Healthcare to monitor human beta-2-microglobulin (b2m) with a covalently bound anti-beta-2-microglobulin antibody from mouse. The first step of the assay was the direct immobilization of anti-b2m antibody to a CM5 sensor chip. The antibody was covalently bound to the carboxymethylated dextran layer of the sensor chip surface using the amine coupling method. The sensor chip was reusable for several runs. In order to reduce bulk effects all assay solutions have been prepared with the running buffer, a HBS-EP buffer (HEPES Buffer Saline-EDTA Polysorbate 20) pH 7,4 containing 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.15 M NaCl, 3 mM EDTA and 0.005% (v/v) Polysorbate 20. Each day a freshly prepared serial dilution of b2m (2 nM, 4 nM, 8 nM, 16 nM and 32 nM) was employed in single-cycle kinetic measurements. In this assay all five analyte dilutions were sequently measured followed by one regeneration step with glycine hydrochlo-ride 10 mM pH 2.5. All binding experiments were carried out at 25°C and with a flow rate of 30 µL/minute. The sensorgrams for each single-cycle kinetic assay were analyzed by curve fitting based on a 1:1 binding model using the Biacore X100 evaluation software (version 2.0). The parameters that have been analyzed include the maximal theoretical Response Units (Rmax), the dissociation constant (KD), the residuals from the optimal fitted curve and the peak absorption value for every concentration. These parameters were evaluated and then plotted into control-charts. In addition to these data we watched for external influences such as mechanical resilience of the chip and data deviations for example caused by pipeting-errors or material impact. The first series contains 27 cycles and shows a percental relative standard deviation of 4.9% for the KD.

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Synthesis and characterization of ‘clickable’ alkyne-PEGylated FGF2 and its site-directed immobilization to azide modified microspheres via click chemistry Heusler, E.; Jones, G.; Zhao, H.; Lühmann, T.; Meinel, L.

Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany

Growth factors such as vascular endothelial growth factor (VEGF) [1] and bone morphogenetic protein 2 (BMP2) [2] had been covalently immobilized to biomaterials by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) chemistry with the aim to (re-)engineer lost tissues. A remarkable drawback of this EDC/NHS chemistry is that most biologic molecules carry both carboxyl and amino groups yielding crosslinking (intra- and/or intermolecular) and loss of function. Therefore, we used Cu(I) catalyzed azide-alkyne cycloaddition chemistry (click chemistry) to overcome this disadvantage [3]. The suitability of this decoration method for biologics was studied with fibroblast growth factor 2 (FGF2), a heparin-binding growth factor affecting the proliferation, differentiation and migration of many cell types of mesodermal and neuroectodermal origin, stimulating tissue regeneration and having therapeutic potential in wound healing [4]. Firstly, we PEGylated the FGF2 in a site-directed pattern by deploying free thiol groups (four cysteine residues: two pointing outwards, two pointing into the protein core) with thiol reactive 10 kDa PEGs carrying ethinyl groups as described before with modification [3]. Alkyne-PEGylated FGF2 was purified using heparin based affinity chromatography on an ÄKTA purifier system and finally characterized by reducing SDS-PAGE, RP-HPLC and MALDI MS. The bioactivity of alkyne-PEGylated FGF2 was determined by analysis of the proliferation of NIH-3T3 cells. Alkyne-PEGylated FGF2 was coupled to azide modified microspheres by click chemistry and analyzed by flow cytometry (FACS) after incubation with FGF2 primary and FITC labelled secondary antibody. MALDI MS spectra of alkyne-PEGylated FGF2 indicated that either one or two FGF2 cysteine residues were PEGylated, and the potency of alkyne-PEGylated FGF2 was demonstrated by NIH-3T3 cell proliferation assay. The coupling of alkyne-PEGylated FGF2 to the azide modified microspheres was confirmed by FACS analysis. Azide modified microspheres exposed to alkyne-PEGylated FGF2 in presence of copper showed stronger fluorescence than controls in the absence of copper. The functionalization of azide modified microspheres with FGF2 has been successfully demonstrated using click chemistry. This chemical strategy for covalent immobilization avoids the formation of intra- and/or intermolecular covalent aggregates among biologics as well as among biomaterials and consequently may improve the safety profile of functionalized biomaterials compared to EDC/NHS chemical strategies [3]. Acknowledgments: The financial support from the IZKF (Wuerzburg, Germany) with grant number D-218 is gratefully acknowledged.

References: 1. Chiu, L.L.Y. and Radisic, M.: Biomaterials 2010, 31(2): 226-241. 2. Karageorgiou, V. et al.: Journal of biomedical materials research 2004, 71(3): 528–37. 3. Zhao, H., Heusler, E. et al.: Journal of structural biology 2014, 186(3): 420-430. 4. Gospodarowicz, D., Neufeld, G., Schweigerer, L.: Cell differentiation 1986, 19(1): 1–17.

HPLC method development for the determination of linezolid and cefuroxime in synovial fluidsite microdialysis samples of arthritis patients Appelt, A.K.1; Kauzor, D.1; Wicha S.G.1; Brosig, H.1; Zeitlinger, M.2; Kloft, C.1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany 2 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

Objectives: Septic arthritis, a joint destructive infectious disease with the knee as the most frequently involved joint, displays a high morbidity and mortality rate. Cefuroxime is commonly used for perioperative antibiotic prophylaxis. If

septic arthritis caused by gram-positive bacteria resistant to other first-line antibiotics occurs, linezolid is given additionally. A prerequisite for antibiotic efficacy are sufficiently high concentrations at the site of infection, i.e. the synovial fluid. Due to potentially inadequate penetration of the antibiotics into the synovial fluid, insufficient concentrations at the infection site may occur, also accounting for the high morbidity and mortality rate of septic arthritis. Therefore, it is important to measure drug concentrations at the site of infection. A pharmacokinetic study of cefuroxime and linezolid in 10 patients undergoing elective knee arthroscopy for meniscal repair was performed in collaboration with the Department of Clinical Pharmacology, Medical University of Vienna. Concentrations of linezolid and cefuroxime were measured in microdialysis samples obtained in synovial fluid and interstitial, muscular tissue fluid as well as in plasma samples to characterise the pharmacokinetics of both antibiotic agents in combination. For the analysis of study samples an appropriate HPLC assay for the determination of both antibiotics had to be developed. In total, 25 concomitant drugs were given during the trial and needed to be separated from the analytes. Methods: For drug quantification in the biological fluids a Thermo Scientific Dionex Ultimate 3000 HPLC with DAD 3000 detector was used. Detection wavelengths for linezolid and cefuroxime were set based on previous investigations. Separation was achieved by a Thermo Fisher Hypersil GOLD Phenyl column (100 x 4.6 mm, 3 µm) with a Thermo Fisher Phenyl guard column. To isolate cefuroxime and linezolid from the concomitant drugs different modes and mobile phases were assessed. For the isocratic mode, a mobile phase consisting of Milli-Q water and acetonitrile with and without 0.1% trifluoroacetic acid and for the gradient mode, Milli-Q water with 0.1% trifluoroacetic acid as mobile phase A and Milli-Q water and acetonitrile with 0.1% trifluoroacetic acid as mobile phase B were investigated in different ratios. In addition, different injection volumes, flow rates, and column oven temperatures were evaluated. Results: Separation of linezolid and cefuroxime was achieved. Detection wavelengths were set to 251 nm for linezolid and 271 nm for cefuroxime, respectively. A gradient method was performed using Milli-Q water and 0.1% trifluoroacetic acid as mobile phase A and Milli-Q water and acetonitrile 30:70 (v/v) and 0.1% trifluoroacetic acid as mobile phase B were applied at a flow rate of 2.0 mL/min. The injection volume was amounted to 10 µL. The temperature of autosampler and column oven was set to 4° C and 35° C, respectively. The total run time was 20 minutes with retention times of 3.95 and 4.10 minutes for linezolid and cefuroxime, respectively. Except urapidil, all concomitant drugs were successfully separated from the analytes. Conclusion: A successful separation of linezolid and cefuroxime and isolation from 24 of the concomitant drugs was achieved. As next step, an additional HPLC assay has to be developed for the patient receiving urapidil. Both assays shall be extended to plasma as matrix. Thereafter, the methods have to be validated according to the criteria of the EMA Guideline on bioanalytical method validation [1]. In total, concentrations of linezolid and cefuroxime in synovial fluid and interstitial fluid of muscle tissue measured by microdialysis and plasma will be determined and related to each other in order to investi-gate, if current antibiotic regimens will provide effective concentrations at the particular site of infection. Acknowledgement: We thank Dorothea Frenzel, Martin-Luther-Universitaet Halle-Wittenberg for her analytical support.

Reference: 1. European Medicines Agency (EMA): Guideline for bioanalytical method validation 2012

Substituted vinyl sulfones as covalent and reversible cysteine protease inhibitors Kesselring, J.1; Schneider, T.1; Grathwol, C.1; Weickert, A.2; Lee, W.2; Willmes, T.3; Engels, B.2; Sotriffer, C.A.3; Schirmeister, T.1 1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY

2 Institute of Physical and Theoretical Chemistry, Universiy of Würzburg, GERMANY 3 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY

Cysteine proteases play vital roles for the life cycles, nutrition and pathogene-sis of a variety of parasites causing infectious tropical diseases [1,2]. Therefore a promising strategy for the treatment of these diseases is the inhibition of these proteases.

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At present a large number of potent and irreversible cysteine protease inhibitors of different classes, including peptidyl vinyl sulfones, have been reported [3]. The inhibition mechanism of such vinyl sulfones results from the addition of the enzyme’s active site cysteine residue in a Michael type reaction [4]. QM and QM/MM calculations have proposed substituted vinylsulfones which should be able to form a covalent, but reversible bond with the cysteine sulfur of the protease’s active site. Such a reversible reaction should be possible by

- -position of the vinyl sulfone moiety, e.g. nitrile groups, halogens and thiolates, respectively. This leads to a thermoneutral or slightly endergonic vinylic substitution or addition reaction. To confirm these calculations a series of highly functionalized vinyl sulfones with varying peptidic residues were synthesized and reacted with phenylethanethiol as an analogue of the active site’s cysteine residue. The progression of the reaction was monitored by NMR, which allows the determination of rate constants, equilibrium constants, and reaction energies. Additionally the reversibility of the reaction with the enzyme could be proved by dilution and dialysis experiments. Furthermore the inhibitory potencies and binding modes of the synthesized compounds were evaluated by enzyme assays with various cysteine proteases. References: 1. Hanzlik, R.P.: J. Med. Chem. 1984, 27(6): 711. 2. Rosenthal, P.J. and coworkers: Antimicrob. Ag. Chemother. 2003, 47(1): 154. 3. Powers, J.C. et al.: Chem. Rev. 2002, 12: 4639. 4. Palmer, J.T. et al.: J. Med. Chem. 1995, 38 (17): 3193 .

Flavonoid-based compounds as novel selective ABCC1 modula-tors Obreque-Balboa, J.1; Sun, Q.2; Bernhardt, G.1; König, B.2; Buschauer, A.1 1 Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany 2 Institute of Organic Chemistry, University of Regensburg, D-93040 Regensburg, Germany

The superfamily of human ATP-binding cassette (ABC) proteins comprises 48 members divided into 7 subfamilies (ABCA - ABCG) [1]. In addition to physiological functions, a number of these membrane proteins are known as efflux pumps limiting the bioavailability and the access of a wide variety of xenobiotics, including drugs, to the brain. Moreover, the ABCB1 (p-glycoprotein), ABCG2 (BCRP, breast cancer resistance protein) and ABCC1 (MRP-1, multidrug resistance related protein 1) are the most prominent efflux transporters contributing to multidrug resistance. For instance, the expression of these transporters is related with high extrusion levels of chemotherapeutic agent, such as anthracyclines, vinca alkaloids or epipodophyllotoxins, and poor outcome of the treatment [2]. Subtype-selective and potent modulators of these ABC transporters are required as pharmacological tools to investigate their role in health and disease, to improve brain access or to overcome chemoresistance. Previously, potent and selective ABCB1 and ABCG2 modulators have been developed in our laboratory [3-6]. Here we report on the synthesis and characterization of a new class of ABCC1 modulators. The core structure of flavonoids known both, as ABC substrates and inhibitors [7], served as template. The synthesized compounds were investigated in vitro for transporter inhibition in fluorescence based assays using ABCC1- (MDCKII-MRP1 cells), ABCB1- (Kb-V1 cells) and ABCG2- (MCF-7/topo cells) overexpressing cells. The potential to revert drug resistance was explored for selected inhibitors in a kinetic chemosensitivity assay.

Among the synthesized compounds, the most promising modulators were comparable to reversan [8] in potency, and by far superior to the reference substance regarding selectivity. The most potent compound revealed an IC50

value of 10.5 µM (ABCC1) and a maximal inhibitory effect (Imax) of 120% compared to reversan (IC50=4.5 and Imax 100%). In contrast to the latter, which is a combined ABCB1 and ABCC1 modulator, the title compounds were highly selective for ABCC1.]

Acknowledgments: Deutscher Akademischer Austauschdienst, DAAD

References: 1. Dean, M.; Rzhetsky, A.; Alliknets, R.: Genome Res. 2001, 11(7): 1156-66. 2. Leslie, E.M.; Deeley, R.G.; Cole S.P.: Toxicol. Appl. Pharmacol. 2005, 204(3): 216-237. 3. Egger, M. et al.: Eur. J. Org. Chem. 2007, (16): 2643-2649 4. Kühnle, M. et al.: J. Med. Chem. 2009, 52(4): 1190-1197 5. Ochoa-Puentes, C. et al.: ACS Med. Chem. Lett. 2013, 4(4):, 393−396 6. Bauer, S.; et al.: ChemMedChem 2013, 8(11): 1773-1778 7. Leslie, E.M. et al.: Mol. Pharmacol. 2001, 59(5): 1171-1180. 8. Burkhart, C.A., et al.: Cancer Res., 2009, 69(16): 6573-6580


Virtual Screening and Biological Evaluation of new Inhibitors of the ATPase-Domain in GyrB Münsterberg, M.; Fransson, I.; Lemcke, T.

Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany

The development of new antibiotics is essential for the future of the treatment of infection diseases. Although, this fact is well-known [1,2], it has already become of political interest [3]. Gyrase is an important target for antibiotic therapy. It is inhibited by the commonly-used fluoroquinolone family, which interacts with the gyrase-DNA-complex. In contrast, aminocoumarines like novobiocin inhibit the ATPase domain of the gyrase subunit B (gyrB). Currently, no drug acting as gyrB ATPase inhibitor is used in therapy [4]. We performed a high-throughput virtual screening with the “Drug-Like” subset of the ZINC database [5] targeting the ATPase domain of the gyrB-subunit of E. coli. Starting with 11 million compounds we applied several filter criteria (like MW, number of rotatable bonds) for the first substantial reduction of compound numbers. The remaining 3.6 million compounds were docked with the newly developed high-throughput screening tool TrixX [6] which allowed a multi target approach because of its fast algorithm. In this way we were able to dock the compounds in different conformations of the ATPase domain to consider the flexibility of the enzyme. Together with a validated post-processing protocol the amount of compounds could be reduced to several thousands. Subsequently, these compounds were redocked with the glide docking tool of Schrödinger [7]. A crucial visual inspection resulted in 20 compounds which were selected and purchased. Finally, we tested these compounds in a fluorescence quenching and supercoiling assay for their inhibitory activity of the E. coli gyrB. For the fluorescence quenching we adopted the newly developed ener-gy transfer strategy [8,9] in order to obtain a fast, simple and reliable screening method. In this poster we will present a successful, hit search protocol (see scheme below) including the virtual screening and biological evaluation. This work will be continued to further optimize our findings to a possible lead structure for the inhibition of bacterial gyrB ATPase.

Scheme of the Hit Search Protocol

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References: 1. Livermore D.M.: J. Antimicrob. Chemother. 2009, 64(suppl 1): i29. 2. Boucher H.W. et al.: Clin. Infect. Dis. 2009, 48(1): 1–12. 3. Theuretzbacher U.: Int. J. Antimicrob. Agents 2012, 39(4): 295–299. 4. Collin F.; Karkare S.; Maxwell A.: Appl. Microbiol. Biotechnol. 2011, 92(3): 479–497. 5. Irwin J.J. et al.: J. Chem. Inf. Model. 2012, 52(7): 1757–1768. 6. Schlosser J.; Rarey M.: J. Chem. Inf. Model. 2009, 49(4): 800–809. 7. Schrödinger, LLC 2014. 8. Zuck P. et al.: Anal. Biochem. 2005, 342(2): 254–259. 9. Miyata Y. et al.: J. Biomol. Screening 2010, 15(10): 1211–1219.

Bifunctional phosphonates for the functionalization of metal surfaces Klitsche, F.; Maison, W.

Pharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany

The coating of metal surfaces is a valuable method to generate tailor-made materials for a variety of applications. Due to the fact that the formation of biofilms, also known as biofouling, occurs on almost all material surfaces in biological systems it is a crucial issue in medicine, clinical hygiene, food industry and marine technology [1,2]. The process of biofilm formation can be described stepwise. In a first step organic molecules like proteins, polysaccha-rides or glycoproteins attach to the surface reversibly to form a molecular monolayer. This conditioning film forms the soil for the colonization of microorganisms. The next and final step of the so-called microfouling is the formation of an inert bacterial biofilm that is protected by a surrounding mucopolysaccharide layer [3]. At this stage, bacterial biofilms can no longer be removed by antibiotics and cause therefore serious problems in a clinical context and in the food industry. In implant medicine, for example, biofilms can lead to inflammation, impair integration and final rejection of the implant. To prevent biofouling several strategies have been applied which interfere with different steps of the process. Examples include bactericide surfaces generated with metals such as copper or silver. A second approach is the use of repellant nanolayers such as polyethylene glycol (PEG) and other hydro-philic polymers preventing non-specific protein attachment in the first step of biofouling [2,4,5]. A key aspect of this approach is the stable immobilization of hydrophilic polymers on the material surface. We have developed modular tripodal anchor molecules for the stable immobilization of hydrophilic polymers on metal surfaces [6,7]. The synthesis of tripodal phosphonates and their conjugation to PEG is described on the poster. Moreover the immobilization of these conjugates on metal oxides of clinical relevance and the analysis of the resulting antifouling surfaces is reported.

Modular design principle of antifouling coatings [8]

References: 1. Banerjee, I.; Pangule, R.C.; Kane, R.S.: Adv. Mater. 2011, 23(6): 690-718. 2. Lejars, M.; Margaillan, A.; Bressy, C.: Chem. Rev. 2012, 112(8): 4347-4390. 3. Costerton, J.W.; Stewart, P.S.; Greenberg, E.P.: Science 1999, 284(5418): 1318-1322. 4. Fenton, J.W.; Fasco, M.J.: Thromb. Res. 1974, 4(6): 809-817. 5. Sisson, A.L.; Haag, R.: Soft Matter 2010, 6(20): 4968-4975. 6. Pannier, N.; Maison, W.: Eur. J. Org. Chem. 2008, 2008(7): 1278-1284. 7. Maison, W.; Frangioni, J.V.; Pannier N.: Org. Lett. 2004, 6(24): 4567-4569. 8. Franzmann, E. et al.: Chem.-Eur. J. 2011, 17(31): 8596-8603.

Synthesis of oxazinones and oxazinethiones with selective inhibition of NAD+-dependent histone deacetylases (Sirtuins) Beese, K.1; Swyter, S.2; Jung, M.2; Link, A.1 1 Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany 2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr. 25, 79104 Freiburg, Germany

The seven human sirtuins are NAD+-dependent histone deacetylases, which are involved in epigenetic gene expression. The balance of acetylation and deacetylation of histones and non-histone proteins is regarded to be relevant in age-related diseases, e.g. cancer, neurodegenerative diseases like Parkin-son's, Alzheimer's and Huntington's disease. To obtain non-hydrolyzable analogs of the lactone splitomicin, we advanced β-phenylsplitomicin analogs with lactam structure1 and synthesized novel naphtho[1,3]oxazin-3-one and naphtho[1,3]oxazine-3-thione derivatives performing a solvent-free one pot synthesis. The substances were tested in a trypsin-coupled homogeneous assay, which revealed a selective inhibition of Sirt2.

Acknowledgements: Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg. Technical assistance of Dr. A. Bodtke, K. Böheim and J. Technau is gratefully acknowledged.

References: 1. Neugebauer, R.C.; Sippl, W.; Jung, M.: J. Med. Chem. 2008, 51(5): 1203-13

Design, Synthesis and Pharmacological Profiling of Dual Modula-tors of Soluble Epoxide Hydrolase and Peroxisome Proliferator Activated Receptors Blöcher, R.; Wittmann, S.K.; Lamers, C.; Weber, J.; Steinhilber, D.; Schubert-Zsilavecz, M.; Proschak, E.

Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main

The basic idea of this study comprehends the development of polypharmaco-logical agents for the treatment of the metabolic syndrome [1, 2, 3]. Several dual modulators of the peroxisome proliferatior-activated receptors (PPARs) [4] and the soluble epoxide hydrolase (sEH) [5] have been rationally designed, synthesized and evaluated in vitro. It was possible to synthesis a number of active compounds containing the common pharmacophores of both targets (sEH/PPAR) [4, 5, 6]. The potency of sEH inhibition was determined in an in vitro assay with recombinant enzyme [6]. The ability of the PPAR activation was evaluated in a cell-based reporter-gene assay [7]. The sEH inhibtion was achieved at a sub micromolar concentration of the compound. PPAR agonism was reached at micromolar concentration. Relative activations could be demonstrated, from full agonists to partial PPAR modulators. The compounds targeted PPAR in a subtype selective as well as in a nonselective way. Future in vivo studies will show the value of this approach for the therapy of metabolic syndrome related diseases [8].

Acknowledgments: Goethe University, Institute of Pharmaceutical Chemistry, Steinhilber, D.; Schubert-Zsilavecz, M. and Proschak, E. Else Kröner-Fresenius-Foundation

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References: 1. Eckel, R.H.; Grundy, S.M.; Zimmet, P.Z.: Lancet 2005, 365: 1415–1428. 2. Grundy, S.M. et al.: Nat. Rev. Drug Dis. 2006, 5: 295–309. 3. Page Acnp-Bc, J. et al.: J. Am. Acad. Nurse Prac. 2012, 24: 345–351. 4. Pirat, C.et al.: J. Med. Chem. 2012, 55: 4027–4061. 5. Hammock, B. et al.: J. Med. Chem. 2012, 55: 1789−1808. 6. Blöcher, R. et al.: J. Med. Chem. 2012, 55: 10771-10775. 7. Lamers, C.; Schubert-Zsilavecz, M.; Merk, D.: Exp. Opin. Ther. Pat. 2012, 22: 803–841. 8. Imig, J.D. et al.: Experimental Biology and Medicine 2012, 237: 1402-1412.

Biological screening of newly synthesized thiocarbonic/carbonic acid derivatives on isolated guinea pig tissue preparations Hintersteininger, M.1; Erker, T.1; Studenik, C.R.2

1 Department of Pharmaceutical Chemistry, Althanstraße 14, 1090 Vienna, Austria 2 Department of Pharmacology and Toxicology, Althanstraße 14, 1090 Vienna, Austria

The aim of this project was to study the efficacy and the pharmacodynamics of four compounds focusing on their inotropic, chronotropic, vasodilating and spasmolytic action.

1 2 3 4 Compound 1 and Compound 3 are thiocarbonic acid derivatives, compound 2 and compound 4 are carbonic acid derivatives. Force of contraction (fc) on electrically driven papillary muscles (1Hz), spontaneously beating right atria, aortic-rings, arteria pulmonalis-rings (precontracted with 90 mM KCl) and terminal ilea (precontracted with 60mM KCl) of guinea pigs was measured using the method described by Reiter [1]. Compound 1 significantly reduced fc in papillary muscles with an EC50-value of 10 µmol/l. This derivative also caused a negative chronotropic effect (EC50 = 48 µmol/l, right atria) as well as a vasodilating (EC50 = 12.5 µmol/l in aortic rings and 13.7 µmol/l in pulmonalis rings) and spasmolytic effect (EC50 = 9.3 µmol/l in terminal ilea). Compound 2 only showed a significant action on heart muscle preparations with an EC50–value of 12.5 µmol (papillary muscles) and 44 µmol/l (right atria) and on the terminal ilea with an EC50-value of 33.5 µmol/l. Compound 3 significantly decreased force of contraction in heart muscle preparations with an EC50 – value of 5.45 µmol/l (papillary muscle) and 43,5 µmol/l (right atria) as well as in smooth muscle preparations with an EC50 of 91.5 µmol/l in aortic rings, 50 µmol/l in pulmonalis rings and 20.5 µmol/l in terminal ilea. Compound 4 did not show significant effects on any preparations with exception of a negative inotropic one on papillary muscles (EC50 = 30 µmol/l). We demonstrated that compound 1 and 3 had the most potent effects on all tissue preparations possibly caused by H2S/COS release followed by opening of KATP-channels. Compound 2 showed a negative inotropic, negative chronotropic and spasmolytic effect, while compound 4 only excerted a negative inotropic effect. Our results suggest that derivatives with a thiocar-bonyl moiety possess a strong efficacy in all isolated tissues, while compounds with a carbonyl moiety are less potent but more tissue selective. References: 1. Reiter, M.: Drug. Res. 1967, 17:1249-1253.

The mitochondrial Amidoxime Reducing Component (mARC) is involved in energy metabolism Jakobs, H.1; Mikula, M.2; Havemeyer, A.1; Ostrowki, J.2; Clement, B.1

1 Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University, Gutenbergstraße 76, 24118 Kiel, Germany

2 Department of Oncological Genetcs, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland

mARC is the fourth mammalian molybdenum enzyme and was recently discovered in our lab [1]. It reduces N-oxygenated structures and is a metabolic counterpart of P450s and FMOs [2]. However, mARC is not solely active but requires cytochrome b5 and NADH-cytochrome b5 reductase for electron transport [3]. The enzyme system is involved in N-reductive drug metabolism and in the activation of amidoxime prodrugs [4,5]. Yet, little is known about the endogenous functions of the enzyme system. Besides other functions [6,7], links to energy metabolism were demonstrated like an association with diabetes in animal models [8]. Moreover, a mARC1 SNP is associated with altered blood lipids [9,10] and involvement in lipid synthesis in 3T3 adipocytes was proven [11]. Based on the latter findings, we tested whether the enzyme system undergoes changes due to glucose in hepatoma cell lines and due to diet in mice. We tested mRNA and protein levels as well as N-reductive activity. Indeed the studied proteins alter due to glucose in cell culture and both fasting and high fat diet in mice. Taken together with other recent studies [9,10,11,12], it is evident that the mARC protein is involved in energy and lipid metabolism. In continuation, we tested the influence of fasting on N-reductive metabolite concentrations in vivo and thus the potential impact on prodrug activation. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable. References: 1. Havemeyer, A.; Bittner, F.; Wollers, S.: J. Biol. Chem 2006, 281(46): 34796-34802. 2. Grünewald, S.; Wahl, B.; Bittner F.:J. Med. Chem. 2008, 51(24): 8173-8177. 3. Havemeyer, A.; Lang, J.; Clement, B.: Drug Metab. Rev. 2011, 43(4): 524–539. 4. Havemeyer, A. et al.: Drug Metab. Dispos. 2010, 38(11): 1917–1921. 5. Jakobs, H. et al.: ChemMedChem. 2014, DOI: 10.1002/cmdc.201402127. 6. Krompholz, N. et al.: Chem. Res. Toxicol. 2012, 25(11): 2443–2450. 7. Kotthaus, J. et al.: Biochem. J. 2011, 433(2): 383–391. 8. Malik, A.N. et al.: Biochem. Biophys. Res. Commun. 2007, 357(1): 237–244. 9. Teslovich, T.M. et al.: Nature 2010, 466(7307): 707–713. 10. Aslibekyan, S. et al.: PLoS ONE 2012, 7(10): e48663. 11. Neve, E.P.A. et al.: J. Biol. Chem. 2012, 287(9): 6307–6317. 12. Brown, S.D.M.; Moore, M.W.: Genome 2012, 23(9-10): 632–640.

Development and validation of a new analytical method to characterize PEG-asparaginase by flow field-flow fractionation John, C.1; Herz, T.2; Hempel, G.2; Langer, K.1 1 Department of Pharmaceutical Technology and Biopharmacy - University of Münster, Corrensstr. 48, 48149 Münster, Germany 2 Department of Pharmaceutical and Medicinal Chemistry - Clinical Pharmacy, University of Münster, Corrensstr. 48, 48149 Münster, Germany

Background: PEG-asparaginase (PEG-ASNASE) is widely used in combina-tion therapy for the treatment of acute lymphoblastic leukemia (ALL). The PEGylated form is designed to reduce immunogenicity due to formation of antibodies against the foreign protein, reduce its degradation process and to decrease the clearance of the drug. Besides the physiological advances PEGylated peptides also show better solubility thus making PEGylation attractive to pharmaceutical companies and therefore increase the need for suitable analytical methods. Method: An AF 2000 MT-system (Postnova Analytics, Landsberg/Lech, Germany) was used to perform an asymmetrical flow field-flow fractionation (AF4) of PEG-ASNASE (Oncaspar®). Detection was performed by a dual wavelength UV-VIS detector set to 220/280 nm, a refractive index detector (RI) and a multi angle light scattering (MALS) detector. Additionally, a fraction collector was connected to the AF 2000 MT-system gathering analyte of 5 min periods for subsequent activity estimation using the well-established microplate reader-based assay by Lanvers et al.[1]. Moreover, a protein determination was carried out using a Pierce® BCA Protein Assay Kit (ThermoScientific, Waltham, Massachusetts). Validation was assessed by determining within and between run accuracy and precision, stability tests including room temperature, fridge and freeze/thaw cycles according to FDA guidelines (Guidance for Industry, Bioanalytical Method Validation, May 2001). Results: The method showed valid results for all measured parameters. Linearity was achieved over the range of 15-750 IU/mL. Accuracy was 86.7% to 102.2% for within run series with a precision of 1.2 to 10.2%. Between run series showed an accuracy of 85.3% to 109.9% with a precision of 2.5 to 6.3%.

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The results were verified by activity measurements. Here, accuracy for within run and between run series was 88.3% to 137.0% and 97.4% to 114.3%, respectively. The according precision was 2.4 to 9.3% and 0.1 to 12.3%, respectively. The samples stored at room temperature were measured after 24h, 7d, and 14d resulting in a recovery of 86.7±0.9%, 82.1±1.4%, and 77.3±4.8% (mean ± SD), respectively. In an analogous manner analyzed samples stored at fridge temperature presented a recovery of 101.2±5.2%, 92.6±7.9%, and 90.5±0.3% (mean ± SD), respectively. Analyzing the collected AF4 fractions for activity and protein content made it possible to correlate them. The correlation showed that protein fragments lacking enzymatic activity were formed in stressed samples whereas residual unimpaired molecules (> 350 kDa) were characterized by high activity. Conclusion: AF4 represents a useful tool for the analysis of PEG-ASNASE. The successful validation according to FDA guidelines and also verification by an established, clinically and routinely used activity assay could make it an attractive method for product evaluation by pharmaceutical companies. References: 1. Lanvers, C. et al.: Analytical Biochemistry 2002, 309: 117-126.


Dynamic virtual screening: reducing the search space within a ligand library Telukunta, K.K.; Lucas, X.; Günther, S.

Institute of Pharmaceutical Sciences, Research Group Pharmaceutical Bioinformatics, Hermann-Herder-Strasse 9, 79104, Germany

In the field of drug discovery, virtual screening has become an important technique which may replace time consuming in vitro assays to obtain an inhibitor for a protein. Current molecular libraries which can be used for virtual screening may contain over 35 million drug like molecules [1]. Present methods mainly apply brute force techniques i.e. all compounds are docked one after the other. To optimize this technique and accelerate the virtual screening process we have developed a more rational approach called dynamic virtual screening.

In this method we first obtain all the physicochemical properties of the complete compound library with the help of the tool QikProp [2]. Subsequently some arbitrary representative candidates of the complete library are taken. The representative small molecules are scored using the Standard Precision (SP) algorithm of Glide and sorted by their Glide score. From the sorted set of compounds we take the top-ranked few compounds for the process of learning appropriate physicochemical properties of putative binders. These learned properties are used to filter the main compound library and generate a reduced compound library space. The carved compound library is then applied for virtual screening. The complete method can be reapplied to reduce the library. We benchmarked the presented technique on existing virtual screening campaigns and analysed if dynamic virtual screening technique could speed up the search for inhibitors.

References: 1. John, J.I. et al.: J. Chem. Inf. Model. 2012, 52(7): 1757-1768.

2. Small-Molecule Drug Discovery Suite. 2014-1: QikProp, version 4.0: Schrödinger, LLC. 2014, NY.

Development of partial farnesoid X receptor (FXR) agonists Merk, D.1; Carrasco-Gomez, R.1; Flesch, D.1; Gabler, M.1; Lamers, C.1; Schneider, G.2; Schubert-Zsilavecz, M.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany 2 Institute of Pharmaceutical Sciences, ETH Zürich, Wolfgang-Pauli-Str. 10, CH-8093 Zürich, Switzerland

The ligand activated transcription factor nuclear farnesoid X receptor (FXR) acts as a regulator in many metabolic pathways including lipid and glucose homeostasis and pharmacological activation of FXR seems a valuable therapeutic approach to treat several pathophysiological conditions. FXR activation holds promise for the treatment of liver diseases such as primary biliary cirrhosis and non-alcoholic fatty liver disease, metabolic disorders linked to insulin resistance and certain forms of cancer. However, full FXR agonism as it is exhibited by known FXR ligands can evoke undesirable effects which hinder the clinical development of many known FXR agonists. FXR activation may e.g. cause cholestasis and impaired lipid homeostasis. For long-term treatment of metabolic disorders partial FXR agonists with balanced, moderate FXR activating activity are therefore required, that avoid disadvantageous FXR over-activation. We report the development and SAR of anthranilic acid derivatives as potent FXR modulators in a reporter gene assay and on mRNA level in liver cells. The most potent compound 1 of this scaffold exhibits partial FXR agonism with an EC50 value of 8±3 nM.

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References: 1. Düfer et al.: Diabetes. 2012, 6: 1479–1489. 2. Fiorucci, S. et al.: Future Med Chem. 2012, 7: 877–891. 3. Fiorucci, S. et al.: Curr Opin Gastroenterol 2009, 3: 252-259. 4. Lee, J.Y. et al.: Br J Cancer. 2011, 6: 1027–1037. 5. Merk, D. et al.: Bioorg. Med. Chem. 2014, 8: 2447–2460. 6. Merk, D. et al.: J Med Chem, submitted 7. Mudaliar, S. et al.: Gastroenterology. 2013, 3: 574-82.e1. 8. Richter, H.G. et al.: Bioorg. Med. Chem. Lett. 2011, 1: 191–194.

Pyridinol/Pyridinon-tautomerism determining activity at Far-nesoid X Receptor: new agonistic or antagonistic ligands of FXR Lamers, C.1; Merk, D.1; Flesch, D.1; Gabler, M.1; Carrasco-Gomez, R.1,2; Schneider, G.2; Schubert-Zsilavecz, M.1

1 Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt a.M., Germany 2 Swiss Federal Institute of Technology (ETH), Institute of Pharmaceutical Sciences, Valdimir-Prelog-Weg 4, 8093 Zürich, Switzerland

Nuclear Farnesoid X Receptor (FXR) is a ligand-activated transcription factor that is highly expressed in liver, intestine and kidney.[1] After activation it binds

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to a DNA response elements either as monomer or as heterodimer with Retinoid X Receptor (RXR) and regulates target gene expression. These genes are involved in bile acid metabolism, lipid and glucose homeostasis. Endogenous ligands are bile acids (most active: chenodeoxycholic acid) [2, 3, 4] as well as their metabolites and polyunsaturated fatty acids. Intense research has been made to find potent synthetic ligands since FXR has gained interest as potential pharmaceutical target [5, 6] due to its role in metabolic pathways as well as in inflammation and tumorgenesis. Presently, FXR agonists (e.g. 6-ECDCA, 6α-ethylchenodeoxycholic acid) are in clinical development for treatment of primary billiary cirrhosis (PBC), non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Besides FXR agonists the development of FXR antagonists has come into focus in recent years since FXR knockout studies suggested positive impact of FXR inhibition on insulin sensitivity and atherosclerosis in obese conditions.[7, 8] Here we present the development of new potential ligands of FXR starting from

a dual PPAR/γ ligand previously developed in our workgroup which showed slight FXR activity in our transactivation assay. Further improvement in activity and selectivity at FXR was achieved by introduction of a substituted pyridinol moiety. Therfore tautomerism occurs between pyridinol/pyridinon and interestingly it distinguished between agonistic, antagonistic activity or inactivity of the derivatives. We investigated the different binding modes of the agonistic and antagonistic derivatives in docking studies and furthermore optimized the reaction conditions to manage tautomerism.

References: 1. Forman, B.M. et al.: Cell 1995, 81(5): 1866-1870. 2. Wang, H. et al.: Mol Cell 1999, 3(5): 543-553. 3. Makishima, M. et al.: Science 1999, 284(5418): 1362-1365. 4. Parks, D.J. et al.: Science 1999, 284(5418): 1365-1368. 5. Pellicciari, R. et al.: J. Med. Chem. 2005, 48(17): 5383-5403. 6. Modica, S. et al.: FEBS Lett. 2006, 580(23): 5492-5499. 7. Prawitt, J. et al.: Diabetes 2011, 60(7): 1861-1871. 8. Zhang, Y.: Arterioscler. Thromb. Vasc. Biol. 2006, 26(10): 2316-2321.

Revival of an ancient tyrphostine scaffold: Computer-guided design, synthesis and biological evaluation of quinoxa-linebisarylureas as FLT3 inhibitors. Bensinger, D.; Göring, S.; Schmidt, B.

Clemens Schöpf - Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, 64287 Darmstadt, Germany.

Activating mutations of FLT3 are present in ~30% of patients with acute myeloid leukemia (AML) and associated with poor prognosis.[1] Point mutations in the Tyrosine kinase domain (TKD) are observed as primary mutations or acquired as secondary mutations in FLT3 with internal tandem duplications (FLT3-ITD) after treatment with Tyrosine kinase-Inhibitors (TKIs). Although dozens of potent inhibitors against FLT3-ITD are reported in literature, activating TKD point mutations especially at residues F691 and D835 remain the leading cause for clinical resistance and failed studies showing the steady need of new potent Inhibitors targeting specific resistance patterns. [2] Here we report on the discovery and characterization of novel quinoxaline based FLT3 inhibitors. The known TK family III inhibitor AG1295 represents one of the first “tyrphostins” showing activitiy in AML disease models but has not been developed to clinical studies due to low inhibitory strength. We discuss the pharmacophore features of a diverse set of known inhibitors as starting point for a new optimization algorithm for type II TKI following pharmacophore filtering and induced-fit docking of an in silico library to homology models from different related kinases. This lead to the design of a

set of diverse quinoxalinebisarylurea compounds that have been synthesized and assayed for FLT3 kinase activity. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay.

References: 1. Tiesmeier, J. et al.: Leukemia Res. 2004, 28: 1069-1074. 2. Smith, C.C. et al.: Nature 2012, 485: 260-263. 3. Levis, M. et al.: Blood 2001, 98: 885-887.


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Lack of evidence for the expression of the histamine H4-receptor on human monocytes Werner, K.; Neumann, D.; Seifert, R.

Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

The histamine H4-receptor (H4R) plays an important role in inflammation and immune response. The H4R has been unequivocally identified in human eosinophils [1,2], but the expression on human monocytes has only been poorly studied so far [2,3]. The H4R is a Gi/o-protein-coupled receptor [1]. In human myeloid cells, activation of Gi-coupled receptors canonically leads to activation of phospholipase C (PLC) and subsequent increases in intracellular calcium [Ca2+]i concentrations [4]. The aim of our present study was to clarify whether the H4R is expressed and functionally active on human monocytes. Therefore, we isolated monocytes from peripheral blood of healthy human volunteers via density gradient centrifugation and magnetic activated cell sorting. Additionally, we conducted our experiments with U937 promonocytes. To assess H4R expression at the RNA level, we performed quantitative real-time PCR. However, we did not examine protein expression of H4R since serious concerns regarding specificity of H4R antibodies were raised [5]. In order to investigate functional activity of the H4R, we determined changes in [Ca2+]i concentrations using the Fura-2AM method. Monocytes were stimulated with different concentrations of HA and H4R agonists (5-methylhistamine and 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine (UR-PI376)), whereas U937 promonocytes were stimulated with HA alone and in combination with HxR antagonists. We did not obtain evidence that the H4R is expressed at the RNA level on human monocytes and U937 promonocytes. hH4R-transfected HEK-293 cells were used as positive control cells. In human monocytes, adenosine 5’-triphosphate (ATP) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induced large increases in [Ca2+]i concentrations, but stimulation with HA and H4R agonists did not cause any increase in [Ca2+]i indicating that the H4R is not present in monocytes. In U937 promonocytes, HA increased [Ca2+]i. However, the effect was blocked by the H1R antagonist mepyramine, but not by the H2R antagonist famotidine and the H4R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) proofing that in these cells expression of the H1R is exclusively responsible for changes in [Ca2+]i caused by HA. In conclusion, our study shows that the H4R is not expressed on human monocytes and U937 promonocytes. References: 1. Reher, T.M. et al.: Biochem. Pharmacol. 2012, 84(2): 192-203. 2. Seifert, R. et al.: Trends Pharmacol. Sci. 2013, 34(1): 33-58. 3. Dijkstra, D. et al.: J. Allergy Clin. Immunol. 2007, 120(2): 300-307. 4. Dillon, S.B. et al.: Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1988, 55(2): 65-80. 5. Beermann, S. et al.: Naunyn. Schmiedebergs Arch. Pharmacol. 2012, 385(2): 125-135.

Influence of increased levels of the transcriptional ca-activator CRTC1 in cardiac hypertrophy Morhenn, K.1,2; Quentin, T.1; Schroeder, S.1; Pahl, A.1; Schlossarek, S.2,3; Carrier, L.2,3; Eschenhagen, T.2,3; Cardinaux, J.-R.4; Lorenz, K.5; Lutz, S.6; Zimmermann, W.H.6; Steinmetz, M.7; Kaul, A.8; Hasenfuss, G.8; Laufs, U.9; Guo, Z.10; Oetjen, E.1,3,6,11 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck 3 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 4 Center for Psychiatric Neuroscience, Site Cery, 1008 Prilly-Lausanne, Switzerland 5 Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany 6 Department of Pharmacology, University Medical Center Göttingen, Robert Koch Straße 40, 37075 Göttingen, Germany

7 Department of Pediatric Cardiology and Intensive Medicine, University Medical Center Göttingen, Robert Koch Straße 40, 37075 Göttingen, Germany 8 Department of Cardiology and Pneumology, University Medical Center Göttingen, Robert-Koch-Staße 40, 37075 Göttingen, Germany; 9 Clinic for Internal Medicine III, University Medical Center Saarland, Kirrberger Straße 100, 66421 Homburg/Saar, Germany 10 University of Kentucky School of Medicine, Department of Physiology, 900 South Limestone, Lexington, Kentucky 40536, USA 11 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany

Maladaptive cardiac hypertrophy leads to heart failure, one of the common causes for hospitalization in the western world. Chronic β-adrenergic signalling contributes to the pathogenesis of cardiac hypertrophy, as evidenced by the therapeutic success of β-adrenoceptor antagonists. The cAMP Regulated Transcriptional Coactivator 1 (CRTC1) is regulated by increases in cAMP and calcineurin, as elicited by β-adrenergic signalling, both known to participate in the development of cardiac hypertrophy. An elevated protein content of CRTC1 was found in heart tissue of humans and mice under two conditions of maladaptive hypertrophy by immunoblotting. The CRTC1 protein content was neither elevated in hearts of humans under conditions of dilated cardiomyopathy nor in hearts of mice under conditions of physiological or dilated hypertrophy. Treatment of cardiomyocytes with the β-adrenoceptor agonist isoprenaline resulted in the activation i.e. dephosphorylation of CRTC1, as revealed by immunoblot analysis. This effect was prevented by the addition of the β-adrenoceptor antagonist propranolole. To study the role of CRTC1 in the heart, mice deficient in CRTC1 were investigated. These mice showed signs of hypertrophy compared to their wildtype siblings, indicated by the increased ratio of heart weight to tibia length and increased myocyte size. In addition, they showed reduced CRTC1-mRNA levels but unchanged mRNA levels of CRTC2 and CRTC3 as measured by quantitative real-time PCR. In CRTC1 deficient mice the mRNA and protein level of the anti-hypertrophic regulator of G-protein Signalling 2 (RGS2) was reduced. RGS4 and RGS5 showed no reduction in mRNA levels. Transient transfection assays of a luciferase reporter gene under the control of the murine RGS2 promoter (-867 bp to +1 bp) showed that overexpressed CRTC1 stimulated RGS2 promoter transcriptional activity. Mutation of the CRTC1-binding site, the cAMP-response element, within the promoter prevented CRTC1-induced transcrip-tional activity. Our data show that CRTC1 is elevated under conditions of maladaptive cardiac hypertrophy and is activated through beta-adrenergic signalling. Also, CRTC1 stimulates RGS2 gene expression and is thereby presumably decreasing Gαq-mediated hypertrophic signalling. Thus, the increased CRTC1 protein levels in cardiac hypertrophy might represent a protective mechanism and the loss of CRTC1 augments cardiac hypertrophy.

Investigations of biased signaling of the chemokine receptor CXCR3 with small allosteric modulators Brox, R.; Bernat, V.; Admas, T.H.; Tschammer, N.


Anomalies in the regulation and response of the chemokine receptor CXCR3, a rhodopsin-like G protein-coupled receptor that is mainly expressed on activated T cells, are associated with various pathologies including autoim-mune diseases, cancer, vascular diseases and transplant rejection. Therefore CXCR3 is an attractive pharmacological target [1]. In the contrast to chemo-kines small synthetic ligands bind to the chemokine receptors at the allosteric site(s) inside the hydrophobic pocket formed by transmembrane helices. While allosteric compounds mediate their effects at sites that are topographically distinct from the orthosteric binding site they gain advantages like probe-dependence or saturability of effect [2]. Furthermore it is well established that a given ligand is able to induce signalling bias by the activation of G proteins or β-arrestin-mediated pathways only. These ligands have a potential clinical relevance by blocking harmful and maintaining beneficial stimuli [3]. Biased allosteric modulation of G protein-coupled receptors represents thus an attractive approach in drug design. For the investigation of the molecular mechanisms of biased allosteric modulation we used a small library of novel negative allosteric modulators

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(NAMs), which were built on the 8-azaquinzolinone scaffold. Design of these compounds and subsequent mutagenesis studies were based on predictions from the CXCR3 homology modelling and docking. To estimate the binding affinity of the NAMs, we performed an allosteric radioligand (RAMX3) [4] displacement assay. This assay revealed that some of our novel compounds bind to a second allosteric binding pocket in CXCR3, distinct from the binding pocket occupied by RAMX3. In the next step the probe-dependent inhibition of CXCR3 by novel NAMs was determined. The ability of NAMs to inhibit CXCL11 and CXCL10 mediated activation of CXCR3 was determined in the [35S]GTPγS incorporation assay, which monitors the activation of G proteins, and in the β-arrestin 2 recruitment, which detects the recruitment of β-arrestin

2 to the activated receptor. Within our novel set of compounds we identified two biased NAMs named BD064 and BD103, which display probe-dependent inhibition of CXCR3. BD064 preferentially inhibit the CXCL11-mediated recruitment of β-arrestin 2 to CXCR3 (pKb = 7.46 and αβ =0) over the

activation of G proteins (pKb = 6.05 and αβ = 0.11). BD103 preferentially inhibits the activation of G proteins (pKb = 6.43 and αβ = 0.14) over the β-arrestin 2 recruitment (pKb = 8.73 and αβ = 0.09). In contrast, the inhibition of CXCL10-mediated activation of CXCR3 no pathway was preferred by mentioned compounds. To identify amino acid residue(s) important for the allosteric modulation of CXCR3 and its interactions with ligands, we mutated ssveral residues within the putative binding pocket. We identified the residue Lys300 as a molecular switch that constrains the activation of G proteins. The mutation K300M releases this molecular switch and increases the CXCR3-mediated coopera-

tivity between CXCL11 and the G protein, but not between CXCL11 and the β-

arrestin 2. Overall, we conclude that our novel class of compounds can serve as a useful tool for the investigation of key receptor-ligand interactions and induction of ligand-biased signaling. Furthermore the residue Lys300 is an important anchor amino acid that can be explored for the design of biased allosteric modulators of CXCR3.

References: 1. Wijtmans, M. et al.: ChemMedChem, 2008, 3: 861-872. 2. Christopoulos, A. et al.: Pharmacol Rev, 2002, 54: 352-354. 3. Rahmeh, R. et al.: PNAS, 2012, 109: 6733 . 4. Bernat, V. et al.: ChemMedChem, 2012, 7: 1481-1489.

Monovalent cations influence muscarinic M2 receptor spontane-ous activity and agonist-dependent signaling De Min, A.; Mohr, K.

Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany

G protein-coupled receptors (GPCRs) comprise the largest superfamily of cell-surface receptors, with key functions in physiology and drug targeting. Recently, several class A GPCRs were crystallized and the presence of a sodium binding site in the middle of the 7 transmembrane helical bundle was identified in adenosine A2A, adrenergic β1, protease activated receptor 1 and δ-opioid receptors. The main residue involved in the coordination of the ion is a highly conserved aspartate (D2.50) in the transmembrane helix 2 [1]. The muscarinic acetylcholine receptors also belong to the family A of GPCRs, and there are five different subtypes (M1-M5) expressed in many regions of the central nervous system and in various peripheral tissues. The structures of M2 and M3 receptors were crystallized, but no sodium binding pocket was described so far, even though all the subtypes contain the D2.50 residue. The aim of this study was to investigate whether sodium chloride (NaCl), had an influence on M2 receptor activation, both in the presence or absence of an agonist. The influence of potassium chloride (KCl) was studied for comparison. Membrane homogenates prepared from CHO cells stably expressing the human M2 receptor were used in [35S]-GTPγS binding studies to investigate the influence of increasing concentrations of NaCl and KCl on basal G protein activity, activation by the endogenous agonist acetylcholine (ACh) and inhibition by the inverse agonist atropine. Besides, [35S]-GTPγS binding assays were used to obtain dose-response curves of ACh and the superagonist iperoxo [2] under control conditions and in the presence of 200 mM of NaCl or KCl. Both salts had the same influence on receptor-mediated G-protein activation: [35S]-GTPγS basal binding and binding induced by the agonist and the inverse agonist gradually decreased; furthermore, the spontaneous activity of the M2

receptor progressively diminished and was then completely abolished at the highest salt concentration. Data from the dose-response assays showed that both salts shifted the inflection point of the curves (pEC50) to lower values, indicating that these monovalent cations reduced the agonist potency. Additionally, the effect of NaCl was stronger than that of KCl in decreasing the pEC50 values, in the case of both ACh and iperoxo. Taken together, M2 constitutive activity and agonist signaling were negatively influenced by KCl and NaCl. The finding that NaCl had a greater effect on the pEC50 of the compounds might suggest the presence of a sodium binding site in this receptor. References: 1. Katritch, V. et al.: Trends Biochem. Sci. 2014, 39: 233-244. 2. Schrage, R. et al.: Br. J. Pharmacol. 2012, 169: 357-370.

Discovery of a Highly Potent Biased Allosteric Agonist of the Human Chemokine Receptor CXCR3 Milanos, L.; Brox, R.; Tschammer, N.


The human chemokine receptor CXCR3 is a rhodopsin-like Gi protein coupled receptor, found on the surface of T–cells, natural killer cells and astrocytes. Its endogenous agonists are soluble proteins called chemokines CXCL9, CXCL10 and CXCL11. The activation of CXCR3 upon binding of CXCL9, CXCL10, and CXCL11 guides the trafficking of leukocytes during infection and inflammation. Moreover CXCR3 and its chemokines seem to be involved in numerous inflammatory and autoimmune diseases; therefore is a promising pharmaceuti-cal target. Although the development of CXCR3 allosteric antagonist was the main focus of medicinal chemists, recently the interest in the allosteric agonists arose. Previously reported series of the non-peptidic allosteric agonists VUF10661 and PS372424 namely showed anti-inflammatory activity [1, 2]. Furthermore it was shown that the endogenous agonists CXCL10 and CXCL11 promote wound healing properties [3]. This implies that the development of small-molecule CXCR3 allosteric agonist might be of particular therapeutic interest. We have chosen VUF10661 as a starting point for our library of small-molecule allosteric agonists. VUF10661 was reported to promote the CXCR3-mediated β-arrestin2 recruitment with an EC50 of 1 μM and 160% efficacy compared to CXCL11, and an activation of G proteins with an EC50 of 600 nM and the efficacy comparable to CXCL11 (measured in the [35S]GTPγS incorporation assay) [4]. With the aim to improve the potency and bias of VUF10661, we synthesized a series of analogues based on this non-peptidic agonist. Our efforts led us to the discovery of a very potent and strongly biased allosteric partial agonist LMT36. LMT36 activates the CXCR3-mediated recruitment of β-arrestin2 with an EC50 of 0.04 nM (25.000 fold increase compared to VUF10661), an efficacy of 60% (compared to CXCL11) and has no effect on the CXCR3-mediated activation of G proteins as detected in the [35S]GTPγS incorporation assay. This unique highly potent and biased allosteric agonist will be used as a chemical tool to dissect the complex molecular mechanisms governing the allosteric modulation of CXCR3. The discovery of this allosteric ligand that induces highly biased signaling expands the ways in which the function of CXCR3 can be manipulated.

References: 1. Stroke, I.L. et al.: Biochem. Biophys. Res. Commun. 2006, 349: 221–228. 2. O’Boyle, G. et al.: PNAS 2012, 109(12): 4599-4603.

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3. Davidson, J.M.: The Am. J. Pathol. 2010, 176(4): 1588-1591. 4. Scholten, D.J. et al.: Br. J. Pharmacol. 2012, 166: 898-911.

Synthesis of photoactivatable probes for labeling of the human chemokine receptor CXCR3 Admas, T.H.; Bernat, V.; Brox, R.; Tschammer, N.

Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer center, Friedrich Alexander university, Schuhstraße19, 91052 Erlangen, Germany

The chemokine receptor CXCR3, which belongs to the family of rhodopsin-like G protein-coupled receptors, is mainly expressed on activated T lymphocytes [1]. CXCR3 is involved in various autoimmune and inflammatory diseases, which make this receptor an attractive therapeutic target. Although intense efforts were dedicated to the development of small molecule antagonists of CXCR3, the precise binding mode of these molecules remains to be elucidat-ed. One of the most commonly used technique for the determination of structural elements in the binding pocket of a receptor is photoaffinity labeling followed by mass spectrometry. This method involves the design and synthesis of ligands, which carry an appropriate photoactivatable functional group (such as azide, diazirine or benzophenone). Upon the exposure to the UV light, these functional groups yield reactive intermediates. Formed reactive intermediates can than interact with amino acids in the binding pocket and form covalent bond. The fragments can be later detected by appropriate mass spectrometry technique. This project aims at mapping of the allosteric binding site of CXCR3 receptor with photoaffinity labeling followed by mass spectrometry. For this purpose compound 1, a negative allosteric CXCR3 modulator with an azide functional group was designed and synthesized first. As determined in a radioligand RAMX3 [2] displacement assay, compound 1 and its non-biotinylated analogue had comparable binding affinities of 0.8 nM and 9.8 nM respectively. This assay indicated that the biotinylated group and the PEG linker are well tolerated by CXCR3. Unfortunately, the azide derivative 1 was unstable upon the UV light exposure at 254 nm, yielding undesired short fragments of the compound. Hence we designed and synthesized another photoaffinity probe that incorporated diazirine as a photoactivatable moiety and well characterized 8-azaquinazoline scaffold. The small size of diazirines and the possibility to irradiate the probe at a longer UV wavelength (365 nm) makes diazirines preferable choice over other photoactivatable moieties. In a similar fashion to compound 1, the newly synthesized diazirine based photoaffinity probe 2 and its non-biotinylated analogue have shown nanomolar affinity towards CXCR3 in the RAMX3 displacement assay. The tendency of the photoactivatable probe 2 to form the corresponding reactive carbene intermediate upon UV irradiation was assessed by a phenomenon called carbene trapping. Unlike compound 1, the photoaffinity probe 2 provides the desired reactive intermediate on UV exposure. Studies on the irreversible binding of the photolabeled ligand with the receptor and identification of the residues, which directly interact with the ligand, are ongoing.

References: 1. Baggiolini, M.: Nature. 1998, 392(6676): 565-568. 2. Bernat, V. et al.: ChemMedChem. 2010, 7(8): 1481-1489.

Synthesis of Quinazoline Derivatives as Adenosine A3 Receptor Antagonists Briel, D.1; Schäke, F.1; Schwan, G.1; Müller, C.E.2; Vielmuth, C.2; Lang, M.1 1 University of Leipzig - Institute of Pharmacy, Bruederstraße 34, 04103 Leipzig, Germany 2 Rheinische Friedrich-Wilhelms-Universität Bonn, Pharmazeutisches Institut, An der Immenburg 4, 53121 Bonn, Germany

Diversely aliphatic and aromatic-substituted quinazoline derivatives were synthesized to evaluate their structure-activity relationships on the four subtypes (A1, A2A, A2B and A3 [1]) of G-protein-coupled adenosine receptors (ADOR).

The ADORA3 is a potential drug target [1,2], antagonists have been developed and pharmacologically evaluated [3,4]. Apparently this receptor is involved in the progression of several inflammatory diseases, as for instance plasma extravasation [4], asthma bronchiale, chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis [5]. Moreover, ADORA3 antagonists may be useful in the treatment of cancer to support chemotherapy [6]. The A3 receptor is coupled to Gi proteins mediating inhibition of adenylate cyclase [7]. The development of selective antagonists is required to achieve selective pharma-cological effects. In the present study two different types of quinazoline derivatives were investigated. For type I different aromatic amines were coupled by nucleophilic substitution with a 2,4-chloroquinazoline bearing hydrogen- or methoxy in position 6 and 7. In order to obtain type II derivatives different alkyl- and aryl substituents were attached to a 6,7-dimethoxyquinazoline core. In a second step the hydroxyl function in position 4 could be transformed into a chloride. The obtained quinazoline derivatives were assayed for their potency and selectivity on the four ADOR subtypes in vitro. Compound 1 was found to be potent at human A3: Ki = 30.6 ± 7.7 nM and showed high selectivity.

Thanks are due to J. Ortwein for HPLC analysis and Dr. L. Hennig for recording and analysis of NMR data. References: 1. Fredholm, B.B. et al.: Pharmacol. Rev. 2001, 53: 527–552. 2. Gessi, S. et al.: Pharmacology & Therapeutics 2008, 117:123–140. 3. Lee, J. et al.: Am. J. Pathol. 2013, 183: 1488-1497. 4. Mikus, E.G. et al.: Eur. J. Pharmacol. 2012, 699(1-3): 62–66. 5. Della Latta, V. et al.: Pharm. Res. 2013, 76: 182–189. 6. Merighi, S. et al.: Pharmacology & Therapeutics 2003, 100: 31–48. 7. Birnbaumer, L.: Biochim. Biophys. Acta 2007, 1768(4): 772–93.

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Structural comparison of the allosteric binding pocket in musca-rinic acetylcholine receptors. Bermudez, M.; Wolber, G.

Institute for Pharmacy; Free University Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

G-protein coupled receptors (GPCRs) trigger multiple signal-switching mechanisms, not only G-protein activation but also binding of ß-arrestin proteins and activation of kinases [1]. Despite recent advances in GPCR x-ray crystallography the understanding of conformational changes resulting in these activations is still incomplete and remains a major challenge for the design of specific GPCR modulating drugs. Due to the highly conserved orthosteric binding pocket (figure 1) other binding sites are highly interesting for the design of subtype selective drugs. The recently published crystal structures of the M2 and the M3 muscarinic acetylcholine receptor (PDB: 3UON [2], 4DAJ [3], 4MQS [4] and 4MQT [4]) provides the possibility to rationalize and understand the binding of ligands to muscarinic acetylcholine receptors and to search for novel binding subsites. We present the results of homology modeling for all 5 muscarinic acetylcholine receptor subtypes based on the available crystal structures. The obtained 3D-models represent active and inactive receptor conformations and indicate differences in their allosteric binding pockets. These differences give new mechanistic insights in terms of subtype selectivity and allosteric receptor modulation.

Figure 1: Superimposition of the crystal structures of M2 AChR (yellow, first number) and M3 AChR (blue, second number) with key residues for ligand binding.

References: 1. Jacoby, E., et al.: Chemmedchem, 2006, 1(8):760-782. 2. Haga, K. et al.: Nature 2012, 482(7386): 547-551. 3. Kruse, A. et al.: Nature 2012, 482(7386): 552-556. 4. Kruse, A. et al.: Nature 2013, 504(7478): 101–106.


GPR17: the elusive dual uracil nucleotide/cysteinyl-leukotriene receptor or still an orphan?

Simon, K.1; Hennen, S.1; Merten, N.1; Schröder, R.1; Peters, L.1; Schrage, R.2; Vermeiren, C.4; Mohr, K.4; Müller, C.E.3; Gillard, M.4; Gomeza, J.1; Kostenis, E.1 1 Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany 2 Pharmacology and Toxicology, University of Bonn, Gerhard-Domagk 3, 53121 Bonn, Germany

3 Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 4 CNS Research, UCB Pharma sprl, 1420 Braine l’Alleud, Belgium

GPR17 is an orphan G protein coupled receptor (GPCR), which is predomi-nantly expressed in the oligodendrocyte-lineage cells of the central nervous system. [1] Recently, inhibition of this receptor has been proposed as a novel and promising therapeutic strategy for the treatment of demyelinating diseases such as multiple sclerosis. [1, 2] In spite of its attractivity as novel target to foster repair of demyelinated neurons, the nature of its true endogenous ligands is still under debate. [3] While uracil nucleotides and cysteinyl-leukotrienes have been proposed as endogenous activators, [4] a number of independent studies did not confirm the original deorphaning report. [2-3, 5-7] We further investigated whether GPR17 represents the elusive orphan receptor indeed responding to two ligand classes or whether it may still require pairing with a yet unknown endogenous modulator. To this end we employed a number of different functional assay platforms based on label-free dynamic mass redistribution and label-free bioimpedance, but also classical assays capturing defined intracellular events such as Gαi, Gαq, and Gαs activation, β- arrestin recruitment, activation of extracellular signal-regulated kinases 1 and 2, and binding of [35S]GTPγS to Gα proteins. Notably, we were unable to demonstrate activation of GPR17 by any of the purported endogenous ligands. Yet, robust activation was obtained across all assays when the receptor was stimulated with the small molecule MDL29,951. Based on these data and the current literature, which does not support the notion that GPR17 has been successfully matched with its correct endogenous partners, we propose that GPR17 must still be regarded as orphan, awaiting the identification of the true native ligands.

References: 1. Chen, Y et al.: Nat. Neurosci. 2009, 12(11): 1398–1406. 2. Hennen, S. et al.: Sci. Signal. 2013, 6(298):ra93. 3. Qi, A.D.; Harden, T. K.: Nicholas, R. A.: J. Pharmacol. Exp. Ther. 2013, 347(1):38-46. 4. Ciana, P. et al.: EMBO J. 2006, 25(19):4615-4627. 5. Heise, C.E. et al.: J. Biol. Chem. 2000, 275(39):30531-30536. 6. Maekawa, A. et al.: Proc. Natl. Acad. Sci. USA. 2009, 106(28):11685-11690. 7. Benned-Jensen, T.: Rosenkilde, M. M.: Br. J. Pharmacol. 2010, 159(5):1092-1105.

Molecular insights into the high constitutive activity of the human histamine H4 receptor

Wifling, D.1; Löffel, K.1; Nordemann, U.1; Bernhardt, G.1; Dove, S.1; Seifert, R.2; Buschauer, A.1 1 Institut für Pharmazie, University of Regensburg, D-93040 Regensburg, Germany 2 Institut für Pharmakologie, Hannover Medical School, D-30625 Hannover, Germany

The histamine H4R belongs to class A of G-protein coupled receptors and is considered as a promising drug target for the treatment of inflammatory diseases like allergic asthma [1]. The validation of the H4R in translational animal models is hampered by species-dependent differences. There are considerable discrepancies in terms of ligand efficacies, potencies and affinities, in particular, between the hH4R (human) and the rodent orthologs, i.e. the mH4R (mouse) and rH4R (rat) [2,3]. In contrast to the mouse and rat H4R, the human H4R shows a high degree of constitutive activity. Aiming at more detailed insights into the molecular determinants of ortholog-dependent ligand-receptor interactions, we generated and expressed (Sf9 cells) a series of H4R mutants to determine radioligand binding and functional data ([35S]GTPγS assay) [2]. Apart from F169, which was identified by Lim et al. as a key amino acid for distinct ligand binding affinities at H4R orthologs [5], we mutated, based on molecular modeling studies, S179, S330 and R341 to the corresponding amino acids of the rodent H4Rs, resulting in hH4R F169V, hH4R S179M, hH4R S179A, hH4R F169V+S179M, hH4R F169V+S179A, hH4R S330R and hH4R R341S [2]. Besides, the reciprocal mH4R mutants, mH4R V171F and mH4R V171F+M181S served as control [2]. Moreover, to study the role of the F168/F169 motif, which is also found in, e.g., the β2AR, H3R and the M2R, we expressed the hH4R F168A mutant in Sf9 cells. Whereas changes in ligand affinity and potency were only minor, the constitu-tive activity of the hH4R-F169V and the double mutants was significantly reduced compared to the wild-type hH4R [2]. By contrast, an exchange of S179 by M or A alone did not significantly affect constitutive activity. Strikingly,

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the double mutants were comparable to the mH4R and to the rH4R, which are devoid of constitutive activity. The inverse agonism of thioperamide decreased from the hH4R via the hH4R F169V mutant to the hH4R F169V+S179M and hH4R F169V+S179A double mutants, respectively [2]. The data for the hH4R-F168A mutant revealed a major contribution of F168 to ligand binding with a concomitant, up to over 100-fold decrease in ligand potencies and a complete loss of constitutive activity, compared to the wild-type hH4R. Thioperamide acted as a neutral antagonist and JNJ7777120 turned to partial agonism. The results suggest that, in particular, F168 and F169 alone or F169 in concert with S179 favor the switch from the inactive to the active state of the human H4R. Acknowledgments: This work was supported by the Graduate Training Programme (Graduiertenkolleg) GRK 760 of the DFG and by the European Cooperation in Science and Technology, COST Action BM0806.

References: 1. de Esch, I.J. et al.: Trends Pharmacol. Sci. 2005, 26: 462-469. 2. Wifling, D. et al.: Br. J. Pharmacol. 2014, in press, doi: 10.1111/bph.12801. 3. Schnell, D. et al.: Naunyn. Schmiedebergs Arch. Pharmacol. 2011, 383: 457-470. 4. Nordemann, U. et al.: PLoS ONE 2013, 8(9): e73961. 5. Lim, H.D. et al.: J. Pharmacol. Exp. Ther. 2008, 327: 88-96.

Label-free dynamic mass redistribution analysis of 5-oxo-ETE receptor signaling via Gαi and Gβγ signaling routes Büllesbach, K.1; Bautista, O.2; Blättermann, S. 1; Schröder, R.1; Weaver, C.D.3; Guetschow, M.2; Kostenis, E.1 1 Molecular-, Cellular- and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany 2 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany 3 Vanderbilt Institute of Chemical Biology, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States

Dynamic mass redistribution (DMR) is a cutting edge label-free optical biosensor technology to capture cellular responses in a holistic manner upon exposure to extra- and intracellular stimuli [1]. This method has proven to be particularly valuable for dissecting complex signaling behaviors in living cells triggered via the family of G protein-coupled receptors, the largest family of membrane signaling proteins. GPCRs respond to a wide array of stimuli and serve to translate extracellular information to the inside of the cell via coupling to heterotrimeric αβγ G proteins. G proteins are classified into four major families, Gαq/11, Gαs, Gαi/o, and Gα12/13, and both Gα and free Gβγ subunit complexes regulate diverse intracellular effectors. Most GPCRs couple to more than one G protein heterotrimer, a feature referred to as pleiotropic signaling. Therefore compounds that selectively interfere with individual G protein heterotrimers are of great value to decipher contribution of defined signaling branches to complex signaling networks. Among such compounds are pertussis toxin (PTX), an invaluable probe to analyze Gαi-mediated cell responses and FR900359 (UBO-QIC) as well as YM-254890 [2], two cyclic depsipeptides that specifically silence signaling of Gαq family members. PTX and FR900359 interdict signaling of entire heterotrimers, i.e. abrogate both Gα and Gβγ–mediated cellular signaling. We have recently identified a small molecule, Gue1654, that specifically dampens Gβγ but not Gαi signaling of the chemoattractant receptor OXE-R, thereby uncovering a novel mechanism for selective inhibition of Gβγ signaling downstream of specific receptors [3]. Herein we wanted to take advantage of this novel biased ligand to answer the following questions: 1) Do holistic DMR recordings visualize the contribution of Gβγ signaling? 2) To which extent do Gα and Gβγ effectors impact on complex whole cell activation profiles? To this end, signaling of OXE-R forcibly expressed in human embryonic kidney (HEK293) cells and primary human neutrophils was investigated in cells pre-treated with a) receptor-specific, βγ-biased inhibitor Gue1654, b) small molecule Gβγ inhibitor Gallein [4], c) Gαiβγ inhibitor PTX, and d) novel indole OXE-R antagonists recently disclosed in ref. [5] using DMR technology along with classical endpoint assays. Results show that 5-oxo-ETE-mediated DMR responses were entirely abolished in cells pre-treated with PTX, partly diminished in cells pre-treated with Gβγ inhibitor Gallein but essentially unaffected by OXE-R specific, Gβγ biased Gue1654. However, when intracellular inositolmonophosphate (IP1) accumulation was recorded to assess receptor activity both Gue1654 and Gallein completely blunted the receptor-mediated IP1 response. The resulting

disparity between these assay results leads us to posit the intriguing concept to target Gβγ-effector interaction in an effector- and receptor-specific way potentially offering unprecedented precision in the control of cellular signaling originating from cell surface GPCRs.

Acknowledgements: K.B. is a member of the DFG-funded Research Training Group RTG 1873

References: 1. Schröder, R. et al.: Nat. Protoc. 2011, 6(11): 1748-1760. 2. Nishimura, A. et al.: PNAS 2010, 107(31): 13666-13671. 3. Blättermann, S. et al.: Nat. Chem. Biol. 2012, 8(7): 631-638. 4. Lehmann, D.M. et al.: Mol. Pharmacol. 2008, 73(2): 410-418. 5. Gore, V. et al.: J. Med. Chem. 2014, 57(2): 364-377.

Comparison of Label-Free Methods with Conventional Assays for the Functional Characterization of GPCRs: The Human Histamine H1 Receptor as an Example Lieb, S.1; Bernhardt, G.1; Wegener, J.2; Buschauer, A.1

1 Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany 2 Institute of Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany

Optical- and impedance-based assays, as label-free techniques, exploiting a holistic readout, enable the non-invasive study of cellular responses, e.g., upon binding of a ligand to a transmembrane receptor. For instance, dynamic mass redistribution (DMR) utilizes the refraction index as readout, whereas electric cell-substrate impedance sensing (ECIS) records alterations of the impedance signal caused by changes in the cell morphology [1,2]. Signals are recorded in real time. In principle both holistic approaches are applicable to the study of G-protein coupled receptors (GPRs), and the construction of concentration-response curves is possible, regardless of the involved signalling cascade(s). With respect to deconvolution of these complex signals, we used the afore-mentioned label-free methods and compared the results with those obtained from established functional assays, namely mobilization of intracellular calcium, GTPase activity and luciferase gene reporter technology. Here we present the results of investigation on the human histamine H1 receptor (hH1R), a Gαq protein coupled GPCR, the stimulation of which results in phospholipase C (PLC) activation, an increase in inositol trisphosphate (IP3) and subsequent mobilization of intracellular Ca2+. The endogenous ligand, histamine, and the H1R agonist KUM530 (N-[2-(1H-imidazol-4-yl)ethyl]-2-[2-(3-bromophenyl)-1H-imidazol-4-yl]ethan-1-amine) [3] as well as the selective hH1R antagonists mepyramine, diphenhydramine and azelastine were studied on U-373 MG cells, which constitutively express the hH1R and on genetically engineered HEK293T cells, which stably express the hH1R and the firefly luciferase under the control of a cyclic AMP responsive element (CRE). Generally, both agonists showed a 10 to 100-fold higher sensitivity when studied with label-free methods compared to conventional assays (Fura-2/AM calcium, GTPase, luciferase assay). The pEC50 values determined with both label-free methods were in good agreement. The investigated antagonists revealed comparable pKB values in all functional assays. As method-dependent differences became obvious from the investigations of H1R agonists, it may be speculated that, in addition to the calcium signal, other cellular signalling mechanisms contribute to the holistic readout. The dissection of these pathways with appropriate molecular tools will be indispensable for deconvolution.

Acknowledgments: A PhD fellowship by the Hanns-Seidel-Stiftung to S. L. is gratefully acknowledged. The authors thank N. Kagermeier, Dr. A. Strasser and Dr. R. Robelek for fruitful co-operation, as well as Dr. H.-P. Steffens (Perkin-Elmer) for kindly providing the EnSpire multimode plate reader.

References: 1. Scott, C.W.; Peters, M.F.: Drug Discovery Today 2010, 15(17-18): 704-716. 2. Fang, Y., et al.: Comb. Chem. High Throughput Screening. 2008, 11(5): 357-369. 3. Strasser, A., et al.: Mol. Pharmacol. 2009, 75: 454-465.

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Adenosine A2A receptors heterodimerize with A2B receptors Hinz, S.1; Seibt, F.B.1; Schiedel, C.A.1; Franco, R.2; Müller, C.E.1 1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, 53121 Bonn, Germany 2 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain

It is now well accepted that G protein-coupled receptors are able to form dimers or oligomers in intact cells, which may consist of identical receptor proteins (“homomeric”) or of different receptors (“heteromeric”). Because of the unique pharmacological properties of these complexes, in particular in the case of heteromers, they represent novel targets for drug development. Different biophysical techniques such as resonance energy transfer (biolumi-nescence or fluorescence energy transfer, BRET or FRET) or fluorescence complementation techniques have been used to identify these complexes in living cells. It is of great interest to analyze their structure, including different possible interfaces that might be involved in receptor dimeriza-tion/oligomerization.[1] In the present study we examined potential heteromer formation between the adenosine A2A and A2B receptor subtypes, which play important (patho)physiological roles and are co-expressed in various cell types and tissues, e.g. in heart.[2] Radioligand receptor-binding assays at membranes of stably co-transfected CHO cells were performed to examine pharmacologi-cal properties of potential heteromers. Receptor function was investigated by measuring agonist-induced cAMP production of the Gs-coupled receptors. Furthermore, we used confocal laser scanning microscopy to show that the ECFP and EYFP tagged receptor subtypes were co-localized at the plasma membrane and in the ER/Golgi apparatus in stably co-transfected CHO cells. Moreover with FRET studies in transiently transfected CHO cells expressing both, A2A and A2B receptor subtypes, we were able to show that A2A/A2B receptor heteromers were formed. Further studies using a C-terminal deletion mutant of the A2A receptor showed that the C-terminus only plays a negligible role for interaction of the receptors within heterodi- or oligomers. We report here for the first time, that human A2A and A2BARs form heterodimers with altered receptor pharmacology. References: 1. Ferré, S. et al.: Pharmacol. Rev. 2014, 66(2): 413-434. 2. Zhan, E. et al.: Am. J. Physiol. Heart Circ. Physiol. 2011, 301(3): H1183-1189.

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Discovery of new protease inhibitors from nature Oli, S.1; Grün, J.1; Abdelmohsen, U.R.2; de Sousa, L.R.F.3, Wu, H.1, Hentschel, U.2, Efferth, T.1, Schirmeister, T.1 1 Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099 Mainz, Germany 2 Julius-von-Sachs-Institute for Biological Sciences, University of Würzburg, Julius-von-Sachs-Platz 3,Würzburg 97082, Germany

3 Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luís, Km 235, 13565-905 São Carlos, SP, Brazil

Proteases catalyze the breakdown of proteins via catalytic hydrolysis of peptide bonds [1]. Malfunction in the control of protease activity leads to undesired and unregulated proteolysis which causes many diseases. Therefore, inhibitors of proteases have the potential to provide successful therapeutics for a wide range of diseases [2,3]. The abundance of compounds with different chemical functional groups has renewed the interest in nature as a source for new leads for the next genera-tion of drugs. In this study, bioactive compounds with protease inhibiting properties were discovered from sponges, plants and sponge-associated bacteria following bioactivity-guided fractionation. For detection of protease inhibiting properties we used fluorometric enzyme assays and microscale thermophoresis. The bioactivity-guided fractionation of the crude extracts and the isolation, identification and structural elucidation of novel and biologically active secondary metabolites from marine sponge-associated bacteria, sponge biomass and plants yielded e.g. plakortide E [4] from the sponge Plakortis halichondroides and flavonoids from the Brazil plant Byrsonima coccolobifolia Kunth.[5] as new protease inhibitors.

References: 1. Turk, B . et al.: Nat. Rev. Drug Discov. 2000, 6 (5): 785–799. 2. Powers, J.C . et al.: Chem. Rev. 2002, 102: 4639–4750. 3. Rawlings, N.D . et al.: Biochemie 2008, 90: 243–259. 4. Oli, S. et al.: Mar. Drugs. 2014, 12: 2614-2622. 5. Lorena R.F.de Sousa . et al.: J. Nat. Prod. 2014, 77 (2): 392–396.

The war of invention: how the necessities of World War I pro-moted chemical drug development Helmstädter, A.1,2; Siebenand, S.2

1 Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main 2 Govi Verlag Pharmazeutischer Verlag GmbH, Eschborn, Germany

Political crisis and the outbreak of World War I a hundred years ago let to severe supply shortfalls in almost every economic area, including pharmacy. So Germany soon fell into short supply of natural products imported from abroad, including most alkaloid containing plant materials. So to give only one example, Merck’s atropine production stopped in October 1916, as Hyoscya-mus species usually imported from Africa were no longer available. As chemical synthesis had already made some progress in the early 20th century, it was obvious to look for synthetic routes based on domestic materials to substitute natural products. Several of these substitution products and procedures were abandoned after the war, but, as shown by Guy Hartcup [1], there are also examples where necessities of war katalysed developments also highly useful in times of peace. This study is intended to explore, if and how far this approach also holds true for pharmaceutical substances. So the well-known antipsoriatic agent Cignolin is a WW I development. It was synthesized to substitute Chrysarobin, a compound derived from the South American plant Andira araroba which was not available in times of war. Cignolin was developed by the German Dermatologist Eugen Galewski (1864-1935) and the Bayer company in 1916 and turned out to have several advantages over the mother compound. In the early 20th century, extracts from the North-American medicinal plant Lobelia inflata were frequently used as antiasthmatic agent and breathing stimulant. Shortage of plant material promoted research regarding isolation of the active ingredients and developing a synthetic procedure. Heinrich Wieland (1877-1957) and two of his PhD students investigated possibilities of a lobelin

total synthesis but succeeded not before 1927. However, a technically suitable synthesis procedure was not developed before 1937 when Boehringer started to market synthetic lobelin right before World War II. The product, which was still largely used as a medicine in the German army soon became the company’s blockbuster and was marketed until the year 1980, being recom-mended as an agent for smoking cessation in the last decades. One of the most important natural products unavailable in wartime was quinine and the need for quinine substitutes clearly stimulated the active search for synthetic antimalaria medicines which, however, did not succeed before the 1920s, when the Bayer company launched plasmoquine followed by chloro-quine in 1934. In his study on the history of synthetic antimalarials, David Greenwood stated: “During the war, the shortage of quinine impaired the efficiency of the German forces especially in the East Africa Campaign. It is doubtful if any of the synthetic antimalarials in use have been developed had not Germany been deprived of all sources of quinine during the First World War” [2].

