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Fourth World Conference on Physico-Chemical Methods in Drug Discovery and Development

Programme &

Book of Abstracts Red Island, Croatia, September 21-24, 2015

Fourth World Conference on Physico-Chemical Methods in Drug Discovery and Development Red Island, Croatia, September 21-24, 2015

Programme & Book of Abstracts Published by

International Association of Physical Chemists E-mail: [email protected], URL: http://www.iapchem.org For Publisher Zoran Mandić Editor Zoran Mandić Design, Page Making and Computer Layout Aleksandar Dekanski Circulation 200 Copies

mailto:[email protected]

http://www.iapchem.org/

The Scientific and Organizing Committee:

Alex Avdeef, In-ADME Research, USA

Elena Boldyreva, Russian Academy of Sciences, Russia

Biserka Cetina-Čižmek, PLIVA, Croatia

Mario Grassi, University of Trieste, Italy

Ulrich Griesser, University of Innsbruck, Austria

Rolf Hilfiker, Solvias, Switzerland

Josef Jampilek, University of Veterinary

and Pharmaceutical Sciences, Czech Republic

Zoran Mandić, University of Zagreb, Croatia

Sibylle Neuhoff, Simcyp, UK

Christos Reppas, University of Athens, Greece

Marti Rosés, University of Barcelona, Spain

Stanko Srčič, Universty of Ljubljana, Slovenia

Kiyohiko Sugano, Toho University, Japan

Krisztina Takács-Novák, Semmelweis University, Hungary

Kin Tam, University of Macau, Macau

Klara Valko, GSK, UK

Hong Wan, Shanghai Hengrui Pharmaceutical, China

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CONTENTS

CONFERENCE PROGRAMME _______________________________ XI

ORAL PRESENTATIONS ____________________________________ 1

Models and methods to study drug transport accross barriers Péter Krajcsi ___________________________________________________________ 3

Physicochemical and biomimetic properties to guide lead optimization to improve in vivo efficacy

Klara Valko ____________________________________________________________ 4

Studying interplay of dissolution, solubility and permeability in formulation development using in situ concentration monitoring

Konstantin Tsinman _____________________________________________________ 5

The measurement of physicochemical properties of orally inhaled drugs at 37 °C in Simulated Lung Fluid – methods and formulations

Antonio Llinas, Rebeca Ruiz, John Comer _____________________________________ 6

Determination of the acidity of poorly water-soluble drugs by the Internal Standard - Capillary Electrophoresis method

Martí Rosés, Elisabet Fuguet, Joan Marc Cabot _______________________________ 7

Strategies for the development of liquid, semisolid and solid lipid-based drug delivery systems

Abu Serajuddin _________________________________________________________ 8

Predictive in vitro tests for locally acting oral medicinal products? Christos Reppas _________________________________________________________ 9

In vitro evaluation of 8-substituted 6,9-diazaspiro[4.5]decan-7,10-diones as skin permeation modifiers

Josef Jampilek, Aneta Cernikova, Katarina Tabiova, Pavel Bobal _________________ 10

Early biopharmaceutical characterization to predict eye-related bioavailability Jasmina Lovrić _________________________________________________________ 11

Terfenadine solubility studies Vesna S. Živanović, Miloš P. Pešić, Viola Horváth, János Madarász, Ilija N. Cvijetić, Gordana V. Popović, Tatjana Ž. Verbić, Alex Avdeef ___________________________ 12

Data Mining for Equilibrium (Non-Amorphous) Solubility of Druglike Molecules Alex Avdeef ___________________________________________________________ 13

Best practices for saturation shake-flask measurements of thermodynamic solubility Krisztina Takács-Novák, Gergely Völgyi _____________________________________ 14

Solubility–pH profiles of some drugs determined by the shake-flask method Elisabet Fuguet, Elham Shoghi, Elisabeth Bosch, Clara Ràfols ___________________ 15

Red Island, Croatia, September 21-24, 2015

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The solubility challenge Antonio Llinàs _________________________________________________________ 16

Antibody-drug conjugate (ADC) drug development from ADME perspectives Hong Wan ____________________________________________________________ 17

Molecular imaging of fluorescent ligand-DNA interaction in live cells for guiding the development of anti-cancer agents

Ta-Chau Chang ________________________________________________________ 18

Dynamic prediction of hydration structures of biomolecular complexes Csaba Hetényi, Norbert Jeszenői, Mónika Bálint David van der Spoel, István Horváth 19

The role of uptake, cellular or tissue distribution, and the target on the efficacy of tyrosine kinase inhibitors

G. J. Peters, R. J. Honeywell ______________________________________________ 20

Permeability assessment and pharmacokinetics of some β-amyloid aggregation inhibitors for the treatment of Alzheimer’s disease

Kin Tam, Wei Zhou, Ken Yung, Ricky Wong, Hung Wing Li ______________________ 21

The predictors of drug effects on Alzheimer’s disease: shed new light from EEG parameters to brain connectomics

Yin Tian, Zechao Ding, Zhongyan Wang, Huiling Zhang ________________________ 22

the role of material science in the research and development of drug substance and drug product

Ernest Meštrović _______________________________________________________ 23

Non-ambient conditions in drug forms discovery and development Elena V. Boldyreva _____________________________________________________ 24

The impact of molecular mobility and molecular interaction patterns on physical stability of amorphous anti-inflammatory agents

Marzena Rams-Baron, Zaneta Wojnarowska, Mateusz Dulski, Katarzyna Grzybowska, Justyna Knapik, Marian Paluch ________________________ 25

Quantification of amorphous content in drug substance and drug product Rolf Hilfiker ___________________________________________________________ 26

Quantitative mechanical measurements at the nano-scale for the crystalline pharmaceutical ingredients

Biljana Janković, Stane Srčič ______________________________________________ 27

Toward better understanding of crystallization of amorphous drugs under compression: Isochronal crystallization kinetics approach

Marian Paluch, Karolina Adrjanowicz ______________________________________ 28

Application of titanate nanotube composites for the modification of the solubility, dissolution kinetic and processability of drugs

Tamás Sovány, Barbara Sipos, Zoltán Kónya, Klára Pintye-Hódi, Géza Regdon jr. ___ 29

4th World Conference on Physico-Chemical Methods in Drug Discovery and Development

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QsarDB: chemo-informatics solution for preserving and efficient use of in silico physicochemical, toxicological, biochemical and human health endpoint models

Uko Maran, Sulev Sild, Villu Ruusmann, Geven Piir, Alfonso T. García-Sosa, Kalev Takkis, Mare Oja, Elmar Laigna, Priit Ahte, Iiris Kahn, Andre Lomaka ________ 30

Simulation of in vitro Caco-2 Papp from molecular structure (Kubinyi vs. QSAR) and estimation of intracellular Km for efflux transporters

Michael B. Bolger, Viera Lukacova, James M. Mullin, Ke Xu-Szeto ________________ 31

Structure-activity relationship analysis of new antipseudomonal gemini-imidazolium chlorides

Łukasz Pałkowski, Jerzy Błaszczyński, Andrzej Skrzypczak, Jan Błaszczak, Alicja Nowaczyk, Roman Słowiński, Jerzy Krysiński ____________________________ 32

Antibacterial drug release from a biphasic polymeric system: mathematical modelling

Mario Grassi, Samuel Golob, Fabio Pontelli, Michela Abrami, Gianluca Chiarappa, Gabriele Grassi, Beatrice Perissutti, Dario Voinovich __________________________ 33

The role of mathematics in mass spectrometry driven species identification Ena Melvan, Antonio Starcevic, Jurica Zucko, Mario Cindric, Ivo Ugrina, Daslav Hranueli ________________________________________________________ 34

LC-SPE-(cryo)NMR/MS as routine analysis technique C. Daolio _____________________________________________________________ 35

Exploration of tetrahelical DNA structures as dynamic drug targets by NMR J. Plavec ______________________________________________________________ 36

The Application of NMR within Drug Discovery: Overview and Case Studies K. Embrey ____________________________________________________________ 37

Fluorine NMR in pharma development: from drug substance to drug product T. Biljan ______________________________________________________________ 38

POSTER PRESENTATIONS _________________________________ 39

Small-molecule inhibitors of influenza A virus that act by disrupting PB2 cap-binding of the viral polymerase

Shuofeng Yuan, Jie Zhou, Richard Y. Kao, Bo-Jian Zheng ________________________ 41

Mast cell inhibitors with potential benefits in tumor angiogenesis Stefana Avram, Anca M. Cimpean, Marius Raica _____________________________ 42

Triterpene derivatives effects on angiogenesis and melanoma Stefana Avram, Anca M. Cimpean, Camelia Oprean, Ionut Ledeti, Adriana Fulias, Corina Danciu, Ioana Z. Pavel, Sorin I. Avram, Cristina A. Dehelean_______________ 43

Pharmacokinetics, in vitro and in silico assessment of anti-inflammatory alkaloids from Isatis tinctoria

Evelyn A. Jähne, Mouhssin Oufir, Veronika Butterweck, Daniela E. Eigenmann, Maxime Culot, Roméo Cecchelli, Fruzsina Walter, Mária A. Deli, Martin Smiesko, Matthias Hamburger ___________________________________________________ 44


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Antifungal styrylquinolines probably act as a cellular razor blade Jacek Mularski, Wioleta Cieślik, Joanna Szczepaniak, Anna Krasowska, Robert Musioł _________________________________________________________ 45

Controlled suspensions as a method for small scale dissolution profiling of poorly soluble compounds

Sara Andersson, Caroline Alvebratt, Johan Gråsjö, Christel Bergström ____________ 46

High throughput lipophilicity determination of novel amidino substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives

V. Kelava, M. Ilijaš, S. Dragojević, S. Koštrun, M. Hranjec, V. Gabelica Marković ____ 47

Lipophilicity determination of zwitterionic compounds: a comparative study Clara Ràfols, Xavier Subirats, Martí Rosés, Elisabeth Bosch _____________________ 48

Microemulsion Electrokinetic Chromatography as a very suitable tool for lipophilicity determination of acidic and basic compounds

Xavier Subirats, Alejandro Carrillo, Martí Rosés ______________________________ 49

Modelling tadpole narcosis by means of chromatographic systems M. Rosés, E. Fuguet, S. Amézqueta, A. Fernandez-Pumarega ____________________ 50

Performance of chromatographic systems to model aquatic toxicity Elisabet Fuguet, Martí Rosés, Susana Amézqueta, Alejandro Fernández-Pumarega, Sandra Farré, Laura Muñoz-Pascual _______________________________________ 51

Critical comparison of several methods for lipophilicity determination Adriana Port, Magda Bordas, Raquel Enrech, Martí Rosés, Clara Ràfols, Elisabeth Bosch, Xavier Subirats __________________________________________________ 52

Medium-throughput determination of lipophilicity and dissociation constant by liquid chromatography–mass spectrometry

Paweł Wiczling, Łukasz Kubik, Wiktoria Struck-Lewicka, Danuta Siluk, Michał Jan Markuszewski, Roman Kaliszan ___________________________________________ 53

Implementation of Analytical Quality by Design (AQbD) approach to RP-HPLC method for dissolution testing

Maja Bartolec, Josip Pranjić, Senka Radošević, Maja Lusina Kregar ______________ 54

Filtration as phase separation technique in saturation shake-flask solubility determination method

Gergely Völgyi, Krisztina Takács-Novák _____________________________________ 55

Chitosan/xanthan multilayer films as a prospective vehicle for buccal administration Bissera Pilicheva, Yordanka Uzunova, Ivan Bodurov, Ivanka Vlaeva, Asya Viraneva, Ginka Exner, Sotir Sotirov, Tsenka Grancharova, Maria Marudova, Temenuzhka Yovcheva __________________________________________________ 56

Permeation of betamethasone esters through excised pig skin Tina Jarc, Katja Berginc, Mateja Erdani Kreft, Katja Kristan _____________________ 57

Introduction of Golem v2, a new model for biorelevant dissolution studies Ivan Stupák, Lucie Gruberová, Jakub Vysloužil, Jiří Dohnal, Martin Čulen __________ 58


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Furosemide solvates: can they serve as precursors to different polymorphs? A. A. Beloborodova, V. S. Minkov, V. A. Drebushcak, E. V. Boldyreva ______________ 59

Mannitol as co-milling agent for the preparation of theophyline inhalable nanocrystal agglomerates

Maria Malamatari, S. Somavarapu, M. Bloxham, G. Buckton ___________________ 60

The composites of betulin and its diacyles with improved biological activity Tatyana P. Shakhtshneider, Mikhail A. Mikhailenko, Ilia V. Eltsov, Vladimir V. Boldyrev, Elena V. Boldyreva, Svetlana A. Kuznetsova, Yury N. Malyar, Anna S. Zamay, Alexander S. Kozlov __________________________ 61

Novel synthons in sulfamethizole cocrystals: Structure−property relations and solubility

Kuthuru Suresh, Vasily S. Minkov, Kranthi Kumar Namila, Elizaveta Derevyannikova, Evgeniy Losev, Ashwini Nangia, Elena V. Boldyreva ___________________________ 62

Amorphous protic ionic systems as promising active pharmaceutical ingredients: The case of sumatriptan drug

Z. Wojnarowska, J. Cielecka-Piontek, M. Paluch ______________________________ 63

Measuring and modelling pH-dependence of passive transport in artificial membrane for wide range of pH

Mare Oja, Uko Maran ___________________________________________________ 64

Exploring and predicting in QsarDB repository: membrane permeability QSAR model over gastrointestinal tract pH-range

Mare Oja, Sulev Sild, Uko Maran __________________________________________ 65

Exponential repulsion for molecular docking Václav Bazgier,

Karel Berka, Michal Otyepka, Pavel Banáš _____________________ 66

Human serum albumin binding of certain antimalarials Olivera S. Marković, Ilija N. Cvijetić, Mario V. Zlatović, Igor M. Opsenica, Nataša V. Terzić-Jovanović, Bogdan A. Šolaja, Tatjana Ž. Verbić ________________ 67

Physico-chemical analysis of new synthetic derivatives from triterpenoid class Adriana Fuliaş, Ionuţ Ledeţi, Gabriela Vlase, Lenuţa-Maria Şuta, Titus Vlase _______ 68

Comparative thermal stability of statins Ionuţ Ledeţi, Gabriela Vlase, Titus Vlase, Lenuţa-Maria Şuta, Ionel Ciucanu, Adriana Fuliaş _________________________________________________________ 69

Employment of hyphenated techniques for analysis of betulin-type compounds Ionuţ Ledeţi, Gabriela Vlase, Lenuţa-Maria Şuta, Titus Vlase, Adriana Fuliaş _______ 70

Spectroscopic characterisation of newly synthesised bile acid derivatives Srđan Bjedov, Dušan Škorić, Marija Sakač, Mihalj Poša, Janoš Čanadi ____________ 71

A Quality by Design (QbD) based automated analytical method development for determination of impurities in new pharmaceutical drug product

Ana Georgieva, Irena Brašnarska, Sonja Ugarkovič ___________________________ 72


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Rapid and Green Chemistry Catalysis in Synthesis of Hexahydroquinazolinones and Cyclopenta, Cyclohepta[d]pyrimidinones with Prediction of Biological Activity via PASS INET

Ahmed M. M. El-Saghier, Eman A. Ahmed, Ahmed M. A. Mahmoud ______________ 73

Identification, structural caracterization and qualification of unidentified impurity in Bisoprolol film-coated tablets

Ivana Mitrevska, Ema Kikovska, Gjorgji Petruševski, Ana Georgieva, Sonja Ugarkovič, Aneta Dimitrovska _______________________________________ 74

Chemical stability and in vitro and clinical efficacy of bis-retinamido methylpentane, a novel hybrid retinoid derivative

Ki Hyun Kim, Nam Ah Kim, So-Hyun Park, Jin Hun Cho, Seong Hoon Jeong _________ 75

Synthesis of PLGA /nano-ZnO composite particles for biomedical application A. Stanković, M. Lukić, M. Jović, M. Sezen, M. Milenković, M. Stevanović __________ 76

Effect of expression system on assay performance of human ABC transporters of pharmacological importance in vesicular transport assays

Balázs Vaskó, Beáta Tóth, Viktória Juhász, Ildikó Nagy, Anita Kurunczi, Krisztina Herédi-Szabó, Joseph K. Zolnerciks, Emese Kis, Rémi Magnan, Kent K. Grindstaff, Peter Krajcsi, László Szilágyi ______________________________________________ 77

Selenium nanoparticles as a potential candidate in cancer treatment Nenad Filipović, Jana Nunić, Metka Filipič, Milos Filipović, Magdalena Stevanović __ 76

AUTHOR INDEX ________________________________________ 79

Conference Programme

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Sunday, September 20th

15:00 – 19.30 Participant registration, Room Marie

20:00 – Welcome party, The castle, restaurant Lanterna terrace

All sessions will be held in Huetterott lecture hall

Monday, September 21st

08:00 Poster mounting, Lounge bar in front of the Huetterott lecture hall

Morning session

09:00 – 09:15 Opening Ceremony

Moderator: Biserka Cetina Čižmek

09:15 – 10:00 OP 01

Peter Krajcsi, Solvo Biotechnology, Hungary Models and methods to study drug transport accross barriers

10:00 – 10:45 OP 02

Klara Valko, GSK, UK Physicochemical and biomimetic properties to guide lead optimization to improve in vivo efficacy

10:45 – 11:15 Coffee break

Moderator: Kin Tam

11:15 – 11:50 OP 03

Konstantin Tsinman, Pion Inc, USA Studying interplay of dissolution, solubility and permeability in formulation development using in situ concentration monitoring

11:50 – 12:25 OP 04

Antonio Llinàs, Rebeca Ruiz, John Comer, Sirius Analytical Ltd., UK The measurement of physicochemical properties of orally inhaled drugs at 37 °C in Simulated Lung Fluid – methods and formulations

12:25 – 13:00 OP 05

Martí Rosés, Elisabet Fuguet, Joan Marc Cabot, University of Barcelona, Spain Determination of the acidity of poorly water-soluble drugs by the Internal Standard - Capillary Electrophoresis method

13:00 – 15:00 Lunch


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Afternoon session

Moderator: Klara Valko

15:00 – 15:45 OP 06

Abu Serajuddin, St. John’s University, USA Strategies for the development of liquid, semisolid and solid lipid-based drug delivery systems

15:45 – 16:30 OP 07

Christos Reppas, University of Athens, Greece Predictive in vitro tests for locally acting oral medicinal products?


Moderator: Christos Reppas

17:00 – 17:35 OP 08

Josef Jampilek, Aneta Cernikova, Katarina Tabiova, Pavel Bobal, University of Veterinary and Pharmaceutical Sciences, Czech Republic In vitro evaluation of 8-substituted 6,9-diazaspiro[4.5]decan-7,10-diones as skin permeation modifiers

17:35 – 18:10 OP 09

Jasmina Lovrić, University of Zagreb, Croatia Early biopharmaceutical characterization to predict eye-related bioavailability

18:10 – 18:30 OP 10

Vesna S. Živanović, Miloš P. Pešić, Viola Horváth, János Madarász, Ilija N. Cvijetić, Gordana V. Popović, Tatjana Ž. Verbić, Alex Avdeef, University of Belgrade, Serbia Terfenadine solubility studies

Evening solubility session Moderator: Alex Avdeef

20:30 – 20:50 OP 11

Alex Avdeef, in-ADME Research, USA Data mining for equilibrium (non-amorphous) solubility of druglike molecules

20:50 – 21:10 OP 12

Krisztina Takács-Novák, Gergely Völgyi, Semmelweis University, Hungary Best practices for saturation shake-flask measurements of thermodynamic solubility

21:10 – 21:30 OP 13

Elisabet Fuguet, Elham Shoghi, Elisabeth Bosch, Clara Ràfols, University of Barcelona, Spain Solubility–pH profiles of some drugs determined by the shake-flask method

21:30 – 21:50 OP 14

Antonio Llinàs, AstraZeneca R&D, Sweden The solubility challenge

21:50 – Solubility session closing remarks


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Tuesday, September 22nd

Morning session

Moderator: Yin Tian

09:00 – 09-45 OP 15

Hong Wan, Shanghai Hengrui Pharmaceutical, China Antibody-drug conjugate (ADC) drug development from ADME perspectives

09:45 – 10:20 OP 16

Ta-Chau Chang, National Taiwan University, Taiwan Molecular imaging of fluorescent ligand-DNA interaction in live cells for guiding the development of anti-cancer agents

10:20 – 10:50 OP 17

Csaba Hetényi, Norbert Jeszenői, Mónika Bálint, David van der Spoel, István Horváth, Hungarian Academy of Sciences, Hungary Dynamic prediction of hydration structures of biomolecular complexes


Moderator: Hong Wan

11:15 – 11:50 OP 18

Godefridus J. Peters, R.J. Honeywell, VU University Medical Center, The Netherlands The role of uptake, cellular or tissue distribution, and the target on the efficacy of tyrosine kinase inhibitors

11:50 – 12:25 OP 19

Kin Tam, Wei Zhou, Ken Yung, Ricky Wong, Hung Wing Li, Unviersity of Macau, Macau Permeability assessment and pharmacokinetics of some β-amyloid aggregation inhibitors for the treatment of Alzheimer’s disease

12:25 – 13:00 OP 20

Yin Tian, Zechao Ding, Zhongyan Wang, Huiling Zhang, ChongQing University of Posts and Telecommunications, China The predictors of drug effects on Alzheimer’s disease: shed new light from EEG parameters to brain connectomics

13:00 – 15:00 Lunch

Afternoon session

Moderator: Rolf Hilfiker

15:00 – 15:40 OP 21

Ernest Meštrović, Pliva Croatia Ltd., Croatia The role of Material Science in the Research and Development of Drug Substance and Drug Product

15:40 – 16:20 OP 22

Elena Boldyreva, Novosibirsk State University, Russia Non-ambient conditions in drug forms discovery and development

16:20 – 16:40 OP 23

Marzena Rams-Baron, Zaneta Wojnarowska, Mateusz Dulski, Katarzyna Grzybowska, Justyna Knapik, Marian Paluch, University of Silesia, Poland The impact of molecular mobility and molecular interaction patterns on physical stability of amorphous anti-inflammatory agents



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Moderator: Elena Boldyreva

17:00 – 17:45 OP 24

Rolf Hilfiker, Solvias AG, Switzerland Quantification of amorphous content in drug substance and drug product

17:45 – 18:15 OP 25

Biljana Janković and Stane Srčič, University of Ljubljana, Slovenia Quantitative mechanical measurements at the nano-scale for the crystalline pharmaceutical ingredients

18:15 – 18:30 OP 26

Marian Paluch and Karolina Adrjanowicz, University of Silesia, Poland Toward Better Understanding of Crystallization of Amorphous Drugs under Compression: Isochronal Crystallization Kinetics Approach

18:30 – 18:45 OP 27

Tamás Sovány, Barbara Sipos, Zoltán Kónya, Klára Pintye-Hódi, Géza Regdon jr, University of Szeged, Hungary Application of titanate nanotube composites for the modification of the solubility, dissolution kinetic and processability of drugs

18.45 Poster session (until 23:00)


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Wednesday, September 23rd

08:00 Poster dismounting

Morning session

Moderator: Mario Grassi

09:00 – 09:45 OP 28

Uko Maran, Sulev Sild, Villu Ruusmann, Geven Piir, Alfonso T. García-Sosa, Kalev Takkis, Mare Oja, Elmar Laigna, Priit Ahte, Iiris Kahn, Andre Lomaka, University of Tartu, Estonia QsarDB: chemo-informatics solution for preserving and efficient use of in silico physicochemical, toxicological, biochemical and human health endpoint models

09:45 – 10:30 OP 29

Michael B. Bolger, Viera Lukacova, James M. Mullin, Ke Xu-Szeto, Simulations Plus Inc., USA Simulation of in vitro Caco-2 Papp from molecular structure (Kubinyi vs. QSAR) and estimation of intracellular km for efflux transporters


Moderator: Uko Maran

11:00 – 11:35 OP 30

Łukasz Pałkowski, Jerzy Błaszczyński, Andrzej Skrzypczak, Jan Błaszczak, Alicja Nowaczyk, Roman Słowiński, Jerzy Krysiński, Nicolaus Copernicus University, Poland Structure-activity relationship analysis of new antipseudomonal gemini-imidazolium chlorides

11:35 – 12:10 OP 31

Mario Grassi, Samuel Golob, Fabio Pontelli, Michela Abrami, Gianluca Chiarappa, Gabriele Grassi, Beatrice Perissutti, Dario Voinovich, University of Trieste, Italy Antibacterial drug release from a biphasic polymeric system: Mathematical modelling

12:10 – 12:40 OP32

Ena Melvan, Antonio Starcevic, Jurica Zucko, Mario Cindiric, Ivo Ugrina, Daslav Hranueli, University of Zagreb, Croatia The importance of mathematics in proteomics sequencing

12:40 Lunch

14:00 Excursion + Dinner


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Thursday, September 24th

Joint PCMDDD-4 & met4pharm session

Moderator: Predrag Novak

10:00 – 10:45 OP 33

Cristina Daolio, Bruker BioSpin GmbH, Germany LC-SPE-(cryo)NMR/MS as routine analysis technique

10:45 – 11:30 OP 34

Janez Plavec, National Institute of Chemistry, Slovenia Exploration of tetrahelical DNA structures as dynamic drug targets by NMR


Moderator: Csaba Szantay

12:00 – 12:45 OP 35

Kevin Embery, AstraZeneca R&D, UK The Application of NMR within Drug Discovery: Overview and Case Studies

12:45 – 13:30 OP 36

Tomislav Biljan, Pliva Croatia Ltd., Croatia Fluorine NMR in pharma development: from drug substance to drug product

13:30 Conference closing

Oral Presentations


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Models and methods to study drug transport accross barriers

Péter Krajcsi

SOLVO Biotechnology, Budaörs, Hungary Transporters play an important role in drug influx, efflux and transcellular flux. To study drug transport physiologically relevant models are preferred. Holistic in vitro cellular systems such as primary cultures as well as immortalized cells are often good options. However, a vailability, stability of transporter expression and specificcity issues often impact data. Transfected/infected cells or membranes derived from these overcome the above issues provided relevance of the expression system is established (e.g. the lipid environment as well as transporter trafficking should mimic physiological). In vivo methods are the best tools to establish relevance if species specificity issues are considered. Physchem properties of drugs (e.g. passive permeability, protonation/deprotonation) should be considered during assay selection. High solubility and high permeability compounds are often thought to overcome limitations posed by transporter specificity. Nevertheless, transporters contribute to transmebrane flux of drugs with high passive permeability. Barriers are different. Therefore, a specific set of models should be used to study drug permeability through a specific barrier. Example will be given to study drug transport accross the blood –brain barrier.

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Physicochemical and biomimetic properties to guide lead optimization to improve in vivo efficacy

Klara Valko

Analytical Chemistry, Platform Technology Sciences, GSK, Stevenage, United Kingdom Measurements and calculations of physcicochemical (solubility, lipophilicity, pKa) and biomimetic (protein binding, phospholipid binding) properties in early drug discovery help the lead optimization process to design compounds that are in a better property space reducing the risk of later stage failures. We can use these properties to estimate in vivo distribution and drug efficiency of the compounds

1. The Drug Efficiency, introduced by

Braggio et al2

in 2010, is defined as the free concentration of the drug at the site of action relative to the dose. It has been demonstrated that 70% of marketed drugs have higher than 1% drug efficiency; by contrast failed clinical candidates generally have less than 1% drug efficiency. The concept was extended by incorporating the in vitro potency (pXC50). Thus the sum of the logarithmic drug efficiency and the pXC50 is called the Drug Efficiency Index (DEI)

3 and has been shown to be proportional to the in vivo receptor occupancy

relative to the dose. The DEI parameter represents the PK/PD properties of a potent compound in a single number highlighting the inter-relationships of several properties of the molecules, such as solubility, permeability, absorption, volume of distribution, metabolism, half life, etc. During lead optimization when more in vitro and in vivo data become available, more accurate measurements of drug efficiency are possible. It is demonstrated how the simultaneous optimization of potency and drug efficiency can help guide candidate selection toward compounds of increased quality and with reduced chance of later stage drug development failures. 1. Valko et al., Journal of Pharmaceutical Science, 2012, 101(11) 4155-4169 2. Braggio et al., Expert Opinion in Drug Discovery, 2010, 5(7) 609-618 3. Montanari et al., Expert Opinion in Drug Discovery, 2011, 6(9) 913-920

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Studying interplay of dissolution, solubility and permeability in formulation development using in situ concentration monitoring

Konstantin Tsinman

Pion Inc., 10 Cook Str., Billerica, MA 01821 USA, [email protected] Development strategy for insoluble compounds requires not only measurements of the solubility enhancement from formulations but also the assessment for the effect formulations having on permeability. An introduced dissolution-permeability (µFLUX) measurement platform allows simultaneous monitoring for both effects enabling in vitro setup for early in vivo predictive formulations testing. Ability to measure concentration of free (solubilized) drug in situ is critical necessity in formulation research because any off-line solution handling can disturb quasi-stable (kinetic) phase that low soluble compounds often form in the presence of excipients. Case studies involving two different detection techniques based on fiber-optic UV measurements and potentiometric free drug sensors (FDS) will be presented. These case studies will highlight:

Combining dissolution and permeability assays for better formulation design, understanding the food effect on bioavailability and more realistic IVIVC;

Studying if solubility enhancement in the bio-relevant media leads to the same gain in the absorption and bioavailability;

Real time concentration monitoring of free drug in the presence of lipid passed formulations, nanoparticles and binding proteins;

Monitoring in real time the free API fraction released from nanoparticles and predicting absorption enhancement from nanoparticle formulations;

Developing of predictive in vitro method to monitor powder/formulation dissolution and concomitant precipitation processes in dynamically changing biorelevant media.