References: 1. Hartcup, G.: The War of Invention. Scientific Developments, 1914-18, London 1988. 2. Greenwood, D.: J. Antimicrob. Chemother. 1995, 36: 857-872.

Tripterygium wilfordii: how a Taiwanese medicinal plant found its way to the West

Helmstädter, A.

Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main

Tripterygium wilfordii Hook. f. is a traditional medicinal plant originally found in Taiwan. It has been known in Europe since the late 1850s but has not been widely investigated in the West before the 1970’s. In the last few decades increasing efforts have been made to elucidate its value as an anti-inflammatory and immunosuppressive agent, in particular for the treatment of rheumatoid arthritis. The plant is said to be already mentioned in the Chinese herbal “Dian Nan Ben Cao” written in the 15th century by Mao Lan (1397-1476), and the ‘Compendium of Materia Medica” compiled by Li Shi-Zheu in 1578. The species later called Tripterygium wilfordii (German: “Wilfords Dreifügelfrucht”) has been collected in Taiwan in June 1858 and the name refers on the one hand to the the unusual three-winged shape of the fruits, on the other hand to the man who had harvested the plant. “Pterygium” is said to be the diminutive of “pteryx”, the Greek word for “wing”. The specific epithet honours Charles Wilford, a botanist working for Kew Gardens, London, from 1857 to 1859. He was sent as botanical collector on the “splendid steam yacht The Emperor” to India “for making botanical researches among the numerous islands of the Japanese territories” and embarked May 2nd, 1857 to Hong Kong [1]. The later “Tripterygium wilfordii” was collected in June 1858 and labelled as has been found “on banks of the river Sanar, Formosa”. Wilford did not care a lot about the instructions issued by his employer, according to which the medicinal use of indigenous plants should be carefully monitored and reported. This might be one of the reasons why the medicinal virtues of T. wilfordii remained largely unknown in Western science until the end of the 20th century. Early interest focused on the insecticidal use of T. wilfordii root powder in the 1930’s. Around 1940 as well, first reports about the chemical nature of active ingredients appeared in the literature (Schechter and Haller 1942), systematic investigations were undertaken in the early 1950’s. Most of the work has been done by Morton Beroza (1917-2011), who did his PhD thesis on the subject and eventually became a member of the Agricultural Research Service's Science Hall of Fame. Further interest was triggered by the cultural changes taking place in China after the World War II leading – among many other developments – to an integration of Western style medicine into Chinese healthcare. Western-trained and oriented physicians influenced medical practice even in rural areas and vice versa, became familiar with local traditions. As reports on beneficial, but also toxic effects of T. wilfordii were manifold, trials in a number of autoimmune and inflammatory diseases were organized and rapidly performed which led to a re-discovery of the plant’s medicinal virtues. In 1982, already experiences with more than 2000 patients with rheumatoid arthritis were reported [2]. Triptolide is now believed to be responsible for most of the biological activities of T. wilfordii extracts [3] and also serves as lead compound in drug development [4,5].

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References: 1. Bretschneider, E.V.: History of European Botanical Discoveries in China. St. Petersburg 1898, pp. 539-544. 2. Lipsky, E., Tao, X.L.: Sem. Arthr. Rheum. 1997, 26: 713-723. 3. Brinker, A.M. et al.: Phytochemistry 2007, 68: 732-766. 4. Zhou, Z.L. et al.: Nat. Prod. Rep. 2012, 29: 477-475. 5. Werz, O.: Pharm. Ztg. 2012, 157: 404-415.

Effectiveness of bulb extracts of Allium species on some selected plant pathogenic fungi Samadi, S.; Keusgen, M.

Institute of Pharmaceutical Chemistry, Philipps-Universität Marburg D-35037, Germany

The antimicrobial activity of Allium species has long been recognized, with allicin, other thiosulfinates, and their transformation products having antimicro-bial activity [1]. The sulfur chemistry of Allium species located in South West and Middle Asia is much more complex and diverse than the chemistry of those species which were traditionally used as vegetable, spice or medicine in the Western World. Allium volatile compounds of the species from Asia seem to be an excellent source for new sulphur compounds and aroma constituents. This variety of sulphur compounds should also lead extracts with unknown biological activities [2]. Fungal infections have increased over the past few decades. There has been an intensive search for new drugs more effective and less toxic than those already in use [3]. In vitro susceptibility testing of filamentous fungi is becoming increasingly important because of the frequency and diversity of infections caused by them [4]. Antifungal activities (MIC) of A. ampeloprasum L., A. atroviolaceum Boiss., A. cepa L., A. cepa L. var. aggregatum G. Don, A. darwasicum Regel, A. hollandicum R.M.Fritsch, A. jesdianum Boiss. & Buhse, A. jesdianum Boiss. & Buhse subsp. angustitepalum (Wendelbo) F.O.Khass. & R.M.Fritsch, A. karataviense Regel, A. macleanii Baker, A. moly L., A. nevskianum Vved., A. oschaninii B.Fedtsch., A. rosenorum R.M.Fritsch, A. rotundum L., A. sativum L., A. scorzonerifolium Redouté, A. stipitatum Regel, A. talassicum Regel and miconazole (positive control) on Aspergillus flavus, A. niger, Penicillium digitatum, P. italicum and Mucor hiemalis were investigated. Dilution series of ethyl acetate extracts obtained from Allium bulbs were tested on all the above mentioned fungi using PDA micro-dilution susceptibility testing method, disk diffusion method and double-dish chamber. Extracts of A. stipitatum showed the highest antimicrobial effect against all the tested fungi (MIC ≥ 0.53g/ml; related to the weight of the fresh bulbs) followed by A. sativum (MIC ≥ 0,54g/ml), A. ampeloprasum (MIC ≥ 0,85g/ml) and then A. karataviense (MIC ≥ 1,53g/ml). The MIC of miconazole as a control was ≥0.27mg/ml. From the fungal point of view, P. italicum showed the highest susceptibility, while M. hiemalis and A. flavus demonstrated more resistancy towards Allium extracts and miconazole. The results indicate that extractions of Allium spp. have antifungal activity and might be promising, at least, in ‘biological’ treatment of fungal-associated plant diseases. Raw extracts were investigated. Identification of active substances is ongoing.

Acknowledgments: Many thanks to Dr Mehrdad Abbasi, Head of department of Botany at Iranian Research Institute of Plant Protection, for providing us with fungal specimens, and to Prof. Reinhard M Fritsch, Das Leibniz-Institut für Pflanzengenetik und Kulturpflanzen-forschung (IPK), for the wild Allium samples. References: 1. Kyung, K.H.. Current Opinion in Biotechnology, 2012, 23(2): 142–147. 2. Keusgen, M. Volatile Compounds of the Genus Allium L. (Onions). In Volatile Sulfur Compounds in Food (pp. 183–214). American Chemical Society. 2011. 3. Pinto, E. et al.: Industrial Crops and Products. 2007, 26(2): 135–141. 4. Meletiadis, J.; Meis, J.F.G.M.; Mouton, J.W.: Journal of Clinical Microbiology. 2001, 39(2): 478–484.

Characterization of Haemanthus coccineus extract and its bioactive component: a novel anti-inflammatory approach Fuchs, S.1,2; Hsieh, L.T.4; Saarberg, W. 3; Schaefer, L.4; Erdelmeier, C.A.J.3; Koch, E. 3; Fürst, R.1

1 Institute of Pharmaceutical Biology, Goethe-University Frankfurt, Biocenter, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany 2 Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 3 Preclinical Research, Dr. Willmar Schwabe Pharmaceuticals, Willmar-Schwabe-Str. 4, 76227 Karlsruhe, Germany 4 Institute of Pharmacology and Toxicology, Division of Nephropharmacology, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany

Haemanthus coccineus (Amaryllidaceae) extracts (HCEs) have been used in traditional African medicine against febrile colds and asthma. The main ingredient of HCE, the non-basic alkaloid narciclasine, was recently reported to induce apoptosis in tumor cell lines [1]. Beyond this anti-cancer action, we hypothesized that HCE and narciclasine could exhibit an anti-inflammatory potential. Dried bulbs of H. coccineus were extracted using 60 % (w/w) ethanol. The ethanol was largely removed and the remaining solution was partitioned with ethyl acetate. The organic phase was separated and dried (DER 50:1). The resulting HCE contained 2.2 % narciclasine. In vitro, HCE (3 ng/ml to 10 μg/ml) concentration-dependently inhibited the proliferation of lymphocytes and the synthesis of pro-inflammatory cytokines (TNF-α, IL-6, IL-1 β) in murine macrophages without inducing cytotoxicity. Moreover, HCE decreased the TNF-α-induced adhesion of leukocytes to human endothelial cells (ECs) and the surface expression of EC adhesion molecules (ICAM-1, VCAM-1, E-selectin) without affecting EC viability. Extract fractions containing basic alkaloids did not display any activity, whereas the non-basic main alkaloid narciclasine (1 nM to 10 µM) clearly reduced adhesion molecule expression. HCE as well as narciclasine attenuated the TNF-α-triggered expression of NF-κB-dependent genes (reporter gene assay) without influencing NF-κB DNA-binding activity (gel shift assay), IκB-α degradation (Western blot), or p65 nuclear translocation (microscopy). In vivo, HCE (450 mg/kg, orally) was found to clearly reduce edema formation and leukocyte infiltration in a dermal ear edema model by croton oil or arachidonic acid (AA). Also in a renal injury model we could demonstrate that HCE (50 µg/animal, i.p.) strongly attenuates leukocyte infiltration and cytokine expression. In conclusion, our study highlights that the use of HCE/narciclasine could represent a novel interesting anti-inflammatory approach.

Acknowledgments: HCE and narciclasine were kindly provided by Dr. Willmar Schwabe GmbH & Co. KG. This work was supported by the German Research Foundation (DFG, SFB 815, project A5, SFB 1039, project B2) to L.S. and by GRK1172 (cooperation contract with Merck Serono) to L.T.H. References: 1. Ingrassia, L. et al.: J. Transl Oncol. 2008, 1(1): 1-13.

Antischistosomal activity of derivatives of caffeic acid bornyl ester isolated from Valeriana wallichii rhizomes Glaser, J.1*; Schurigt, U.2*; Suzuki, B.3, Caffrey, C.R.3#; Holzgrabe, U.1# 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 2 Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2/D15, 97080 Würzburg, Germany 3 Center for Discovery and Innovation in Parasitic Diseases, Department of Pathology, University of California, San Francisco, California, United States of America *shared first authorship #shared senior authorship

Schistosomiasis is a parasitic disease infecting over 200 million people that continues to pose a public health hazard in developing countries. Schistosoma blood fluke parasites are prevalent in Africa, Asia and South America, and utilize amphibious or aquatic snails as vector hosts. Infection of humans occurs during contact with contaminated water bodies. Free-swimming larvae (cercariae) penetrate the skin to eventually reach the bloodstream where they develop into adult worms [1]. The parasites can infest the human body for decades and their eggs induce various organ pathologies leading to a range of chronic and morbid physical and psychological consequences.

Treatment and control of schistosomiasis relies on just one drug, praziquantel [2-3], and the danger of resistance by the parasite to the drug cannot be ignored [4-5]. Research needs to concentrate on finding new and better drugs to fight this disease.

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We recently discovered caffeic acid bornyl ester with antileishmanial activity from the rhizomes of Valeriana wallichii and subsequently synthesized a small compound library of systematically varied derivatives [6]. The library was screened for schistosomizidal activity against Schistosoma mansoni post-infective larvae (schistosomula) at a 5 µM concentration. Especially eugenyl cinnamate (1) and thymyl cinnamate (2) showed good activity with compound 1 leading to the death of the schistosomula after 72 hours. Additionally both compounds induced the formation of vacuoles, an interesting new phenotype.

Thanks are due to the SFB 630 for financial support.

References: 1. Gryseels, B. et al.: Lancet 2006, 368: 1106-1118. 2. WHO: Schistosomasis Fact sheet N°115. 3. WHO: Weekly epidemiological record 2014, 21-28. 4. Doenhoff, M.J., Pica-Mattoccia L.: Expert Rev. Anti Infect. Ther. 2006, 4(2): 199-210. 5. Doenhoff, M.J., Cioli D., Utzinger J.: Curr. Opin. Infect. Dis. 2008, 21: 659-667. 6. Glaser, J. et al.: Molecules 2014, 19: 1394-1410.

Humulus lupulus derived bitter acids - potential antidepressant and anxiolytic effects mediated by TRPC6 channel activation Freigang, M.1,2; Pischetsrieder, M.2; Friedland-Leuner, K.1

1 Department of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany 2 Department of Chemistry and Pharmacy, Emil-Fischer-Center, Henriette Schmidt-Burkhardt Chair of Food Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

Humulus lupulus extracts are used in folk medicine to treat anxiety, depression and sleeping disorders. In animal models, sedative, anxiolytic and antidepres-sant-like effects could be demonstrated. However, the molecular mechanism of action and the active constituents of the extract are still discussed. Interesting-ly, the hop bitter acid lupulone and its analogs have strong structural resem-blances with hyperforin, the antidepressant active constituent of St. John’s wort extract. Both show acyl- and prenyl-modifications on a phloroglucinol core structure. Results from in vivo tests on rats suggest an anti-depressant effect of this class of hop bitter acids. Hop β-acid fractions (5mg/kg) reduced immobility time in the forced swimming test without achieving a sedative effect. The purpose of this study was to investigate if hop ß-acids might also activate the molecular target of hyperforin, the classical transient potential channels TRPC6. TRPC6 channel activation by hyperforin leads to an influx of sodium and calcium ions. This shift of the electrochemical gradient indirectly inhibits monoamine transporters and directly improves synaptic plasticity, two important mechanisms involved in antidepressant activity. First, we investigat-ed if hop ß-acids lead to a non-selective cation influx in PC12 cells. Hop β-acids showed a hyperforin-like activity profile in PC12 cells. Based on our findings we next wanted to elucidate if hop ß-acids also activate TRPC6 channels. To address this question, several pharmacological tools known to interfere with non-selective cation channels were used such as SK&F 96365, 2-APB, flufenamic acid, lanthanum, gadolinium and ACA as well as ruthenium red, which blocks TRPV, TRPM6, TRPM8, and TRPA1 channels. All inhibitors except ruthenium red strongly impaired hop ß-acid induced calcium influx in PC12 cells. These results suggest that hop ß-acids might also activate TRPC6 channels and thereby mediate their antidepressant effects. However, further experiments are needed to further support this hypothesis.

Targeting virulence: Inhibition of a streptococcal key pathogenic mechanism by bacterial natural compounds Rox, K.1,2,3; Gerth, K.4; Rohde, M.1,3; Chhatwal, G.S.3; Müller, R.2 1 Central facility for microscopy, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany 2 Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Saarland University Campus, Building C2.3, 66123 Saarbrücken, Germany 3 Department of Medical Microbiology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany 4 Department of Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany

Every year millions of people all over the world are suffering from streptococcal diseases. Whereas common throat infections can be treated and cured quite effectively with penicillin, sepsis or necrotizing fasciitis can become life-threatening.[1] Moreover, post-infection sequelae represent a serious health hazard especially in developing countries.[2] Antibiotic resistance is emerging – therefore, it is inevitable to find new antibiotics targeting key pathogenic mechanisms.[3],[4] Such a mechanism is invasion of eukaryotic cells: after invasion of streptococci dissemination is possible as well as persistence which can cause recurrent infections.[5] Consequently, several compounds and extracts derived from myxobacteria were tested with the aim to block the invasion process. Two different Group A streptococcal strains representing two important invasion mechanisms were used for cell infection assays.[6] In every assay compounds or extracts were added to analyse possible inhibitory effects. To estimate the range of inhibition of invasion, double-immunofluorescence staining and examination via electron microscopy after an infection experiment were used. By using a bacterial invasion assay intracellular surviving bacteria were determined. Moreover, cytotoxicity was assessed by using an MTT assay under infection conditions. Two compounds inhibit invasion of Group A streptococci into two different types of epithelial cells. As two different invasion mechanisms are addressed in the infection assays, it can be shown that these different pathways can be inhibited quite effectively. Additionally, the compounds do not show cytotoxic effects under infection conditions. Inhibition of invasion will be the first step to encounter persistence and dissemination: if streptococci are disabled to invade cells, the reservoir required to cause recurrent infections is reduced. Moreover, dissemination will be hampered as streptococci will only be able to adhere to cells. Additionally, killing of streptococci using antibiotics such as penicillin will be facilitated as the bacteria cannot hide in an intracellular compartment anymore.

Acknowledgments: Katharina Rox acknowledges the President’s Initiative and Networking Fund of the Helmholtz Association of German Research Centers (HGF) (contract number VH-GS-202) for supporting her research project. References: 1. Lamagni, T.L. et al.: J. Clin. Microbiol. 2008, 46(7): 2359-2367. 2. Carapetis, J.R. et al.: Lancet. Infect. Dis. 2005, 5(11): 685-694. 3. Cantón, R. et al.: J. Antimicrob. Chemother. 2002, 50(S1): 9-24. 4. Capoor, M.R. et al.: Jpn. J. Infect. Dis. 2006, 59(5):334-336. 5. Rohde, M., Chhatwal, G.S.: Curr. Top. Microbiol. Immunol. 2013, 368:83-110. 6. Nitsche-Schmitz, D.P., Rohde, M., Chhatwal, G.S.: Thromb. Haemost. 2007, 98(3):488-496.

Rice Bran Extract administration improves age-related brain mitochondrial dysfunction in NMRI mice Hagl, S.1; Berressem, D.1; Grebenstein, N.2; Frank, J.2; Eckert, G.P.1

1 Department of Pharmacology, Biozentrum Niederursel, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany 2 Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstr. 28, 70599 Stuttgart, Germany

In the last few decades, life expectancy was rising constantly due to improve-ments in health care and technology which lead to increased incidence of age-related diseases including Alzheimer’s disease. Research revealed that

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mitochondria are significantly involved in aging processes that ultimately lead to neurodegeneration. A healthy lifestyle including a diet rich in antioxidants and polyphenols represents one strategy to protect the brain and to prevent neurodegeneration. Key components of Rice Bran Extract (RBE) are tocopherols, tocotrienols and γ-oryzanol. RBE has been reported to have anti-inflammatory, antioxidant, cholesterol-lowering and anti-diabetic activities. Since we could recently show that RBE feeding increased brain mitochondrial function in young guinea pigs [1] we now tested the effect of RBE administration on brain mitochondrial function in aged mice. Aged (18 months old) NMRI mice were fed with RBE (340mg RBE/kg body weight) via oral gavage once a day for 3 weeks. 3 and 18 months old NMRI mice fed with vehicle served as control groups. We assessed mitochondrial function by measuring mitochondrial respiration in isolated brain mitochondria and mitochondrial membrane potential (MMP) and ATP levels in dissociated brain cells (DBC). Additionally, we determined levels of mitochondrial marker proteins using Western Blot analysis and examined blood plasma and brain tissue concentrations of key components of RBE. ATP levels, complex I respiration, the respiratory control ratio and protein levels of PGC1α were significantly decreased in aged NMRI mice. RBE administration was able to entirely compensate this age-related mitochondrial dysfunction by elevating PGC1α protein levels and citrate synthase activity and by improving the resistance of DBC against sodium nitroprusside induced nitrosative stress. According to these results, RBE is a very promising candidate neutraceutical in the prevention of age-related neurodegenerative diseases like Alzheimer’s disease. Reference: 1. Hagl, S. et al.: Pharmacol. Res. 2013, 76: 17-27.

Effect of polyphenol-rich grape extract on mitochondrial dys-function in PC12-cells and memory and motor performance in C57BL/6 mice Asseburg, H.1; Plank, C.1; Borchiellini, M.2; Mueller, M.3; Pohland, M.1; Berressem, D.1; Eckert, G.P.1. 1 Department of Pharmacology, Biozentrum Niederursel, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Department of Pharmaceutical Science, University of Perugia,Via del Liceo 1, 06123 Perugia, Italy 3 Provadis School of International Management and Technology, Industriepark Höchst, 65926 Frankfurt am Main, Germany

The increasing life expectancy is accompanied by several age-related neurological changes which may lead to neurodegenerative diseases. One key factor of aging and neurodegenerative diseases is mitochondrial dysfunction (MD) which includes decreased mitochondrial membrane potential (MMP) and ATP levels [1,2]. Polyphenol-rich fruit like grapes and berries have been shown to have neuroprotective potential in rodents and human pilot studies [3,4], however, information on the effects on MD is scarce. Therefore, this study aims to investigate the effects of polyphenol-rich grape extract (PGE) on mitochondrial function and behavioral performance in aging. PC12 cells were pre-incubated with PGE (50µg/ml) for 1 h and subsequently incubated with 0.5mM sodium nitroprusside (SNP) leading to MD. Parameters of mitochondrial function in vitro and ex vivo included ATP levels (luciferase-catalysed bioluminescence), MMP (rhodamine 123 fluorescence) and activity of mitochondrial respiratory complexes (Oxygraph-2k). SNP-treatment of PC12 cells decreased ATP levels and MMP to 40.±2.4% and 83.4±1.3% of untreated controls, respectively. PGE pre-incubation reduced the SNP-induced MD resulting in ATP levels of 54.6±1.3% and MMP of 90.0±2.0% of untreated controls. In vivo effects of PGE were assessed in C57BL/6 mice. For short-term treatment in aged mice (19-22 months), PGE (200mg/kg b.w.) was administered via oral gavage over a period of 3 weeks. Mitochondrial function was measured in isolated brain mitochondria and dissociated brain cells. Short-term PGE treatment did not have an effect on mitochondrial function. The reduced ATP levels and impaired activity of the mitochondrial respiratory chain in aged mice compared to young mice were not ameliorated by PGE treatment. However, as mitochondrial function was assessed after short –term treatment in aged mice, a longer treatment period starting at middle age might be more effective to protect mitochondria und therefore prevent or delay the age-related decline in brain function. For long-term treatment of mice, PGE dissolved in water (2g/L, eq. to 200mg/kg b.w.) served as sole source of liquid from the age of 6 months. Motor performance (Rotarod) and short-term

memory (Social Recognition Test) were assessed in a 3-month interval. PGE treatment for 6 months but not for 9 months led to an improvement in motor performance and short-term memory. The long-term effect of PGE on MD needs further investigation.

References: 1. Schaffer, S. et al.: Mol. Neurobiol. 2012, 46(1): 161-178. 2. Asseburg, H.; Hagl, S.; Eckert, G.P.: Pharma Nutrition - An Overview (Springer) 2014, in press. 3. Cherniack, E.P.: Br. J. Nutr. 2012, 108(5): 794–800. 4. Joseph, J.A.; Shukitt-Hale, B.; Willis, L. M.: J. Nutr. 2009, 139(9): 1813S-1817S.

Nematicidal activity of some Allium spp extracts against root-knot nematode Meloidogyne incognita Jivishova, S.; Jivishov, E.; Keusgen, M.

Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps University of Marburg, Marbacher Weg 6, 35037, Marburg, Germany

To investigate nematicidal activity against root-knot nematode Meloidogyne incognita, 17 ethyl acetate extracts of Allium species were tested. 8 Concentra-tions of bulb and flower extracts were prepared and nematicidal activity was determined after 48 hours of exposure. Responses varied with test material and concentration. Good nematicidal activity against infective second-stage juveniles (J2) was achieved with A. sativum, A. karataviense, A. zebdanense, A. aflatunense and A. stipitatum bulb extracts. The highest nematicidal activity was shown by A. sativum and A. karataviense at LD50 concentrations of 0.07425 mg ml-1 and 0.1891 mg ml-1, respectively (values related to amount of extract). Lannate 20 L was used as a positive control with LD50 of 0.1249 mg ml-1. Other bulb extracts of Allium spp showed weak nematicidal activity, while nematicidal activity of flower extracts was not significant.

Acknowledgments: Institut für Pflanzenschutz in Freising Mr. Andreas Hermann, Julius Kühn-Institut Dr. Johannes Hallmann, Institute of Pharmaceutical Chemistry, Philipps University of Marburg, Mr. Floris van Elsäcker, DuPont de Nemours (Deutschland) GmbH, Dr. Martin Lechner.

References: 1. Nguyen, D.-M.-C. et al.: Nematology 2009, 11(6): 835-845. 2. Park, I.-K. et al.: Nematology, 2005, 7(5): 767-774. 3. Danquah, W.B. et al.: Nematology, 2011, 13(7): 869-885. 4. Choi, I.-H. et al.: Nematology, 2007, 9(2): 231-235. 5. Amaral, D.F. et al.: Nematology, 2003, 5(6): 859-864.

Qualitative and quantitative analysis of cysteine sulfoxides in flowers of some Allium species Jivishov, E.; Neumann, S.; Keusgen, M.

Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, D-35037, Marburg, Germany

Since ancient times, onions, garlic and some other species of the genus Allium L. (onions) have been used as phyto-pharmaceutics, seasonings, and vegetables. Most prominent are common onion (A. cepa L.) and garlic (A. sativum L.). The medicinal benefits of these two species were intensely investigated during the last decades and lipid lowering, antibiotic, anti-atherosclerotic and anti-diabetic effects were described. Also, a canceroprotec-tive effect was proven by a number of ethnic studies. The health benefits of Allium vegetables are mainly related to sulphur containing compounds as well as saponins. The species-rich genus Allium has a main centre of distribution reaching from Southwest Asia to the high mountains of Central Asia. In this area, several wild species are used by the local population, as one can be concluded from casual remarks in some floras. The so-called cysteine sulphoxides of these plants are believed to be mainly responsible for these health benefits. These compounds are converted to thiosulphinates like allicin, when plant material is disrupted. This reaction is catalysed by the action of the enzyme alliinase.

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Flowers of 22 Allium species, most of them for the first time, were analyzed for their cysteine sulfoxide(CSO) content. While all of the flowers possessed methiin, only a few of flowers carried any of the following CSOs: propiin, isoalliin, alliin, marasmin, S-(2-pyrrolyl)-cysteine sulfoxide, S-(2-pyridyl)-cysteine N-oxide and butiin (Figure). Total CSO content of analyzed flowers varied from 0.01% to 0.5% relatively to fresh material weight. A few com-

pounds, which possibly can be γ-glutamyl derivatives of some aminoacids

were observed in mass chromatograms. No homoisoalliin, penthiin, ethiin or

known γ-glutamyl derivatives of CSOs were detected in any of the flowers.

Figure: Most important cysteine sulphoxides as well as an N-oxide of the genus Allium

Acknowledgements: Institute of Pharmaceutical Chemistry, University of Marburg, van Elsäcker F, Brauschke M, Gercke N, Pipp K References: 1. Kusterer, J., Keusgen, M.: J. Agric. Food Chem. 2010, 58: 1129–1137. 2. Kusterer, J. et al.: J. Agric. Food Chem. 2011, 59: 8289–8297. 3. Kubec, R. et al.: J. Agric. Food Chem. 2011, 59: 5763–5770.

Stereochemistry of the cysteine sulphoxide marasmin in the South African plant Tulbaghia violacea Harv. Neumann, S.; Keusgen, M.

Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6-10, D-35032 Marburg, Germany

Cysteine sulphoxides (CSO) are well known sulphur-containing aroma precursors of plants belonging to the family of Alliaceae. When a cell is disrupted, cysteine sulphoxides deposited in the cytoplasm are cleaved by an alliinase stored in the cell vacuole. This enzymatic reaction results in the formation of volatile sulphenic acids, which condensate spontaneously to thiosulphinates. These substances do have a strong alliaceous smell and interesting bioactivities like antimicrobial effects [1]. The thiosulphinate derived from the CSO marasmin was named marasmicin and has been shown to be the main flavour compound of some fungi and plants mentioned below. Biological activity against fungi and tuberculosis has been recently demonstrated [2]. The first record of the CSO marasmin was for some basidiomycota of the genus Marasmius [3]. These fungi contain the γ-glutamylised form of marasmin with (S) being the absolute configuration at the sulphur atom [(SS,RC)-marasmin; Figure]. The first detection in a plant was 11 years later in the Malaysian tree Scorodocarpus borneensis Becc.. Its fruits contain the free sulphoxide with converse orientation at the sulphur atom ((RS,RC)-marasmin) [4]. From then on marasmin was found in numerous genera of the Alliaceae especially Tulbaghia, Allium, Ipheion and Leucocoryne [5,2,6]. The investigation of the CSO content of T. violacea, known as ‘sweet garlic’ or ‘society garlic’, showed something very unusual. All parts of the investigated plant did not only contain (RS,RC)-marasmin), even the (SS,RC)-marasmin) could be detected in relevant amounts. These findings are rather unusual, because the appearance of both stereoisomers of the same CSO in a plant has been rarely reported so far and previous research did not reveal the (SS,RC)-marasmin in T. violacea nor in any other plant.

Acknowledgements: Philipps-Universität Marburg, Floris van Elsäcker References: 1. Kusterer, J.: Neue Erkenntnisse der Schwefelchemie und Chemotaxonomie in Arten des Genus Allium. (Universitätsbibliothek Marburg) 2010 2. Kusterer, J.; Fritsch, R.M.; Keusgen M.: J. Agric. Food Chem. 2011, 59(15):8289–8297. 3. Gmelin, R. et al.: Phytochem. 1976, 15(11):1717–1721. 4. Kubota, K.; Hirayama H.; Sato Y.: Phytochem. 1998, 49(1):99–102. 5. Kubec, R.; Velísek, J.; Musah, R.A.: Phytochem. 2002, 60(1):21–25. 6. Kubec, R. et al.: J. Agric. Food Chem. 2013, 61(6):1335–1342.

Biological active compounds from Streptomyces living in symbiosis with Arnica montana Wardecki, T.1; Brötz, E.2; Ebeling, S.1; von Löwenich, F.3; Häcker, G.3; Luzhetskyy, A.2; Merfort, I.1 1 Institute of Pharmaceutical Science, Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, 79104 Freiburg, Germany 2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C 2.3, 66123 Saarbrücken, Germany 3 Institute of Microbiology and Hygiene, University Hospital Freiburg, 79104 Freiburg, Germany

Arnica montana L. is a well-known traditional medicinal plant. In modern therapy extracts of Arnica montana flowerheads are used externally to treat several injuries as bruises, distortions, sprains, hematoma and inflamed insect stings. Moreover, the extracts are used as supportive treatment of rheumatic diseases. As active compounds, the sesquiterpene lactones helenalin, 11α,13-dihydrohelenalin and their esters have been identified [1]. Beside their pronounced anti-inflammatory effects these sesquiterpene lactones also showed antibacterial activity against gram-positive bacteria [2]. Streptomyces are gram-positive bacteria that are frequently found in soil and also in plant roots. They have come into focus due to their ability to produce important biological active compounds, such as antibiotics and antitumorals [3]. Endophytic Streptomyces have already been discovered in other members of the Asteraceae family, for example in the medicinal plant Artemisia annua L. [4]. Therefore, it was attractive to search for Streptomyces in Arnica montana as well. We observed that the roots of these plants indeed were inhabited by Streptomyces. Intriguingly these organisms are able to grow in such a hostile environment, where antibacterial sesquiterpene lactones are present. We cultivated these bacteria and analyzed their extracts. Several secondary metabolites were identified. The extracts were shown to exhibit cytotoxic activity and antibacterial properties against gram-negative bacteria.

References: 1. Merfort, I.: Z. Phytother. 2010, 31: 188-192. 2. Lee, K.-H. et al.: Phytochemistry 1977, 16(8): 1177-1181. 3. Procópio, R.E. et al.: Braz J Infect Dis. 2012, 16(5): 466-471. 4. Lin, L. et al.: Planta 2012, 236(6): 1849-1861.

Inhibition of SDF-1-induced tumor cell activation and migration by purified fucoidan fractions Ehrig, K.; Schneider, T.; Alban, S.

Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany

Fucoidans from brown algae exhibit numerous biological effects relevant for anti-tumor activity. Currently, the SDF-1(CXCL12)/CXCR4 axis is considered a promising target for tumor therapy, since recent results showed that this chemokine/receptor axis plays a central role in tumor progression, angiogene-sis, metastasis and therapy resistance [1]. The aim of this study was to characterize the fucoidan from Saccharina latissima (S.l.) and to investigate whether it influences the SDF-1/CXCR4 axis. Extraction of S.l. from Faroe Islands provided > 5% alginic acid-free sulfated glycans, which were purified and separated into two distinct fractions by anion exchange chromatography. The more active one was identified as a sulfated branched galactofucan (SGF) with a degree of sulfation of 0.8.

S

O NH2

COOHS

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NO

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COOH

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COOH

Methiin Alliin Isoalliin Propiin

Butiin

Homoisoalliin Marasmin

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SGF exhibited no cytotoxic activity, but showed a variety of effects relevant for anti-metastatic and anti-angiogenic activity such as inhibition of heparanase, elastase and chemotaxis of tumor cells in migration and scratch assays. Investigations by flow cytometry, Western blotting, ELISA and fluorescence microscopy revealed that SGF interferes with the SDF-1/CXCR4 axis in Raji cells (Burkitt lymphoma) by inhibiting the SDF-1-induced CXCR4 activation. Compared to fucoidan from Sigma and heparin, it was much more active. In contrast to the CXCR4 receptor antagonist AMD3100, SGF antagonizes the chemokine. In gene expression analysis, SGF additionally reduced the expression of SDF-1 and several further genes important for tumor progression and metastasis. In conclusion, SGF from S.l. is obtained in reproducible quality by an optimized isolation method. The pharmacological profile of SGF indicates potent anti-tumor effects. Based on our results, SGF should now be examined in vivo. Acknowledgement: This study is part of the project “Algae against cancer”, financially supported by the Federal Ministry of Research and Education (BMBF). Reference: 1. Cojoc, M. et al.: OncoTargets and Therapy 2013, 6: 1347–1361.

Triterpenes from birch bark and their influence on cutaneous cells derived from healthy and diabetic patients Werner, P.1,4; Wardecki, T.1,4; Thomas, M.2; Zanger, U.M.2; Brandner, J.3; Merfort, I.1

1 Institute of Pharmaceutical Science, Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, 79104 Freiburg, Germany 2 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, 70376 Stuttgart, Germany 3 Clinic for Dermatology and Venereology, University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany 4 These authors contributed equally to this work

One of the major health problems in industrial countries is diabetes mellitus [1]. The prevalence in Germany has been estimated at 7.2 % and is predicted to increase further. This high number of patients suffering from diabetes mellitus leads to enormous costs in public health system [2]. One problem which many diabetic patients have to deal with is impaired wound healing. Therefore, efficient remedies are urgently needed. Besides conventional drugs phytomed-

icines may be an interesting alternative. An extract from birch bark (Betula

pendula Roth), named as TE1 and enriched in triterpenes with betulin as the main constituent, has been clinically proven to benefit wound healing [3]. Previously we have shown that TE1 and betulin transiently upregulate pro-inflammatory mediators, such as cytokines and COX-2 in keratinocytes and promote keratinocyte migration by altering the actin cytoskeleton [4]. In continuation of our studies we here focus on the effects of TE1 and betulin on human dermal fibroblasts from healthy and diabetic donors. TE1 and betulin treatment led to increased levels of IL-6, IL-8, Rantes, and TNF-α mRNA expressions which were higher compared to those ones in keratinocytes. Additionally, some factors were only upregulated in fibroblasts, e.g. CCL2. Presumably fibroblasts are more susceptible to the used stimuli than keratino-cytes, although it has to be considered that a high variability was observed between the different donors. Expression of these genes were similar in fibroblasts from healthy and diabetic patients. Moreover, we concentrated on cytoskeletal changes of migrating fibroblasts from healthy patients. Treatment with TE1, betulin, and also with the minor constituents of TE1, lupeol and betulinic acid, promoted the reorganization of actin cytoskeleton in fibroblasts, a prerequisite for migration into the wound site and creation of granulation tissue [5].

Acknowledgement: Funding by Aif and providing of TE1 and the triterpenes by Birken AG

is gratefully acknowledged. References: 1. Rothenbacher, D. Diabetes aktuell 2011, 9(8):340–343. 2. Heidemann, C. et al.: Bundesgesundheitsbl 2013, 56:668–677. 3. Metelmann, H. et al.: J Craniomaxillofac Surg. 2012, 40(5):e150-4. 4. Ebeling, S. et al.: PLoS One 2014, 9(1):e86147. 5. Gurtner, G.C. et al.: Nature 2008, 453(7193):314-321.

Expeditious Syntheses of Rutaecarpine and Analogs and Preliminary Evaluation for Cytotoxic Activity Huang, G.1; Roos, D.1; Heilmann, J.2; Decker, M.1 1 Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany 2 Institut für Pharmazie, Universität Regensburg, Universitätstrasse 31, D-93053 Regensburg, Germany

Rutaecarpine (1) is a natural quinazolino carboline-type alkaloid first isolated from fruits of Evodia rutaecarpa (Juss.) Hook. f. et Thoms (Rutaceae) which are used in Traditional Chinese Medicine as herbal remedy [1]. Subsequently, 1 and derivatives bearing hydroxy and methoxy groups at ring A or E were isolated also from other plant genera. Accumulating research has proven useful biological properties of 1 and its natural and synthetic analogs, such as anti-platelet aggregation, vasorelaxing properties, and cyclooxygenase-2 inhibition [2], often exhibiting in-vitro activity in the lower micromolar range [3]. Rutaecarpine and derivatives were described as templates for anti-cancer drugs [4, 5]. Its multiple biological activities intrigued many efforts on the syntheses of 1 and its analogs [6 and references therein]. During our search for new inhibitors of cholinesterases, we have developed a novel and expeditious procedure for preparation of rutaecarpine and its analogs. Two starting materials, isatoic anhydride and 3,4-dihydroisoquinoline or 4,9-dihydro-3H-pyrido[3,4-b]indole, respectively, are mixed and heated overnight. Afterwards, after simple precipitation good to excellent yields of the heterocycles are obtained (69 - 96%). This novel, short and versatile method gives high yields while devoid of tedious purification procedures [7]. Unlike previously described procedures, neither multi-step syntheses nor special reagents are necessary, and no special starting materials have to be prepared prior to the synthesis [6, 7]. Applying this reaction, 20 novel heterocyclic compounds were obtained. In a preliminary in vitro study, selected compounds were evaluated for cytotoxicity on a HeLa cell line. Several derivatives exhibit pronounced potency compared to rutaecarpine.

References: 1. Wang, L.; Argade, N.P.: Planta Med. 2005, 71(5): 416–419. 2. Mhaske, S.B. et al.: Tetrahedron 2006, 62(42): 9787–9826. 3. Baruah, B. et al.: Bioorg. Med. Chem. 2004, 12(9): 1991–1994. 4. Yang, L.-M.: Bioorg. Med. Chem. Lett. 1995, 5(5): 465-468. 5. Dong, G. et al.: J. Med. Chem. 2012, 55(17): 7593-7613. 6. Richers, M.T. et al.: Synthesis 2013; 45(13): 1730-1748. 7. Huang, G. et al.: Tetrahedron Lett. 2014, 55(26): 3607-3609.

Horse chestnut fruit extract: Criteria for cell established use in chronic venous insufficiency are met Kraft, K.1; Kelber, O.2; Müller, J.2

1 Chair of Natural Medicine, University of Rostock, Rostock, Germany; 2 Scientific Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany

Purpose: In the therapy of chronic venous insufficiency (CVI) compression treatment is the most popular option, despite it often causes discomfort and has been associated with poor compliance. Oral drug treatment with standard-ized extracts from horse chestnut seed (HCSE) is therefore an attractive treatment option. For an overview a systematic evaluation of the evidence

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(available clinical studies, Cochrane reviews, monographs, medical guidelines in this indication) concerning standardization, efficacy and safety was conducted. Results: Standardized HCSE leads to a clear improvement in CVI related signs and symptoms compared with placebo. This is reflected in all reviews and monographs as well as in clinical guidelines. Adverse events were usually mild and infrequent. Study durations of 12 weeks were appropriate and sufficient to demonstrate clinical efficacy in comparison to compression treatment. Adher-ence was 90%, compared to 47% with compression treatment. Conclusion: HCSE is an efficacious and safe treatment for CVI, as studies of up to 12 weeks duration have shown. Long-term compliance is key factor in CVI treatment and is higher for treatment with HCSE than for compression therapy. As long-term tolerability is good according to pharmacovigilance data, HCSE can be recommended for long-term application in CVI. The therapeutic usefulness of HCSE is therefore well established also in-long term treatment.