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The measurement of physicochemical properties of orally inhaled drugs at 37 °C in Simulated Lung Fluid – methods and formulations

Antonio Llinas, Rebeca Ruiz*, John Comer*

AstraZeneca, Sweden *Sirius Analytical Ltd., UK

The absorption of drugs administered by oral inhalation is influenced by their physicochemical properties. In an effort to increase understanding, we measured the pKa, log P, solubility and dissolution rates of 41 compounds under physiologically relevant conditions. 18 of these were generic drugs that are frequently administered by inhalation; the remaining 23 were experimental compounds. We first characterised the compounds by measurements in aqueous solution, and then repeated the measurements in simulated lung fluid (SLF), having first evaluated several formulations of SLF to determine their ease of preparation, storage characteristics and compatibility with analytical procedures. All measurements were made at 37 °C. Dissolution experiments were run in 2.0 mL volumes. Significant challenges were met and overcome during this project. The results are likely to provide a valuable basis for future research.

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Page 7

Determination of the acidity of poorly water-soluble drugs by the Internal Standard - Capillary Electrophoresis method

Martí Rosés*, Elisabet Fuguet*,**, Joan Marc Cabot*

*Departament de Química Analítica – Institut de Biomedicina, Universitat de Barcelona Martí i Franquès 1-11, 08028 Barcelona, Spain

**Serra-Húnter Programme, Generalitat de Catalunya, Spain

The Drug Discovery and Development process requires the high-throughput screening of drug activity promising compounds. Physicochemical properties, such as acidity, lipophilicity and solubility, must be measured for a large number of compounds in a short time in order to select the ones with the most favorable ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties for further development. Among the different measurement techniques, capillary electrophoresis offers many advantages in selectivity, versatility, economy, automatization and low sample and solvent consumption for the high throughput screening of drug candidates. The internal standard (IS) is a fast high-throughput method for determination of acidity constants by capillary electrophoresis (CE). The IS-CE method is based on the use of internal standards of pKa similar to that of the test compound. The pKa of the test compound is determined from the effective mobilities in pH-buffered solutions where it is partially ionized, as usual. However, the pH of these solutions is measured in situ inside the capillary from the mobilities of the internal standards of known pKa, avoiding external potentiometric pH measurement. The simultaneous measurement of pH and mobilities and the use of several internal standards at the same time decrease the time and number of measures and increase the precision. This communication describes further advantages of the method regarding pKa determination of drugs sparingly soluble in water, a common problem in drug discovery and development. Several strategies based on the rapidity of the method, which allows mobility and pH measurement when the solution is still supersaturated, have been developed [2]. These strategies allow pKa measurement for drugs for a log S value (S in mol L

-1) down to -6.2 which cannot be directly measured by the most common

potentiometric and CE methods. More insoluble drugs require the use of a cosolvent, such as methanol, for pKa measurement and extrapolation to pure water. Even in this case, the IS-CE method requires cosolvent concentration lower than in the classical methods and thus, lower errors in the extrapolation to water value. Furthermore, specific CE instrumentation with automatic and fast buffer and cosolvent preparation implementation has been developed, converting the IS-CE method in a really high-throughput method for pKa determination in drug discovery and development [3]. References: [1] J.M. Cabot, E. Fuguet, M. Rosés, ACS Comb. Sci. 16 (2014) 518-525. [2] J.M. Cabot, E. Fuguet, M. Rosés, Electrophoresis 35 (2014) 3564-3569. [3] J.M. Cabot, E. Fuguet, M. Rosés, P. Smejkal, M.C. Breadmore, 87 (2015) Anal. Chem. 6165-6172.

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Strategies for the development of liquid, semisolid and solid lipid-based drug delivery systems

Abu Serajuddin

St. John’s University, New York, USA In recent years, there has been a great interest in the pharmaceutical field on lipid-based drug delivery systems for the oral delivery of poorly water-soluble drugs. They are usually developed as solutions in mixtures of lipids, surfactants and/or co-surfactants that form microemulsion or nanoemulsion upon dilution with the aqueous media of the gastrointestinal tract and thereby promote drug absorption. The formulations are often called self-microemulsifying or self-nanoemulsifying drugs delivery systems (SMEDDS/SNE-DDS). However, no systematic approach of selecting appropriate lipids for SMEDDS and SNEDDS has been reported in the literature. This presentation will discuss various strategies of formulating them by selecting suitable lipids or mixtures thereof. In particular, effects of mono-, di- and tri-glycerides (or propylene glycol mono- and di-esters) of medium chain fatty acids on the performance of various formulations will be presented. Additionally, strategies for converting liquid lipid formulations into solid dosage forms by entrapment of liquids into solid polymeric excipients (e.g., poloxamer 188) or adsorption onto porous excipients (e.g., silicates) will be discussed.

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Predictive in vitro tests for locally acting oral medicinal products?

Christos Reppas

Faculty of Pharmacy, National and Kapodistrian University of Athens, Greece

In this presentation the focus will be on the in vitro evaluation of bile acid sequestrants and on the in vitro evaluation of modified release (MR) products of mesalamine. Although some guidelines for the in vitro assessment of bioequivalence (BE) of bile acid binding resins exist, it is interesting to note that intragastric conditions can affect the ability of cholestyramine to sequester bile salts in the fed small intestine. After incubating cholestyramine for ninety minutes in milk gradually digested with pepsin, the binding of taurocholates from fed state simulating intestinal fluid onto the resin becomes non-specific and the affinity constant is reduced. These data are consistent with the comparatively poor performance of cholestyramine products when administered in the fed state [1]. BE testing of two modified release mesalamine products is typically evaluated by combining human pharmacokinetic (PK) data and in vitro dissolution data. However, although PK data are requested to be collected under both fasting and fed state conditions, there are not clear directions for collecting the in vitro data. In recent years the physicochemical environment of colonic contents which orally administered MR products will face, after administration in the fasted and in the fed state, has been characterized [2-4]. This has allowed the collection of biorelevant in vitro dissolution data corresponding to fasted state and fed state conditions by using commercially available apparatus [5]. However, more work is needed in order to better address the importance of hydrodynamics in the region, especially near the ileocecal valve. [1] Kalantzi L, Page R, Nicolaides E, Digenis G, Reppas C. In vitro methods can forecast the effects of

intragastric residence on dosage form performance. Eur. J. Pharm. Sci. 33 (2008) 445-451. [2] Diakidou A, Vertzoni M, Goumas K, Söderlind E, Abrahamsson B, Dressman J, Reppas C.

Characterization of the contents of ascending colon to which drugs are exposed after oral administration to healthy adults. Pharm Res. 26 (2009) 2141-2151.

[3] Vertzoni M, Goumas K, Söderlind E, Abrahamsson B, Dressman JB, Poulou A, Reppas C. Characterization of the Ascending Colon Fluids in Ulcerative Colitis. Pharm Res. 27 (2010) 1620-1626

[4] Reppas C, Karatza E, Goumas C, Markopoulos C, Vertzoni M. Characterization of Contents of Distal Ileum and Cecum to Which Drugs/Drug Products are Exposed During Bioavailability/Bioequivalence Studies in Healthy Adults. Pharm Res. (2015)

[5] Markopoulos C, Vertzoni M, Symillides M, Kesisoglou F, Reppas C. Two-Stage Single-Compartment Models to Evaluate Dissolution in the Lower Intestine. J Pharm. Sci. (2015)

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In vitro evaluation of 8-substituted 6,9-diazaspiro[4.5]decan-7,10-diones as skin permeation modifiers

Josef Jampilek, Aneta Cernikova, Katarina Tabiova, Pavel Bobal

Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic,

[email protected]

Transdermal therapeutic systems are drug delivery systems designed for systemic administration. Due to their pharmacokinetic advantages they are a good alternative to traditional formulations with systemic administration. To reach the blood capillaries and achieve systemic therapeutic effect, a drug must be able to cross the skin barrier, especially the outermost layer, stratum corneum. Unfortunately transdermal drug delivery often faces the problem of insufficient or no permeation of drug substances through the skin. The use of chemical permeation enhancers (CPEs) is an approach for facilitating drug delivery through the skin [1]. Small polar molecules containing a characteristic fragment of heteroatoms X−CO−N=, where X is −CH2−, −NH2 or −NH−, may break the intermolecular H-bonds that hold the ceramides together in the stratum corneum [2]. Based on the hypotheses of the mechanism of action of CPEs, 8-substituted 6,9-diazaspiro[4.5]decan-7,10-diones were prepared and evaluated as a potential CPEs. First of them, 8-methyl derivative, demonstrated the excellent enhancement activity in a number of in vitro tests [3–6]. All other new 8-substituted 6,9-diazaspiro[4.5]decan-7,10-diones were tested for their in vitro transdermal permeation enhancement effect using a vertical Franz diffusion cell and full-thickness pig ear skin. They showed promising enhancement effect (especially very fast start of action) with respect to various model drug substances. They also expressed no toxicity or skin irritability. This study was supported by the IGA VFU Brno 302/2015/FaF. [1]. J. Jampilek, Transdermal application of drugs and techniques affecting skin barrier, J. Bioequiv.