Isothiocyanate Sulforaphane inhibits expression of the central iron regulator hepcidin in an inflammatory cell model Martin, J.; Fischer, B.; Steinhilber, D.; Stein, J.; Ulrich-Rückert, S.

Institute of Pharmaceutical Chemistry Goethe-University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt a.M., Germany

Hepcidin, a regulatory peptide of the iron metabolism, is regarded as the central mediator of anaemia of chronic inflammation (ACI), a common complication in chronic inflammatory disease [1]. Via the activation of STAT3 signal transduction, inflammatory stimuli invoke an increase in hepcidin expression, leading to the impairment of intestinal iron absorption [2]. The anti-inflammatory properties of the isothiocyanate sulforaphane (SFN), primarily found in cruciferous vegetables, have been confirmed in numerous in vitro and in vivo models [3, 4] . The aim of this experiment was therefore to achieve hepcidin regulation using SFN. Caco-2, Huh7 and HepG2 cells were cultivated in standard conditions and incubated for 16 hours with SFN [10µM]. An inflammatory situation was imitated by stimulation of the cells with Oncostatin M [10ng/ml] (OncM). Quantitative real-time PCR was performed to determine mRNA expression. For reporter gene assays, calcium chloride was used to transfect plasmid and luciferase activity was measured luminometrically. Proteins were detected by Western blot analysis. SFN significantly inhibits not only OncM-induced hepcidin promoter activation (***p<0.001), but also hepcidin mRNA expression (HepG2 *p<0.05; Huh7 **p<0.01). Furthermore, in the co-culture model, an increase of intracellular ferritin levels in intestinal cells was achieved (**p<0.01). Whereas OncM-induced effects are mediated via activation of the transcription factor STAT3, which was confirmed using a specific STAT3 inhibitor [50µM] (***p<0.001), the effects of SFN are apparently not induced by inhibition of this signal transduc-tion pathway, since it was not possible to achieve a reduction of OncM-induced STAT3-phosphorylation (**p<0.01). The study data show for the first time that SFN counteracts the induction of hepcidin by proinflammatory stimuli, although further research is necessary to discover the exact mechanisms involved. SFN may therefore be an option for improving iron absorption in patients with chronic inflammatory disease.

References: 1. Means, R.T.Jr.: Am J Med Sci 2013, 345(1): 57-60. 2. Poli , M. et al. : Front Pharmacol 2014, 28(5): 86. 3. Li, B. et al.: Exp Neurol 2013, 250: 239-249. 4. Lin, W. et al.: Biochem Pharmacol 2008, 76(8): 967-973.


Joint action of herbal components influence the effects of STW 5 on inflammatory processes and disturbed motility Hoser, S.1; Winkelmann, V.1; Baumgärtel, A.1; Mishenzon, N.1; Abdel-Aziz, H.2; Weiser, D.2; Okpanyi, S.N.2; Nieber, K.1; Kelber, O.2 1 University of Leipzig, Institute of Pharmacy, Pharmacology, Leipzig, Germany; 2 Steigerwald Arzneimittelwerk GmbH, Scientific Department, Darmstadt, Germany

The herbal drug STW 5 (Iberogast®) contains nine individual plant extracts which affect inflammatory processes and disturbed motility in the gut. The aim of the study was to investigate the effect of individual ethanolic extracts (in concentration used in STW 5) and selected combinations on targets related to inflammation. The cytotoxicity was examined in CaCo-2 cells using the lactate dehydrogen-ase (LDH) assay whereas, THP-1 cells were used to determine the TNFα release using an ELISA. ACh-induced isometric contractions of a rat small intestinal preparations were measured in an commercial organ bath setup. STW 5 (500.05μg/ml), STW 5-II (5.11μg/ml, containing only 6 of the 9 extracts) and lemon balm (59.9μg/ml) reduced LPS (10ng/ml)-induced LDH release from CaCo-2 cells by 74-75%. Iberis amara (29.1μg/ml), peppermint (38.9μg/ml), chamomile (116.3μg/ml), angelica (84.7μg/ml) and milk thistle (15.2μg/ml) inhibited LDH release by 25-37%. Liquorice (84.9μg/ml), caraway (30.9μg/ml) and celandine (61.9μg/ml) had no effect. The combinations of Iberis amara and peppermint as well as peppermint and milk thistle revealed synergistic or additive effects, whereas the combination of chamomile and angelica evoked additive or antagonistic effects depending on their compositions. STW 5, STW 5-II, Iberis amara, peppermint, liquorice, caraway, milk thistle and lemon balm

in concentration used in LDA assay inhibited LPS (100ng/ml)-induced TNF release to 51-67%. The combinations of Iberis amara and peppermint as well as chamomile and liquorice had additive or antagonistic effects depending on their compositions. STW 5 and STW 5-II reduced the acetylcholine (ACh)-induced contractions in rat ileum preparations to 81-83%. The individual extracts inhibited the contractions to 83-91% except lemon balm. The combination of Iberis amara and peppermint as well as liquorice and caraway had additive or antagonistic effects depending on the ratios. Our results allow insight into the interactions between the extracts of STW 5 and confirm the concept of multi-target actions. Disclosure: Funding for experimental studies was provided by Steigerwald Arzneimittelwerk GmbH.

Reference: 1. Hoser, S. et al.: Planta Med 2013, 79: 1258.

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N-phenyl substituted thiazolidine-2,4-diones as neuroprotective agents Kraus, A.L.1; Oppermann, S.2; Elsässer, K.2; Schrader, F.C.1; Wegscheid-Gerlach, C.1; Culmsee, C.2; Schlitzer, M.1 1 Institut für pharmazeutische Chemie, Fachbereich Pharmazie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Deutschland 2 Institut für Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, Philipps-Universität Marburg, Karl-von-Frisch-Str. 1, D-35032 Marburg, Deutschland

Mitochondrial damage is a result of glutamate-induced excitotoxicity or oxidative stress and plays an important role in neuronal death, e.g. after cerebral ischemia or in neurodegenerative diseases. In the underlying intrinsic death pathway of programmed cell death, the protein Bid is a well-established key factor in the process of mitochondrial demise, characterized by loss of mitochondrial membrane potential, mitochondrial fission and increased mitochondrial ROS formation. Therefore, Bid was chosen as target for neuroprotection. A range of N-phenyl substituted thiazolidine-2,4-diones were synthesized and tested for their neuroprotective effects in a model of Bid-dependent neural cell death. Some of the initially synthesized compounds showed pronounced protection in vitro and are basis for further modification and development of novel structures.

The synthesis was carried out using thioglycolic acid methyl ester and either substituted phenylisocyanates or substituted aniline and 1,1’-carbonyldiimidazole. The most promising structures contained ortho-substitutents on the aromatic moiety, leading to a rotational barrier and reduced flexibility.

References: 1. Becattini, B. et al.: Proc. Natl. Acad. Sci. U.S.A. 2006, 103: 12602-12606. 2. Becattini, B. et al.: Chem. Biol. 2004, 11: 1107-1117. 3. Oppermann, S. et al.: J. Pharmacol. Exp. Ther. 2014, 350: 273-289.

Interaction between TRPC6 channels and FKBP51 – implications for the pathophysiology of mood disorders Ye, L.1; Hausch, F.2, Friedland, K.1 1 Department of Molecular and Clinical Pharmacy, University of Erlangen, 91052 Erlangen, Germany 2 Max Planck Inst. for Psychiatry, 80804 Munich, Germany

The FK506 binding protein 51 (FKBP51) is a member of the immunophilin superfamily. FKBP51 acts as an Hsp90-associated co-chaperone regulating responsiveness of steroid hormone receptors. Genetic association studies revealed an association of FKBP51 with emotional processing and affective disorders such as major depression. Until now, its molecular effects were mainly considered to be mediated via steroid hormone receptors. Here, we demonstrate that part of its effects in neuronal cells might be induced by the interaction with the classical transient receptor potential channel TRPC6. TRPC6 channels are non-selective cation channels permeable for mono- and divalent cations such as calcium. In the CNS, TRPC6 channels are discussed to be involved neuronal differentiation and proliferation, in axon pathfinding and synaptogenesis during development but also in the adult brain. Furthermore, TRPC6 channels are the molecular target of hyperforin, the antidepressant

active constituent of St. John’s wort extract, which is used to treat mild to moderate depression. These findings might contribute to a better understand-ing how FKBP51 and TRPC6 channels are involved in the pathogenesis of mood disorders.

Nutritive Modulators of Mitochondrial Function in Neurodegener-ative Diseases Denzer, I.1,2; Pischetsrieder, M.2; Friedland-Leuner, K.1 1 Department of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Cauerstr. 4, 91058 Erlangen, Germany 2 Department of Chemistry and Pharmacy, Emil Fischer Center, Henriette Schmidt-Burkhardt Chair of Food Chemistry,Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schuhstr. 19, 91052 Erlangen, Germany

In the wake of increasing life span of humans, aging-associated neurodegen-erative diseases have become a major health problem in society [1]. Age-related mitochondrial dysfunction is an early event in the pathology of many common neurodegenerative diseases, including Alzheimer’s disease (AD) [2,3]. Diet and lifestyle seem to have a major impact on neuronal function and are considered to be important preventive key-players in healthy aging [4]. Therefore, mitochondria seem to be an interesting pharmacological target for nutritive modulators of brain functionality and activity in aging and AD. Several constituents of food such as L-sulforaphane (SFN) from broccoli, S-allyl-L-cysteine (SAC) from garlic or isoliquiritigenin (iso) from licorice are known to activate the nuclear factor erythroid-2-related factor 2 (Nrf2) [5]. Importantly, Nrf2 expression is downregulated in AD and this might result in reduced mitochondrial biogenesis and finally in impaired production of ATP [6]. The purpose of this study was the screening of food items for mitochondrial protection. Therefore, we first investigated the effects of iso, SAC and SFN in a neuronal cell model on basal mitochondrial function and cell viability and afterwards looked for protective effects in a cell model of aging and AD. 20-h preincubation with iso (1 µM), SAC (5 µM) or SFN (1 µM) showed an increase of the mitochondrial membrane potential of PC12 cells, which were harmed with the complex I inhibitor of the respiratory chain rotenone (3 µM, 24 h) as a cellular model of aging. Similar results could be observed for the mitochondrial membrane potential and the cell viability of PC12 cells which were preincubat-ed with iso (1 µM), SAC (5 µM) or SFN (1 µM) for 20 h and stressed with the nitric oxide donor sodium nitroprusside (SNP) (350µM or 400 µM, 24 h), a generator of nitrosative stress and a complex IV inhibitor. These results show that the nutritive Nrf2 activators iso, SAC and SFN improve mitochondrial function and cell viability in a cell model for aging and AD.

Funding Acknowledgments: This study is implemented within the framework of the Neurotrition Project, which is supported by the FAU Emerging Fields Initiative.

References: 1. Hampel, H. et al.: Prog. Neurobiol. 2011, 95: 718-728. 2. Leuner, K. et al.: Front. Neurosci. 2010, 4: 44. 3. Leuner, K. et al.: Antioxid. Redox Signaling 2012, 16 (12): 1421-1433. 4. Eckert, G.P. et al.: Mol. Neurobiol. 2012, 46 (1): 136-150. 5. Surh, Y. J. et al.: Planta Med. 2008, 74 (13): 1526-1539. 6. Tufekci, K.U. et al.: Parkinsons‘s disease 2011, 2011: 314082.


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Novel analogues of strychnine as potent inhibitors of glycine receptors Zlotos, D.P.1; Mohsen, A.Y.1; Holzgrabe, U.2; Jensen, A.A.3 1 The German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City, 11835 Cairo, Egypt 2 Institut für Pharmazie and Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany 3 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

Strychnine (1), the major alkaloid from the plant Strychnos nux vomica, exhibits pharmacological activity at several neurotransmitter receptors, including a number of ligand-gated ion channels. Its most pronounced action is a strong antagonistic activity at glycine receptors (GlyRs), often referred to as ‘strychnine-sensitive glycine receptors‘, which are anionic chloride channels linked to hyperpolarisation and inhibition of neuronal firing [1-3]. In the course of our studies on Strychnos alkaloids, we have recently identified the lactam group and the C19-C20 double bond of strychnine as essential structural features required for strong activity at glycine α1 and α1β receptors [4]. Moreover, the only structure modification that did not impair the activity of strychnine was the (E)-configurated hydroxyimino group at C22 (compound 2) [5]. Here, we describe the synthesis and pharmacological evaluation of a series of oxime ethers 3a-f obtained by O-alkylation of 2. Interestingly, most of the new ligands 3a-f display high antagonistic potency at glycine α1 and α1β receptors, comparable to that of strychnine.

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Acknowledgments: Deutscher Akademischer Austauschdienst (DAAD), The Novo Nordisk Foundation

References: 1. Lynch, J.W.: Physiol. Rev. 2004, 84: 1051. 2. Laube, B. et al.:Trends Pharmacol Sci., 2002, 23: 519. 3. Jensen, A.A.; Kristiansen, U.: Biochem. Pharmacol. 2004, 67: 1789. 4. Mohsen, A.M.Y. et al.:Chemistry & Biodiversity, 2014, accepted 5. Jensen, A.A. et al.:Eur. J. Pharmacol. 2006, 539: 27.

Activation of SK channels prevents ER stress-induced neuronal cell death. Richter, M.1, 2; Dolga, A.M.1; Dodel, R.2; Culmsee, C.1 1 Institute for Pharmacology and Clinical Pharmacy, University Marburg, 35032 Marburg, Germany 2 Department of Neurology, University Marburg, 35043 Marburg, Germany

Stress of the endoplasmic reticulum (ER stress) is involved in the pathogene-sis of various neurodegenerative diseases. Contribution of the unfolded protein response (UPR) in Parkinson’s and Alzheimer’s disease has been shown in post-mortem human brains and different rodent models in vivo. Thus, drugs that cope with neuronal ER stress are supposed to have broad therapeutic potential. Recent studies emphasized the importance of small conductance calcium-activated potassium (SK) channels in neuronal calcium homeostasis. Cytoprotection by activation of mitochondrial SK channels has been described in conditions of mitochondrial dysfunction in neuronal cells. Here, we ad-dressed the question whether pharmacological activation of SK channels can affect the ER unfolded protein response, and protect against ER stress-associated cell death. Pharmacological activation of SK2 channels by use of CyPPA (N-Cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine) rescued HT-22 neurons from apoptosis induced by the ER stress evoking compound brefeldin A as analyzed by AnnexinV/PI staining and flow cytometry. Brefeldin A treatment affected intracellular calcium levels, which was compensated by SK channel activation. Furthermore, cleavage of caspase 12 and caspase 3 was attenuated as detected by Western Blot. SK channel activation altered the level of UPR proteins, i.e. further increased CHOP levels and reduced ATF4 levels compared to brefeldin A-treated cells. Knockdown of ATF4 by siRNA approaches showed similar protective effects like SK channel activation, pointing for an important role of ATF4 protein attenuation in CyPPA-mediated protection against brefeldin A. Oxidative stress (assessed by H2DCFDA and MitoSOX), as a late result of ER stress, was diminished by positive modulation of SK channels. In the absence of extracellular calcium as well as in the presence of the extracellularly acting SK channel blocker apamin, pharmaco-logical activation of SK channels was still protective against ER stress. Therefore, we concluded that intracellularly located SK channels are important for CyPPA-mediated protection. Overall, these data show that pharmacological SK channel activation restores disturbed calcium levels and reduces neuronal cell death associated with ER stress. Intracellularly located SK channels are essential mediators of protection against ER stress. Collectively, it is proposed that SK channels could be a therapeutic target in neurodegenerative diseases related to ER stress.

Bid links ferroptosis to mitochondrial cell death pathways in neurons Neitemeier, S.; Laino, V.; Oppermann, S.; Ganjam, G.K.; Dolga, A.; Culmsee, C.

Institut für Pharmakologie und Klinische Pharmazie, Biochemisch-Pharmakologisches Centrum Marburg, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg Germany

Ferroptosis is considered to be an iron-dependent form of nonapoptotic cell death first described in cancer cells induced by the small-molecule erastin which inhibits the cystine/glutamate antiporter (Xc

-). The subsequent increase in lipid peroxides is a major hallmark in the death pathways of ferroptosis in contrast to other forms of apoptotic cell death [1]. The inhibition of the Xc

- is also the initiator of a cell death pathway called oxytosis in which toxicity is achieved by high extracellular glutamate concentrations. Oxytosis plays a pivotal role in neurodegeneration [2]. Besides the formation of reactive oxygen species (ROS) the activation of the proapoptotic BH3-only protein Bid is crucial in this cell death mechanism and leads to mitochondrial demise [3]. In the present study we investigated whether pathways of ferroptosis and oxytosis may be linked through the activation of Bid and according mitochondrial damage in neural cells. In order to elucidate the role of Bid in ferroptosis we induced cell death with erastin in a mouse hippocampal HT-22 cell line and investigated the effects of the Bid-inhibitor BI-6c9 on cell viability and mitochondrial integrity and function. Further, we analyzed the effects of ferrostatin-1 in glutamate-induced mitochondrial damage and neural cell death. The inhibition of Bid by BI-6C9 prevented erastin-induced morphological changes of HT-22 cells and the loss of cell viability detected by the MTT assay. Similar protective effects were observed by ferrostatin-1 in the glutamate model of oxytosis. Because lipoxygenation is crucial for erastin-dependent cell death lipid peroxidation was further investigated by Bodipy staining and subsequent FACS analysis. BI-6c9 fully abolished lipid peroxide formation in the erastin model, and also ferro-statin-1 inhibited lipid peroxidation after the glutamate challenge. Mitochondrial ROS production was also significantly decreased by BI-6c9 and ferrostatin-1 in

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the particular model systems assessed by MitoSOX staining and FACS analysis. Next, mitochondrial morphology and function were analyzed to gain more insights into the changes in mitochondrial integrity after the respective challenges associated with oxytosis and ferroptosis. BI-6c9 significantly reduced mitochondrial fission and also preserved the mitochondrial membrane potential in erastin-treated cells detected by flow cytometric analysis after TMRE staining. Further, luciferase-based measurements of ATP revealed that BI-6c9 prevented erastin-induced loss of ATP. Ferrostatin-1 reduced mito-chondrial fission, preserved mitochondrial membrane potential and ATP levels after glutamate exposure as well. To further confirm the role of Bid in both cell death pathways HT-22 cells were co-transfected with a red-labeled Bid and a mitochondrial-targeted GFP vector to investigate the translocation of Bid to the mitochondria in response to erastin and glutamate-induced stress, respective-ly. Both BI-6c9 and ferrostatin-1 were able to inhibit the translocation of Bid to mitochondria as detected by fluorescent confocal microscopy. Overall, these results show that ferroptosis and oxytosis share common features and that Bid links both pathways to intrinsic mechanisms of mitochon-drial demise.

References: 1. Dixon, S.J. et al.: Cell 2012, 149(5): 1060-1072. 2. Tan, S.; Schubert, D.; Maher, P.: Curr. Top. Med. Chem. 2001, 1(6): 497-506. 3. Tobaben, S. et al.: Cell Death Differ. 2011, 18(2): 282-292.

Sustained neuroprotective effects after pharmacological inhibi-tion of p53 compared to siRNA-mediated p53 silencing Neitemeier, S.; Diemert, S.; Ganjam, G.K.; Culmsee, C.

Institut für Pharmakologie und Klinische Pharmazie, Biochemisch-Pharmakologisches Centrum Marburg, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg Germany

The tumor suppressor p53 is regarded as the guardian of the genome playing crucial roles in sensing DNA damage and cell cycle regulation. Depending on the nature and the extent of cellular stress and associated DNA damage, p53 can either mediate DNA repair or cell death. The inhibition of p53 by the drug pifithrin-alpha (PFT) has been shown to be protective against oxidative stress and subsequent mechanisms of pro-grammed cell death in neuronal cells. This suggests that the selective inhibition of p53 is a potential target to mediate neuronal survival. Therefore, in the present study we used a model of glutamate induced cell death in a mouse hippocampal HT-22 cell line to further investigate p53-dependent pathways in neuronal cell death. Specific siRNA (20 nM) targeting p53 was applied in the HT-22 cells for silencing of the tumor suppressor protein. The p53 knock down was confirmed at mRNA levels and protein levels by RT-PCR and Western blotting, respec-tively. Gene silencing of p53 protected HT-22 cells against glutamate induced damage detected by the MTT assay. However, in contrast to the sustained protective effects of PFT, the neuroprotection by the p53 siRNA was transient and lasted only for two hours as detected by real time impedance measure-ments (xCelligence system, Roche). Both, PFT and p53 siRNA blocked the transcriptional activity of p53 after glutamate treatment and under control conditions as shown by a luciferase reporter assay. Notably, p53 siRNA did not preserve mitochondrial morphology and integrity, as detected in fluorescent microscopic analysis of mitochondrial fission and measurements of the mitochondrial membrane potential at 7 h and 14 h after onset of the glutamate challenge by FACS analysis. In contrast, PFT significantly reduced mitochon-drial fission and also preserved the mitochondrial membrane potential in glutamate treated cells. Overall, these results suggest that p53 siRNA mediated neuroprotective effects via transcriptional effects while PFT may exert additional effects at the level of mitochondria, which was essential to provide sustained neuroprotection.

Improvement of mitochondrial dysfunction by modulators of the human γ-secretase modulators/PPARγ agonists with a dual mechanism of action Pohland, M.1; Hagl, S.1; Wurglics, M.2; Schubert-Zsilavecz, M.2; Eckert, G.P.1 1 Department of Pharmacology, Goethe-University Frankfurt am Main, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany 2 Instiute of Pharmaceutical Chemistry, Goethe-University Frankfurt am Main, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder leading to dementia. Deposits of beta amyloid protein (Aβ) and intracellular neurofibril-lary tangles are pathological hallmarks of AD. Increasing evidences indicate mitochondrial dysfunction as an early event in AD pathogenesis. Mitochondria are essential for the supply of energy but are also involved in oxidative stress and apoptosis. Current drugs act merely symptomatic and new disease modifying drugs against AD have almost failed in human clinical trials recently. We investigated the efficacy of a novel class of acidic γ-secretase modula-tors/PPARγ agonists with a dual mechanism of action against mitochondrial dysfunction in HEK293-APP695 cells expressing neuronal APP. Dimebon, DAPT, and Pioglitazone were used as controls. The basic structure of the new compounds, derivated of pirinixic acid, is thiobarbituric acid. Optimization of pharmacological activities leaded to new molecules with a significantly increased activity at both pharmacological targets. All examined compounds were active and influenced the cell biology of the studied HEK293-APP695 cells. First, the cell viability was measured by MTT assay in the range of 0,03 - 30 µM. For the non-toxic concentrations we measured changes in mitochondrial membrane potential (MMP) and levels of adenosine triphosphate (ATP) to determine alterations of mitochondrial efficiency and function. The compounds MH49, MH73, MH84 and MH163 were able to exhibit significant protective effects against NO-releasing drugs like sodium nitro prusside (SNP). Aβ-induced changes in mitochondrial enzyme activities are leading to oxidative stress and enhanced apoptosis. The rate of mitochondrial respiration was investigated and especially in the complex-IV respiration, which is decreased by Aβ, we were able to show a significant improvement after treatment with compound MH84 compared to control group. To get a closer look insight, we measured the citrate synthase (CS) activity, used as a marker enzyme for the mitochondrial mass. CS activity in HEK293-APP695 cells was increased by the GSM MH84, indicating an increased mitogenesis. In summarizing review substances MH84 and MH73 in our in-vitro experiments were the most convincing compounds in the nM range. Further experiments will reveal the molecular consequences of γ-secretase/PPARγ modulation and to test the compounds in mouse models of Alzheimer`s disease.

This project is supported by the Doktor Robert Pfleger-Stiftung

Caspase inhibition prevents alpha synuclein-toxicity in human dopaminergic neurons Ganjam, G.K.1; Dolga, A.M.; Neitemeier, S.1; Bolte, K.4; Höllerhage, M.2; Oertel, W.E.3; Hoeglinger, G.U.2,3; Culmsee, C.1 1 Institut für Pharmakologie und Klinische Pharmazie, Biochemisch-Pharmakologisches Centrum Marburg, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg, Germany 2 German Center for Neurodegenerative Diseases (DZNE), Max Lebsche Platz 30, 81377 Munich, Germany. 3 Department of Neurology, Philipps-Universität Marburg, Rudolf-Bultmann-Strasse 8, 35033 Marburg, Germany. 4 Department of Biology, Faculty of Biology,Philipps-University Marburg, Karl-von-Frisch-Straße 8, 35043 Marburg, Germany.

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Parkinson’s disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. However, the molecular mechanisms underlying alpha-synuclein-mediated toxicity in human dopamin-ergic neuronal loss are not well defined. The goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. Therefore, we have generated neuron specific, adeno associated virus type 2 (AAV2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and EGFP expressing viruses used as respective controls. Overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular ATP, enhanced mitochondrial ROS production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. In addition, real-time analysis of mitochondrial bioenergetics using the Seahorse Bioscience system following AAV infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. Our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. In addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells express-ing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. In addition, by pharmacological approaches, we found that inhibition of caspases by QVD significantly ameliorated alpha synuclein-induced dopamin-ergic neuronal death. Interestingly, inhibition of caspases preserved neuronal network integrity, ATP levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. Overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity. Thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in Parkinson’s syndromes that are associated with alpha synuclein toxicity.

Dynamic changes in extracellular ATP levels during status epilepticus as monitored by a novel microdialysis probe Lietsche, J.; Imran, I. ; Hardt, S. ; Klein, J.

Department of Pharmacology, Biocenter N260, Max-von-Laue Str. 9, Goethe University Frankfurt, 60438 Frankfurt, Germany

In the neuroscience research environment microdialysis is a widely applied technique to collect extracellular fluid of many tissues, including brain. Samples of the extracellular space are proteinfree and can be used for chemical analysis without any further purification [1]. The major element of microdialysis is the probe which contains a semi-permeable membrane. Substances in extracellular fluid surrounding the probe pass the dialysis membrane by passive diffusion along their concentration gradient [2]. In our lab a custom-made microdialysis probe with a 10 kDa molecular weight cut-off (MWCO) is applied. The assembly was developed by Santiago and Westerink in 1990 [3]. This probe is equipped with a polyacrylonitrile mem-brane (AN69 HF; Hospal-Gambro, Planegg-Martinsried, Germany) which is no longer available. Therefore a newly developed probe containing a semiperme-able membrane (FXCorDiax 600; Fresenius Medical Care, Bad Homburg, Germany) with a MWCO of 30 kDa had to be constructed with new parameters because the diameter of the dialysis membrane was smaller than that of polyacrylonitril membrane (210 µm vs. 280 µm). In the course of probe manufacture, several parameter such as tubing diameters, silica diameters and type of glue had to be optimized. The 30 kDa custom-made probe (exchange length 2 mm) yields significantly higher recoveries for ATP (22.4 ± 0.7 %) than the 10 kDa custom-made probe (13.1 ± 0.7 %). The improvement of ATP recovery allowed measurement of ATP in the extracellular space in distinct brain areas with the method of microdialysis. Here, we show dynamic changes of hippocampal ATP release during lithium-pilocarpine induced status epilepticus in rats. Administration of pilocarpine (30mg/kg s.c.) to rats pretreated with litium chloride (127 mg/kg i.p.) caused a decrease of ATP levels when rats start to develop tonic seizures. The present study reveals that basal ATP levels (1.12 nM; recovery corrected) decrease under detection limit (0.1 nM) immediately with the beginning of status

epilepticus and remain low when seizures were stopped by administration of diazepam (10mg/kg i.p.). Various studies have shown that the purinergic system is involved in the pathophysiology of epilepsy. During epileptic seizures adenosine concentra-tions are increased which are considered to have an anticonvulsant effect [4]. Adenosin originates from the catabolism of ATP by ectonucleotidases and ecto-5´-nucleotidase [5] which leads to the assumption that, when adenosine concentration increases, ATP concentration has to decrease. This hypothesis is confirmed by our findings described above.

Acknowledgments: The authors are grateful to G. Barka (SunChrom, Friedberg, Germany) who provided the epoxy glue, and to Fresenius Medical Care (Bad Homburg, Germany) for the supply of the Capillary Haemodiafilter FX CorDiax 600. Funding was obtained from Goethe University, Frankfurt. References: 1. Benveniste, H.: J. Neurochem. 1989, 52(6): 1667-1679. 2. Bourne, J.A.: Clin. Exp. Pharmacol. Physiol. 2003, 30(1): 16-24. 3. Santiago, M., Westerink, B.H.: Naunyn-Schmiedebergs Arch. Pharmacol. 1990, 342, (4): 407-414. 4. Boison, D.: Neuroscientist. 2005, 11(1): 25-36. 5. Zimmermann, H.: Neurochem. Int. 2008, 52(4): 634-648.

A kinetic study of triheptanoin fed mice in a middle cerebral artery occlusion model Koch, K.A.; Konietzka, J.; Berressem, D.; Eckert, G.P.; Klein, J.

Department of Pharmacology, Goethe University, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

Stroke is a major medical emergency and the second most frequent cause of death in the world. While drug treatment of stroke remains unsatisfactory, dietary approaches to stroke prevention and regeneration, such as anaplerotic diet, have recently attracted much interest. Our anaplerotic approach is triheptanoin, which is cleaved into glycerol and three heptanoate moieties. In the liver, heptanoate is metabolized via β-oxidation in two mol of acetyl-CoA and one mol of propionyl-CoA. Our group hypothesized that propionyl-CoA could act as an anaplerotic agent being metabolized to succinyl-CoA which feeds into the citric acid cycle. In the present study, we have used a common experimental stroke model, middle cerebral artery occlusion (MCAO), in triheptanoin-fed mice to investigate the levels of odd chain fatty acids before and after brain ischemia. In female CD-1 mice, ischemia was induced in the left hemisphere with the MCAO method. After 90 min of ischemia, mice were sacrificed, blood was withdrawn and liver and brain tissue were harvested and extracted for odd-chain fatty acid measurements by GC-MS. Brain and liver homogenates were extracted in a biphasic manner with metha-nol/water/chloroform. The residue of the water soluble phase was derivatized with BSTFA/TMCS (99:1). In liver homogenate of mice fed triheptanoin ad libitum for two weeks, odd chain fatty acids were: propionate (sham group) 25.66 ± 8.65 µM, propionate (stroked group) 37.18 ± 10.69 µM, pentanoate (sham group) 14.70 ± 4.28 µM and pentanoate (stroked group) 29.52 ± 8.41 µM (data ± SEM, N=6) (all data calculated for intracellular water). In the control group, which was fed with soja oil instead of triheptanoin, the odd-chain fatty acids were below limit of detection. In brain homogenates, the following data were obtained: propionate (sham group) hemisphere 32.67 ± 5.64 nM, propionate (stroked group) left hemisphere 60.52 ± 6.40 nM, right hemisphere 70.05 ± 18.76 nM; pentanoate (sham group) 60.10 ± 13.04 nM; pentanoate (stroke group) left hemisphere 109.20 ± 23.00 nM, right hemisphere 100.8 ± 21.92 nM. Plasma levels were: propionate (sham group) 5.79 ± 1.47 µM and propionate (stroked group) 6.51 ± 2.08 µM; pentanoate (sham group) 7.59 ± 4.31 µM and pentanoate (stroked group) 4.31 ± 0.30 µM; heptanoate (sham group) 15.45 ± 5.43 µM and heptanoate (stroked group) 2.24 ± 0.21 µM. In conclusion, we found a characteristic pattern of distribution in triheptanoin-fed mice. While control mice, which were fed with soja oil, did not show any detectable levels of odd-chain fatty acids in plasma or brain, triheptanoin-fed mice had levels of propionate and pentanoate in the micromolar range. Odd-chain fatty acids in triheptanoin-fed mice were highest in the liver, lower in in plasma and even lower in brain homogenates. Current projects are aimed at measuring extracellular levels of these metabolites in the brain by microdialy-sis.

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Dynamics of acetylcholine and metabolites release during lithium-pilocarpine-induced status epilepticus in rats: a microdi-alysis study

Imran, I.; Hillert, M.; Klein, J.

Department of Pharmacology, Max von Laue Str. 9, Goethe University Frankfurt, Germany

Status epilepticus (SE) is a life threatening condition which requires an intensive therapeutic management. The lithium-pilocarpine model is an epilepsy model in rats inducing status epilepticus with low mortality rate. In this study we measured changes of acetylcholine (ACh) and metabolites released in the hippocampus before, during and after status epilepticus as monitored by microdialysis in unanesthetized rats. After 90 minutes of status, rats were decapitated for the isolation of mitochondria from brain to evaluate the respiratory functions. Administration of pilocarpine (30 mg/kg s.c.) to rats which were pretreated with lithium chloride (127 mg/kg) caused a massive, six-fold increase of hippocampal ACh release concomitant with the development of tonic seizures. When seizures were terminated with diazepam (10 mg/kg) or ketamine (75 mg/kg) after 90 minutes, ACh levels returned to normal. Administration of atropine (1 mg/kg) 2 h after pilocarpine caused a further increase of ACh but did not affect seizures whereas injection of mecamylamine (5 mg/kg) reduced ACh levels. Local infusion of tetrodotoxin (TTX, 1 µM) or hemicholinium (HC-3, 10 µM) strongly reduced ACh release, but had only a minor and delayed effect on seizures. Among the metabolites quantified; glucose remained unchanged, lactate increased up to 4-6 fold. Glycerol an indicator of membrane damage increased up to 6-10 folds. Surprisingly, mitochondrial respiration was found to be absolutely normal after 90 minutes of SE. Taken together, our results demonstrate that seizure development in SE model is accompanied by massive increases of extracellular ACh, lactate and glycerol whereas glucose levels and mitochondrial integrity remain unaltered. Inhibition of ACh release or blockade of cholinergic receptors, however, does not terminate seizures. I am thankful to DAAD (German academic exchange service) for stipend and other financial support.

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BIOPHARMACEUTICS (BP01-BP09)

How to address the variety of eating habits in children in the development of biorelevant dissolution test methods simulating fed state conditions in the paediatric GI tract Kersten, E.; Blank, S.; Klein, S.

University of Greifswald, Department of Pharmacy, Institute of Biopharmaceutics & Pharmaceutical Technology

Eating and drinking habits of children can be very heterogeneous and especially in the first years of life, they differ significantly from adult eating behaviour [1]. Since food and fluid consumption may have a tremendous impact on drug dissolution in the upper gastrointestinal tract, it is of vital importance to address the special eating habits in children when developing of biorelevant dissolution methods mimicking the fed state in children of different age groups. We performed a survey on the food and fluids consumed by children at the age of 1 - 6 years. Parents of infants and pre-school children were asked to document the respective data over 4 days in a questionnaire. 97 completed questionnaires were received back. The reported meals and fluids were grouped according to the age of the child. Since the morning is the most important time of drug intake, the focus of the present study was set on the composition of the breakfast. When evaluating the data set, it became obvious that there are huge inter-, but also intraindividual differences when comparing the meal and fluid composi-tion, as can be seen in table 1. This is particular true for children at the age of 1 – 2 years. The table shows the breakfast consumed by 3 individuals at four days. Whereas one of the infants still consumed formula milk without any solid food, breakfast composition of the other two children was similar to that of other children or adults and also varied from one day to another. This observation clearly indicates that there will not be a “one-fits-all” test design but that rather the variability observed in our survey with extreme conditions whereas food was administered without any fluid or meals with high osmolality and/or fat content were administered will need to be properly addressed to obtain predictive in vitro results

Reference: 1. Mesch, C. et al.: Appetite, 2014, 76: 113-119.

Structural Dynamics of Novel Gastrointestinal Model Fluids by combined SANS and DLS Khoshakhlagh, P.1a; Johnson, R.1a; Nawroth, T.1a; Langguth, P.1a; Schmueser, L.1b; Decker, H.1b; Hellmann, N.1b; Szekely, N.2 1 Gutenberg-University, a) Pharmacy; b) Molecular Biophysics; Staudingerweg 5, D-55099 Mainz 2 JCNS Outstation at FRM II, Instrument KWS2, Lichtenbergstr.1, D-85747 Garching

Oral administration of drugs is considered as the preferred route in drug delivery. Solubility and bioavailability of drugs are depicted by the Biopharma-ceutics Classification System (BCS). Lipophilic drugs of the BCS classes II and IV are subject of drug formulation development during the last decade. However, many of these drugs do not have sufficient solubility and that makes them undesirable for in vivo studies. In vivo, the chyme containing the drug from stomach emptying undergoes in the first half of the duodenum a pH shift. After subsequent addition of bile and pancreatic juice it shows a slightly acidic pH (6.5), while the concentration varies depending on the composition of the meal and fluid intake. In parallel intestinal nanoparticles (micelles and liposomes) develop from the bile components, food and drug in a dynamic process. These native nanoparticle systems can be used and modified for lipophilic drug solubilization and uptake. In vitro biorelevant media such as fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) are established tools to predict the absorption of several drugs in vivo. We have developed physiologically similar intestinal fluids, which differ from the former FaSSIF/FeSSIF by the presence of cholesterol. The lipid composi-tion in the novel FaSSIF-C fluids was systematically varied, according to the lipids occurring in the physiological composition of the human bile: FaSSIF-7C modelled female, FaSSIF-10C male, and FaSSIF-13C people with diseases leading to gall stones. Time resolved Dynamic Light Scattering (DLS) and Small Angle Neutron Scattering (SANS) were used to investigate the structural development (kinetics) of the fluids with and without Fenofibrate (BCS class 2) in a gastrointestinal simulator device (GIMod). The development of micelles to liposomes with intermediate size structures was observed. In the time regime 1-60 min after the modelled bile influx to the intestine (FeSSIF to FaSSIF dilution), two micellar intermediates were detected, which could influence the resolution and delivery of the hydrophobic drug.

Acknowledgments: We are thankful for the funding by the German ministry of science and education BMBF, grant 05KS7UMA; the Forschungszentrum Jülich FZJ, Jülich Centre of Neutron Science JCNS, outstation at the FRM2 reactor Munich Garching; and support by the Dr. Georg-Scheuing Stiftung, Mainz. This work was contributed to the OrBiTo project (http://www.imi.europa.eu/content) as side ground. References: 1. Amidon, G. L. et al.: Pharmaceut. Res. 1995, 12(3): 413-420. 2. Nawroth, T. et al.: Mol. Pharm. 2011, 8,(6): 2162-2172. 3. Kleberg, K. et al.: J. Pharm. and Pharmacol. 2010, 62(11): 1656-1668.