Availab. 5 (2013) 233–235. [2] J. Jampilek, K. Brychtova, Azone analogues: Classification, design, and transdermal penetration

principles, Med. Res. Rev. 32 (2012) 907–947. [3] J. Jampilek, R. Opatrilova, L. Coufalova, A. Cernikova, J. Dohnal (FaF VFU Brno), Utilization of

alaptide as transdermal penetration modifier in pharmaceutical compositions for human and veterinary applications containing anti-inflammatory drugs and/or antimicrobial chemotherapeutics, WO/2013/020527 A1, 2013.

[4] R. Opatrilova, J. Jampilek, Rapid screening of mupirocin skin permeation modification by micronized and nanonized alaptide, ADMET 2 (2014), 56–62.

[5] A. Cernikova, R. Opatrilova, J. Jampilek, Rapid informative screening of nano-alaptide as potential transdermal permeation enhancer of acetylsalicylic acid and paracetamol, Mil. Med. Sci. Lett. 83 (2014) 34–39.

[6] A. Cernikova, R. Opatrilova, P. Bobal, J. Jampilek, Rapid screening of permeation of rutin through skin using alaptide enantiomers, ADMET 2 (2014) 248–253.

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Early biopharmaceutical characterization to predict

eye-related bioavailability

Jasmina Lovrić

University of Zagreb, Faculty of Pharmacy and Biochemistry Department of Pharmaceutical technology, Zagreb, Croatia

Early prediction of the eye-related bioavailability is critical to efficient development of topical ophthalmic drugs. Intraocular absorption of topically applied drugs is a complex process determined by dynamic and static barriers of the anterior parts of the eye, the physicochemical properties of the drug and characteristics of the ophthalmic formulation. To advance the understanding of formulation impact and accurately predict the drug product in vivo performance it is necessary to assess formulation candidates in a biorelevant and mechanistic manner. Therefore, there is an urgent need for the development and standardization of nonclinical models of eye barriers enabling safe and efficacious screening of ophthalmic formulation candidates. Here, the results we obtained in the optimization of nonclinical eye-related bioavailability screening procedure based on in vitro and ex vivo models of the cornea will be presented. Significance of such characterization to identify the formulation components and physicochemical characteristics that are crucial in the development of either innovative or generic ophthalmic formulations will be discussed.

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Terfenadine solubility studies

Vesna S. Živanović, Miloš P. Pešić, Viola Horváth*, János Madarász*, Ilija N. Cvijetić**, Gordana V. Popović***, Tatjana Ž. Verbić, Alex Avdeef****

Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Serbia, [email protected]

*Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szt. Gellert ter 4, Hungary

**Innovation Center of the Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Serbia

***Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Serbia ****in-ADME Research, 1732 First Avenue, New York, USA; [email protected]

Determination and prediction of solubility of highly insoluble molecules is quite a difficult process. The solubility–pH profile of drug substances is affected by pKa values, ionic strength, buffer effects, aggregation, time of stirring and sedimentation, etc. A novel approach, based on the saturation shake-flask method, was described to address many of the challenges of solubility determinations [1]. Terfenadine (Tf), formerly used antihistaminic drug, was used as a model compound as there is a lack of reliable data in the high pH region [2] to pin down the value of the intrinsic solubility. So far, published estimates for the intrinsic solubility range from 0.6 to 1400 ng/mL [3]. At high pH, Tf seems to form saturated solutions that are meta-stable, where the apparent solubility keeps decreasing with time, even after 68 h. Many of the mea-surements reported for Tf were done in pure water, with pH not reported. Such mea-surements are completely unreliable, because the ambient level of carbon dioxide in water can easily alter the pH of the solution, so that attempts to calculate the intrinsic solubility from the apparent water solubility can be in error by as much as 3 orders of magnitude. We have attempted to address the challenge by carefully measuring the pH dependence of the Tf solubility in HEPES and ethylenediamine dihydrochloride/lactic acid buffers, with values taken at as high a pH as analytical methods permit (pH range 0.6-11.8). Several techniques were used to measure Tf concentrations and study the characteristics of the solid residues (HPLC-MS-MS, powder XRD, SEM, elemental analysis, potentiometric titrations). The computer program pDISOL-X was used for data processing and refinement of equilibrium constants. There are many other druglike molecules with similarly challenging properties, which have not been adequately characterized. What we learn from Tf may shed relevant insights about other molecules.

Acknowledgements: The Ministry of Education, Science, and Technological Development of Republic of Serbia supports this work (Grant No. 172008).

References: 1. G. Völgyi, A. Marosi, K. Takács-Novák, A. Avdeef, ADMET&DMPK 1 (2013) 48-62. 2. W.H. Streng, S.K. Hsi, P.E. Helms, H.G.H. Tan, J. Pharm. Sci. 73 (1984) 1679-1684. 3. Avdeef A. Absorption and drug development, 2

nd Ed, John Wiley & Sons, Inc., Hoboken, New

Jersey, 2012.

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Data Mining for Equilibrium (Non-Amorphous) Solubility of Druglike Molecules

Alex Avdeef

in-ADME Research, 1732 First Avenue, New York, USA; [email protected]

PURPOSE For typical druglike molecules, it had been often assumed that the interlaboratory reproducibility in measured solubility is about 0.6 – 0.7 log unit. It will be shown that much better results can be produced in data mining for training sets used in the in silico prediction of solubility. Two ways of improving the usefulness of published solubility-pH measurements will be described, consisting of normalizing the data for ionization and temperature effects. Also, ways to improve the quality of future measurement of equilibrium solubility will be briefly discussed. METHODS The “gold standard” shake-flask (SF) and related methods (e.g., DTT, CheqSol) used to measure equilibrium (non-amorphous) solubility of ionizable drug-like compounds as a function of pH were examined, by studying hundreds of publications. The solubility-pH analysis computer program, pDISOL-X

TM, has been extended to calculate (a) intrinsic

solubility (S0) and saturation pH (pHsat) from solubility measurements done in pure water with unspecified pH, and (b) to transform S0 values determined at various temperatures to that at a single reference temperature (25 or 37

oC).

RESULTS Useful methods to improve the quality of mined solubility data have been identified. A few examples of interlaboratory/intertemperature log S0

25 (molarity scale) values include

acetylsalicylic acid: -1.69 ± 0.09 (n=13); ampicillin: -1.62 ± 0.13 (n=10); barbital (3 polymorphs): -1.42 ± 0.04 (n=11); caffeine: -0.96 ± 0.08 (n=10); ciprofloxacin: -3.64 ± 0.15 (n=11); cyclosporin A: -5.00 ± 0.14 (n=5); dexamethasone: -3.65 ± 0.08 (n=4); diclofenac: -5.36 ± 0.19 (n=26); ethisterone: -5.66 ± 0.06 (n=4). CONCLUSION With the aid of the new data analysis algorithm and improved methodology, it becomes evident that the currently expected poor interlaboratory reproducibility can be reduced to near 0.1 log unit (and possibly less in many cases).

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Best practices for saturation shake-flask measurements

of thermodynamic solubility

Krisztina Takács-Novák, Gergely Völgyi

Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary

Solubility is an essential physico-chemical parameter of compounds both in original drug research and in generic drug development. Its experimental determination is a daily-routine in pharma. Nowadays various methods are available for the measurement but the saturation shake-flask (SSF) technique has still remained the “gold standard” approach. The SSF is a very simple nevertheless time consuming method which does not require special, expensive instrumentation, it can be easily performed in a standard analytical lab. However to obtain high-quality, reliable and precise thermodynamic (equilibrium) solubility data one has to pay attention on several critical experimental conditions and aspects. It is especially valid when solubility of very slightly soluble (So: 100-1000 µg/ml) or practically insoluble (So < 100 µg/ml) compounds have to be measured. In the presentation the main factors affecting the accuracy of SSF solubility measurements are surveyed and recommendations for the best practices are committed. The following points will be discussed:

the importance of experimental design

the knowledge of sample properties: physical (crystallinity, polymorphism, particle size...) and chemical (ionization, stability, capability for aggregation ...) features, purity, available amount etc.

buffer type and concentration, pH control

the optimal incubation time

the aspects of phase separation method selection

when identification of the final form of the solid excess is needed

the most frequent reasons of inaccuracy

the good practice how to express the solubility results (So, SpH, Ssalt, logS/pH profile)

Using the examples of some selected challenging compounds (maprotiline, aripiprazole, deramciclane, venlafaxine, ...) the critical points and the limitations of SSF method are also presented.