Use of Simulated Intestinal Fluid Solutions in Integrated Dissolu-tion/Permeation Models for Poorly Soluble Drugs with Rat Intestine Meinhardt, K.1; Khoshakhlagh, P.1; Konerding, M.2; Nawroth, T.1; Langguth, P.1 1 Johannes Gutenberg University Mainz, Staudingerweg 5, 55099, Germany 2 University Medical Center of Johannes Gutenberg University Mainz, Johann-Joachim Becher Weg 13, 55099, Germany

Biopredictive media as simulated intestinal fluids (SIFs) are often used in dissolution experiments of poorly soluble drugs, making it reasonable to also apply them in integrated dissolution/permeation models. Until now they are considered incompatible with rat intestine1). This incompatibility has only been shown for rat ileum2). The attempted explanation as to why SIFs are compati-ble with Caco-2 cells but not with rat ileum has been the different experimental

Individual 1

Individual 2 Individual 3

Food/Drink Food Drink Food Drink

Day 1

235 mL formula milk Pre

1 roll with strawberry jam ½ roll with spread meat 1 clementine 1 toast with spread meat

20 mL water

30 g cornflakes 150 mL milk (3.8 % fat) 4 cherry tomatoes 2 mini-salami

50 mL fennel tea

Day 2

235 mL formula milk 1

1 toast with butter 1 toast with liver sausage 1 yoghurt (strawberry flavour)

-

15 g cornflakes 50 mL milk (1.8 % fat) 2 cherry tomatoes 1 mini-salami

100 mL milk (1.8 % fat)

Day 3


1 toast with liver sausage ½ roll with liver sausage 1 piece of cucumber 1 clementine

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½ rolls with butter and strawberry jam 2 small tomatoes

150 mL fennel tea with apple juice

Day 4


1 toast with spread meat 1 toast with liver sausage 1 toast with margarine 1 clementine

-

1 toast 1 slice of salami 2 small tomatoes

100 mL milk (3.5 % fat)

Table 1: Breakfast of three children at the age of one year on four different days

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set-up: Rat ileum has been used in the Ussing Chamber where the abrasion could have been higher than in the experimental set-up used for the Caco-2 cell system. We suggest that the incompatibility is rather region specific and can be circumvented using upper parts of the intestine as excised sheets. The viability of three parts of rat intestine (duodenum, jejunum and ileum) was investigated in various media: Hank’s Balanced Salt Solution (HBSS), Modified Fasted-State Simulated Intestinal Fluid (FaSSIFmod 6.5

3), Fasted-State Simulat-ed Intestinal Fluid including 7 % Cholesterol (FaSSIF-7C), Modified Fed-State Simulated Intestinal Fluid (FeSSIFmod 6.5

3) and Fed-State Simulated Intestinal Fluid including 7 % Cholesterol (FeSSIF-7C). The potential difference across the excised sheet was constantly measured for 2 hours as indicator of the intestinal viability. At the end of the experiment the morphological state of the corresponding intestinal region was monitored via scanning electron micro-graph. The results indicate the viability of duodenum in all of the investigated media throughout the duration of the experiments. This applies to some extent to the jejunum. The ileum, as the lowest investigated part of the intestine, is not viable in simulated intestinal fluids. This study shows that an integrated dissolution/permeation model using both, simulated intestinal fluids as dissolution media and rat intestine as permeation model, is feasible provided that the intestinal segment is chosen adequately. Acknowledgments: We thank Mrs Kerstin Bahr for her great technical assistance with the scanning electron micrograph.

References: 1. Kleberg, K. et al.: Journal of Pharmacy and Pharmacology. 2010, 62(11): 1656–1668.

2. Patel, N. et al.: Drug Development and Industrial Pharmacy. 2006, 32(2): 151–161.

3. Kataoka, M. et al.: Journal of Pharmaceutical Sciences. 2006, 95(9): 2051–2061.

Ionic liquid versus prodrug strategy – overcoming biopharma-ceutical challenges Wiest, J.; Balk, A.; Meinel, L.; Holzgrabe, U.

Institute of Pharmacy and Food Chemistry, Am Hubland, University of Würzburg, DE-97074 Würzburg, Germany

The main problems of drug discovery and development are compounds of high molecular weight and high lipophilicity combined with poor water solubility [1]. A poorly water soluble acidic active pharmaceutical ingredient (API) against migraine attacks is prepared as an ionic liquid (IL) and compared to a prodrug strategy. An ionic liquid is defined as a salt with a melting point below 100°C. The IL approach leaded to a significantly longer duration of API supersatura-tion, a 700 fold faster dissolution rate, an 8 fold faster reach of the maximum plasma concentration and a twofold increased bioavailability as compared to both, the free acid and the prodrug. The underlying mechanism was studied by NMR spectroscopy, X-ray crystallography and Sirius experiments.

Acknowledgements: 2 Novartis Pharma AG, Lichtstraße 35, CH-4002 Basel, Switzerland, Widmer, T; Berghausen, J.; Galli, B. 3 Institute for Inorganic Chemistry, Am Hubland, University of Würzburg, DE-97074 Würzburg, Germany, Matthes, P; Müller-Buschbaum, K., Bertermann, R. This study was funded by Novartis Pharma AG, Basel.

References: 1. Lipinski, C.A et al.: Advanced Drug Delivery Reviews 1997, 23: 3-25.

A combined doxorubicin and doxorubicinol pharmacokinetic model to evaluate dosing regimens in infants and older children - results of the EPOC-MS-001 study Völler, S.1; Boddy, A.V.2; Boos, J.3, Kontny, N.E.1; Krischke, M.4; Würthwein, G.4.; Hempel, G.1 1 Westfälische Wilhelms-Universität, Institut für pharmazeutische und medizinische Chemie, Klinische Pharmazie, Corrensstraße 48, 48149 Münster, Germany 2 Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK 3 Universitätsklinikum Münster, Klinik und Poliklinik für Kinderheilkunde, Pädiatrische Hämatologie und Onkologie, Funktionsbereich Pädiatrische Hämatologie und Onkologie, Albert-Schweitzer-Straße 33, 48149 Münster, Germany 4 Universitätsklinikum Münster, Centre for Clinical Trials, ZKS Münster, Münster, Germany

Objectives: Although almost 60% of children diagnosed with cancer receive anthracyclines as part of their treatment, the knowledge on the pharmacokinet-ics (PK) of the drug in children, especially in the very young, is limited. Empirical dose reduction in infants is performed in almost all clinical trial protocols. However, dose reduction strategies differ widely across studies due to the unavailability of clinical PK data. As doxorubicin was included in the European Medicines Agency priority list for studies on off-patent paediatric medicinal products, a multicentre, multinational phase II PK study investigating a possible age-dependency in the clearance (CL) of doxorubicin in children with solid tumours and leukaemia was conducted. The data were analysed using a population PK model generated in NONMEM® 7.2. Methods: Samples from 2 doxorubicin administrations in 101 patients treated according to the tumour-specific national or European therapeutic trial were collected with a particular focus on recruiting children less than 3 years. PK data of doxorubicin and its main metabolite doxorubicinol from 94 patients were analysed using NONMEM® 7.2, R, Xpose4 and a predefined stepwise strategy. A large number of covariates including patient characteristics, laboratory values (e.g. bilirubin, serum creatinine, alanine aminotransferase, serum albumin, haematocrit) and 17 single nucleotide polymorphisms were available in the study. Covariate modelling was performed using the stepwise covariate modelling strategy (SCM) integrated in PsN® with the linearisation option. Results: A three compartment model for doxorubicin and one additional compartment for doxorubicinol was most suitable to characterise the PK of doxorubicin and its metabolite. All parameters of the model were scaled to body surface area. The inclusion of age on the CL of doxorubicin yielded a significant improvement of the model. No other patient-related covariate, including liver function, was found to influence the parameters of the model. Pharmacogenetic variants, including those in transporters and drug metabolis-ing enzymes, had no influence on pharmacokinetic parameters. Using the mean estimated CL value for each individual, children less than 3 years had a lower CL (21.1 ± 5.8 l/h/m2) than older children (26.6 ± 6.7 l/h/m2) (p=0.0004), even after correcting for body size. These results indicate that the empirical dose reduction of doxorubicin in infants is justified. Conclusion: This study demonstrates an age-dependency in the clearance of doxorubicin in children. The results may be useful for refining dosage regimens in this patient group.

Acknowledgments: This project was funded by the European Community’s Seventh Framework Programme (FP7/2009-2013) under grant agreement n° 222910.

Investigation of the hydrodynamics in the Ph. Eur. disintegration test device Kindgen, S.1; Wachtel, H.2; Langguth, P.1 1 Institute of Pharmacy and Biochemistry, Staudingerweg 5, 55128 Mainz, Germany 2 Boehringer Ingelheim Pharma GmbH & Co.KG, Binger Straße 173, 55216 Ingelheim am Rhein, Germany

Disintegration of oral solid dosage forms is a key step in drug liberation. In vitro disintegration testing according to the Ph. Eur. is indispensable in the

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development of solid oral dosage forms. For such in vitro tests it is essential to simulate the in vivo conditions accurately to predict the in vivo performance appropriately. The main factors influencing in vivo disintegration of solid oral dosage forms are food composition, fluid viscosity, water diffusivity, film precipitation on tablet surface, hydrodynamics around the dosage form and mechanical stress on the dosage form [1]. Recently, Radwan et al. studied the impact of food composition, viscosity and water diffusivity on tablet disintegra-tion and found a delayed disintegration rate and decreased water uptake in viscous media [1, 2, 3]. But to our knowledge, there is only limited information available on the hydrodynamics and mechanical stress in the disintegration test device and the impact of these factors on disintegration. The aim of this work was to experimentally and computationally characterize the hydrodynamics in the disintegration test device using Particle Image Velocimetry (PIV) and Computational Fluid Dynamics (CFD), respectively. PIV is an optical method to determine velocity fields. Therefore the medium is enriched with tracer particles which are photographed on small timescales. The velocity vectors can be calculated from the particle position at the photo series. The results show, that when entering the tube the particles follow a laminar pattern. The tablet forces the fluid aside and right above the tablet some turbulences occur. At a certain height the fluid flow returns to a laminar pattern. CFD uses numerical methods and algorithms to solve fluid flow problems. Using the CFD software SolidWorks, the geometry of the basket rack assembly and the beaker were reconstructed. A tablet was fixed at the bottom of one of the tubes of the basket rack assembly and the simulations were run for media with different viscosities to examine the influence of viscosity on fluid flow and velocity magnitude. The fixed tablet represents an obstacle for the fluid which is forced aside. Although, the basket rack assembly is moved in a sinusoidal profile with maximum speed of 80 mm/s, the calculated maximum speed in the liquid is much higher, increasing with increasing viscosity. From our experiments and CFD calculations we conclude that the flow velocity in the tubes of the disintegration test device is much too fast compared to the in vivo situation. The hydrodynamics are depending on the viscosity of the test medium and do not reflect the in vivo situation. Thus, the compendial disintegration test device and the test conditions are not suitable to predict the in vivo performance of solid oral dosage forms and need to be modified.

This work was contributed to the OrBiTo project (http://www.imi.europa.eu/content/) as sideground. References: 1. Radwan, A. et al.: European Journal of Pharmaceutical Science. 2014, 57: 273-279. 2. Radwan, A., Amidon, G.L., Langguth, P.: Biopharm. Drug Dispos. 2012, 33: 403-416. 3. Radwan, A. et al.: Mol. Pharmaceutics. 2013, 10: 2283-2290.

Systemic and pulmonary pharmacokinetics of inhaled olodaterol Borghardt, J.M.1,2; Weber, B.2; Staab, A.2; Kunz, C.2; Schiewe, J.3; Kloft, C.1 1 Dept. of Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany 2 Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397 Biberach, Germany 3 Respiratory Drug Delivery, Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Str. 173, 55216 Ingelheim am Rhein, Germany

Objectives: Up to now, the quantitative, mechanistic understanding about pulmonary deposition, dissolution and absorption of inhaled drugs is limited and based on many assumptions, such as fast absorption of dissolved drugs [1,2]. With the aim to increase the quantitative understanding about these processes and to evaluate several assumptions about the pharmacokinetics

(PK) after drug inhalation, clinical data of inhaled olodaterol (a long-acting -sympathomimetic drug approved for the treatment of COPD) was analysed with a mathematical modelling approach. Methods: Plasma concentration-time profiles after inhalation and intravenous (IV) administration and urine data after IV administration were available for population PK modelling from three trials in healthy volunteers. In addition to single dose profiles, one trial also contained plasma concentration-time profiles after once daily inhalation of an olodaterol solution with the Respimat® over two weeks. The PK analyses were performed using NONMEM 7.2.0 and R 2.14.2. As a first step, a systemic PK disposition model was developed with IV data only. Subsequently this IV model was assumed constant for further analyses to

assess pulmonary processes independent of systemic distribution characteris-tics. As a second step, various absorption models were investigated to characterise the absorption process. Oral bioavailability of swallowed drug was considered negligible due to virtually no bioavailability of olodaterol after oral dosing. Results: A PK model that characterised the pulmonary absorption of olodaterol with three parallel absorption processes best described the clinical data after drug inhalation. The three pulmonary absorption processes were characterised by different first-order absorption rate constants (ka) and different fractions that were associated with one of the three ka values (fast, intermedi-ate, slow). Only a small fraction of 3.90% of the lung dose was fast absorbed with an absorption half-life of 18.9 min and 8.35 min in non-smokers and smokers, respectively. This fast absorbed fraction mainly contributed to the early maximum plasma concentration. The slowly absorbed fraction of 74.8% was absorbed with an absorption half-life of 27.1 h and mainly contributed to the terminal shape of the plasma concentration-time profiles after drug inhalation and also contributed to higher plasma trough concentrations after multiple dose inhalation compared to trough concentrations after single dose inhalation. The lung dose was estimated with 51.7% of the nominal dose with a wide range of individual estimates ranging from 21.5% to 83.5%. Discussion/Conclusions: The PK characteristics of inhaled and intravenously administered olodaterol solution were successfully described by a population PK model with three parallel absorption processes. The determined lung dose of approximately 50% was in agreement with in vitro deposition data indicating a lung dose of 67% [3]. The large range of individual estimates for the lung dose emphasised the high variability of inhaled drugs. Although in vitro assays and deposition models indicated that the Respimat has a pronounced deposition in the peripheral airways and the alveoli [3], where absorption of dissolved drug is assumed to be fast, an unexpected small fast absorbed fraction of the lung dose was determined. In addition it was unexpected that a large fraction of the dissolved drug was slowly absorbed to the plasma with a half-life of approximately one day. Both findings raised the question if the frequently used assumption of fast absorption of dissolved drug especially in the alveolar space holds true. For inhaled olodaterol, the results indicate that additional aspects than the pulmonary deposition patterns determine the absorption characteristics. One mechanistically plausible aspect for olodaterol might be lysosomal trapping.

References: 1. Ruge, C., Kirch, J., Lehr, C.: Lancet Respir Med. 2013, 1(5): 402-413 2. Patton, J.S., Byron, P.R.: Nat Rev Drug Discov. 2007, 53(1):67–74. 3. Ciciliani, A.M., Wachte, l.H., Langguth, P.: Respiratory Drug Delivery 2014, 2: 453-456.

For abstract see short lectures SL.12.

Structural Dynamics of Liposomal Amphotericin B in FaSSIF by combined SANS and DLS Johnson, R.1a; Khoshakhlagh, P.1a; Nawroth, T.1a; Langguth, P.1a; Schmueser, L.1b; Decker, H.1b; Hellmann, N.1b; Szekely, N.2 1 Gutenberg-University, a) Pharmacy; b) Molecular Biophysics; Staudingerweg 5, D-55099 Mainz 2 JCNS Outstation at FRM II, Instrument KWS2, Lichtenbergstr.1, D-85747 Garching

Amphotericin B remains the drug of choice for the treatment of systemic fungal infections. However, it is still administered parenterally due to its poor solubility and permeability (BCS IV) as well as instability in gastric fluid. Current research is directed towards the development of an appropriate oral dosage form with improved aqueous solubility and intestinal permeability. Particle size plays a crucial role in the solubilisation and uptake of drugs in the gastrointes-tinal tract. The release of bile acids into the duodenum influences the size and structural development of dosage forms thus affecting the extent to which drugs permeate the mucosa of the small intestines(1). Additionally, intestinal lymphatic transport has been considered a minor pathway of drug absorption except for highly lipophilic molecules and other lipoidal molecules. For these molecules, of which Amphotericin B is part, the intestinal lymphatics represent

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an alternate route to the portal blood by which they can gain access to the systemic circulation [2]. With this insight, small unilamellar vesicles of amphotericin B were prepared by the film method, swelling, vortexing and subsequent sonication using DOPC with and without cholesterol. The nanoparticle size (100 nm (MLV), 30 nm (SUV)) and structure were monitored by dynamic light scattering DLS and neutron small angle scattering SANS. The resolution and kinetics of Amphoter-icin B nanoparticles in gastrointestinal model fluid (FaSSIF) was estimated with stopped flow technology for SANS and DLS in a simulator of the human gastrointestinal system GIMod [3]. According to SANS (fig.1) the sonified PC-Cholesterol-AmB liposomes (SUV) were unilamellar depicting a size of 34 nm and evidence of a lateral phase separation. This investigation gives an insight into whether the drug would be in an uptake competent form for efficient permeation.

Fig.1: Neutron small angle scattering SANS of liposomes (SUV) from lecithin (DOPC), cholesterol and Amphoter-icin B. The evaluation by a Guinier plot above yields a radius of Gyration Rg = 14.51 ± 0.07 nm; i.e a size of s = 34.02 ± 0.2 nm (with a membrane span d = 5 nm).

Acknowledgements: We are grateful for the funding by the German ministry of science and education BMBF, grant 05KS7UMA, The Forschungszentrum Jülich FZJ, outstation at the MLZ, FRM2 reactor Munich Garching, the government of Ghana and the DAAD. This work was contributed to the OrBiTo project.

References: 1. Dangi, J.S. et al.: Drug Dev. Ind. Pharm. 1995, 21 (17):2021-2027. 2. Charman, W.N. et al.: Adv. Drug Dev. Rev. 1991, 7: 1-14. 3. Nawroth, T.et al.: Mol. Pharm. 2011, 8(6), 2162-2172.

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PHARMACEUTICAL TECHNOLOGY AND DRUG DELIVERY (PT01-PT34)

Advanced Formulation for Solid Crystal Suspensions Reaching Supersaturation Reitz, E.; Thommes, M.

Institute of Pharmaceutics and Biopharmaceutics, University of Duesseldorf, Universi-taetsstrasse 1, 40225 Duesseldorf, Germany

Solid dispersions are one approach to enhance the dissolution rate of poorly water soluble drugs. These systems are frequently characterised by a physical instability, which is not found in terms of solid crystal suspensions. Thereby a crystalline drug is dispersed in a crystalline carrier, consisting of a sugar alcohol mixture. In this study sodium lauryl sulphate (SLS) added as solubilizer to the formulation in hot melt extrusion process using different quantities between 0.1 and 10 %. The final tablets were characterized with respect to its solid state properties by powder x-ray diffraction (XRPD), differential scanning calorimetry (DSC), dissolution and confocal laser scanning microscopy (CLSM). The solid crystal suspensions were prepared by hot melt extrusion (Leistritz Micro GL 27 – 28D). The tableting was performed on a rotary die press (IMA pressima) using the tableting mixture introduced by Reitz et al. (2014). The extrudate as well as the tablets were characterized by DSC (DSC 1, Mettler Toledo) and by XRPD (X’Pert Pro, Panalytical). Drug dissolution was determined in accordance to the Ph.Eur. using a paddle apparatus and water as dissolution media. In CLSM the drug (405 nm, griseofulvin) and carrier (559 nm, sugar alcohol mixture) were excited specifically and the fluorescence were detected at 436-486 nm (griseofulvin) and 573-633 nm (sugar alcohol mixture). The solid state investigation by XRPD and DSC indicate no amophization or polymorphic change of the drug in the formulations, which is consistent with the concept of solid crystal suspensions. However supersaturating was obtained in dissolution studies and also found after storage for several weeks in stability tests (figure 1). These observations were unexpected because it is in contrast to the general concepts of solid crystal suspension. Therefore, different reasons were investigated. The formulations containing SLS showed a higher dispersity, found in CLSM investigations (figure 2). Presumably, the SLS supports a drug desagglomeration in the carrier melt during extrusion at levitated temperatures. In conclusion a physically stable solid dispersion was prepared allowing supersaturation during dissolution. This could be a general approach for several drugs with low aqueous solubility.

Figure 1: Drug release of tablets containing a SCS before and after storage of 6 weeks (av ± CI, n = 6).

Figure 2: CLSM picture of the surfactant free (left) and surfactant containing extrudate (right).

Reference: 1. Reitz, E. et al.: Pharm. Ind., 2014, 76(2): 286-296.


Cationic nanoparticles as a promising drug delivery system? In detail characterization of surface properties Gossmann, R.1; Mulac, D.1; Hummel, M.2; Brockmeyer, J.2; Langer, K.1

1 Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Corrensstr. 48, 48149 Muenster, Germany 2 Institute of Food Chemistry, University of Muenster, Corrensstr. 45, 48149 Muenster, Germany

The preparation of cationic nanoparticles for intracellular drug delivery enters the focus of many research groups, because of their promising increased cellular uptake based on the electrostatic interaction between the cationic surface and the negatively charged lipid membrane [1]. The aim of the present study was to establish an analytical characterization of cationic didodecyldime-thylammonium bromide (DMAB) stabilized poly(lactic-co-glycolic acid) (PLGA) nanoparticles based on examination of size, zeta potential, electrolyte sensitivity, and cellular uptake. The preparation of DMAB-stabilized nanoparticles was performed using emulsion-diffusion method leading to nanoparticles with a diameter of approximately 80 nm, a PDI below 0.1, and a positive zeta potential of about +50 mV [2]. Modification of the nanoparticles was performed by altering the surface using the hydrophilic polymers poly(vinyl alcohol) (PVA) and polyeth-ylene glycol (PEG). The DMAB-stabilized nanoparticles appeared to be sensitive to electrolyte influence due to compression of the electrical double layer in conjunction with a decrease in zeta potential. This resulted in particle agglomeration which was shown by increased size and PDI. In contrast the modifications with PVA or PEG enabled steric stabilization and avoided agglomeration. Uptake studies performed with Caco-2 cells showed that compared to negatively charged nanoparticles both modified nanoparticle systems were taken up more effectively, confirming the expected effect of cell mebrane interaction. In addition to the characterization of the particle system and cellular uptake, the identification of the protein corona, which occurs by applying the nanoparti-cles into biological systems, was a special focus of this research project. Identification of adsorbed serum proteins was performed by in solution digestion and subsequent high resolution LC-MS analysis. The adsorbed protein corona gives the nanoparticles a kind of new biological identity, which determines the physiological response including agglomeration, cellular uptake, circulation lifetime or toxicity [3]. In conclusion this study offers a closer and critical point of view in preparation, in-vitro and analytical evaluation of DMAB-stabilized nanoparticles for the physiological use. Furthermore this work succeed in identifying the adsorbed serum proteins, which could give a first hint on possible physiological response.

References: 1. Xu, A., et al.: Int. J. Nanomedicine 2012, 7: 3547–3554. 2. Bhardwaj, V. et al.: Pharm Res 2009, 26(11): 2495–2503. 3. Rahman, et al.: Protein-Nanoparticle Interactions (Springer Berlin Heidelberg) 2013.

The Effect of Different Salts on the Disintegration Properties of Enteric-Coated Soft Gelatin Capsules Al-Gousous, J.1; Penning, M.2; Langguth, P.1

1 Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany 2 Pennconsult, Mainz, Germany

The purpose of this investigation was to characterize the effect of using different salts of shellac on the properties of enteric-coated soft gelatin capsules. Oval size 4 placebo capsules were coated with four shellac-based enteric coating solution formulations differing from each other by the salt of shellac used. One formulation was based on an ammonium salt, one the sodium salt, one on the potassium salt and one on a composite ammonium-sodium salts containing ammonium and sodium ions in a 1:1 mole:mole ratio.

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The coating was performed to the same level for all formulations using a Glatt® GC-300 drum coater (Glatt, Germany). Disintegration testing was performed according to the European Pharmacopoeia (Ph Eur) and disintegration times in the pH 6.8 buffer were noted. The disintegration testing results have shown that alkali metal salts promote faster disintegration compared to ammonium salts. The reasons behind this phenomenon were investigated by comparing the solubility of dried films of the different shellac salts and measuring their FTIR, NMR, DSC and MALDI-TOF spectra. It was shown that ammonium-containing films are water-insoluble while alkali metal salt films are water-soluble which can explain the differences in the disintegration behaviour. The explanation behind that can be offered by the spectral findings which suggest that, in the presence of ammonium, the degree of ionization of the shellac carboxyls is lesser due to the protonation of the carboxylate ions by the ammonium ions and loss of ammonia during drying. In addition, in the presence of ammonium ions, oxidation of the shellac’s aldehyde groups into carboxylic acid groups was promoted leading to stronger solute-solute interactions. In addition, the potentially greater extent of the partial hydrolysis of the high molecular weight fractions of shellac in the case of sodium and potassium salts may be a contributing factor. Therefore, the choice of the salt used to prepare a shellac-based enteric coating solution is an important formulation parameter that can affect the release properties of the product. Acknowledgments: We would like to thank the German Academic Exchange Service (DAAD) for their support, Catalent Pharma Solutions for providing us with the placebo capsules, the group of Professor M. Karas at University of Frankfurt for performing the MALDI-TOF measurements, and the group of Professor T. Schirmeister at University of Mainz for performing the NMR measurements.

Hyperbranched thermosensitive polyglycerol nanogels – new tools for efficient skin delivery Obst, K.1; Witting, M.2; Soler, M.M.3; Friess, W.2; Calderon, M.3; Küchler, S.1 1 Institute for Pharmaceutical Sciences, Freie Universität Berlin, 14195 Berlin, Germany 2 Department of Pharmaceutical Sciences, Ludwig-Maximilians-Universität, 80539 Munich, Germany 3 Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany

To enhance the efficiency and to reduce systemic side effects, new delivery systems for topical applications particularly for biomacromolecules such as proteins are needed. In this study, we investigated the suitability of hyper-branched polyglycerol (hPG) based nanogels as new potential protein delivery system [1, 2]. Thermosensitive (TS) polyglycerol based nanogels are able to maintain the stability of proteins and allow for a triggered drug release at temperatures higher than 32°C [1, 2]. To assess the efficacy of the nanogels for topical drug delivery, we studied the particle properties, protein release and activity as well as skin absorption in reconstructed skin models using the model proteins bovine serum albumin and L-asparaginase. For characterisation, the particle size and dispersity were measured by dynamic light scattering (DLS) applying a temperature ramp from 25 – 40°C (1 C°/min). In addition, the protein release was evaluated by size exclusion HPLC. To determine the enzymatic activity of L-asparaginase after loading and release, a specific asparaginase activity assay was performed [3]. To assess the dermal drug delivery efficiency, we applied asparaginase-loaded nanogels onto reconstructed skin models (normal and barrier-impaired) [4] applying a temperature ramp from 32°C to 37°C for 3 hours. Subsequently, the protein amount in the epidermis was determined with an asparaginase activity assay or skin constructs were embedded in tissue freezing medium, cryosections were prepared and immunostaining with a monoclonal antibody against asparaginase was performed. The particle size of BSA-loaded TS hPG nanogel was 207 ± 12 nm (PDI 0.5). Following temperature increase to 34°C – 35 °C, the TS hPG-nanogel exhibited a sharp decrease in particle size to 170 ± 3 nm (PDI 0.1), but the nanogels remained unchanged at temperatures below the trigger point of 34°C. At temperatures ≥ 35°C the TS hPG nanogel instantly released 85% of the protein payload. Furthermore, the activity of L-asparaginase was preserved after loading and release (specific activity 93.2 ± 4.6 %). In terms of protein delivery efficiency, skin absorption studies in reconstructed skin models showed significantly enhanced amounts of asparaginase (9.5 ± 0.1 µg) in the epidermal layers of the skin models following the application of protein-loaded nanogels. In contrast, the application of the aqueous asparagi-nase solution resulted in significantly lower amounts (6.3 ± 0.1 µg). Furthermore, we studied the penetration of L-asparaginase into normal and barrier-deficient skin constructs by immunohistochemistry. Data analysis

showed significantly enhanced skin absorption of L-asparaginase in the viable epidermis after application of the thermoresponsive nanogels. This effect was most pronounced in barrier-deficient skin models. The application of the control solution did not result in dermal penetration of L-asparaginase. In conclusion, our data indicate that thermosensitive hPG-nanogels are suitable and promising carrier systems for labile drugs such as biomacromole-cules. Despite harsh chemical conditions efficient protein encapsulation and release from the hPG nanogels were achieved with good preservation of protein activity. Furthermore, our results clearly indicate skin penetration enhancing effects which are most pronounced in barrier-impaired skin. In the next step, the model protein will be replaced by the therapeutic protein transglutaminase-1 in order to provide the proof-of-concept that topical protein substitution might be an interesting therapeutic approach for the treatment of severe skin diseases. Acknowledgments: This work was supported by a grant from the German Research Foundation (DFG; KU 2904/2-1 S.K.).

References: 1. Calderón et al.: Journal of Controlled Release 2011, 151: 295-230 2. Cuggino et al. Soft Matter 2011, 7:11259-11266. 3. Mashburn, L.T. and Wriston, J.C.: BBRC 1963, 12(1): 50-55. 4. Küchler et al.: ATLA 2011, 39:471-480.

Preparation and characterization of solid adsorbates based on semisolid SNEDDS Hassan, T.H.; Metz, H.; Mäder, K.

Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, Martin Luther University, Wolfgang-Langenbeckstr. 4, D-06120 Halle (Saale), Germany.

Self-nanoemulsifying drug delivery systems (SNEDDS) have created considerable interest and ultimately therapeutical and commercial success in the oral delivery of poorly water-soluble drugs. They provide the drug in the form of solubilized nanodispersions. Accordingly, the rate-limiting step of drug dissolution is bypassed. However, SNEDDS are typically filled in soft gelatine capsules, which might cause the following problems: interaction with the capsule shell, instability, higher production cost and possible drug precipitation [1,2]. Therefore, alternative formulation strategies, e.g. the inclusion of SNEDDS into a solid dosage form are desirable; nevertheless, very challeng-ing. Neusilin® US2 (N-US2) is an amorphous, synthetic, neutral grade of magnesi-um aluminium-metasilicate. It occurs in nanoporous, ultra-light granules prepared by spray drying. N-US2 is non-toxic and do not form gel upon contact with aqueous fluids. Furthermore, N-US2 has an excellent flowability, very high specific surface area and good compressibility [3]. N-US2 was recently used to solidify liquid self-emulsifying systems [4,5]. Semisolid SNEDDS was incorporated (20 % - 90 %) in N-US2. The physical appearance and flow properties were evaluated. Differential scanning calorimetry (DSC) and benchtop nuclear magnetic resonance (BT-NMR) were used to study the interaction between the semisolid SNEDDS and the nanoporous carrier. Furthermore, the adsorbates were moulded or com-pressed into tablets and their dispersibility was evaluated in 0.1 N HCl and phosphate buffer pH 6.8 USP. In addition, Lumogen® F305 florescence dye was incorporated in the SNEDDS to help visualizing the release process. At least 40 % of N-US2 was required to obtain a freely flowable solid. At lower level, gummy solids to plastic pastes were obtained. DSC study showed a shift in the melting behaviour of the SNEDDS with the increase of N-US2 level. BT-NMR study showed a strong interaction between SNEDDS and N-US2 with the presence of different mobility states. Moulded adsorbates showed complete dispersibility within 2 - 3 hr, while compressed ones were non-dispersible. A disintegrant was required to provide a fine dispersion of the compressed adsorbates. Tablets with 10 % disintegrant gave a fine dispersion with good SNEDDS release.

Acknowledgments: We would like to thank the Egyptian Ministry of Higher Education and Scientific Research and the Deutscher Akademischer Austauschdienst (DAAD) for the financial support as a PhD scholarship to Mr Tamer H. Hassan, Assistant Lecturer, Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. We would like to thank Mrs. Kerstin Schwarz for the DSC measurements. Abitec Corporation, BASF AG, Croda GmbH, and Fuji Chemical Industry Co. Ltd are gratefully acknowledged for the excipient donation.

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References: 1. Pouton, C.W.: Eur. J. Pharm. Sci. 2000, 11: S93–S98. 2. Rahman, M.A., et al.: Drug Dev. Ind. Pharm. 2013, 39(1): 1–19. 3. Sander, C. and Holm, P.: AAPS PharmSciTech 2009, 10(4): 1388–1395. 4. Williams, H.D., et al.: J. Pharm. Sci. 2014, 103(6): 1734–1746. 5. Gumaste, S.G., Dalrymple, D.M. and Serajuddin, A.T.M.: Pharm. Res. 2013, 30(12): 3186–3199.


Small scale production of nanocrystals in drug discovery and development phase Romero, G.B.1; Keck, C.M.2; Müller, R.H.1 1 FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr.31, 12169 Berlin, Germany 2 Fachhochschule/University of Applied Sciences Kaiserslautern, Applied Pharmacy, Campus Pirmasens, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany

The process of drug discovery and development has increasingly become more costly and longer during the last decades. New drugs approvals are interrupted due to lack of efficacy, toxicity, poor absorption. Some of these issues are related to poor solubility and dissolution velocity, which can be overcome by nanonization [1]. However, during pre-formulation stage and late discovery, the new chemical moiety is normally available at very low amounts. Therefore, in this study, the miniaturization of nanocrystals production was evaluated using an accessible, cheap, simple approach. Milling was performed in a 2 mL glass vial and the grinding media was yttria stabilized zirconium oxide beads (sizes varying from 0.05 mm to 0.6 mm). The trick of the set up was the use of a triple arrangement of 3 magnetic stirrers (one above the other). Various model drug were used, e.g. cyclosporin A, ascorbyl palmitate, hesperidin. The coarse suspension was composed of 5% (w/w) drug powder and 1% (w/w) stabilizer. Stirring was performed on a magnetic stirring plate RCT basic (IKA-Werke GmbH & Co. KG, Germany) at 1,200 rpm. Particle size and polydispersity index (PI) were assessed by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instru-ments, UK) and light microscopy (LM). Samples were drawn from 1 to 120 hours. Nanonization of drug crystals can be industrially achieved by different techniques and different equipments. Some equipments on the market are able to produce nanosuspensions in a small scale, such as the Bühler PML-2 (150 g batch) or the APV LAB 40 homogenizer (40 mL batch). Even smaller batches can be obtained with the Nanomill System® (10 mL batch) or the Avestin EmulsiFlex-B3 (3.5 mL batch). However, this is still not sufficiently small to perform screening tests. Therefore, the batch size of nanosuspension in the present study was 0.5 g (25 mg of drug). For all tested drugs, particle size reduced as a function of time until it reached a lower size limit from where further milling either did not lead to particle size reduction or resulted in crystal growth or aggregation. It was also observed that the smaller the milling beads size, the more efficient particle size reduction occurred. The smallest PCS diameter was about 90 nm and polydispersity index was 0.14 for cyclosporin A after 72 hours milling (0.05 mm milling beads). Moreover, the weight of the magnet stirrers was assessed before and after milling and no corrosion was observed (unlike other setups in which corrosion occurred). Although very simple and unexpensive, this method can successfully produce sub-micron crystals and nanocrystals in the lower nanorange (<100 nm). This is an accessible method since it requires commonly used equipments found in every laboratory, unlike previously described methods which require specific, costly equipments. It allows the screening of different stabilizers/stabilizer concentrations, as well as production parameters i.e. milling beads size, using minute amounts of the drug. Acknowledgements: the authors would like to thank PhamaSol GmbH and the Brazilian ministry of education through the CAPES/DAAD doctoral program (Grant no. 12416/12-6) for R&D and financial support.

Reference: 1. Shegokar, R., Müller, R.H.: Int. J. Pharm. 2010, 399: 129-139.

Pig ear skin penetration of azithromycin nanocrystals for Lyme Borreliosis infection Jin, N.1; Staufenbiel, S.1; Keck, C.M.1,2; Müller, R.H.1 1 FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany 2 FH Kaiserslautern, Applied Pharmacy Division Campus Pirmasens, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany

For more than one century, many people in North America and Europe are predisposed to Lyme Borreliosis infection after tick bite. Antibiotic treatment has proven to be effective in clearing the infection when used early. However, bacterial resistance to administered antibiotic or malaise and toxicity has to be considered when using antibiotics systematically. These side effects can be reduced when drug concentrations are kept at a minimum and the antibiotic is administrated dermally. Azithromycin is preferred in this study due to its lower minimum inhibitory concentration, fewer adverse effects and quicker onset time compared to other popular antibiotic agents. Considering the difficult diagnosis in the early stage of Lyme Borreliosis and serious clinical signs in its late stage, quick initiation of a dermal azithromycin treatment after tick exposure could pave the way for preventing Lyme Borreliosis. Even though there is a dermal formulation, applying ethanol to dissolve the raw drug powder [1], to overcome solubility problems, according to Fick’s first law, nanocrystals are assumed to have improved skin penetration. Thus the object of this study was to develop a dermal gel with azithromycin nanocrystals.

10% azithromycin nanosuspension was produced by bead milling (Bühler; PML 2), stabilized with 1% tocopheryl polyethylene glycol succinate (TPGS). The saturation solubility of the nanosuspension compared to raw drug powder formulation including TPGS was determined in water by shaking for 8 hours in vials. The obtained concentrations of dissolved drug were determined by HPLC. Nanocrystals and raw drug powder with 0.5% TPGS was incorporated into 5% hydroxypropylcellulose (HPC) to get 5% azithromycin-nanocrystal gel and 5% azithromycin-raw drug powder gel, respectively. 10% azithromycin-ethanol-solution gel (azithromycin raw drug powder 10%; (94%) ethanol 77.5%; polyacrylate 0.5%; HPC 5%; mygliol 7%) was selected as a compari-son which demonstrated effectiveness in clinical studies [1]. Subsequently a penetration study (n=3) via tape stripping was performed in the pig ear skin model and the AZ amount in the different strips was measured by HPLC.

Azithromycin nanocrystals with particle size of 189 nm (z-ave) were produced by bead milling in only 10 minutes. The nanosuspension had an about 2 times higher saturation solubility in water (227 µg/ml) compared to the raw drug powder. 5% azithromycin-nanocrystal gel showed higher penetration ability compared to raw drug powder, penetration was even higher than for the “ethanol-solution gel” with 10% azithromycin. This can be seen by considering e.g. the azithromycin amounts found in the strips of the 6th and the 7th layer: 10% ethanol solution gel (12.3 µg±2.2 / 10.7 µg±2.0), azithromycin-raw drug powder gel (19.9 µg±1.9 / 13.3 µg±2.4) and azithromycin-nanocrystal gel (47.8 µg± 4.3 / 27.3 µg± 4.9). This may be due to both the volatility of ethanol [2] leading to re-crystallization in the solution formulation, and increased saturation solubility of nanocrystals.

In summary, a 5% azithromycin-nanocrystal gel was developed in this study. It showed higher saturation solubility and higher penetration than 5% raw drug powder and penetration was even higher than the reported 10% ethanol azithromycin dermal formulation.

Acknowledgments: PharmaSol GmbH Berlin, Germany and China Scholarship Council.

References: 1. Knauer, J. et al.: J. Antimicrob. Chemother. 2011, 66 (12): 2814-2822. 2. Belsey, N.A.; Rojas, L.R.C.; Guy, R.H.: Clinical Dermatology (Springer Ber-lin/Heidelberg) 2014.

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Azithromycin nanocrystals for i.v. drug targeting – effect of stabilizers on surface hydrophobicity and protein corona Staufenbiel, S.; Jin, N.; Müller, R.H.

FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany

The use of nanocrystals as drug carriers for i.v. administration is a promising approach to overcome application problems of poorly soluble drugs, whereby adsorbed blood proteins determine their biodistribution. Beside others (e.g. size, shape, charge etc.) surface hydrophobicity is one of the key parameters determining the protein corona, whereby hydrophilic surfaces can lead to a decreased liver uptake due to a decreased protein (opsonin) adsorption and subsequently to an increased blood circulation [1]. Thus one can select nanocrystal coatings to reduce surface hydrophobicity. Besides analyzing hydrophobicity, additionally the protein adsorption pattern has to be analyzed, because the adsorption of some specific proteins can lead to drug targeting, e.g. into the brain. Thus it was the aim of this study to analyze hydrophobicity and protein corona from differently coated azithromycin nanocrystals, because cerebral infections are still challenging until today. Azithromycin nanosuspension (10% w/w) was produced by bead milling (PML 2, Bühler) with 4 different stabilizers (1% w/w; Poloxamer 188, Poloxamer 407, Tween 80, TPGS) leading to approx. 250 nm particles (photon correlation spectroscopy, Zetasizer Nano ZS). Surface hydrophobicity was determined by hydrophobic interaction chromatography (HIC) and two phase partitioning (TPP). HIC was performed using a C 10/20 column (Pharmacia Biotech) filled with octyl-agarose. Elution (2 ml/min) profiles were recorded by UV reading. TPP was performed by partitioning particle suspensions in a system containing a 12% (m/v) polyvinyl alcohol phase and a 16% (m/V) dextran phase. Furthermore, samples were incubated with pooled human plasma and total amount of adsorbed protein was determined with the bicinchoninic acid (BCA) assay. Subsequently, the protein adsorption pattern was analyzed by two-dimensional-polyacrylamide-gel-electrophoresis (2D-PAGE) and spots were quantified with the MELANIE software. All results are the mean of three independent measurements. The HIC elution time (peak maxima) from the TPGS, Tween 80 and Poloxamer 188 sample were close to the void volume (6.4 min, 6.5 min, 7.4 min). This indicates a low hydrophobicity whereby polypropylene glycol (PPG) blocks in the Poloxamer 188 sample lead to a slight increase in elution time. The Poloxamer 407 sample was even eluted at 18.7 min reflecting the increased PPG content. TPP results confirming these observations, because large amounts of those samples were found in the more hydrophilic dextran phase ([c dextran]/[c PVA] = 3.08; 1.03; 0.69 / TPGS; Tween 80; Poloxamer 188), whereby this ratio of the Poloxamer 407 sample was only 0.18. The TPGS sample seems to be even more hydrophilic here compared to HIC results, reflecting the fact that this is the only investigated stabilizer with only one polyethylene glycol (PEG) chain increasing dextran interactions due to decreased intra-molecular PEG/PEG interactions. Additionally the Tween 80 and the TPGS sample showed low BCA amounts (8 and 8.5 mg protein/g particles), whereby the Poloxamer samples had approx. 17 mg of total adsorbed protein, reflecting the avoiding of protein adsorption by hydrophilic surfaces, whereby also other parameters (like e.g. steric orientation of the stabilizer) influence protein adsorption. This can be seen from the Poloxamer 188 sample. 2D-PAGE results revealed that the Poloxamer samples had a high adsorption of the opsonin fibrinogen (about 50% of all detected protein) whereby apolipoprotein-AI (Apo-AI) adsorption was only about 10%. This was changed on the TPGS samples where fibrinogen was decreased to 37.4% and Apo-AI was increased to 31.8%. In contrast especially to the Poloxamer samples, the Tween 80 sample showed only 14.5% fibrinogen and 52.3% Apo-AI. In summary, azithromycin nanocrystals with different coatings were produced whereby HIC results were in accordance with the structures of these stabilizers (respectively with their HLB values) and these results were additionally confirmed and supplemented by TPP. The low surface hydrophobicity of the TPGS and Tween 80 samples was in agreement with a low amount of total adsorbed protein and simultaneously with a low amount of detected opsonins. This can lead to an increased systemic circulation. Additionally the Tween 80 sample showed a particularly high adsorption of Apo-AI, which opens the option for a certain brain targeting potential [2]. Acknowledgments: The authors would like to thank PharmaSol GmbH for financial and scientific support.

References: 1. Owens Iii, D.E. and N.A. Peppas: International Journal of Pharmaceutics, 2006, 307(1): p. 93-102. 2. Petri, B., et al.: Journal of Controlled Release, 2007, 117(1): p. 51-58.

ARTcrystal®-technology for nanocrystal production: Compari-son of continuous and discontinuous premilling step Scholz, P.1,2; Müller, R.H.2; Keck, C.M.1,2 1 University of Applied Sciences Kaiserslautern, Campus Pirmasens, Germany 2 Free University of Berlin, Germany

Introduction: The ARTcrystal®-technology is a combination process aiming for an economi-cally production of drug nanocrystals. It combines a high speed pre-milling step and high pressure homogenization (HPH) at reduced pressure and cycle numbers [1]. The pre-milling step is performed with an ART MICCRA D27 rotor-stator system, allowing a continuous high speed stirring, i.e. pre-milling, at up to 36,000 rpm. After pre-milling, the suspension is subjected to HPH at 300-500 bar for up to 5 cycles in order to form a nanosuspension [2]. Aim: In order to further understand and to optimize the pre-milling step, pre-milling was performed in continuous mode as well as in discontinuous mode. In a discontinuous process every particle is forced to pass the milling chamber while continuous processing allows faster processing, but turbulences in the product container can lead to an unequally number of passages of every particle through the milling chamber. Materials and Methods: Coarse material (rutin, Denk Ingredients, Munich, Germany) was used as testing material. Pre-milling was performed in an ART MICCRA D27 (ART Prozess- und Labortechnik, Müllheim, Germany) at 24,000 rpm for 30 minutes (continuous) or 100 cycles (discontinuous). Cooling at -10°C was provided. Particle size was analyzed by laser diffraction (Mastersizer 2000, Malvern, UK) and light microscopy (DM 1000, Leica, Wetzlar, Germany). Results and Discussion: The continuous process provided an efficient reduction in particle size. After only 5 minutes of processing a d(v) 0.50 of 3.74 µm and a d(v)0.99 of 26.51 µm was obtained. Further processing of up to 30 minutes led to a further diminution of the larger particles (d(v)0.50: 3.03 µm, d(v)0.99: 15.07 µm). Within the discontinuous process similar results to 5 minutes of processing in continuous mode where obtained after 20 cycles (d(v) 0.50: 3.47 µm, d(v)0.99: 20.81 µm). 60 cycles led to sizes similar to processing in continuous mode for 30 min (d(v)0.50: 3.09 µm, d(v)0.99: 15.52 µm). Further processing to up to 100 cycles did not lead to a further size reduction (d(v)0.50 3.02 µm, d(v)0.99 16.27 µm). Thus 60 cycles represent the processing condition at which the maximum dispersity of the material is reached. Since one cycle in discontinu-ous mode takes about 6 seconds, it can be followed that by discontinuous processing (60 cycles) the optimum could theoretically be reached after 6min, while this optimum was only reached after 30 minutes (i.e. about 300 passages) by continuous processing. Results, i.e. lower milling efficacy with continuous processing, can be explained by turbulences inside the product container, which lead to an inhomogeneous number of passages for each single particle. Conclusion: Both process modes lead to a similar dispersity of the material, either after 30 minutes (continuous mode) or after 60 cycles of processing (discontinuous mode). While processing in discontinuous mode was more effective, the processability in continuous mode is much better. Thus, further developments will focus on combining the advantages of each processing mode. References: 1. Keck, C.M., US20130095198 A1, 2011. 2. Scholz, P., Keck, C.M., ARTcrystal®-technology: Influence of starting material size on final particle size. DPhG - Tag der Pharmazie, July 4th, 2014, Berlin.

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Dermal delivery systems – nanoemulsion vs. nanostructured lipid carriers (NLC) Pyo, S.M.1; Meinke, M.C.2; Keck, C.M.3; Müller, R.H.1 1 FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr.31, 12169 Berlin, Germany 2 University Hospital Charité Berlin, Department of Dermatology, Venerology and Allergology, Charitéplatz 1, 10117 Berlin, Germany 3 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany

Couperosis is a hereditary skin disorder which is characterized by the weakness of the connective tissues. Topically applied vitamin A1 allows a causal therapy due to its new collagen forming properties. Many products on the market containing the active formed as an emulsion. NLC are a well-known dermal delivery system to improve the penetration of actives by forming an occlusive film, also called the “invisible patch” [1]. An ex vivo penetration study was performed to compare these delivery systems with the aim to identify the delivery system better suited for achieving higher concentrations in deeper skin layers. Vitamin A1 loaded NLC suspension (6.0 % retinol 50C, 6.0 % carnauba wax, 2.0 % Miranol Ultra 32, 86.0 % distilled water w/w) was produced by rotor-stator homogenization (30 s, 8,000 rpm) (Ultra-Turrax T25, Jahnke und Kunkel, Germany) followed by hot high pressure homogenization (HPH) (2 cycles, 800 bar, 85°C) (Micron LAB 40, APV, Germany). Vitamin A1 nanoemul-sion (NE) with same active agent and lipid content (6.0 % retinol 50C, 6.0% MCT, 2.0 % Miranol Ultra 32, 86.0 % distilled water w/w) was prepared as reference by HPH (1 cycle, 500 bar, 85°C). The particle size distributions were analysed and compared by laser diffraction analysis (LD, Mastersizer 2000, Malvern Instruments, UK). An ex vivo tape stripping test on pig ear skin was performed with tesa Film No. 5529 (Beiersdorf, Hamburg, Germany) after 20 and 60 minutes penetration time. Before starting the penetration study, the nanoemulsion showed LD diameter 95 % of 0.227, 99 % of 0.274 and 100 % of 0.345 µm. NLC suspension possessed similar particle size distribution with LD diameter 95 % of 0.210, 99 % of 0.250 and 100 % of 0.305 µm. The relative concentrations of the penetrated vitamin A1 from NE and NLC suspensions in respective layers of the stratum corneum (SC) were compared among each other (Table 1). After 20 minutes in the depth of 3 % of SC a high amount of 46 % of the active agent could be found from NE whereas just 3 % of active agent reached the same depth applied as NLC. Also in a depth of 12 % of the SC a higher relative concentration of vitamin A1 could be found from NE (4.7 %) than NLC (0.5 %). Table 1: The relative concentration of vitamin A1 at different depths of SC as a function of delivery system and penetration time.

afte

r

20 m

in. SC NE NLC

afte

r 60

min

. SC NE NLC

depth [%]

rel. c(A1)

[%]

rel. c(A1)

[%]

depth [%]

rel. c(A1)

[%]

rel. c(A1)

[%]

3 46.0 3.0 22 2.5 12.0

12 4.7 0.5 36 0.8 2.4

81 0.2 0.0 50 0.5 1.8

However, after 60 minutes penetration time, the concentrations of vitamin A1 in the layers of the SC show an obvious change of the penetration profiles for the benefit of NLC. In the depth of 22 % of the SC just 2.5 % of active could be detected from NE whereas 12 % (fivefold higher) reached the same depth with NLC. Considering the SC in the depth of 36 and 50 %, more than a three times higher concentration of active could be detected from NLC (2.4 and 1.8 %) compared to NE (0.8 and 0.5 %). The observed profiles can be explained by a faster release from the nanoemulsions (liquid lipid droplets) compared to the NLC (solid particle matrix). The occlusive effect of NLC takes some time to develop, which finally leads to higher penetration into the stratum corneum. In summary, NE provide faster initial release, NLC act as prolonged delivery systems with better penetration. Reference: 1. Müller, R.M. et al.: Euro Cosmet. 2013, 6: 20-22.

Glass transition temperature of poly(D,L-lactic-co-glycolic acid) nanoparticles Lappe, S.; Langer, K. Institute of Pharmaceutical Technology and Biopharmacy, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany

Nanoparticles based on poly(D,L-lactic-co-glycolic acid) (PLGA) and stabilised with poly(vinyl alcohol) (PVA) are widely used in pharmaceutical research [1,2], but little is known about the glass transition temperature of the resulting polymer matrix and the possible influences on this. The glass transition temperature Tg is a characteristic of amorphous polymers like PLGA. Within this temperature range the material changes from being hard and brittle to a more soft and reactive manner. For Resomer® RG 502 H, the PLGA polymer used in this study, depending on the measurement conditions Tg is between 38°C and 45°C. This temperature is easily achieved during particle preparation by homogenisation or subsequent purification of the nanoparticles. The aim of this study is to examine the reasons for the differences of glass transition of pure PLGA (38.6 ± 1.6°C), freeze dried PLGA nanoparticles (35.6 ± 3.4°C), and PLGA nanoparticles in suspension (32.2 ± 0.9°C). For examination of the glass transition temperature a differential scanning calorimetry (DSC) method was established. In order to exclude the influence of the ingredients a physical mixture of PLGA, PVA, and mannitol (used in the freeze drying process for stabilisation) as well as a solid solution of these substances were tested. Neither the presence of the stabiliser nor an influence of different preparation methods (emulsion diffusion method, solvent displacement) could be observed. But when preparing the nanoparticles with emulsion diffusion evaporation method using a rotary evaporator a shift of Tg to lower temperatures was observed. This was probably due to residual organic solvent in the nanoparticle formulation. In all yet mentioned cases PVA was used in one concentration, expecting that other concentrations of the polymer might have an influence on Tg, which couldn´t be confirmed. Residual moisture content might explain the differences between the values for glass transition of pure PLGA, freeze dried PLGA nanoparticles, and PLGA nanoparticles in suspension, respectively. Blasi et al. [3] confirmed the existence of different kinds of water whereat non-freezable water is closely associated to the polymer chains and lowers the Tg. This observation could be confirmed, with the result that for freeze dried nanoparticles measured under moisture displacing conditions a glass transition temperature of 47.0 ± 0.7°C could be achieved.

References: 1. Vandervoort, J., Ludwig, A.: Int. J. Pharm. 2002, 238(1-2): 77-92. 2. Mundargi, R.C. et al.: J. Control. Release 2008, 125(3): 193-209. 3. Blasi, P. et al.: J. Control. Release 2005, 108(1): 1-9.

Strategies to improve colloidal stability of lysozyme-loaded nanoparticles Thoma, F.; Langer, K.

Institute of Pharmaceutical Technology and Biopharmacy, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany

Proteins become more and more important for the treatment of several diseases. Due to the high advantages of nanoparticles used as drug carrier, a focus of current research is on the entrapment of therapeutic proteins into nanoparticles.

The aim of our work was the entrapment of model compound lysozyme, a small protein of 14 kDa, into a nanoparticle system based on poly(lactic-co-glycolic acid) (PLGA) by solvent displacement method [1].

During nanoparticle preparation the characteristics of lysozyme caused problems in nanoparticle stability. Lysozyme exhibits a comparatively high isoelectic point (IEP), which results in a positively charged model protein under the conditions of PLGA nanoparticle preparation. This led to a high protein adsorption on PLGA nanoparticles. Hence, before purification lysozyme-loaded PLGA nanoparticles showed a positive zetapotential, that changed to negativ

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values during particle purification. This was due to a removal of adsorptive bound lysozyme during the purification procedure. Passing the point of neutral surface charge induced stability problems of the colloidal system. Additionally, the adsorptive binding of the positively charged lysozyme to a negatively charged PLGA-matrix is responsible for ionic interactions which results in strong particle agglomeration and sedimentation.

Within the study we developed different strategies for stabilization of this drug delivery system during preparation and purification, like different steric stabilisators, buffers, and coatings.

Reference: 1. Niu et al.: Drug Dev. Ind. Pharm. 2009, 35(11): 1375-1383.

Nanocapsules - Is the preparation of core-shell structured nanosystems that simple? Wessels, L.1; Tacke, S.2; Mulac, D.1; Langer, K.1 1 Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster , Corrensstr. 48, 48149 Muenster 2 Institute of Medical Physics and Biophysics, University of Muenster, Robert-Koch-Str. 31, 48149 Muenster

Nanocapsules seem to have many advantages as a drug delivery system in comparison to nanospheres. The polymeric shell shields the active pharma-ceutical ingredient (API) from degradation as well as it protects tissue from hazardous effects of the API. Another advantage over nanospheres is the high drug encapsulation efficiency due to optimized solubility of the API in the core material in contrast to low polymer content [1]. There are many publications linked with the keyword nanocapsule, which describe different methods for nanocapsule preparation [1] but often without any differentiation between spheres and capsules and without a confident proof of a core-shell structured particle. Furthermore, some groups use the terms nanocapsules and nanospheres interchangeably. We investigated solvent displacement method (nanoprecipitation method) to gain an oily core nanocapsule formulation [2] and double emulsification method to build nanocapsules with an aqueous core [3]. The resulting particle systems were analyzed by dynamic light scattering (DLS), scanning electron microscopy (SEM), and cryo- scanning transmission electron microscopy (cryo-STEM) for size, size distribution, and morphology. Nanocapsules could be identified in the samples by the analytical methods used. However in most cases we observed that the multitude of particles was in shape of nanospheres instead of capsules. Therefore we conclude that a quantitative formation of nanocapsules by the methods described in literature is difficult to obtain.

The authors want to acknowledge Ulrike Keller (Institute of Medical Physics and Biophysics, University of Muenster, Robert-Koch-Str. 31, 48149 Muenster) for her skilful supportive SEM measurements.

References: 1. Mora-Huertas, C.E.; Fessi, H.; Elaissari, A.: Int. J. Pharm. 2010, 385 (1-2): 113-142. 2. Rübe, A. et al.: J. Control Release 2005, 107 (2): 244-252. 3. Zhu, Y. et al.: J. Surfactants Deterg 2005, 8 (4): 353-358.

Mucoadhesive polymyxin B-dexamethasone acetate nanocrys-tals for ocular delivery Romero, G.B.1; Keck, C.M.2; Müller, R.H.1; Bou-Chacra, N.A.3 1 FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr.31, 12169 Berlin, Germany 2 Fachhochschule/University of Applied Sciences Kaiserslautern, Applied Pharmacy, Campus Pirmasens, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany 3 University of Sao Paulo, Faculdade de Ciências Farmacêuticas, Departamento de Farmácia, Av. Lineu Prestes 580 - Bloco 15, Caixa-Postal: 6608, 05508-900 Sao Paulo, Brazil

The conventional ophthalmic preparations have limited residence time in the ocular region as a result of the protective mechanisms of the eye. In addition, the cornea acts as a barrier contributing to the reduced concentration of the drug in the vision organ. As a consequence, ophthalmic products show poor bioavailability. These limitations require patient adherence to treatment regimen with multiple administrations per day and substantial loss of the instilled material. These drawbacks can be overcome by nanonization of the drug powder, resulting in drug nanocrystals with increased saturation solubility, dissolution velocity and additionally, increased mucoadhesion, which is specially desired for this application site [1]. In this study, the production of positively charged nanocrystals of dexamethasone acetate in combination with polymyxin B was evaluated. The production method was a super small scale bead milling performed in a 2 mL glass vial with 0.05 mm yttria stabilized zirconium oxide beads as the grinding media. The coarse suspension was composed of 5% (w/w) dexame-thasone acetate, 1% (w/w) stabilizer and optionally 1% polymyxin B. Stirring was performed on a magnetic stirring plate RCT basic (IKA-Werke GmbH & Co. KG, Germany) at 1,200 rpm during 24 hours. Subsequently, the 5% concentrated suspension was diluted to the final desired dexamethasone acetate and polymyxin B concentration. Particle size and polydispersity index (PI) were assessed by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, UK) and light microscopy (LM). The conjunctiva and the cornea are protected by the tear film by secreting mucin, electrolytes and fluid. Mucin, the mucus layer that coats the corneal surface, is negatively charged. Therefore, the ideal carrier system for the eye would be a cationic particle with high adhesion to the mucosa. The electrostat-ic interaction between the opposite charges of the mucin and the cationic nanocrystals would allow adequate concentration of drug at the site of action. This strategy can dramatically improve the drug ocular bioavailability. Polymyxin B as the only stabilizer resulted in positively charged nanocrystals with zeta potential of + 11 mV (in original medium) after 24 hours milling, but the stabilization provided only by polymyxin B was not sufficient to avoid agglomeration, which could be detected by PCS and light microscopy. Dilution of this concentrate with traditional nonionic stabilizers at a concentration of 0.1% allowed the disintegration of the agglomerates and revealed nanocrystals of 491 nm and 687 nm for poloxamer 188 and Tween® 80, respectively. However, after dilution, the zeta potential reduced to around 0 mV for both stabilizers, which is not desired. In contrast, when the nanosuspensions were first produced using a cationic stabilizer such as cetylpyridinium chloride or benzalkonium chloride and subsequently diluted with polymyxin B solution, the final zeta potential had a positive value of around +30 mV (in original medium). Also, for this case, the particle size remained in the sub-micron range, being 130 nm and 176 nm for the nanosuspensions stabilized with cetylpyridinium chloride and benzalkonium chloride, respectively. This is an elegant strategy to stabilize such particles, since these two molecules are preservatives, which are added to a multi-dose topical ophthalmic preparation to prevent the growth of, or to destroy the microorganisms introduced inadvertently during the treatment interval. Positively charged nanocrystals with increased mucoadhesion have been successfully produced. These formulations have the potential to overcome the drawbacks of the conventional ophthalmic preparations providing an innovative ocular drug delivery system by increasing their therapeutic efficacy and safety. Polymyxin B-dexamethasone acetate nanocrystals can be used to treat superficial infections such as conjunctivitis, keratoconjunctivitis and bacterial blepharitis with improved performance and patient compliance compared to standard formulations. Acknowledgements: the authors would like to thank PhamaSol GmbH and the Brazilian ministry of education through the CAPES/DAAD doctoral program (Grant no. 12416/12-6) for R&D and financial support. References: 1. Shegokar, R., Müller, R.H.: Int. J. Pharm. 2010, 399: 129-139.

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Tetrahydrocannabinol-loaded NLC for nasal drug delivery against break-through pain in cancer patients Hommoss, G.; Keck, C.M.; Zhai, X.; Müller, R.H.

Institute of Pharmacy - Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmet-ics, Kelchstr. 31, 12169 Berlin, Germany

Tetrahydrocannabinol (THC) is a lipophilic molecule, binding non-specifically to a variety of receptors in the brain and body with analgesic medical property. For treating permanent pain in cancer patients, regularly fixed doses of drug are used. But administration of additional dose for treating the high peaks pain would be a hyper medication. Fast onset of action and easy administration is highly desired to cover these peaks. Nasal drug delivery systems as spray or aerosols based on nanoparticles are ideal due to their fast drug release and can be designed to provide adhesion to the nasal mucosa. Nanostructured lipid carriers (NLC) loaded with THC were produced for the development of a nasal delivery system. Due to the lipophilic property of THC, it is highly suitable to be incorporate into a lipid matrix. Therefore the solubili-ty/miscibility of various lipids with oily THC was investigated. Compritol ATO 888 and cetyl palmitate were identified as optimal lipids. THC-loaded NLC formulated with 1.0 % lipid blend (0.125 % THC and 0.875 % lipid), 0.05 % surfactant and water up to 100 % were produced by hot high pressure homogenization using a Micron LAB 40 homogenizer (APV Deutschland, Germany). The produced THC-loaded NLC were characterized for particle size by photon correlation spectroscopy (PCS, Zetasizer Nano ZS) and laser diffraction (LD, Mastersizer 2000, both from Malvern Instruments, UK). Zeta potential was measured in conductivity adjusted water (50 µS/cm) by using again Zetasizer Nano ZS. First a screening was performed producing unloaded NLC to save costly THC material. Screened was for optimal composition and production parameters. With about 300 nm, cetyl palmitate yielded distinctly smaller NLC than Compritiol ATO 888 and it was chosen as optimal solid lipid. Cetylpyridinium chloride proved being superior to benzalkonium chloride, yielding NLC being about 100 nm smaller. Final production parameters were only 1 homogeniza-tion cycle at 800 bar, being industrially friendly. Loading with THC decreased the particle size of the NLC. A small particle size of about 220 nm was obtained, no large particles or agglomerations were observed. Cetylpyridinium chloride as cationic surfactant reversed the negative charge of the NLC; the zeta potential was close to +50 mV. First this high zeta potential provides sufficient physical stability in long-term storage and during the spraying process in application to the nose. In addition it increases adhesion to the negatively charged mucosa. Spray-ability test was performed using a commercial spray nasal bottle and the PARI BOY inhalator. The particle size of the THC-loaded NLC changed little after spraying with both delivery systems. The particle size was 280 nm after spraying from the nasal spray bottle and 247 nm after nebulizing from the PARI BOY (n=3), indicating good physical stability of produced THC-loaded NLC. Presently the formulation is in a long-term stability study at different temperatures.

Characterisation of PEGylated nanoparticles comparing the nanoparticle bulk to the particle surface using UV-vis spectros-copy, SEC, 1H NMR spectroscopy and X-ray photoelectron spectroscopy Spek, S.1; Haeuser, M.1; Schaefer, M.2; Langer, K.1 1 Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Corrensstraße 48, 48149 Muenster, Germany 2 nanoAnalytics, Heisenbergstraße 11, 48149 Muenster, Germany

Nanoparticles prepared of poly(lactic-co-glycolic acid) (PLGA) were assembled using an emulsion-evaporation method. Different mixtures of PLGA and PEGylated PLGA polymer were used for particle preparation and led to nanoparticles containing up to 15 wt.% PEG. For all polymeric mixtures the preparation technique required the use of polyvinyl alcohol (PVA) as a stabilising agent. Overall, 4 nanoparticle species of different PEGylation degree, each of about 200 nm in diameter, were characterised with respect to residual PVA and PEG content. This work focusses on the quantitative determination of all utilised polymeric compounds using different analytical

approaches, comparing the polymer content of the particle bulk to the particle surface. For polymer quantification of the nanoparticle bulk, PVA was determined using a historically well-established UV-vis spectroscopic method [1] as well as size exclusion chromatography (SEC), and 1H nuclear magnetic resonance (NMR) spectroscopy. The need for different PVA quantification methods was due to the fact that the photometric quantification of a coloured PVA complex using Lugol’s solution and boric acid cannot be analysed as soon as PEG was present in the nanoparticle sample. This was due to the fact that PEG itself forms a water insoluble complex with the chemicals used. In this work, all three quantification methods for PVA were compared to each other under standard-ised conditions. Additionally, PEG determination of the nanoparticle bulk was carried out using quantitative 1H-NMR spectroscopy. The amount of PLGA in particle bulk was calculated indirectly by subtraction of PVA and PEG from the overall solids content. Furthermore, all prepared nanoparticle samples were investigated by X-ray photoelectron spectroscopy (XPS), giving reliable information about the relative polymer composition of the nanoparticle surface. In conclusion, the comparison of nanoparticle bulk to nanoparticle surface showed an increase of hydrophilic polymers at the surface with increasing PEGylation degree. Furthermore, interesting information about the arrangement of a PEG corona can be extracted from the data comparing the results of the nanoparticle surface and the nanoparticle bulk. Reference: 1. Pritchard, J.G., Akintola, D.A.: Talanta 1972, 19: 877-888.

“Nano-Curry” for improved health Rostamizadeh, K.1,2; Gerst, M.2,3; Scholz, P.2,3; Arntjen, A.2; Keck, C.M.3,4

1 Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran 2 Applied Pharmacy, University of Applied Sciences Kaiserslautern – Campus Pirmasens, Pirmasens, Germany 3 Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany 4 PharmaSol GmbH, Berlin, Germany

Introduction: Antioxidants are important to improve health as it has been shown in vivo and in vitro. Nowadays, isolated antioxidants are not recom-mended anymore, because they can also show pro-oxidative effects (e.g. isolated vitamin E). Thus natural antioxidants in natural combinations are of great interest. Curry is composed of several spices, possessing a high antioxidant capacity (AOC). However, at least some compounds of curry are poorly soluble, e.g. curcumin. Therefore, to further increase the antioxidant capacity, the solubility of curry should be increased. Thus, the production of nanocrystals which increase solubility might be a good strategy to increase the AOC of curry powder. Therefore the aim of this study was to produce nanosized curry powder and to compare its AOC to the original powder. Materials and Methods: Curry was purchased from the local supermarket and was homogenized by high pressure homogenization (HPH). The AOC was measured by a modified DPPH method [1]. Particle size was analyzed by photon correlation spectroscopy (PCS), laser diffractometry (LD), and light microscopy. Results: LD measurements indicated that the original powder possessed a large particle size of about 210µm (d(v) 50%). Light microscopy revealed that the original sample contained large crystals and needles. During the homoge-nization process, coarse curry microparticles were disintegrated into nanopar-ticles. Subsequently, the mean particle size decreased with an increase in homogenization cycles. The lowest mean particle size of the curry nanopowder obtained after 10 homogenization cycles at 1500 bar was 333 nm (PCS). Further homogenization to up to 20 cycles lead to a slight increase in particle size, probably due to heat increase. The AOC of original curry powder and the curry nanoparticles were determined via the DPPH method. The results revealed that nanosized curry possessed a much higher AOC than the original powder. In addition to the high value of AOC, the decrease in absorption was faster for the homogenized curry, when compared to the original curry powder. Conclusion: Nanosized curry could be obtained by high pressure homogeniza-tion. The decrease in particle size led to an increase in the AOC. Thus the production of “Nano-Curry” might be a successful strategy to further improve the health benefit of curry powder for either oral or dermal application. Further studies will focus on the development of an optimized production method to obtain optimized “Nano-Curry”.

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Acknowledgments: The authors are grateful for the financial support of this project by Germany academic exchange Service (DAAD).

References: 1. Pisoschi, A.M.; Cheregi, M.C.; Danet, A.F.: Molecules 2009, 14: 480-493.

Gene Delivery and Knockdown using Novel Lipopolyplexes Pinnapireddy, S.R.; Bakowsky, U.

Institute for Pharmaceutical Technology and Biopharmacy, Philipps University, 35037 - Marburg, Germany.

This study was aimed at formulating nucleic acid carriers which are composed of a Liposomal shell encapsulating a Polymer and Nucleic acid complex together termed Lipopolyplex. These are intended for transfection and for knockdown or down-regulation of a particular gene. In this current study we investigated the in-vitro efficacy of DOPE (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine) and Cholesterol based liposomes which were used to encapsulate a PEI (Polyethylenimine) based polymer and Nucleic acid complex. The physical characteristics of the Lipopolyplexes were analysed using Dynamic Light Scattering, Laser Doppler Micro-electrophoresis and Scanning Electron Microscopy. Complex stability analysis was done using Complex Stability Assay and Heparin Competition Assay. Transfection efficiency was done using Luciferase reporter gene assay, GFP expression and RNAi. Cytotoxicity was evaluated using MTT assay. With a size of 237 ± 5 nm and Zeta potential of 6 ± 2 mV, the complexes were well within the suitable range for use in Transfec-tion. Luciferase expressions in the range higher than 1000000 RLU/mg protein were achieved. Comparison against other transfection agents showed a better cytotoxicity profile which was relatively lower. Acknowledging the results of the study, it can be concluded that Lipopolyplexes are by far a better alternative to the conventional transfection reagents bearing in mind their cytotoxicity profiles.

Stability testing of medical devices – estimation of shelf life for Reactive Skin Decontamination Lotion (RSDL®) Bogan, R.; Klaubert, B.; Zimmermann, T.

Central Institute of the Bundeswehr Medical Service Munich, Ingolstädter Landstr. 102, 85748 Garching-Hochbrück, Germany

For regulatory reasons stability studies are mandatory for drugs. As medical devices do not pass identical procedures during the authorization and certification process, stability data are not always strictly collected. German Armed Forces stockpile medical devices over a long period and use them in all climatic zones. Therefore stability is an essential matter of concern. In this work we describe the estimation of shelf life for the Reactive Skin Decontami-nation Lotion (RSDL®), a class IIa medical device with CE certification for the European market, which is currently procured. RSDL® contains polyethylene glycol monomethylether and water as solvent system and, in a proprietary formulation, 2,3-butandione monoxime (= diacetylmonooxime, DAM) as active ingredient (AI) for the decontamination of chemical warfare agents and toxic industrial compounds on the skin [1,2]. RSDL® was exposed to temperatures of 40°C, 53°C and 70°C up to 4 months. We analysed the contents of DAM, of the putative degradation product dimethylglyoxime (DMG) and of unknown degradation products by means of reversed phase high pressure liquid chromatography (RP-HPLC) with diode array detection (DAD). Based on the gathered concentration-time curves the kinetics of degradation was determined and the temperature dependent rate constants were calculated according Arrhenius equation [3]. These rate constants were used to calculate the time span until defined threshold values for DAM, DMG and unknown degradation products were exceeded. These data were basis for estimation of shelf life at different storage conditions.

The decline of DAM followed 1st order kinetics while formation of DMG as well as one unknown degradation product could by described by zero order kinetics. The degradation rates were distinctive temperature dependent. Based on an acceptable DAM-content of 90% (w/w) RSDL® proofed to be stable for several years at the recommended storage conditions from 15°C to 30°C. Calculated shelf life was 38.3a at 15°C and 7.6a at 30°C. For the degradation product DMG a valid threshold value of 0.1% (w/w) was considered. Time span to meet 0.1% was 22.6a at 15°C and 5.0a at 30°C. In both cases higher temperatures led to a remarkable shortening of shelf life. An additional unknown degradation product arose during our experiments. For unknown degradation products ICH Q3B(R2) gives threshold values (percentage of AI) of 0.05% (reporting threshold), 0.1% (identification threshold) and 0.15% (qualification threshold) [4]. If these values established for drugs were applied analogously for the medical device RSDL®, even the highest value of 0.15% was reached after 1.0a at 15°C and 0.5a at 30°C. The quality of RSDL® is warranted for a period of 4 years. According to our calculations in that timespan the content of the AI as one crucial parameter of quality will not fall under an acceptable limit if RSDL® is stored correctly. For specified or unknown degradation products an obvious limitation of shelf live could be shown even if stored at the recommended conditions. Thus a short retest-period is necessary due to a risk orientated quality monitoring of drugs and medical devices being stockpiled or used in missions abroad.

References: 1. Schwartz, M.D. et al.:Curr Pharm Biotechnol. 2012, 13 (10): 1971-1979. 2. http://www.rsdecon.com/pages/aboutUS.htm 3. Grimm, W. in: Drug Stability, Principles and Practices (Taylor & Francis) 2000. 4. ICH Harmonised Tripartite Guideline Q3B(R2), Impurities in New Drug Products, 2006.

Are polymeric nanoparticles able to cross the gastrointestinal barrier? Söbbing, J.; Grünebaum, J.; Mulac, D.; Langer, K.

Institute of Pharmaceutical Technology and Biopharmacy, University of Münster, Corrensstraße 48, 48149 Münster

Oral delivery of active pharmaceutical ingredients (API) is the most popular and most patient-friendly form of drug administration. However, there are several challenges, which have to be overcome. In case, APIs of the Biophar-maceutical Classification System (BCS) classes II or IV, the poor solubility and the poor permeability are the biggest problems.1 Therefore drug carrier systems like nanoparticles (NP) are discussed to increase the bioavailability of these drugs due to an enhanced delivery of pharmaceuticals over the gastrointestinal (GI) barrier. In literature many groups described that nanopar-ticles are able to deliver insulin over the GI barrier.2 However, in most of these studies only secondary effects e.g. the blood glucose response after insulin administration were measured. The real transport of NP through the barrier was not detected until now. The aim of this study was to develop an improved drug delivery system for an oral application. In detail, 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (mTHPP), a second-generation photosensitizer, was embedded into polymeric NP to enhance the bioavailability in comparison to the free drug. NP based on polymers like poly(DL-lactide-co-glycolide) (PLGA) or poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) (Eudragit® E) were formed using the emulsion-diffusion-method.3 Different stabilizing agents, such as chitosan hydrochloride or didodecyldime-thylammonium bromide (DMAB) were used for particle preparation in order to influence the transcellular pathway. Furthermore surface modification of these particles with ligands should enable specific transport mechanisms. The photosensitizer mTHPP was successfully embedded into the different NP formulations. Furthermore a surface modification of the NP could be achieved. Dynamic light scattering analysis showed an average diameter ranging from 200 to 300 nm and a polydispersity index < 0.1. All formulations showed no cytotoxicity concerning Caco–2 cells and could be tested in the GI barrier model. The model was validated regarding barrier integrity, tight junction formation and permeability of control compounds. Barrier integrity was controlled continuously with an online measurement of the transepithelial electrical resistance. Previously described NP formulations were investigated over a period of 24 hours, where no permeation of particles could be detected. The obtained results are in contrast to the literature, which is why we see the

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need for further investigations to differentiate between particle permeation and carrier-mediated drug transport.

Nanoparticles for gene delivery Look, J.1; Wilhelm, N.2; Gorjup, E.2; von Briesen, H.2; Noske, N.3; Rodriguez, J.R.4; Prosper, F.4; Serra, M.5; Carrondo, M.5; Alves, P.5; Langer, K.1 1 Institute of Pharmaceutical Technology and Biopharmacy, University of Münster, Corrensstraße 48, 48149 Münster, Germany 2 Fraunhofer Institute for Biomedical Engineering, Ensheimer Str. 48, 66386 St. Ingbert, Germany 3 apceth GmbH & Co. KG, Max-Lebsche-Platz 30, 81377 München, Germany

4 Clinica Universidad de Navarra, Av. Pio XII 36, 31008 Pamplona, Spain 5 Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal

Gene therapy is a promising tool for treating many serious diseases like cancer or genetic disorders. The difficulty is to develop a safe and effective DNA vector, which is able to transport the DNA to the nucleus without prior degradation and with a controlled DNA release at the target location. Until now only viral gene transfer vectors work efficiently but are still plagued with safety concerns. Aim of this study is the development of a nanoparticular gene transfer system as a safer alternative to the risky use of viral gene transfer vectors. The obtained nanoparticles should be biocompatible, biodegradable, cell specific by ligand modification and should enable receptor mediated uptake and a controlled DNA vector release. Therefor DNA vector loaded nanoparticles based on the protein human serum albumin were prepared by a well-established desolvation method [1]. Different stabilisation methods were tested to check the influence on DNA vector release. To enable receptor mediated uptake the nanoparticles were surface-modified. Different cell penetrating peptides were coupled to the primary amino groups on the nanoparticle surface using a bifunctional PEG-based crosslink-er. Nanoparticles with monodisperse size distribution and an effective DNA incorporation were obtained. Preservation of plasmid functionality during particle preparation was shown by plasmid extraction from the final nanoparti-cle suspensions followed by Lipofectamine transfection. In vitro release studies showed a stable entrapment of the DNA vector under storage conditions. The surface modification worked successfully which could be revealed by peptide quantification using C18-RP-HPLC analytics. Cell uptake and binding studies proved a higher cell uptake for the surface-modified compared to unmodified nanoparticles. Transfection studies showed gene expression for the surface-modified nanoparticles.

Acknowledgements: This work was financially supported by the BMBF (13N115391, 13N115402, 13N115413).

Reference: 1. Steinhauser, I. et al.: J. Drug Target. 2009, 17 (8): 627-637.

Influence of cross-linker type and content on oligonucleotide deposition and delivery from gelatine-based hydrogels Schwabe, K.1; Ewe, A.2; Kascholke, C. 1; Aigner, A.2; Hacker, M.C.1; Schulz-Siegmund, M.1

1 Universität Leipzig, Institute of Pharmacy, Pharmaceutical Technology, Eilenburger Str 15a., 04317 Leipzig, Germany 2 Universität Leipzig, Faculty of Medicine, Rudolf-Boehm-Institute of Pharmacology and Toxicology | Clinical Pharmacology, Härtelstraße 16-18, 04107 Leipzig, Germany

With the objective to improve upon current treatment of severe bone defects, that are typically addressed with implants containing unphysiologically large amounts of Bone Morphogenetic Protein 2 (BMP-2), we strive to suppress BMP-2 antagonist locally by the controlled delivery of specific siRNA from implant materials and thus increase the osteogenic potential of low doses of BMP-2 [1]. In this context, we focus on hydrogels as one possible cell carrier and delivery system due to their similarity to natural extracellular environments and stabilizing effects on embedded nanoparticles and therapeutic proteins. Through the use of natural polymers, hydrogel formulations with good biocompatibility and optimized degradation kinetics can be obtained. Polypep-

tides as found in gelatine preparations are an interesting example of such materials, as they promote cell adhesion, are non-immunogenic, economic and comparably easy to process. For the required cross-linking we utilized a recently established oligomeric cross-linker and are able to engineer a variety of hydrogels with different physic-chemical properties [2, 3]. Hydrogels fabricated from Collagel® (type B, Gelita AG, Germany) and different anhydride-containing cross-linkers were investigated as controlled release systems for Polyethylenimine (PEI)-complexed nanoparticles for target gene knockdown. To investigate the distribution of oligonucleotides in the hydrogels, a model plasmid DNA (pDNA) was covalently linked to a fluorophore using the mirusbio Lable IT(R) kit. The pDNA was then complexed by branched PEI F25 for half an hour4, diluted to an appropriate volume to rehydrate the lyophilized, preformed hydrogel and finally cryo-sectioned and analysed using fluorescence micros-copy. In order to determine the release of functionally active nanoparticles a model system was established. Hydrogels were loaded with PEI-based complexes containing siRNAs against luciferase and stably luciferase-expressing SKOV-3-Luc were cultured on the hydrogels. The biological activity was determined by the luciferase knockdown of the released siRNA-containing nanoparticles. Our results show that complexed oligonucleotides can by loaded to hydrogels with varying modes of deposition dependent on pore structure and surface characteristics. We could verify that immortal as well as primary cells grow and spread on various gel formulations. Cell number, distribution and penetration depend on physical and chemical properties of the gels. Cells, cultured in osteogenic medium, formed extracellular matrix. To evaluate the hydrogel’s performance for bone regeneration, human adipose tissue-derived stem cells (hASC) were proliferated and differentiated along the osteogenic lineage on hydrogels. hASC proliferation was observed via CLSM imaging. Calcium as a late marker of osteogenic differentiation was determined using a colorimetric method with cresolphthaleine as complexing reagent. Moreover, we evaluated the knockdown of luciferase activity in stable luciferase transfected SKOV cells via RNAi as a proof of concept. These results are promising towards the development of a local siRNA release platform for osteogenic regeneration. In addition, the gels appeared to be well osteocompatible as they allowed for adhesion and differentiation of adult stem cells.