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Solubility–pH profiles of some drugs determined by the shake-flask method

Elisabet Fuguet*,**, Elham Shoghi*, Elisabeth Bosch*, Clara Ràfols*

*Departament de Química Analítica – Institut de Biomedicina, Universitat de Barcelona Martí i Franquès 1-11, 08028 Barcelona

**Serra-Húnter Programme, Generalitat de Catalunya

The equilibrium solubility, i. e., the concentration of a compound in a saturated solution when an excess of solid is present and solution and solid are at equilibrium, is generally used to define the solubility of a compound. There is a large list of methods to measure it, being the classical one the shake-flask (S-F) method [1]. This method is based on the measurement of the concentration of the compound of interest in a saturated solution. In this work, solubility vs. pH profiles of five ionizable drugs of different nature (a monoprotic acid, a monoprotic base, a diprotic base and two amphoteric compounds showing a zwitterionic species each one) have been determined through the classical S-F method using the appropriate Henderson-Hasselbalch (H-H) or derived relationships [2]. Nevertheless, in this work a critical revision of some factors that affect the obtained solubility values has been done: the pH change of several buffers when the compounds are solubilized up to saturation, which can shift up to 2 pH units, has been evaluated; the time to reach equilibrium has been sistematically monitored; and finally, the influence of the electrolyte used as buffering agent on solubility values has been checked. Related to this last point, it has been observed that some deviations of the experimental points with respect the H-H profiles can be attributed to specific interactions between the buffering electrolyte and the drug due to the hydrotrophic character of citric and lactic acids. However, in some other cases, the observed deviations are independent of the buffers used since they are caused by the formation of new species such as drug aggregates (cefadroxil) or the precipitation of a salt from a cationic species of the analysed compound (quetiapine)[3]. References: [1] EPA (1998). EPA Product Properties Test Guidelines, OPPTS 830.7840, Water Solubility: Column

Elution Method; Shake Flask Method. OPPTS 830.7840. [2] A. Avdeef, 59 (2007) Adv. Drug Deliv. Rev. 568-590. [3] E. Shoghi, E. Fuguet, E. Bosch, C. Ràfols, 78 (2013) Eur. J. Pharm. Sci. 291-300.

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The solubility challenge

Antonio Llinàs

R&D AstraZeneca, Sweden Reviews of the performance of ADMET prediction methods and models often end with thestatement that is “relatively simple to develop models that fit the entire data set, but that,typically, such models do not predict new data sets well”, what is needed, all reviewersagree, is more and better data. It doesn’t matter how good a new model is, because in theend a model is as good as the data it is based on.A criteria‐based evaluation of aqueous solubility shows that 95‐100 percent of thedatabase literature is of poor or unevaluable quality. The accuracy and reliability of thevast majority of the data are unknown due to inadequate documentation of the methodsof determination used by the authors (for example, estimates of precision have beenreported for only 20% of experimental Sw data).To understand the current state of our predictive capabilities we challenged the scientificcommunity by launching an open prediction “Solubility Challenge”. We accuratelymeasured the intrinsic solubility of 100 druglike molecules using the CheqSol approach,which will be also described in this talk. This technique is a highly reproduciblepotentiometric technique that ensures the thermodynamic equilibrium is reached rapidily.We then challenged researchers to predict the intrinsic solubility of 32 other druglikemolecules, measured with the same technique but yet to be published. The major findingsof the Solubility Challenge will be discussed.

[1] A. Llinas, R.C. Glen, J.M. Goodman, Solubility Challenge: Can You Predict Solubilities of 32

Molecules Using a Database of 100 Reliable Measurements?, J. Chem. Inf. Model. 48 (2008) 1289‐1303.

[2] A.J. Hopfinger, E.X. Esposito, A. Llinas, R.C. Glen, J.M. Goodman, Findings of the Challenge to Predict Aqueous Solubility, J. Chem. Inf. Model. 49 (2009) 1‐5.

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Antibody-drug conjugate (ADC) drug development from ADME perspectives

Hong Wan

Shanghai Hengrui Pharmaceutical CO., LTD., Dept. of DMPK/Tox, Shanghai, P.R. China, [email protected], Tel: +86(0)21-54759078; Fax: +86(0)21-54759072

Biologics or large molecules (LMs), primarily monoclonal antibodies (mAbs) and antibody-drug conjugate (ADC) as main stream therapeutics continue to grow in number of new approvals and targets recently. This talk will be intended to provide an overall comparison between small molecules (SMs) and biologics or large molecules (LMs) concerning drug metabolism and pharmacokinetic (DMPK) or associated with absorption, distribution, metabolism and elimination (ADME) testing, which will help design and conduct relevant ADME testing for biologics such as mAbs and ADCs. In particular, ADC drug development as an example will be discussed to understand its complexity and challenges from extensive in vitro characterization to in vivo animal PK studies and bioanalytical strategies as well as nonclinical safety evaluations including drug metabolites and potential drug-drug interaction (DDI) risk assessment. Due to complex target-mediated clearance and additional CYP-enzymes based metabolism involved in ADC’s disposition pathways, the prediction of human PK and translation of preclinical animal PK/PD data to clinic appear challenging, which will be highlighted from the preclinical and human PK data of two marketing ADC drugs (ADCETRIS, SGN-35 and KADCYLA, T-DM1). As a result, the conduct of relevant ADME testing and fronting loading PK/PD studies for a quantitative understanding of the PK/PD of ADCs and their components in both serum and tissue as well as the key safety evaluations of lead biologics as early as possible are necessary to ensure safe and efficacious preclinical candidates.

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Molecular imaging of fluorescent ligand-DNA interaction in live cells for guiding the development of anti-cancer agents

Ta-Chau Chang

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan, Department of Chemistry, National Taiwan University, Taipei, Taiwan, [email protected]

Molecular imaging with fluorescent anti-cancer agent (FACA) provides a fantastic tool to visualize cellular uptake, localization, and distribution of FATA for better understanding FACA-target interactions in live cells. Here an emerging target for cancer treatment will be illustrated based on the support of fluorescent imaging. Compelling evidence suggests that formation of guanine-quadruplex (G4) can protect the integrity of chromosome ends in eukaryotes, and regulate the activity of some gene promoters. Consequently, G4 has become a novel therapeutic target. We have synthesized a FACA, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), which can be used not only as a fluorescent probe to map endogenous and exogenous G4 in live cells, but also as therapeutic agent that arrests cancer growth by inhibiting telomerase activity and regulating gene expression. Thus, the fluorescence of a G4 anti-cancer agent is an invaluable tool to detect G4 in cells, investigate ligand-G4 interaction in live cells, examine the biological function of G4, and guide the development of new anti-cancer agents. Mitochondria are important not only in producing metabolic energy for the live cells but also in regulating the process of cell death. Mitochondrial targeting has gained much attention for many diseases including cancer. Since we have established the design principles for chemical engineering of BMVC to selectively target intracellular organelles, a number of BMVC derivatives are synthesized to selectively target to mitochondria of cancer cells with strong fluorescence, but is distributed in cytoplasm of normal cells with weak fluorescence. Of importance is that fluorescent studies of these FACAs in live cells and in isolated mitochondria from HeLa cells indicate that their major target is mitochondrial DNA (mtDNA). Fluorescent imaging verifies the presence of mtDNA G4 in live cells. Further bioassays suggest that the interaction of these molecules with mtDNA G4 can suppress mitochondrial gene expression and eventually induce cancer cell death without harming normal cells. Here we provide convincing evidence for the existence of mtDNA G4s in live cells and illustrate the emerging target of mtDNA G4 for drug development.

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Dynamic prediction of hydration structures of biomolecular complexes

Csaba Hetényi, Norbert Jeszenői*, Mónika Bálint** David van der Spoel***, István Horváth****

MTA-ELTE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Pázmány sétány 1/C, 1117 Budapest, Hungary

*Department of Genetics, **Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, 1117 Budapest, Hungary

***Uppsala Center for Computational Chemistry, Science for Life Laboratory, Department of Cell and Molecular Biology, University of Uppsala, Box 596, SE-75124 Uppsala, Sweden,

****Chemistry Doctoral School, University of Szeged, Dugonics tér 13 6720 Szeged, Hungary.

Hydration structure has a fundamental role in biomolecular interactions, and its knowledge is essential for drug esign [1]. Experimental determination of water positions has numerous limitations [2,3] such as crystallographic artefacts, assignation problems, etc. Although various computational methods have been published in this field, the prediction of hydration structure of biomolecules remains a great challenge [4]. Our presentation features recent results and future work on MobyWat [5], a program for calculation of hydration structures using molecular dynamics trajectories (www.moby-wat.com). The discussion focuses on prediction of water positions of protein-ligand interfaces important in drug discovery and also describes possible applications of our method in both theoretical and experimental research. Acknowledgements: Simulations were carried out on resources provided by the Swedish National Infrastructure for Computing (SNIC) at the ’Abisko’ supercomputer of the High Performance Computing Center North (HPC2N, Sweden, grant SNIC2013-26-6) and in Hungary at NIIF National Information Infrastructure Development Institute. The work was supported by the Hungarian Scientific Research Fund (OTKA K112807) and the MedinProt project of HAS. We are thankful to the Gedeon Richter Pharmaceutical Plc. for a scholarship to N.J. References [1] A.T. García-Sosa, Hydration Properties of ligands and drugs in protein binding sites: tightly-

bound, bridging water molecules and their effects and consequences on molecular design strategies, J. Chem. Inf. Model. 53 (2013) 1388-1405.

[2] C.X. Weichenberger, P.V. Afonine, K. Kantardjieff, B. Rupp, The solvent component of macromolecular crystals, Acta Cryst. D Biol. Cryst. D71 (2015) 1023–1038.

[3] B. Halle, Biomolecular cryocrystallography: structural changes during flash-cooling, Proc. Natl. Acad. Sci. USA 101 (2004) 4793-4798.

[4] E. Nittinger, N. Schneider, G. Lange, M. Rarey, Evidence of water molecules - a statistical evaluation of water molecules based on electron density, J. Chem. Inf. Model. 55 (2015) 771-783.