Funded by Sächsisches Staatsministerium für Wissenschaft und Kunst (SMWK) and DFG-Transregio 67 A1

References: 1. Schneider et al.: Tissue Eng Part A 2014, 20: 335-345. 2. Loth, T. et al.: React. Func. Polym. 2013, 73: 1480–1492. 3. Loth, T. et al.: Biomacromolecules 2014, 15: 2104–2118. 4. Ewe, A. et al.: Acta Biomaterialia 2014, 10, 2663-2673.

Oral drug delivery of therapeutic gases- carbon monoxide release for gastrointestinal diseases Steiger, C.; Lühmann, T.; Meinel, L.

Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany

Purpose: Carbon monoxide (CO) has therapeutic effects in various gastroin-testinal diseases [1] yet clinical use today is challenged by inappropriate delivery modes [2]. Consequently, we developed a tablet referred to as oral carbon monoxide release system (OCORS) (Figure 1) providing precise, controlled and targeted CO delivery for the treatment of gastrointestinal injury and inflammation, respectively. Methods: OCORS is an oral tablet based on sulfite induced CO release from the CO releasing molecule 2 (CORM-2) [2]. OCORS performance was detailed as a function of the presence of buffer within the tablet core and characteristics of a water-insoluble cellulose acetate coating, forming a semipermeable shell around the tablet core. Amperometric detection was deployed for recording CO release profiles throughout 10 hours. Results: OCORS was tuned for environmental pH insensitivity by appropriate buffer systems blended within the tablet core. OCORS delivered CO for up to 10 hours, contrasting CORM-2 suspensions delivering the gas within 1.5

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hours. Zero order CO release was observed within approximately 30 to 240 minutes in different release media and was tuned by the thickness of the semipermeable shell. Conclusion: OCORS is a readily available tablet for oral use. The controlled release system reliably delivered CO independent of environmental pH, such that the therapeutic gas can be safely generated at gastric, intestinal or colonic sites. In vivo experiments of OCORS are required to demonstrate the pharmacokinetics and clinical potential of this oral delivery platform for therapeutic gases.

Figure 1: Schematic drawing of the oral carbon monoxide release system (OCORS). Left part of the cartoon: Water permeates the semi-permeable cellulose actate shell, dissolving the swelling coating layer around the sodium sulfite crystals which readily dissolve. Right part of the cartoon: The Na2SO3

interacts with CORM-2, thereby causing CO release. References: 1. Motterlini, R.; Otterbein, L.E.: Nature Reviews Drug Discovery, 2010, 9: 728–743. 2. Steiger, C.; Luhmann, T.; Meinel, L.: Journal of controlled release : official journal of the

Controlled Release Society, (2014).

In Situ Forming Oleogels: In Vitro Investigation of Application, Solidification and Degradation Windorf, M.; Mäder, K.

Martin-Luther-University Halle-Wittenberg, Department of Pharmaceutical Technology and Biopharmaceutics, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany

Introduction Parenteral depot systems provide a sustained drug release over several days, weeks or even months. However, the often used polymers consisting of lactic acid and glycolic acid monomers show a complex and hardly predictable drug release kinetic and furthermore, the accumulation of acidic degradation products often leads to local irritation or influences the stability of the active ingredient [1]. In contrast, liquid and solid lipids more and more turn out to be a promising alternative as matrix forming materials [2]. In situ forming oleogels (ISFO) are composed of a solvent or oil, a low molecular weight organogelator (LMOG) and a small amount of a biocompati-ble organic solvent [3]. After application, the solvent diffuses into the surround-ing tissue and the LMOG self-assembles into aggregates, which build a solid network by inter-molecular physical interactions with incorporated oil droplets. Their relatively simply production, the gentle manufacturing conditions for temperature resp. shear sensitive drugs as well as the ease and painless administration are just some advantages. In this study, the application of the LMOG 12-Hydroxystearic acid (12-HSA) to produce injectable ISFO based on peanut oil is investigated. The solidification of the formulation by extraction of the solvent N-Methyl-2-pyrrolidone (NMP) and the degradation behaviour of the implant in the presence of lipase are presented. Methods The injection force was measured at different injection rates and with different cannula sizes into a beaker or chicken wings. Conductivity measurements were used to determine the extraction velocity of NMP after injection of the ISFO in phosphate buffered saline leading to the solidification of the implant. To determine the degradation rate, ISFO were injected in phosphate buffered saline containing different amounts of Lipoprotein lipase. Every 48 hours the surrounding medium was removed. The time-dependent degradation of the implants was determined by weighing after drying and calculating the mass loss.

Results and Discussion Injectibility measurements indicate a very good syringeability caused by the low viscosity of the ISFO. They show a dependence of the injection rate on the injection force. The higher the injection rate the higher is the required force. For the application into the chicken slightly higher forces are needed compared to the beaker due to the backpressure of the surrounding tissue. By the use of 25G cannulas, very low injection forces of less than 9 N are required for all ISFO and injection rates, providing the possibility of a further reduction in needle size.

The decrease in conductivity of buffered salt solutions by addition of NMP can be used for determining the extraction kinetics out of the applied ISFO. The small amount of the biocompatible solvent NMP is released within 6-10 h, leading to the solidification. This observation is almost independent from the LMOG concentration. The formation of a bulk gel structure causes dissolved molecules to be highly mobile due to the low micro viscosity and to diffuse easily to the interface.

The degradation rate can be controlled by the concentration of the LMOG and ranges from a few days to months. Lowering the LMOG concentration of the ISFO and increasing the lipase activity in the release medium leads to enhanced implant degradation. The implants are degraded layer-by-layer from the surface and mass erosion and rupture does not occur. Therefore, a continuous release of suspended drugs is possible.

The authors would like to thank the BMBF (ProNet-T³; Th-03) for the financial support.

References: 1. Fredenberg, S. et al.: Int. J. Pharm. 2011, 415(1-2): 34-52. 2. Kreye, F. et al.: Expert Opin. Drug Deliv. 2008, 5(3): 291-307. 3. Vintiloiu, A. et al.: J. Control. Release 2008, 125(3): 179-192.

smartLipids® - the next generation of lipid nanoparticles by optimized design of particle matrix Müller, R.H.1; Ruick, R.1; Keck, C.M.2 1 FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany 2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were invented as drug carrier systems about 25 years and 15 years ago, respective-ly [1]. Meanwhile they have established themselves as delivery systems being investigated and developed by many research groups world-wide. Identical to the development of liposomes, entering the cosmetic market before appear-ance of pharmaceutical products, NLC meanwhile are applied in cosmetic products world-wide on the market (e.g., La Prairie, Amore Pacific etc.). The SLN and NLC posses a particle matrix typically made from a single solid lipid (SLN) or a lipid blend (solid lipid plus oil, NLC). To improve the drug loading and to ensure a firm drug incorporation during shelf life, the design of the particle matrix was optimized by “more complex nanostructuring” to yield smarter lipidic nanoparticles (smartLipids®). Many lipids used in SLN and NLC can undergo polymorphic transitions - also known to be influenced e.g., by the stabilizers used [2] – directly after preparation or on storage. This influences the physical stability as well as drug loading properties, i.e., loading capacity and potential resulting drug expulsion (typically more pronounced in SLN, less in NLC). The aim was to develop a new lipid matrix primarily exhibiting an α-modification, which is less densely packed than lower energy β´- and β-modifications and therefore providing a higher drug loading. The aim was also to hinder or delay polymorphic transitions towards higher energy modifications during storage. This should avoid drug expulsion and - from the production side - reduce the dependency on certain stabilizers and processing parameters applied. The developed smartLipids® ideally consist of a multiple mixture (up to 10 or more) of different solid lipids containing mono-, di-, and triglycerides with different chain length (ranging from C10 to C22 or longer) optionally having additional liquid lipids (oils) in the blend. This leads to a polydisperse and less structured matrix with imperfections and hinders the transition to more stable polymorphs (β´- and β-modification), thus increasing the drug loading. A direct comparison to classic SLN formulations containing Dynasan 118 (Tristearin) was made. The lipid matrix consisted either of Dynasan 118 (= SLN) or a smartLipids® blend. Final formulations contained 15 % lipid matrix, different stabilizers i.e., anionic, cationic or nonionic stabilizers (sodium dodecyl sulfate, Lanette E, polyvinyl alcohol, Tween 20, cetylpyridinium chloride or Plantacare 818) and

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Milli-Q water up to 100 %. The formulations were produced by hot high pressure homogenization (3 cycles at 500 bar) and were characterized by photon correlation spectroscopy (PCS) (Zetasizer Nano ZS), laser diffraction (LD) (Mastersizer 2000, both Malvern Instruments, UK), light microscopy (Orthoplan, Zeiss) and wide angle X-ray diffraction (WAXD) (Philips PW 1830, Philips, Netherlands). The polymorphic modifications were determined by WAXD where one single peak located at 2Ө = 21.4° (corresponding 4.15 Å) is indicating an α-modification and the peak at 2Ө = 19.4° (corresponding 4.6 Å) was chosen to identify the presence or absence of the β-modification, because no superposi-tion occurs at this angle. Within the various Dynasan SLN formulations only 1 sample showed a complete α-modification, 2 showed a full β-Modification and 3 a mixture of both depending on the stabilizer. In contrast within the smartLipids® formulations, 3 samples showed a complete α-modification and the other 3 an α-modification with only small amounts of β-modification. The independency from the stabilizer type on the polymorphic state could be shown for smartLipids® formulations in contrast to conventional SLN formulations. Samples which remained stable showed a z-average mainly ranging from 100 nm to 400 nm (polydispersity index mainly between 0.15 and 0.30). By combining multiple lipids a lipid particle matrix could be designed which predominantly forms an α-modification and exhibits polydisperse and less ordered structure. In contrast to the highly ordered β-modification this enables higher drug encapsulation and firmer drug inclusion. The dependency on the applied stabilizer of smartLipids® formulations regarding polymorphic transi-tions is reduced. Therefore smartLipids® represent a “smarter version” of the NLC facilitating drug incorporation, especially when formulating drugs with a priori low solubility in lipids (e.g., dexamethasone acetate) and reducing the problem of drug expulsion.

References: 1. Müller, R.H.; Shegokar, R.; Keck, C.M.: Curr. Drug Discovery Technol. 2011, 8(3): 207-

227. 2. Bunjes, H.; Koch, M.J.; Westesen, K.: Molecular Organisation on Interfaces (Springer

Berlin Heidelberg) 2002.

Skin hydration of NLC due to formation of invisible patch: Influence of vehicle Arntjen, A.1; Maus, A.1; Keck, C.M.1,2 1 Applied Pharmacy, University of Applied Sciences Kaiserslautern – Campus Pirmasens, Pirmasens, Germany 2 Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany

Introduction: Nanostructured lipid carriers (NLC) consist of a solid lipid matrix being composed of a blend of liquid and solid lipid and possess a size typically in the range between 80-500nm [1]. After dermal application they form an “invisible patch” which causes occlusion and thus an increase in skin hydration [2]. For dermal application NLC need to be incorporated into a vehicle, i.e. ointments, creams or gel bases. As different types of vehicle possess different properties, the hydration effect of NLC, i.e. the ability of NLC to form the “invisible patch”, might be affected by the type of vehicle. Therefore, the aim of this study was to evaluate the influence of different vehicles, i.e. water, crema basalis (Basiscreme) and polyacrylate gel on the skin hydration effect of NLC. Material and methods: Fresh pig ears were used as ex-vivo model. The NLC were admixed in 5% and 10% concentration in each base. As controls only the bases were topically applied. Additionally untreated pig ear skin was measured (n=3). Skin hydration was analysed using a Corneometer® CM 825 (Courage & Khazaka Electronic GmbH, Köln, Germany) following the instructions of the manuals. Measurements were performed prior to application and 1h, 4h and 24 h after application of the formulations, respectively. Results: The application of pure water showed no effect in comparison to the untreated control. Water containing 5% or 10% of NLC showed lower skin hydration values in comparison to the controls. The results confirm the formation of the invisible patch upon the addition of NLC, i.e. less skin hydration is measured due to the formation of the lipidic NLC film [3]. However, the standard deviations for 5% NLC are much higher than for the 10% NLC formulations, indicating a less tight film formation for 5% NLC. The application of crema basalis and crema basalis with NLC increased the skin hydration. The hydration effect was more pronounced and longer lasting with NLC 5% and was most pronounced with 10% NLC. In comparison to the NLC in water the skin hydration was detectable for the NLC in crema basalis, indicating that the addition of NLC to a semi solid cream prevents complete film formation of

the NLC. Hence, the structure of the vehicle seems to modify the film formation of NLC. Reasons for this are manifold, e.g. inhomogeneous distribution of the NLC within the vehicle and/or agglomeration of the particles during the application and/or due to drying of the vehicle. The influence of the vehicle structure on film formation and skin hydration of NLC was even more pronounced when polyacrylate gel was used as vehicle. The gel alone led to a strong hydration effect 1h after application, after 4h and 24h the skin hydration was less than the control, indicating a strong drying effect of the hydrogel. Addition of NLC to the gel decreased the strong increase in hydration and the hydration effect after 4h and 24h was also lower than for the gel alone. An explanation for these results is that NLC in hydrogel led to a partial film formation directly after application, i.e. as observed for the NLC in crema basalis. However over time the water of the gel evaporates, the gel structure collapses and the NLC can form a tight film with highly occlusive properties. Conclusions: The type of vehicle used for incorporation of NLC and the concentration of NLC strongly influences the film formation of NLC upon dermal application und thus the occlusive properties of NLC. Corneometer measurements are suitable to identify changes in skin hydration. However, in case of NLC containing formulations the data represent a superposition of effects caused by the film formation of NLC (decrease in measured hydration) and effective skin hydration (increase in measured hydration). Therefore, further studies will focus on the evaluation of additional skin parameters and different bases to identify optimized formulations for the dermal application of NLC with tailor-made dermal properties.

References: 1. Müller, R.H. et al.: Current Drug Discovery Technologies 2011, 8: 207-227. 2. Müller, R.H. et al.: H&PC Today 2014, 9(2): 18-25. 3. Müller, R.H. et al.: EURO COSMETICS 2013, 6: 20-22.

Multi-Targeting Modifiers of Nano-Carriers for individual Therapy and Diagnosis of Cancer Krebs, L.1; Peters, T.1; Langguth, P.1; Goerigk, G.2; Schweins, R.3; Nawroth, T.1

1 Gutenberg University, Inst. Pharmacy and Biochemistry, Pharmaceutical Technology Department, Staudingerweg 5, D-55099 Mainz 2 HZB, Institute of Soft Matter and Functional Materials , BESSY Synchrotron, ASAXS, D-14109 Berlin, Germany 3 Institut Laue Langevin ILL, Group DS/LSS, 71 Avenue des Martyrs, F-38042 Grenoble CEDEX 9, France

The MultiTarget project shall improve therapy and diagnosis of severe diseases, e.g. cancer, by individual targeting of drug-loaded nano-pharmaceuticals towards cancer cells. Specific ligands, which are recognized by the diseased cells, are bound to the nanoparticle surface, and thus capable of directing the drug carriers [1-3]. In the current concept a multiple ligand set is coupled by a fast assembly technique (click link) in the very last step of the formulation. In the clinical application the ligands set (2-5 different) will be selected according to the biopsy analysis of the patient tissue e.g. from tumor.

We synthesize multi-targeting modifiers of metal drug loaded nanoparticles which consist of four structure domains (fig.1). The components are varied and optimized in a case specific manner. The nanoparticles, e.g. biodegradable polymer (PLGA), lipid particles as well as the anchor domain are hydrophobic. The linker binds the ligand in two steps: adsorption and a covalent bond formation as terminal step. The hydrophilic spacer is essential for keeping the distance from the nanoparticle surface. The structure of the modified nanopar-ticles is analyzed by dynamic light scattering DLS, neutron small angle scattering SANS and metal specific X-ray scattering ASAXS, while the effect of the drug is proven in cell culture tests [4]. The multi-targeting modification is applied to lanthanide loaded polymer nanoparticles (PLGA) for radiation therapy [5] and liposomes as a fast available carrier.

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multi-targeting nanoparticle modifiers for individual medicine

Fig.1: Case- and person specific therapy and diagnosis by cell specific nano-pharmaceuticals : The multi-targeting modifiers for drug nanoparticles consist of four domains: 1) hydrophobic anchor, 2) hydrophilic spacer, 3) linker (two parts) and 4) receptor ligand (protein or bioorganic ligand), coupled by fast assembly.

Acknowledgments: We are grateful for the funding by the German ministry of science and education BMBF, grant 05KS7UMA.

References: 1. Ferrara, T.A. et al.: Current Opinion in Molecular Therapeutics 2009, 11, 37-42. 2. Alemdaroglu, F.E. et al.: Adv. Materials 2008, 20, 899-902. 3. Jahn, M.R. et al.: J. Pharm. Pharmacol. 2011, 63, 1522-1530. 4. Buch, K. et al.: Radiation Oncology 2012, 7, 1-6. 5. EU-Patent 11 007 401.0; PCT 13 07 12, 2012 “A particulate system for use in

diminishing cell growth / inducing cell killing”, keyword "Lanthanide", Gutenberg University Mainz, inventors: Buch, K., Nawroth, T., Langguth, P., Schmidberger, H..

Imaging of cardiac infarction by labeling the oil phase of a fluor-containing MRI contrast agent Keller, T.1,2,3; Dietrich, T.3; Bourayou, R.4; Wittstock, K.3; Meyborg, H.3; Licha, K.5; Schnackenburg, B.6; Fleck, E.3; Bunjes, H.1 1 Technische Universität Braunschweig, Institute of Pharmaceutical Technology, Mendelssohnstraße 1, 38106 Braunschweig, Germany 2 B.Braun Melsungen AG, Carl Braun Straße 1, 34212 Melsungen, Germany 3 Deutsches Herzzentrum Berlin,Kardiologisches Forschungslabor, Seestraße 13, 13353 Berlin, Germany 4 www.bourayou.de, Weichselplatz 8, 12045 Berlin, Germany 5 Mivenion GmbH, Robert-Koch-Platz 4, 10115 Berlin, Germany 6 Philips Healthcare, Philipsstraße 14, 20099 Hamburg, Germany

Inflammatory events in the circulatory system can be visualized by Magnetic Resonance Imaging (MRI) using emulsion-based contrast agents containing fluorinated substances as part of the oil phase. In the bloodstream, the oil droplets are phagocytized by cells of the immune system before they enrich especially in inflamed tissues [1]. The aim of the present study was to label the oil phase of such a contrast agent with a Near-Infrared-Fluorescence-dye (NIRF-dye) to allow fluorescence imaging and microscopy in order to follow the in vivo fate of the oil droplets with high sensitivity and resolution [2]. An oil-in-water-emulsion containing perfluorohexyloctane (F6H8) as part of the oil phase was prepared as MRI contrast agent. The NIRF-dye “PM-ITCC”, which was covalently coupled to heptadecafluoroundecylamine to increase its affinity to F6H8, was used as fluorescence label for the oil droplets. The labeled emulsion was incubated with human plasma to ensure that the NIRF-dye was not washed out by contact with plasma components. The contrast agent formulation was tested by intravenous injection to rats with a myocardial infarction as inflammatory model (three rats with an infarction vs. one rat without infarction). Two hours after injection, the rats were sacrificed and the hearts of the animals were characterized with a fluorescence imager ex vivo. In addition, cryosections of the hearts were inspected by brightfield and fluores-cence microscopy using hematoxylin-eosin (H.E.)- and CD68-stainings (macrophages). The ex vivo NIRF images of the infarcted hearts and the corresponding microscopic images displayed a significantly higher fluorescence signal than the non-infarcted heart (see individual values of the scale for each fluores-cence image in Table 1). Furthermore, the infarcted tissues were heavily infiltrated with macrophages (see red-stained cells in the CD68-image in Table 1). The fluorescence-labeled formulation can thus be used to assess the distribution of the F6H8-containing contrast agent with high sensitivity and resolution.

Table 1: Comparison between the fluorescence signals in the heart of two rats two hours after the application of a fluorescence-labeled con-trast agent. The microscopic images show only a fluorescence signal in the infarcted tissue, which is also infiltrated with macrophages.

The authors thank Dr. J. Schmitt, Dr. D. Röthlein, Dr. V. Krüger and W. Schlemermeyer (all B. Braun) for supporting the project.

References: 1. Flögel, U. et al.: Circulation 2008, 118(2): 140-148. 2. Klohs, J. et al.: Mol Imaging 2006, 5(3): 180-187.

Proficiency test on ofloxacin eye drops Maul, K.J.1; Diergardt, T.2; Feldmann, D.3; Wätzig, H.1 1 Technische Universität Braunschweig – Institute of Medicinal and Pharmaceutical Chemistry, Beethovenstraße 55, 38106, Germany 2 Physikalisch-Technische Bundesanstalt – Braunschweig, Bundesallee 100, 38116, Germany 3 Bausch+Lomb, Brunsbütteler Damm 165-173, 13581, Germany

Proficiency testing is a good performance-monitoring procedure and an important part in quality assurance, which should also include validation of analytical methods, the use of certified reference material and a routine internal quality control [1]. This proficiency test is the second of a series in the East African Community (EAC). It is a part of a project called “Establishment of a regional Quality Infrastructure for the pharmaceutical sector in the EAC” initiated by the Physikalisch-Technische Bundesanstalt. It was conducted in April 2014 with 15 participants from 5 different countries. Ofloxacin eye drops were distributed as test material. These were analyzed according to the USP monograph for the amount of active ingredient and the pH-value. The acceptance criteria for the assay are 90.0-110.0% and the pH-value should be between 6.0 and 6.8. Furthermore, the system suitability test was performed. The interlaboratory standard deviation for the pH-value is 0.15 (RSD%=2.41 %) and for the assay using the submitted results 0,622 mg/ml (RSD%=22,1 %). For the evaluation the mean of all laboratories was employed as assigned value. Moreover the z-score for each laboratory was calculated (Figure 1). All laboratories measured a pH-value within the specification. The results for the assay have to be recalculated to eliminate calculation errors.

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Figure 1: Z-score charts. The z-scores of each participant are shown for assay and pH. A z-score between -2 and 2 (white area) is very good. Z-scores between -3 to -2 and 2 to 3 (black area) are acceptable.

References: 1. Thompson, M. et al.: Pure Appl. Chem. 2006, 78(1): 145-196.

Accessing the Nanoparticle Corona in Pulmonary Surfactant Raesch, S.1,2; Tenzer, S.3; Storck, W.3; Ruge, C.1; Schäfer, U.F.1; Lehr, C.-M.1,2 1 Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany 2 Department of Drug Delivery, Helmhotz Institute for Pharmaceutical Research Saarland, 66123 Saarbruecken, Germany 3 Institute for Immunology, University Medical Center of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany

Nanoparticles (NP) are of high interest in medical and pharmaceutical research, especially regarding their toxicological profile, but also potential use as carrier systems. Understanding the outstanding properties of NP´s can not only help assess the possible harm which can arise from them, but it also opens new perspectives for targeted and controlled drug delivery. NP, that come in contact with a biological fluid, are opsonized by biomolecules such as proteins, which build a “corona”. It has generally been accepted that an evolution of such a biomolecule layer occurs, and that this time-dependent layer of adherent biomolecules typifies the actual biological identity of the NP. Considering the many different NP with varying surface modifications which are produced worldwide, differences in resulting corona seem plausible and were identified in the coronas on NP in plasma [2, 3]. The lung is an attractive pharmaceutical target, as the air-blood barrier is a less than 2 µm thin layer with an enormous alveolar surface area larger than 100m2. The enormous amount of potentially polluted air, which passes the lung, makes an effective maintenance system essential. In the alveolar region cells are only covered by a thin pulmonary surfactant (PS) layer and clearance is mainly carried out by alveolar macrophages. PS, secreted by type II alveolar cells, allows gas diffusion and its surface tension lowering effect is essential for stability of the alveoli during breathing cycle. The surfactant layer consists of approximately 90% lipids (mainly phospholipids, especially DPPC) and 10% proteins with about half of them being surfactant specific proteins (SP). Albeit the PS is very thin (~200 nm), it is the first biological barrier an airborne NP comes in touch with. Before NP are either taken up by the alveolar cells or ingested by macrophages, they are coated by the PS building a lipoprotein-“corona”. So far, it remains to be elucidated whether the fate of inhaled NP depends on the coating obtained from the surfactant layer, though there is evidence for the influence of the SP on macrophage uptake [1]. With knowledge about the relationship between the surfaces of NP, the subsequent built coronas, and the recognition of these patterns by cells, an approach for longer residence time of NP or targeted uptake by alveolar or macrophage cells could be exploited. Although understanding the surfactant-NP interaction is fundamental for the fate of NP in the lung, there is, so far, no reproducible method for the analysis of the NP-corona. The unique composition, structure, and properties of the lipid-rich PS require different and more advanced analytical methods for the assessment of the NP-corona in the deep lung. Hence, we used a native pulmonary surfactant preparation, isolated from porcine lungs, for the development of a method, to access the lipid-protein-corona. Magnetic separation of NP was found to be most suitable in compari-son to various commonly used techniques. We present data for three model particles with diverse surface properties and the influence on the adsorbed proteins. Furthermore, future possibilities for the analytical determination of all biomolecules present in the nanoparticle corona in pulmonary surfactant are discussed.

Chiara de Rossi, Jesús Perez-Gil

References: 1. Ruge, C.A. et al.: PLoS ONE 2012, 7(7): e40775. 2. Monopoli, M.P. et al.: J. Am. Chem. Soc. 2011, 133(8): 2525–2534. 3. Tenzer, S. et al.: Nat. Nanotechnol. 2013, 8(Oct): 772–781.

Development of a Bioequivalent Taste Masked Cetirizine HCl 10mg Zydis® Dosage Form using Cyclodextrin Grother, L.; Warren, N.; Cusack, A.

Catalent Pharma Solutions, Frankland Road, Blagrove, Swindon, SN5 8RU, United Kingdom

Purpose Cetirizine HCl is a bitter drug indicated for the treatment of allergies. Due to the nature of allergic episodes, medication is often required at unpredictable times. Consequently dose forms such as orally disintegrating tablets (ODT) are frequently preferred by patients due to the greatly enhanced convenience they provide. In order for the drug to be delivered using an ODT such as the Zydis® dosage form, effective taste masking is required. The aim of this study was to investigate if cyclodextrin could be used to taste mask cetirizine HCl 10mg when formulated in a freeze dried ODT (Zydis®). A chemically stable product with bioequivalence to the reference product was a prerequisite for success. Methods Prototype freeze dried formulations containing cyclodextrin were manufactured using the Zydis® formulation and process1. Dissolution testing was performed on the prototype formulations using USP apparatus II (50rpm) with 900ml purified water. Assay and stability testing was conducted by HPLC. A taste trial was conducted by a panel of 30 volunteers. The probe bioequiva-lence study was a single dose, open label, randomized, two period, two treatment crossover study in which 10 healthy adult subjects received two separate single dose administrations of cetirizine HCl 10mg, following an overnight fast of at least 10 hours. Subjects received Cetirizine HCl 10mg Zydis® ODT and Zirtek® 10mg reference product in a randomized fashion. Results The cetirizine and cyclodextrin were successfully incorporated into the Zydis® dosage form resulting in elegant ODTs with rapid disintegration properties (<2 seconds). Stability and dissolution testing results were comparable or favorable to the Zirtek® control. The results of the 30 subject taste panel were positive, demonstrating that taste masking had been achieved by the use of the cyclodextrin (Figure 1). The probe bioequivalence study in 10 subjects showed that the Zydis® product was bioequivalent to the Zirtek® comparator.

Figure 1. Taste trial questionnaire results Conclusion This study confirmed that cyclodextrin can be successfully incorporated into the Zydis® ODT. The formulation is chemically stable, taste masked and is bioequivalent to the Zirtek® reference product. The work confirms the feasibility of developing a commercially viable ODT formulation of cetirizine HCl 10mg.

Reference: 1. Seager, H.: J.Pharm.Pharmacol. 1998, 50: 375-382.

PT.33 PT.32


Expression and localization of various tight junction-associated proteins in porcine hair follicles and their contribution to trans-dermal barrier function Mathes, C.1; Brandner, J.2*; Hansen, S.3; Schäfer, U.F.1; Lehr, C.-M.1,3* 1 Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, Saarland, Germany, [email protected] 2 Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Hamburg, Germany 3 Department of Drug Delivery, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz-Center for Infection Research (HZI), Saarbrücken, Germany *Correspondence author

Barrier properties of hair follicles gained increasing interest during the last few years, especially with respect to non-invasive antigen delivery via the transfollicular route [1].Tight junctions (TJs), which are barrier-forming paracellular junctions composed of various TJ transmembrane proteins (e.g. claudin-1 to -24, occludin, tricellulin, and junctional adhesion molecules (JAMs)), as well as intracellular scaffold proteins (e.g. ZO-1, -2, -3) [2], have been shown to be localized and expressed in the human hair follicle as was demonstrated several years ago by Brandner et al [3]. In simple epithelia and endothelia their role as a barrier has been well characterized; in stratified epithelia only first barrier properties have been elucidated. Due to difficulty in obtaining human tissue for these types of experiments, we were interested in comparing human and porcine TJ-associated proteins in the hair follicle in order to determine whether the pig ear is suitable for further barrier property investigation. Thus, we performed immunohistochemical staining on paraffin sections (5 µm) of full thickness porcine ear skin using antibodies specific for claudin 1, claudin 4, occludin and ZO-1. Expression and localization of claudin 1, occludin and ZO-1 in pig were in accordance with data reported by Brandner et al. in human [3]. Claudin 1 and 4 revealed a broader distribution in porcine trichocytes than in human tissue, however, after restaining human tissue the same distribution was seen concluding that this effect was most likely caused by higher sensitivity of the new batch of antibody used. Claudin 1 showed the broadest distribution throughout the entire hair follicle, as can also be seen in the epidermis. Co-localizations of at least two proteins were also seen in areas previously reported in the human hair follicle. In conclusion, no difference was seen in the expression and localization of the four TJ-associated proteins making the pig ear model suitable for future investigations. References: 1. Mittal, A. et al.: Vaccine, 2013, 31(34): 3442-3451. 2. Aijaz, S.; Balda, M.S.; Matter, K.: Int. Rev. Cytol. 2006, 248: 261-298. 3. Brandner, J.M. et al.: Arch. Dermatol. Res. 2003, 295(5): 211-221.

PT.34

212

AUTHORS INDEX Abdel-Aziz, H. ................................................................. 98, 151, 188 Abdelmohsen, U.R. ...................................................................... 182 Abe, I. ........................................................................................... 110 Abts, H.F. ........................................................................................ 75 Achenbach, J. ............................................................................... 167 Adler, M. ....................................................................................... 103 Admas, T.H. .................................................................... 72, 176, 178 Ahmad, K. ..................................................................................... 138 Aigner, A. ...................................................... 107, 130, 133, 134, 206 Akakawa, T. .................................................................................. 110 Alasel, M. ...................................................................................... 141 Alban, S. ............................................................................... 115, 186 Albishri, H.M. ................................................................ 144, 162, 168 Al-Gousous, J. .............................................................................. 198 Alhazmi, H.A. ................................................................ 144, 162, 168 Allegretta, G. ................................................................................. 165 Alves, P. ....................................................................................... 206 Andrews, K.T. ................................................................................. 58 Ansari, N. ........................................................................................ 75 Apel, A.K. ...................................................................................... 143 Appelt, A.K. ................................................................................... 170 Argyrou, A. .................................................................................... 157 Arisawa, M. ................................................................................... 109 Arntjen, A. ............................................................................. 204, 208 Asfaw, H. ...................................................................................... 158 Asseburg, H. ................................................................................. 185 Avery, V.M. ..................................................................................... 58 Awwad, K. ....................................................................................... 26 Ayata, K. ....................................................................................... 122 Bacher, A. ............................................................................. 153, 162 Bähre, H. ...................................................................................... 140 Bakowsky, U. ........................................................................ 160, 205 Baleerio, R. ..................................................................................... 64 Balk, A. ......................................................................................... 195 Ball, A.-K. ...................................................................................... 124 Ballell, L. ....................................................................................... 157 Barandun, L.J. .............................................................................. 161 Barho, M.T. ........................................................................... 113, 161 Bartuschat, A.L. ............................................................................ 169 Basavarajappa, D. ........................................................................ 138 Bassett, D.J.P. ................................................................................ 27 Basu, D. ................................................................................ 131, 155 Baudiß, K. ..................................................................................... 122 Bauer, M.R. .................................................................................... 45 Baumeister, S. .............................................................................. 159 Baumgärtel, A. .............................................................................. 188 Bautista, O. ................................................................................... 180 Bechthold, A. .................................................................................. 55 Becker, C. ............................................................................. 129, 131 Beese, K. .............................................................................. 136, 172 Begley, D. ..................................................................................... 154 Behrendt, C.T. ...................................................................... 153, 162 Bendas, G. .................................................................... 128, 129, 130 Bender, A. ....................................................................................... 49 Bensinger, D. ................................................................................ 175 Berger, T. ...................................................................................... 159 Bermudez, M. ......................................................................... 70, 179 Bernat, V. ........................................................................ 72, 176, 178 Bernhardt, G. ........................................................................ 171, 180 Bernhardt, P. ................................................................................ 106 Berressem, D. ....................................................... 123, 184, 185, 192 Betz, M. ........................................................................................ 161 Bhandari, D. .................................................................................... 60 Biel, M. .......................................................................................... 156

Biela, A. ........................................................................................ 142 Bischoff, I. ..................................................................................... 123 Blank, S. ....................................................................................... 194 Blaßhofer, F. ................................................................................. 134 Blättermann, S. ............................................................................. 180 Blöcher, R. .................................................................................... 172 Blohm, A. ...................................................................................... 156 Bock, A. .......................................................................................... 99 Bock, S. ........................................................................................ 159 Boddy, A.V. ................................................................................... 195 Bodem, J....................................................................................... 159 Boeckler, F.M. ................................................................................ 45 Bogan, R. ...................................................................................... 205 Bolte, K. ........................................................................................ 191 Bonnet, D. ....................................................................................... 47 Bonus, M......................................................................................... 53 Boomgaren, M. ............................................................................. 159 Boos, J. ......................................................................................... 195 Borchiellini, M. .............................................................................. 185 Borek, C. ....................................................................................... 128 Borghardt, J.M. ............................................................................. 196 Botermann, L. ............................................................................... 151 Böttcher, M.M. .............................................................................. 160 Bou-Chacra, N.A. ......................................................................... 203 Bourayou, R. ................................................................................. 209 Bracher, F. ............................................................................ 123, 156 Brandner, J. .......................................................................... 187, 211 Braun, A. ............................................................................... 157, 167 Brewster, M.E. ................................................................................ 90 Brezesinski, G. ............................................................................... 77 Briel, D. ......................................................................................... 178 Britz, H. ......................................................................................... 150 Brockmeyer, J. .............................................................................. 198 Brönstrup, M. ................................................................................ 135 Brosig, H. .............................................................................. 148, 170 Brötz, E. ........................................................................................ 186 Brox, R. ................................................................... 72, 176, 177, 178 Brücher, K. .................................................................................... 162 Brück, S. ....................................................................................... 152 Brüggerhoff, A. ............................................................................. 139 Brüßler, J. ..................................................................................... 160 Büchold, C. ................................................................................... 141 Bührmann, M. ............................................................................... 155 Büllesbach, K. ............................................................................... 180 Bunjes, H. ..................................................................................... 209 Burghaus, R. ................................................................................... 44 Busch, D. ...................................................................................... 152 Buschauer, A. ......................................................... 69, 139, 171, 180 Butz, L........................................................................................... 139 Caffrey, C.R. ................................................................................. 183 Calderon, M. ................................................................................. 199 Camacho, C. ................................................................................... 52 Cardinaux, J.-R. ............................................................................ 176 Carlino, L. ..................................................................................... 136 Carlomagno, T. ............................................................................... 51 Carrasco-Gomez, R. ..................................................................... 174 Carrier, L. ...................................................................................... 176 Carrondo, M. ................................................................................. 206 Cheng-Chang, C. .......................................................................... 156 Cheung, S.-Y. ............................................................................... 125 Chhatwal, G.S. ............................................................................. 184 Chirinda, B. ................................................................................... 165 Chung, C....................................................................................... 157 Cieslik, M.B. .................................................................................... 45


Ciglia, E. ............................................................................... 142, 143 Cinatl jr., J. .................................................................................... 134 Clement, B. ................................................................................... 173 Codutti, L. ....................................................................................... 51 Collins, C. ..................................................................................... 142 Cools, F. ....................................................................................... 120 Cristofoletti, R. ................................................................................ 93 Culmsee, C. ...................................... 29, 32, 113, 161, 189, 190, 191 Cusack, A. .................................................................................... 210 Dahse, H.-M. ................................................................................ 159 Dai, B. ........................................................................................... 123 Danhof, M. .................................................................................... 120 Darras, F.H. .................................................................................. 163 Dassinger, N. ........................................................................ 141, 145 De Min, A. ..................................................................................... 177 de Sousa Amadeu, N. .................................................................. 142 de Sousa, L.R.F. ........................................................................... 182 Debaene, F. .................................................................................. 161 Decher, N. ...................................................................................... 32 Decker, H. ............................................................................. 194, 196 Decker, M. ...................................................................... 71, 163, 187 Degenhardt, I. ....................................................................... 113, 159 Denzer, I. ...................................................................................... 189 Diederich, F. ................................................................................. 161 Diederich, W. ........................................................................ 159, 166 Diedrich, A. ..................................................................................... 64 Diedrich, D. ................................................................................... 143 Diehl, O. ........................................................................................ 125 Diemert, S. .................................................................................... 191 Diergardt, T. .......................................................................... 149, 209 Diesel, B. ...................................................................................... 123 Diethelm, S. .................................................................................. 106 Dietrich, T. .................................................................................... 209 Diken, M. ........................................................................................ 77 Dingemanse, J. ............................................................................. 147 Dings, C. ....................................................................................... 147 Dircks, M.G. .................................................................................. 149 Dirksen, U. .................................................................................... 128 Dirsch, V.M. .................................................................................... 54 Dittmar, F. ..................................................................................... 140 Dodel, R. ....................................................................................... 190 Doi, T. ........................................................................................... 111 Dolga, A.M. ..................................................................... 32, 190, 191 Dömling, A. ..................................................................................... 52 Dörje, F. ........................................................................................ 149 Dos Santos Capelo, R. ................................................................. 139 Drescher, S. .................................................................................. 163 Duffy, S. .......................................................................................... 58 Ebeling, S. .................................................................................... 186 Ebert, R. ....................................................................................... 167 Ecke, M. ........................................................................................ 155 Eckert, G.P. .................................................. 123, 184, 185, 191, 192 Efferth, T. ................................................................................ 95, 182 Ehrig, K. ........................................................................................ 186 Ehrlich, S.M. ................................................................................. 132 Eickhoff, C. ................................................................................... 151 Einsle, O. ........................................................................................ 85 Eisenreich, W. .............................................................................. 153 El Deeb, S. ................................................................... 144, 162, 168 Elewa, M. ...................................................................................... 163 Elgaher, W.A.M. ........................................................................... 168 El-Hady, D.A. ................................................................ 144, 162, 168 Elsässer, K. .......................................................................... 113, 189 Empting, M. .......................................................................... 144, 165 Endreas, W. .................................................................................. 160 Engel, F. ....................................................................................... 134 Engel, J. ................................................................ 129, 131, 155, 165 Engels, B. ............................................................................. 128, 170 Erdelmeier, C.A.J. ........................................................................ 183