[5] N. Jeszenői, I. Horváth, M. Bálint, D. van der Spoel, C. Hetényi, Mobility-based prediction of hydration structures of protein surfaces, Bioinformatics, in the press (2015) doi: 10.1093/bioinformatics/btv093.

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The role of uptake, cellular or tissue distribution, and the target on the efficacy of tyrosine kinase inhibitors

G. J. Peters, R. J. Honeywell

Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam

Tyrosine kinase inhibitors (TKI) form a new entity of anticancer drugs that were introduced into the clinic as specific inhibitors of critical signaling pathways in tumors. The first generation TKI, imatinib was a paradigm for development of TKIs, since its inhibition of BCR-ABL changed the treatment of chronic myeloid leukemia. The second generation TKI, gefitinib and erlotinib are relatively specific inhibitors of the Epidermal Growth Factor Receptor (EGFR), while sorafenib and sunitinib targeting the VEGF receptor, act as a cytotoxic drug against tumor cells (e.g. renal cancer) and as an inhibitor of endothelial cell proliferation, preventing formation of new tumor vasculature. Crizotinib is a specific inhibitor of c-Met and the ALK-EML translocation. The third generation TKI, dasatinib is used for patients resistant to imatinib. Most TKIs are highly protein bound and substrates for Phase I and II metabolism. However, these molecules are all slightly polar and hydrophobic in nature, making them excellent substrates for multiple influx and efflux transporters, which ultimately determine their pharmacokinetic profile and efficacy. Steady state plasma levels vary from 0.13 (sunitinib), 0.21 (dasatinib), 0.29 (gefitinib), 2.54 (erlotinib) to 12.1 µM (sorafenib), but are highly variable between patients. Development of TKIs often neglected common pharmacological parameters such as adequate drug uptake in the gut, body distribution and clearance by metabolism. Hence proper evaluation of these properties in suitable model systems will help to predict how drugs will behave in the patient. Several drugs with poor transport characteristics in model systems show a poor distribution in the human body and low uptake in the tumor, leading to decreased inhibition of the target in the tumor of the patients. For several of the most commonly used TKIs we determined their uptake and retention in the CaCo2 epithelial model system as well as in cancer cells and tumors. Gefitinib, erlotinib and dasatinib, uptake in colon cancer cells was partially active, but sunitinib, crizotinib and sorafe-nib uptake was mainly passive, but over time an increased role for active transport was found for sunitinib. Using specific inhibitors it was shown that various organic cation transporters (OCT1,2 & 3) played a role in uptake. P-glycoprotein and ABCG2 (BCRP) played a role in the efflux of dasatinib and sorafenib, respectively. Absolute accumulation over 24 hr (37°C) was very different with erlotinib and dasatinib reaching 20-150 ng/mg protein, while gefitinib was 20 fold higher. Sunitinib, sorafenib and crizotinib accumulation were even higher reaching 2 to 12 µg/mg protein. This pattern matched accumulation of some TKIs observed in patients in the clinical setting. For both sunitinib and to a lesser extent crizotinib, a high lysosomal accumula-tion was found, which masked true cellular uptake, creating a poor dose/effect correlation. These aspects also lead to different pharmacokinetics, with e.g. accumulation in time in red blood cells. In the epithelial gut model system transfer rates from the apical (gut) to the basolateral (blood) site were determined. Transfer rates varied from 43 for sunitinib, 209 for dasatinib, 180 for gefitinib, 223, for sorafenib, and 479 pmol/hr for erlotinib. Transfer was ATP dependent for gefitinib, sunitinib and sorafenib. Unexpectedly transfer from the basolateral to the apical site was higher for most compounds, 685, 4630, 252, 621 and 1623, respectively.

In conclusion, uptake of the various TKIs in tumor cells was highly variable, as well as trans-fer rates in the gut epithelial model, with a very high negative flux for dasatinib. The in vitro models can (partially) explain the variable results which have been seen in clinical studies.

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Permeability assessment and pharmacokinetics of some β-amyloid aggregation inhibitors for the treatment of Alzheimer’s disease

Kin Tam, Wei Zhou, Ken Yung*, Ricky Wong**, Hung Wing Li**

Faculty of Health Sciences, University of Macau, Taipa, China *Department of Biology, Hong Kong Baptist University, Hong Kong, China

**Department of Chemistry, Hong Kong Baptist University, Hong Kong, China

β-amyloid (Aβ) aggregation is thought to be one of the mechanisms leading to Alzheimer’s disease (AD). We previously developed a series of carbazole-based cyanine compounds using various functionalized pyridinium or quinolinium accepting moieties to inhibit the Aβ aggregation. It was found that the binding of some of these inhibitors, namely SLM, SLOH and SLAD-Pr, to Aβ (1-40) were reasonably strong, with binding constants (Kd) in the micromolar regions. To assess the likelihood of central nervous system (CNS) absorption, the in vitro Caco-2 cell model was utilized to study the permeability and efflux ratio of these inhibitors. It was found that the permeabilities (Papp a->b) of these inhibitors were moderate (c.a. 4.0 ×10-6 cm/s), while the efflux ratio of SLAD-Pr was significantly less than that of SLM and SLOH. Pharmacokinetic studies of SLAD-Pr in mice with an intravenous dose of 1 mg/kg revealed a short elimination half-life of 0.37 hr and a moderate clearance (23% of liver blood flow). Our results suggested that SLAD-Pr showed suboptimal CNS absorption, which is due to its short in vivo half-life and moderate Papp a->b value. Clearly, the permeability screening system and the pharmacokinetics studies developed in this study can offer useful insight for further optimization of the carbazole-based compounds. As more potent analogues emerge from the project, in vitro Caco-2 cell model and in vivo pharmacokinetics study will be employed to assess the CNS absorption of these new carbazole-based compounds prior to pharmacodynamics evaluation.

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The predictors of drug effects on Alzheimer’s disease: shed new light from EEG parameters to brain connectomics

Yin Tian, Zechao Ding, Zhongyan Wang, Huiling Zhang

College of Bio-information, ChongQing University of Posts and Telecommunications, ChongQing, 400065, China

Alzheimer’s disease is a neurodegenerative disorder leading to cognitive deficits such as learning and memory impairments, aprosexia, and executive dysfunction. Though the amyloid-β(Aβ) peptides plague aggregation and tau hyperphosphorylation are thought to be two main causal roles in the pathogenesis, the underlying mechanisms remain uncovered. Researchers spent a lot of energy to develop treatments for Alzheimer’s disease (AD). However, the poor translation of drug efficacy data from animals to humans, including the validity of animal models, toxicological issues, and pharmacodynamics (PD) and pharmacokinetics (PK), hampered the success of these therapeutic approaches in human. The pathology of AD could be in part due to synaptic dysfunction. Electroencephalogram (EEG), generating from the result of the postsynaptic potential discharge between cells, could be a potential measure to bridge the gaps between animal and human data. Here we discuss recent evidence on EEG characteristics (i.e. specifical rhythms, amplitude, timecourse changes) and brain connectomics (i.e. small-world network, shortest path length, clustering coefficient, glob effect, and local effect) that could be used for real-timing monitoring of the drug effects on animal models and humans. It is expected that this novel approach will offer a deeper understanding on the drug efficacy at a microcirculatory level, which will be useful to support the development of new AD treatments. Corresponding author: Dr. Yin Tian, email: [email protected], Tel: +86-023-62460536,

Address: No.2, Chongwen Road, ChongQing, China, 400065

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The role of material science in the research and development of drug substance and drug product

Ernest Meštrović

PLIVA Croatia, TAPI R&D, Prilaz baruna Filipovića 25, HR-10 000 Zagreb, Croatia [email protected]

The development of medicines is an elaborative task that demands a great deal of time and expertise in a broad range of physical and medicinal sciences. In this contribution, numerous aspects of drug development and production will be discussed. A particular focus will be laid on solid oral dosage forms. In the last decades there is much different approach and direction of research in Life Science, one of these dimensions is utilisation of Material Science. The field of Materials Science has most commonly been associated with the study of structural or functional materials for engineering and construction applications, such as metals, alloys, semiconductors, ceramics, glass, polymers and composites. Now this discipline put in the focus soft matter, including biopolymers, powders, and biomaterials. From another aspect, in the pharmaceutical science there is many changes and opportunity which can be solved by apply physical principles common in materials science to challenges in such areas as drug delivery, control of drug form, manufacture and processing of nanoscopic and microscopic particle systems, and the structure and properties of bulk powders and their assemblies (e.g., tablets) for use in pharmaceutical applications. The understanding and full control over the solid-state properties of small molecule pharmaceutical agents (which excludes proteins and other macromolecules) plays are central role in the development of solid oral dosage forms. Small molecule drug candidates are known to exist in the form of various polymorphs, solvates or hydrates, as well as in amorphous phases. In addition, a broad range of phase transitions (e.g. polymorph interconversions, desolvation of solvates, etc.) can occur during pharmaceutical processing, thus altering the desired physicochemical properties (e.g. dissolution rate, transport characteristics, morphology) of the drug, All this fact will be point out taking in account relationship among the structure, properties, performance, and processing of a drug.

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Non-ambient conditions in drug forms discovery and development

Elena V. Boldyreva

Novosibirsk State University, Novosibirsk, Russia; 2 – Institute of Solid State Chemistry and Mechanochemistry Siberian Branch of Russian Academy of Sciences,

Novosibirsk, Russia; [email protected]

A drug form is more than a drug substance. To prepare a drug form, one needs to synthesise (or extract from natural resources or wastes) an active pharmaceutical ingredient (API), and then optimise its (crystal/amorphous) structure, particle size, shape, as well as prepare a composite with other components – other APIs or excipients, controlling the composition and the structure of these multi-component systems. Non-ambient conditions – cryogenic temperatures, high temperatures, supercritical conditions, high pressure, mechanical treatment in various machines – are used at all the stages of drug discovery and development. In this lecture I shall give selected examples of using non-ambient conditions, in order to synthesise APIs as single- and poly-component (salts, cocrystals) samples, to modify their crystal structure (polymorphism control, obtaining various amorphous states), to micronize, to control particle shape, to prepare composites and to control their meso-structure. Complexity of “simple systems”, challenges and prospects will be discussed.