Erker, T. ........................................................................................ 173 Eschenhagen, T. .......................................................................... 176 Esparza, I........................................................................................ 77 Ewe, A. ......................................................................................... 206 Exner, T.E. ...................................................................................... 51 Fabian, J. ...................................................................................... 161 Fan, A. .......................................................................................... 140 Fang, Z. ........................................................................................ 129 Faust, A. ....................................................................................... 114 Feeder, N. ....................................................................................... 50 Fehler, S.K. ................................................................................... 169 Feizabad, M.S. ............................................................................. 164 Feldmann, D. ........................................................................ 149, 209 Fenical, W. .................................................................................... 106 Ferreirόs, N. .................................................................................. 124 Fersht, A.R...................................................................................... 45 Fettel, J. ........................................................................................ 126 Fischer, B...................................................................................... 188 Fischer, K...................................................................................... 125 Fischer, M. ............................................................................ 153, 162 Flaßhoff, M.................................................................................... 131 Fleck, E. ........................................................................................ 209 Fleckenstein, K. .............................................................................. 88 Fleming, I. ....................................................................................... 26 Flesch, D............................................................................... 167, 174 Flinspach, K. ................................................................................. 143 Förster, F. ....................................................................................... 56 Fox, D. .......................................................................................... 154 Franco, R. ..................................................................................... 181 Frank, J. ................................................................................ 163, 184 Franke, N. ..................................................................................... 123 Franke, R. ..................................................................................... 135 Fransson, I. ................................................................................... 171 Fredriksson, K. ............................................................................... 51 Freeman, B.A. ................................................................................ 26 Freigang, M................................................................................... 184 Friedland, K. ........................................................... 87, 147, 149, 189 Friedland-Leuner, K. ............................................................. 184, 189 Friedrich, C. .................................................................................. 165 Friess, W....................................................................................... 199 Fritz, D. ........................................................................................... 77 Fröhlich, T. .................................................................................... 132 Fröhling, S. ..................................................................................... 83 Frömel, T. ....................................................................................... 26 Frötschl, R. ................................................................................... 134 Fruth, M. ....................................................................................... 144 Fuchs, S........................................................................................ 183 Fujii, H. ............................................................................................ 40 Fujii, N. ............................................................................................ 41 Fukuda, H. .................................................................................... 109 Funari, S. ........................................................................................ 77 Fürst, R. ................................................................................ 123, 183 Fürtig, M........................................................................................ 149 Gabler, M. ............................................................................. 167, 174 Gajer, M. ......................................................................................... 85 Gallacher, G.J. .............................................................................. 120 Ganjam, G.K. ........................................................................ 190, 191 Garscha, U.................................................................................... 124 Geisslinger, G. ........................................................................ 26, 124 George, S. .................................................................................... 139 Gerber, U. ..................................................................................... 128 Gerbier, R. .................................................................................... 168 Gerhardt, S. .................................................................................... 85 Germershaus, O. .......................................................................... 157 Gerst, M. ....................................................................................... 204 Gerstmeier, J. ............................................................................... 124 Gerth, K. ....................................................................................... 184 Getlik, M........................................................................................ 165 Gieselmann, V. ............................................................................. 117

214

Gillard, M. ..................................................................................... 179 Glaser, J. ...................................................................................... 183 Gmeiner, P. .................................................................................. 169 Gnanapragassam, V.S. ................................................................ 123 Goerigk, G. ................................................................................... 208 Gohlke, H. ....................................................................... 53, 142, 143 Gollos, S. ........................................................................................ 62 Gomeza, J. ................................................................................... 179 Göring, S. ..................................................................................... 175 Gorjup, E. ..................................................................................... 206 Gorzelanny, C. .............................................................................. 129 Gossmann, R. ............................................................................... 198 Gotta, V. ....................................................................................... 120 Grabow, N. ..................................................................................... 36 Grathwol, C. .................................................................................. 170 Gräwert, T. ............................................................................ 153, 162 Grebenstein, N. ............................................................................ 184 Grevelding, C.G. ........................................................................... 156 Griese, N. ..................................................................................... 151 Griesinger, C. ................................................................................. 51 Grimm, C. ..................................................................................... 156 Groh, M. ........................................................................................ 168 Groll, M. ........................................................................................ 153 Grother, L. .................................................................................... 210 Grün, J. ......................................................................................... 182 Grünebaum, J. .............................................................................. 205 Grünweller, A. ............................................................... 107, 130, 133 Grütter, C. ..................................................................................... 165 Guenther, S. ................................................................................... 60 Guetschow, M. .............................................................................. 180 Günther, S. ............................................................................. 68, 174 Guo, Z. .......................................................................................... 176 Guthoff, R. ...................................................................................... 63 Gutmann, M. ................................................................................. 167 Haas, H. .......................................................................................... 77 Haase, C. ...................................................................................... 164 Hachenthal, N. .............................................................................. 123 Hack, C. .......................................................................................... 88 Häcker, G. .................................................................................... 186 Hacker, M.C. ................................................................................. 206 Häckh, M. ....................................................................................... 68 Haefeli, W.E. ................................................................................. 147 Haeuser, M. .................................................................................. 204 Häfner, A.-K. ........................................................................... 26, 124 Hagl, S. ................................................................................. 184, 191 Hähn, S. ........................................................................................ 153 Hahne, M. ....................................................................................... 34 Hamacher, A. .................................................................................. 58 Hammami, M. ............................................................................... 132 Hammamy, M.Z. ........................................................................... 164 Hanefeld, A. .................................................................................... 64 Hanh, B.D. ...................................................................................... 63 Hanke, T. ...................................................................................... 125 Hansen, F.K. ................................................................... 58, 142, 143 Hansen, S. .................................................................................... 211 Harder, M. ....................................................................................... 28 Hardick, J. ..................................................................................... 155 Hardt, S. ....................................................................................... 192 Harenberg, J. ................................................................................ 152 Harloff, M. ..................................................................................... 133 Harmer, N.J. ................................................................................. 154 Hartmann, R.K. ............................................................. 107, 130, 133 Hartmann, R.W. ...................................................... 18, 144, 165, 168 Hasenfuss, G. ............................................................................... 176 Hassan, T.H. ................................................................................. 199 Haupenthal, J. ...................................................................... 144, 168 Hausch, F. .................................................................................... 189 Häussinger, D. ................................................................................ 53 Havemeyer, A. .............................................................................. 173

Hawarden, A. ................................................................................ 156 Heide, H. ......................................................................................... 26 Heilmann, J. .................................................................................. 187 Hein, M. ........................................................................................ 154 Heine, A. ....................................................... 132, 141, 142, 161, 166 Heinrich, M.R. ............................................................................... 169 Held, J............................................................................. 58, 153, 162 Hellmann, N. ......................................................................... 194, 196 Hellwig, M. .................................................................................... 134 Helmstädter, A. ............................................................................. 182 Hempel, G............................................... 86, 128, 154, 161, 173, 195 Hengl, T. ......................................................................................... 75 Hennen, S. .................................................................................... 179 Henrich, A. .................................................................................... 136 Hentschel, U. ................................................................................ 182 Hergenröther, A. ........................................................................... 152 Herrmann, J. ................................................................................... 56 Herz, T. ......................................................................................... 173 Herzog, J. ..................................................................................... 132 Heuckmann, J.M. .......................................................................... 165 Heusler, E. .................................................................................... 170 Hibert, M. ........................................................................................ 47 Hilgenfeld, R. ................................................................................ 164 Hillebrecht, A. ............................................................................... 159 Hils, M. .......................................................................................... 141 Hintersteininger, M. ...................................................................... 173 Hinz, S. ......................................................................................... 181 Hirao, T. ........................................................................................ 109 Hirayama, S. ................................................................................... 40 Höbel, S. ....................................................................................... 134 Hoeglinger, G.U. ........................................................................... 191 Hofmann, B. .................................................... 26, 124, 125, 126, 127 Hohmann, C.................................................................................... 31 Holak, T. ......................................................................................... 52 Holländer, A. ................................................................................. 158 Höllein, L. ...................................................................................... 153 Höllerhage, M. .............................................................................. 191 Holloway, S. .................................................................................. 159 Holzgrabe, U. ................................ 153, 154, 157, 166, 183, 190, 195 Homann, J. ................................................................................... 124 Hommoss, G. ................................................................................ 204 Hönzke, S. .................................................................................... 122 Hoppstädter, J. ............................................................................. 123 Horstkorte, R................................................................................. 123 Hoser, S. ................................................................................. 98, 188 Hoß, S.G. ...................................................................................... 128 Hou, Z. ............................................................................................ 41 Hsieh, L.T. .................................................................................... 183 Huang, G. ..................................................................................... 187 Huber, S.M.................................................................................... 139 Hübner, H. .................................................................................... 169 Hummel, M. .................................................................................. 198 Hüsecken, K. ................................................................................ 144 Hüttel, S. ......................................................................................... 56 Hüwel, D. ...................................................................................... 156 Idzko, M. ....................................................................................... 122 Ihling, C......................................................................................... 116 Ilan, N. .......................................................................................... 128 Ilko, D. ........................................................................................... 157 Illarionov, B. .......................................................................... 153, 162 Imhof, A. ....................................................................................... 136 Imming, P...................................................................... 157, 158, 163 Imran, I.......................................................................................... 192 Irsheid, L. ...................................................................................... 128 Isaak, J. .......................................................................................... 26 Ishida, Y. ....................................................................................... 111 Ishimura, K.................................................................................... 109 Itami, K.......................................................................................... 114 Iwai, T. ............................................................................................ 40


Jaehde, U. ........................................................................ 62, 88, 134 Jakob, F. ....................................................................................... 167 Jakobs, H. ..................................................................................... 173 Jakubzig, B. .................................................................................. 129 Janiak, C. ...................................................................................... 142 Janke, J. ......................................................................................... 64 Jantscheff, P. ................................................................................ 129 Jenner, D. ..................................................................................... 154 Jensen, A.A. ................................................................................. 190 Jiménez-Ruiz, A. .......................................................................... 154 Jin, N..................................................................................... 200, 201 Jivishov, E. ................................................................................... 185 Jivishova, S. ................................................................................. 185 Jockers, R. .................................................................................... 168 Joerger, A.C. .................................................................................. 45 Joerger, M. ..................................................................................... 42 John, C. ........................................................................................ 173 Johnson, R. .................................................................... 81, 194, 196 Jones, G. ................................................................................ 35, 170 Jose, J. ......................................................................................... 134 Juli, C. ........................................................................................... 154 Jung, A. .......................................................................................... 78 Jung, M. .......................................................................... 85, 136, 172 Junker, A. ..................................................................................... 114 Kaever, V. ............................................................................. 140, 160 Kahnt, A.S. ................................................................... 124, 125, 139 Kaiser, A. ...................................................................................... 102 Kaitsiotou, H. ................................................................................ 131 Kalayda, G.V. ......................................................................... 62, 134 Kamal, A. ...................................................................................... 144 Kanitz, M. ...................................................................................... 159 Kapitzke, C. .................................................................................. 143 Karaman, B. .................................................................................... 85 Kascholke, C. ............................................................................... 206 Kassack, M.U. ................................................................................ 58 Katryniok, C. ................................................................................. 138 Kaul, A. ......................................................................................... 176 Kaule, S. ......................................................................................... 36 Kauzor, D. ..................................................................... 148, 149, 170 Kaysser, L. .................................................................................... 106 Keck, C.M. .................................... 200, 201, 202, 203, 204, 207, 208 Kees, F. ........................................................................................ 148 Kelber, O. ................................................................. 97, 98, 151, 188 Keller, M. ...................................................................................... 156 Keller, T. ....................................................................................... 209 Kempin, W. ..................................................................................... 36 Kersten, E. .................................................................................... 194 Kersting, D. ................................................................................... 102 Kesselring, J. ................................................................................ 170 Keßler, V. ...................................................................................... 150 Keusgen, M. ......... 134, 141, 143, 145, 148, 158, 164, 183, 185, 186 Khoshakhlagh, P. ........................................................... 81, 194, 196 Khoury, K. ....................................................................................... 52 Kiefer, W. ...................................................................................... 159 Kiemer, A.K. ................................................................................. 123 Kietz, A. ........................................................................................ 134 Kim, N.H. ........................................................................................ 27 Kindgen, S. ............................................................................. 81, 195 Klaubert, B. ................................................................................... 205 Klebe, G. ............................................... 132, 141, 142, 159, 161, 166 Klein, J. ........................................................................... 30, 123, 192 Klein, S. ........................................................................................ 194 Klemm, M. .................................................................................... 157 Klitsche, F. .................................................................................... 172 Kloft, C. ........................................... 20, 136, 148, 149, 151, 170, 196 Knolle, P. ........................................................................................ 64 Ko, Y.D. .......................................................................................... 88 Koch, E. ........................................................................................ 183 Koch, K.A. ............................................................................. 123, 192

Koeberle, A. .................................................................................... 25 Koeberle, S. .................................................................................... 25 Koes, D. .......................................................................................... 52 Kolb, P. ......................................................................................... 141 Kölbel, K. ...................................................................................... 116 Koling, S. ........................................................................................ 86 Kölln, C. .......................................................................................... 34 Konerding, M. ............................................................................... 194 Konietzka, J. ................................................................................. 192 König, B. ....................................................................................... 171 Kontny, N.E................................................................................... 195 Konzuch, S. .................................................................................. 162 Kornhuber, J. .................................................................................. 87 Kortum, F. ..................................................................................... 156 Kostenis, E............................................................................ 179, 180 Kostewicz, E.S. ............................................................................... 92 Kotz, S. ......................................................................................... 134 Kouretova, J.................................................................................. 164 Kowarz, E. .................................................................................... 137 Kraft, K. ................................................................................. 151, 187 Kramer, C. ...................................................................................... 46 Kranz, L.M. ..................................................................................... 77 Kraus, A.L. ............................................................................ 161, 189 Krause, A. ..................................................................................... 147 Krauth-Siegel, R.L. ....................................................................... 154 Krebs, F. ....................................................................................... 165 Krebs, L. ................................................................................. 81, 208 Kreiter, S. ........................................................................................ 77 Kretschmer, S.B.M. .............................................................. 125, 126 Krischke, M. .................................................................................. 195 Krueger, K..................................................................................... 151 Krüger, M. ..................................................................................... 102 Küchler, S. ............................................................................ 122, 199 Kühn, A. ........................................................................................ 137 Kühnreich, R. ................................................................................ 166 Kullmann, M. ........................................................................... 62, 134 Kunfermann, A. ............................................................................. 153 Kunz, C. ........................................................................................ 196 Kuroda, N........................................................................................ 40 Kurz, T. ........................................................... 58, 142, 143, 153, 162 Ladwein, K.I. ........................................................................... 85, 136 Läer, S. ........................................................................................... 89 Laino, V......................................................................................... 190 Lamers, C. ............................................................................ 172, 174 Lang, M. ........................................................................................ 178 Lange-Grünweller, K. .................................................... 107, 130, 133 Langer, B. ..................................................................................... 102 Langer, K. ..................................... 173, 198, 202, 203, 204, 205, 206 Langguth, P. ............................. 80, 81, 119, 194, 195, 196, 198, 208 Lappe, S. ...................................................................................... 202 Lategahn, J. .................................................................................. 131 Laufer, S. ........................................................................................ 25 Laufs, U. ....................................................................................... 176 Laven, A.......................................................................................... 89 Lechtape, B................................................................................... 128 Lee, W. ......................................................................................... 170 Lehmann, C. ................................................................................. 124 Lehnen, D. .................................................................................... 122 Lehr, C.-M. ...................................................................... 64, 210, 211 Lehr, T. ........................................................................... 43, 147, 150 Leigh, M. ......................................................................................... 91 Lemcke, T. .................................................................................... 171 Lendlein, A. ..................................................................................... 63 Li, L. .............................................................................................. 167 Li, S.-M. ................................................................................ 140, 146 Li, W. ............................................................................................. 117 Licha, K. ........................................................................................ 209 Lieb, S........................................................................................... 180 Liebhold, M. .................................................................................. 146

216

Liebl, J. ........................................................................... 57, 131, 132 Lienau, C. ............................................................................. 153, 162 Lietsche, J. ................................................................................... 192 Lill, A.P. ........................................................................................ 127 Lill, N. .............................................................................................. 26 Lingelbach, K. ............................................................................... 159 Link, A. .................................................................................. 136, 172 Lippert, J. ........................................................................................ 44 Listing, M. ..................................................................................... 125 Löbler, M. ........................................................................................ 63 Loesgen, S. .................................................................................. 106 Look, J. ......................................................................................... 206 Lorenz, K. ..................................................................................... 176 Lorimer, D. .................................................................................... 154 Löscher, D. ................................................................................... 137 Lucas, X. ................................................................................. 68, 174 Lüdeke, S. .............................................................................. 68, 143 Lühmann, T. ........................................................... 35, 167, 170, 206 Lühn, S. ........................................................................................ 115 Lum, L.G. ........................................................................................ 27 Lunter, D.J. ................................................................................... 118 Lutz, S. ......................................................................................... 176 Luzhetskyy, A. .............................................................................. 186 Mäder, K. ...................................................................... 163, 199, 207 Mäder, P. ...................................................................................... 156 Madisch, A. ................................................................................... 151 Maginn, S.J. .................................................................................... 50 Maier, T.J. ............................................................................... 26, 125 Maison, W. .................................................................................... 172 Maiwald, A. ................................................................................... 132 Mandl, M. ........................................................................................ 57 Marek, L. ......................................................................................... 58 Markert, C. .................................................................................... 147 Marschalek, R. ................................................................ 84, 137, 138 Martin, J. ....................................................................................... 188 Massing, U. ................................................................................... 129 Mathes, C. .................................................................................... 211 Matsuda, T. ................................................................................... 109 Matsuda, Y. .................................................................................. 110 Maul, K.J. .............................................................................. 149, 209 Maus, A. ....................................................................................... 208 Mayer-Wrangowski, S. ................................................................. 144 Mazur, A. ........................................................................................ 51 Mederos y Schnitzler, M. .............................................................. 101 Meinel, L. ................................................ 35, 157, 167, 170, 195, 206 Meinhardt, K. ................................................................................ 194 Meinke, M.C. ................................................................................ 202 Meister, S. ...................................................................................... 58 Memmel, E. ............................................................................ 35, 167 Meng, M. ......................................................................................... 77 Merfort, I. .............................................................................. 186, 187 Merk, D. ................................................................................ 167, 174 Merk, H. ........................................................................................ 131 Merkel, O.M. ................................................................................... 27 Merkens, J. ................................................................................... 138 Merten, N. ..................................................................................... 179 Metz, H. ........................................................................................ 199 Metzger, E. ................................................................................... 136 Metzger, S. ............................................................................. 62, 134 Meyborg, H. .................................................................................. 209 Michaelis, M. ................................................................................. 134 Michler, V. ..................................................................................... 165 Mielke, M.G. ......................................................................... 143, 148 Mikula, M. ..................................................................................... 173 Mikulits, W. ................................................................................... 132 Milanos, L. .................................................................................... 177 Milne, T. .......................................................................................... 82 Mishenzon, N. ............................................................................... 188 Miyata, N. ....................................................................................... 39

Mohr, K. ........................................................................ 165, 177, 179 Mohsen, A.Y. ................................................................................ 190 Möller, H.M. .................................................................................... 51 Möllmann, U.................................................................................. 157 Moore, B.S. ................................................................................... 106 Mordmüller, B. ................................................................ 58, 153, 162 Morhenn, K. .................................................................................. 176 Mori, T. .......................................................................................... 110 Morissette, P. ................................................................................ 120 Mozafari, M. .................................................................. 144, 162, 168 Mueller, M. .................................................................................... 185 Muenster, U. ................................................................................... 76 Mulac, D........................................................................ 198, 203, 205 Müller, C. ...................................................................................... 150 Müller, C.E. ........................................................... 117, 178, 179, 181 Müller, G. ...................................................................................... 140 Müller, J. ....................................................................................... 187 Müller, M. ........................................................................................ 68 Müller, R. ........................................................................ 56, 131, 184 Müller, R.H. ........................................... 200, 201, 202, 203, 204, 207 Münsterberg, M. ........................................................................... 171 Murase, H. .................................................................................... 111 Muromoto, R. ................................................................................ 109 Myler, P......................................................................................... 154 Nachbar, M. .................................................................. 144, 162, 168 Nadithe, V. ...................................................................................... 27 Nagase, H. ...................................................................................... 40 Nagel, N. ......................................................................................... 73 Nagel, S. ......................................................................................... 36 Naggi, A.M. ................................................................................... 128 Nakagawa, H. ......................................................................... 39, 111 Nam, S.-J. ..................................................................................... 106 Narkhede, Y. ................................................................................. 164 Nawroth, T. ............................................................. 81, 194, 196, 208 Neffe, A.T........................................................................................ 63 Negri, M. ....................................................................................... 144 Neitemeier, S. ....................................................................... 190, 191 Neochoritis, C. ................................................................................ 52 Netter, M. ........................................................................................ 32 Neumann, D.................................................................................. 176 Neumann, S. ................................................................. 148, 185, 186 Neumann-Haefelin, T. .................................................................... 31 Nguyen, M.A. .................................................................................. 80 Nieber, K. ................................................................................ 98, 188 Nishiyama, S................................................................................. 112 North, N.J........................................................................................ 85 Norville, I.H. .................................................................................. 154 Noske, N. ...................................................................................... 206 Obreque-Balboa, J. ...................................................................... 171 Obst, K. ......................................................................................... 199 Oertel, W.E. .................................................................................. 191 Oetjen, E. ...................................................................................... 176 Ohno, H. ......................................................................................... 41 Ohta, E.......................................................................................... 112 Oishi, S. .......................................................................................... 41 Okamura, H. ................................................................................... 48 Okpanyi, S.N............................................................. 97, 98, 151, 188 Olbrich, C. ....................................................................................... 74 Oli, S. ............................................................................................ 182 Oltmann-Norden, I. ....................................................................... 169 Onila, I. ........................................................................................... 51 Oppermann, S. ..................................................... 113, 161, 189, 190 Ortmann, R. .................................................................................. 159 Ostrowki, J. ................................................................................... 173 Oswald, S. .................................................................................... 152 Ott, I. ............................................................................................. 135 Pahl, A. ......................................................................................... 176 Pannek, M....................................................................................... 85 Parnham, M.J. .............................................................................. 124


Parra-Guillen, Z. ........................................................................... 136 Pasternack, R. .............................................................................. 141 Pauly, A. ......................................................................................... 87 Pawlowska, D. ................................................................................ 77 Pein, M. .......................................................................................... 37 Penning, M. .................................................................................. 198 Peters, J.-U. .................................................................................... 94 Peters, L. ...................................................................................... 179 Peters, T. ...................................................................................... 208 Petersen, S. .................................................................................... 36 Pfankuchen, D. ............................................................................. 130 Pinnapireddy, S.R. ........................................................................ 205 Pischetsrieder, M. ................................................................. 184, 189 Plank, C. ....................................................................................... 185 Platzer, C. ..................................................................................... 128 Plesnila, N. ..................................................................................... 32 Pohland, M. .......................................................................... 185, 191 Popa, A.-L. ...................................................................................... 77 Potratz, J. ..................................................................................... 128 Prante, O. ..................................................................................... 169 Preidl, J.J. ..................................................................................... 123 Prinz, E.-M. ................................................................................... 119 Prochnicka, A. .............................................................................. 135 Proschak, E. ........................................................... 96, 125, 167, 172 Prosper, F. .................................................................................... 206 Prothiwa, M. .................................................................................. 156 Pyo, S.M. ...................................................................................... 202 Quentin, T. .................................................................................... 176 Rademann, J. ............................................................................... 123 Radke, C. ...................................................................................... 161 Rådmark, O. ................................................................................. 138 Radziwill, R. .................................................................................... 31 Raesch, S. .................................................................................... 210 Rauh, D. ....................................................... 129, 131, 144, 155, 165 Raynor, A. ..................................................................................... 129 Rechlin,C. ..................................................................................... 166 Redweik, S. .......................................................................... 144, 168 Reichl, S. ........................................................................................ 34 Reinehr, R. ..................................................................................... 53 Reitz, E. ........................................................................................ 198 Reske, T. ........................................................................................ 36 Reuter, K.C. .................................................................................... 77 Richter, A. ..................................................................................... 157 Richter, F. ....................................................................................... 78 Richter, M. .............................................................................. 32, 190 Richters, A. ................................................................................... 165 Riederer, U. .................................................................................. 153 Riegel, K. ........................................................................................ 75 Rietscher, R. ................................................................................... 64 Rinne, A. ....................................................................................... 100 Roberto, S. ................................................................................... 134 Rödl, C.B. ..................................................................... 125, 126, 127 Rodriguez, J.R. ............................................................................. 206 Roessler, C. .................................................................................... 85 Rohde, M. ..................................................................................... 184 Rohe, A. ........................................................................................ 128 Romero, G.B. ........................................................................ 200, 203 Römpp, A. ....................................................................................... 60 Roos, D. ........................................................................................ 187 Roos, J. .......................................................................................... 26 Rösch, P. ...................................................................................... 154 Ross, T. ........................................................................................ 129 Rostamizadeh, K. ......................................................................... 204 Rothweiler, F. ............................................................................... 134 Rox, K. .......................................................................................... 184 Royer, H.D. ................................................................................... 130 Ruberg, E.-M. ............................................................................... 104 Ruberg, K. ...................................................................................... 88 Rudolph, C. ................................................................................... 105

Rudolph, I. .................................................................................... 157 Ruff, A. ............................................................................................ 92 Ruge, C......................................................................................... 210 Ruhe, D......................................................................................... 149 Ruick, R. ....................................................................................... 207 Rumpf, T. ........................................................................................ 85 Ruth, P. ................................................................................... 16, 139 Rüther, A....................................................................................... 143 Rutherford, T.J. ............................................................................... 45 Saal, C. ........................................................................................... 65 Saarberg, W.................................................................................. 183 Sadek, M.S. .................................................................................. 168 Sahin, U. ......................................................................................... 77 Sahner, J.H. .................................................................................. 168 Saitoh, T. ...................................................................................... 112 Samadi, S. .................................................................................... 183 Sanglier-Cianférani, S. ................................................................. 161 Sannajust, F.................................................................................. 120 Sarin, N. ........................................................................................ 134 Sarkar-Tyson, M. .......................................................................... 154 Sasaki, S......................................................................................... 48 Sato, H. ......................................................................................... 111 Sauer, K. ....................................................................................... 123 Saul, M.J. ...................................................................................... 124 Schader, T. ................................................................................... 125 Schaefer, C. .................................................................................. 128 Schaefer, L. .................................................................................. 183 Schaefer, M. ......................................................................... 156, 204 Schäfer, E.-M. ............................................................................... 159 Schäfer, J........................................................................................ 89 Schäfer, U.F.......................................................................... 210, 211 Schäfer-Korting, M. ....................................................................... 122 Schäftlein, A.......................................................................... 147, 150 Schäke, F...................................................................................... 178 Scheer,F. ...................................................................................... 166 Scheffler, K. .................................................................................... 61 Schepmann, D. ............................................................................. 114 Scherließ, R. ................................................................................... 64 Scherzberg, M.-C. ........................................................................ 138 Schichtel, J. .................................................................................. 119 Schiede, M.l. ................................................................................... 85 Schiedel, C.A. ............................................................................... 181 Schiewe, J. ................................................................................... 196 Schiffmann, R. .............................................................................. 156 Schilcher, P................................................................................... 136 Schiller, S........................................................................................ 64 Schirmeister, T. .................................................... 128, 159, 170, 182 Schlager, H. .................................................................................. 147 Schleithoff, C. ............................................................................... 128 Schlesinger, M. ..................................................................... 128, 129 Schlinzig, K. .................................................................................... 75 Schlitzer, M. .................................................. 113, 156, 159, 161, 189 Schlossarek, S. ............................................................................. 176 Schmetter, R. .................................................................................. 58 Schmidt, B. ................................................................................... 175 Schmidt, C. ................................................................................... 135 Schmidt, C.Q. ................................................................................. 28 Schmidt, I. ..................................................................................... 154 Schmidt, M. ................................................................................... 128 Schmiedel, K................................................................................. 147 Schmitt, M. .................................................................................... 136 Schmitz, B....................................................................................... 53 Schmueser, L. ...................................................................... 194, 196 Schnackenburg, B. ....................................................................... 209 Schneider, G. ................................................................................ 174 Schneider, I................................................................................... 150 Schneider, K. .................................................................................. 37 Schneider, L.S. ............................................................................. 132 Schneider, M................................................................................... 64

218

Schneider, T. ........................................................................ 170, 186 Schober, Y. ..................................................................................... 60 Schoenfeld, A. .............................................................................. 115 Scholl, F. ....................................................................................... 138 Scholz, B. ..................................................................................... 138 Scholz, P. ............................................................................. 201, 204 Schrader, F.C. ...................................................................... 113, 189 Schrage, R. ................................................................................... 179 Schröder, M. ................................................................................... 64 Schröder, R. ......................................................................... 179, 180 Schroeder, S. ................................................................................ 176 Schröpf, S. .................................................................................... 149 Schubert-Zsilavecz, M. ................................. 125, 167, 172, 174, 191 Schüle, R. ..................................................................................... 136 Schulte, F.W. ................................................................................ 133 Schulz, M. ..................................................................................... 151 Schulz-Fincke, J. .......................................................................... 136 Schulz-Siegmund, M. ................................................................... 206 Schurigt, U. ........................................................................... 154, 183 Schutkowski, M. ...................................................................... 85, 128 Schwab, J. ...................................................................................... 24 Schwabe, K. ................................................................................. 206 Schwan, G. ................................................................................... 178 Schwarz, R. .................................................................................. 116 Schweimer, K. .............................................................................. 154 Schweins, R. ........................................................................... 81, 208 Seeber, F. ..................................................................................... 159 Seibel, J. ......................................................................... 35, 128, 167 Seibt, F.B. ..................................................................................... 181 Seidler, S. ..................................................................................... 133 Seidlitz, A. ....................................................................................... 36 Seifert, R. ...................................................................... 140, 160, 176 Sergeev, G. .................................................................................. 135 Serno, P. ......................................................................................... 79 Serra, M. ....................................................................................... 206 Seyfried, S. ................................................................................... 140 Shibasaki, M. .................................................................................. 19 Shimizu, T. ...................................................................................... 25 Shindou, H. ..................................................................................... 25 Shopova, T. .................................................................................. 158 Shuto, S. ....................................................................................... 109 Siebenand, S. ............................................................................... 182 Siegmund, H.-U. ............................................................................. 44 Siegmund, W. ............................................................................... 152 Simmet, T. ...................................................................................... 28 Simon, K. ...................................................................................... 179 Sinclair, D.A. ................................................................................... 85 Singh, O. ....................................................................................... 157 Sinz, A. ......................................................................................... 116 Sippl, W. ......................................................................... 85, 128, 136 Skinner-Adams, T.S. ...................................................................... 58 Školová, B. ................................................................................... 122 Skopp, S. ........................................................................................ 67 Smith, S. ....................................................................................... 155 Snitko, M. ...................................................................................... 159 Söbbing, J. .................................................................................... 205 Sochorová, M. .............................................................................. 122 Soler, M.M. ................................................................................... 199 Sommerfeld, A. ............................................................................... 53 Sorg, B.L. ...................................................................................... 126 Sosio, M. ....................................................................................... 143 Sotriffer, C.A. ........................................................ 154, 163, 164, 170 Spek, S. ........................................................................................ 204 Spengler, B. .................................................................................... 60 Staab, A. ....................................................................................... 196 Stachs, O. ....................................................................................... 63 Stacy, R. ....................................................................................... 154 Stark, H. ................................................................ 124, 125, 126, 127 Stasch, J.-P. ................................................................................. 160

Stauber, R..................................................................................... 128 Staufenbiel, S. ...................................................................... 200, 201 Steegborn, C. ................................................................................. 85 Stegen, B. ..................................................................................... 139 Steiger, C. ..................................................................................... 206 Stein, J. ......................................................................................... 188 Steinbrink, S.D. ............................................................................... 26 Steiner, I.S. ................................................................................... 159 Steinhilber, D. ................. 26, 124, 125, 126, 127, 138, 139, 172, 188 Steinicke, F. .................................................................................. 169 Steinmetz, M. ................................................................................ 176 Steinmetzer, T. ............................................................. 132, 160, 164 Stelzer, E. ....................................................................................... 75 Stenzel, K. ...................................................................................... 58 Steri, R. ................................................................................. 125, 167 Sternberg, K.................................................................................... 63 Steuber, H..................................................................................... 159 Stieler, M....................................................................................... 141 Stölting, D.P. ................................................................................. 130 Storck, W. ..................................................................................... 210 Storr, M. ........................................................................................ 151 Strödke, B. .................................................................................... 123 Strohmeier, J. ............................................................................... 152 Studenik, C.R. .............................................................................. 173 Suess, B. ...................................................................................... 138 Sumanadasa, S.D.M. ..................................................................... 58 Sun, Q........................................................................................... 171 Suzuki, B....................................................................................... 183 Suzuki, T ......................................................................................... 39 Suzuki, Y......................................................................................... 41 Swyter, S. ............................................................................. 136, 172 Syntschewsk, V. ........................................................................... 142 Szekely, N....................................................................... 81, 194, 196 Tacke, S........................................................................................ 203 Tadros, S.A.A. .............................................................................. 168 Takakura, Y. ................................................................................. 109 Takats, Z. ........................................................................................ 22 Tanaka, N. .................................................................................... 162 Taniguchi, Y. ................................................................................... 48 Tänzler, D. .................................................................................... 116 Tasar, A. ......................................................................................... 88 Telukunta, K.K. ............................................................................. 174 Tenzer, S. ..................................................................................... 210 Terpolilli, N. ..................................................................................... 32 Teufel, R. ...................................................................................... 106 Thakur, A. ....................................................................................... 27 Thoma, F. ..................................................................................... 202 Thomas, M. ........................................................................... 133, 187 Thomas, R.K. ................................................................................ 165 Thommes, M. .......................................................................... 33, 198 Thuermann, P.A. .......................................................................... 152 Tillmanns, A. ................................................................................. 128 Tocchetti, A. .................................................................................. 143 Tokimizu, Y. .................................................................................... 41 Tomioka, K...................................................................................... 38 Toth, P. ......................................................................................... 166 Tschammer, N. ....................................................... 72, 176, 177, 178 Tschöp, M. .................................................................................... 140 Tuereli, A.E. .................................................................................. 119 Ulrich-Merzenich, G. ..................................................................... 151 Ulrich-Rückert, S. ................................................................. 138, 188 Umeda, T. ..................................................................................... 162 Umezawa, K. ................................................................................ 112 Urban, N. ...................................................................................... 156 Usman, M. .................................................................................... 154 van Ammel, K. .............................................................................. 120 van der Graaf, P.H. ....................................................................... 120 Vasylyeva, V. ................................................................................ 142 Vávrová, K. ................................................................................... 122


Vermeiren, C. ............................................................................... 179 Verstraelen, J. ................................................................................ 34 Vieira, R.P. ................................................................................... 122 Vielmuth, C. .................................................................................. 178 Vierfuß, S. ..................................................................................... 115 Vinson, B.R. .................................................................................. 151 Visser, S.A.G. ............................................................................... 120 Vlodavsky, I. ................................................................................. 128 Vogt, D. ................................................................................. 125, 126 Voigt, K. ........................................................................................ 157 Völler, S. ....................................................................................... 195 Vollmar, A.M. ............................................................ 56, 57, 131, 132 Volmer, D.A. ................................................................................... 59 Volz, A.-K. ..................................................................................... 147 von Briesen, H. ............................................................................. 206 von Hammerstein, F. .................................................................... 159 von Löwenich, F. .......................................................................... 186 von Schwarzenberg, K. ................................................................ 132 Vornicescu, D. .............................................................. 134, 141, 145 Wachtel, H. ............................................................................. 78, 195 Wada, N. ......................................................................................... 40 Wagner, E. ...................................................................................... 21 Wagner, S. ............................................................................ 114, 132 Wahl, O. ........................................................................................ 153 Wahl-Schott, C. ............................................................................ 156 Wakimoto, T. ................................................................................ 110 Walden, P. ...................................................................................... 64 Wallmeyer, L. ................................................................................ 122 Waltering, I. .................................................................................... 86 Wardecki, T. ......................................................................... 186, 187 Warren, N. .................................................................................... 210 Wätzig, H. ............................................. 144, 149, 162, 168, 169, 209 Weaver, C. .................................................................................... 180 Weber, B. ...................................................................................... 196 Weber, J. .............................................................................. 141, 172 Weckenbrock, W.V. ...................................................................... 134 Wegener, J. .................................................................................. 180 Wegscheid-Gerlach, C. ................................................ 113, 161, 189 Wehle, S. ...................................................................................... 163 Weickert, A. .......................................................................... 128, 170 Weidel, E. ..................................................................................... 165 Weidner, T. ................................................................................... 159 Weigandt, M. .................................................................................. 64 Weigert, A. .................................................................................... 138 Weimer, D. .................................................................................... 142 Weirauch, U. ................................................................. 107, 130, 133 Weischer, M.-L. .............................................................................. 97 Weiser, C. ..................................................................................... 148 Weiser, D. ..................................................................................... 188 Weisner, J. .................................................................................... 129 Weiss, C. ...................................................................................... 152 Weißer, A. ............................................................................. 107, 130

Weitschies, W. ................................................................................ 36 Weiwad, M. ................................................................................... 154 Werner, K...................................................................................... 176 Werner, P.............................................................................. 153, 187 Werz, O........................................................................... 25, 124, 125 Wessels, L. ................................................................................... 203 Wicha, S.G.................................................................................... 170 Wiedemann, B.M. ......................................................................... 155 Wiest, J. ........................................................................................ 195 Wilcken, R....................................................................................... 45 Wilhelm, N. ................................................................................... 206 Willmann, D. ................................................................................. 136 Willmann, S..................................................................................... 44 Willmes, T. .................................................................................... 170 Wilmer, A. ....................................................................................... 88 Windorf, M. ................................................................................... 207 Winkelblech, J. ............................................................................. 145 Winkelmann, V. ............................................................................ 188 Winzeler, E.A. ................................................................................. 58 Winzer, M........................................................................................ 66 Wischke, C...................................................................................... 63 Witting, M. ..................................................................................... 199 Wittmann, S.K. ...................................................................... 125, 172 Wittstock, K. .................................................................................. 209 Wöbke, T.K. .................................................................................. 126 Wolber, G................................................................................ 70, 179 Wolf, C. ........................................................................................... 87 Wolf, M............................................................................................ 64 Wolle, P. ....................................................................................... 144 Woltersdorf, S. ...................................................................... 125, 126 Wu, H. ................................................................................... 159, 182 Wünsch, B. ................................................................................... 114 Wurglics, M. .................................................................................. 191 Würthwein, G. ............................................................................... 195 Wurzel, J. ...................................................................................... 167 Xie, X. ........................................................................................... 146 Xie, Y. ............................................................................................. 27 Yamaguchi, J. ............................................................................... 114 Yamashita, S. ................................................................................. 17 Ye, L. ............................................................................................ 189 Yoshida, M. ................................................................................... 111 Zahler, S. ...................................................................................... 132 Zanger, U.M. ................................................................................. 187 Zech, I. .......................................................................................... 117 Zeitlinger, M. ......................................................................... 148, 170 Zhai, X. ......................................................................................... 204 Zhao, H. ........................................................................................ 170 Zimmermann, T. ........................................................................... 205 Zimmermann, W.H. ...................................................................... 176 Zips, D. ......................................................................................... 139 Zlotos, D.P. ........................................................................... 168, 190

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Geschäftsführer und Leiter der Geschäftsstelle Apotheker Dr. Michael Stein DPhG Geschäftsstelle Hamburger Allee 26 - 28 60486 Frankfurt/Main Tel.: 069-7191596-0 Fax: 069-7191596-29 Email: [email protected]

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trends and perspectives in pharmaceutical sciences

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