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The impact of molecular mobility and molecular interaction patterns on physical stability of amorphous anti-inflammatory agents

Marzena Rams-Baron, Zaneta Wojnarowska, Mateusz Dulski*, Katarzyna Grzybowska, Justyna Knapik, Marian Paluch

Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland and Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1a,

41-500 Chórzow, Poland *Institute of Material Sciences, University of Silesia, 75 Pułku Piechoty 1a,

41-500 Chorzów, Poland The aim of our research was to find possible justification for the substantial differences in the crystallization tendencies of three chemically related amorphous anti-inflammatory agents, etoricoxib, celecoxib and rofecoxib. Since it is well known that the intensified molecular mobility may strongly affect the crystallization behavior of a given material, broadband dielectric spectroscopy (BDS) was used to gain insight into the molecular dynamics of the selected APIs. Interestingly, our experiments did not reveal any significant differences in their relaxation behavior, both in the supercooled liquid as well as in the glassy state. Hence, as a possible explanation for the superior physical stability of etoricoxib, its ability to undergo a tautomerization reaction was recognized. The occurrence of intramolecular proton transfer in the disordered etoricoxib was proven experimentally by time-dependent dielectric and infrared measurements. Additionally, IR spectroscopy combined with DFT calculations pointed out that in the etoricoxib drug, being in fact a binary mixture of tautomers, the individual isomers may interact with each other through a hydrogen bonding network. A possible explanation of this issue was achieved by performing dielectric experiments at elevated pressure. Since compression results in etoricoxib recrystallization, the possible influence of pressure on the observed stabilization effect will be also carefully discussed. Acknowledgment: The authors M.R.B., Z.W. and M.P. are deeply grateful for the financial support by the National Science Centre within the framework of the Opus3 project (Grant No. DEC-2012/05/B/NZ7/03233).

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Quantification of amorphous content in drug substance and drug product

Rolf Hilfiker

Department Solid-State Development, Solvias AG, Römerpark 2, CH-4303 Kaiseraugst, Switzerland, [email protected]

Crystal structure (polymorphism) as well as crystal shape (morphology) and size have a huge practical and commercial impact on active substances all the way from research to manufacture of the final product. Size can in principle be controlled by direct crystallization or by micronization. The micronization process often leads to amorphization of solids in the % range or might lead to (partial) polymorph transformation, making it necessary to develop methods for amorphous content determination. Having a defined application and formulation in mind (e.g., inhalation), several aspects will be discussed, e.g., ways to trace amorphous content in drug substance and drug product.

Ways to trace amorphous content in crystalline solids

Ways to trace amorphous content in formulated products

Micronization and amorphization

Impact on drug substance properties

.

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Quantitative mechanical measurements at the nano-scale for the crystalline pharmaceutical ingredients

Biljana Janković, Stane Srčič

Faculty of pharmacy, University of Ljubljana, Slovenia

Organic crystals are of especial importance in pharmaceutical industry since the majority of drugs are produced and applied in crystalline form. Their properties are strongly dependent from the internal structure (type and strength of intermolecular bonds, molecular packing etc.) as well as crystal habit (morphology of the primary crystals). Mechanical behaviour of materials under stress is of special interest for powder processing by milling, dry granulation and compression. During those processes, particles can respond to stress by elastic, plastic deformation and/or brittle fracture. To understand and elucidate deformational mechanism of molecular crystals used in the pharmaceutical technology, experimental techniques such as an atomic force microscopy (AFM) and instrumented nanoindentation are generally utilized. The improvement of classical nanoindentation method represents dynamic indentation cycling under ultra low loads, known as as DCM-CSM (dynamic contact module- continuous stiffness measurement). The advantages of DCM is related to large sensitivity of the system to surface forces and dynamics due to low indenter column weight (150 mg), improved resolution as a consequence of high resonant frequency functioning as well as a possibility to investigate a pre-contact mechanics. The CSM technique provides the continuous measurement of mechanical properties of materials to be conducted in one sample experiment without the need for discrete unloading cycles. In this case, the contact stiffness can be measured directy during the loading cycle of the indentation test. The results from the nanoindentation cycle represent load vs. displacement curve from which the following parametrs can be calculated: Young’s modulus, indentation hardness as well as the total energy of deformation. For this purpose, Oliver and Pharr method is generally applied [1]. In comparison to nanoindenters, AFM give the opprotunites to gather the images of the morphology with the high resolution and to deform the sample by the AFM tip at the specific areas of the surface. Single-point nanoindentation via AFM is performed in a force mode. In this mode, the tip approaches the surface and indents the sample until the set force is accomplished. After this point, the tip is retracted away from the surface. The data from each cycle is represented in the form of force versus distance graph. By application of Hertz model, it is possible to calculate the parameter of materials elastic property, e.g. Young’s modulus [2]. The lecture will provide concise overlook of listed instrumental techniques as well as examples on pharmaceutical crystals by using AFM and nanoindenter. [1] H. Hertz, Uber die Beruhrung fester elastischer Korper. J. fur die reine and Angew. Mathem. 92

(1826) 156/171. [2] W.C. Oliver, G.M. Pharr, Measurement of hardness and elastic modulus by instrumented

nanoindentation. Advances in understanding and refinements to methodology, J. Mater. Res. 19 (2004) 3-20.

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Toward better understanding of crystallization of amorphous drugs under compression: isochronal crystallization kinetics approach

Marian Paluch, Karolina Adrjanowicz

Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland and NanoBioMedical Centre, Adam Mickiewicz University, ul. Umultowska 85,

61-614 Poznan, Poland

In this presentation, we will show results from the dielectric studies on the effect of different thermodynamic conditions on the physical stability of various amorphous pharmaceuticals [1-3]. By maintaining isochronal condition during measurements, we were able to control the kinetic factor of the crystallization process and untangle purely thermodynamic effects on crystallization from kinetic ones. This cannot be achieved by any other experimental attempts performed at atmospheric pressure. Along with experimental studies, crystallization of the amorphous drugs under pressure was also described theoretically. We have demonstrated within the studied pressure range (up to a few hundreds of MPa) that one should expect an increase of thermodynamic driving force, decrease in melt/crystal interface energy, and critical nuclei size. Therefore, an experimentally observed increase in the overall crystallization rate under isochronal conditions can be exclusively rationalized as due to variations of the thermodynamic factor. References: 1. K. Adrjanowicz, A. Grzybowski, K. Grzybowska, J. Pionteck, and M. Paluch, Cryst. Growth Des. 13,

4648−4654 (2013) 2. K. Adrjanowicz, A. Grzybowski, K. Grzybowska, J. Pionteck, and M. Paluch, Cryst. Growth Des. 14,

2097−2104 (2014) 3. K. Adrjanowicz, K. Kaminski, M. Paluch, and K. Niss, Cryst. Growth Des. 15, 3257−3263 (2015)

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Application of titanate nanotube composites for the modification of the solubility, dissolution kinetic and processability of drugs

Tamás Sovány, Barbara Sipos, Zoltán Kónya*, Klára Pintye-Hódi, Géza Regdon jr.

University of Szeged, Department of Pharmaceutical Technology, Eötvös u. 6., H-6720, Szeged, Hungary

*University of Szeged, Department of Applied and Environmental Chemistry, Rerrich Béla tér 1., H-6720, Szeged, Hungary, and

MTA-SZTE Reaction Kinetics and Surface Chemistry Research Group, Rerrich Béla tér 1, H-6720, Szeged, Hungary,

The biggest challenge of the pharmaceutical industry is to ensure the appropriate bioavailability of the newly developed drugs. The use of micronized or nanosized materials is one of the possible answers to the solubility and permeability issues. Nevertheless, despite of the superior results the processing and stabilization of these materials is still a critical issue of the pharmaceutical industry. The use of naturally nanosized structures, such as carbon nanotubes as vectors for drug delivery is a promising way of the future developments, but some toxic side effect was reported regarding these materials. Other nanostructured materials e.g titanate nanotubes could provide a safe alternative for the production of drug delivery systems. In present study hydrothermally synthesized nanotubes were filled with various APIs (dilthiazem hydrochloride, diclofenac sodium, atenolol, hydrochlorothiazide) from the four different BCS classes to explore how the composite formation with the nanotubes influence the drug release kinetic and material behaviour. The texture of the composites was tested with transmission and scanning electron microscopy, the API carrier interactions were studied with FT-IR, DSC, TG and XRPD investigations. The drug release of capsules and tablets containing the crystalline drugs or composites was compared with the application of USP 2 method. The results suggest that the nanosized carrier may improve the solubility of the drugs, but the release kinetic will be slower, as the API must diffuse from the carrier. The decrease in the speed of drug dissolution is based on the strength of interactions between the API and the nanotubes. Nevertheless, all of the composites exhibited superior mechanical properties over the native crystalline drugs the corresponding tests revealed excellent flowability, compactibility and compressibility for the composites compared with the poor results of the native drugs. In summary, the titanate nanotubes are promising vectors for the oral delivery of various drugs, and may widely improve the physicochemical properties and bioavalability of those APIs. Acknowledgement: The publication is supported by the European Union and co-funded by the European Social Fund. Project numbe: TÁMOP-4.1.1.C-13/1/KONV-2014-0001

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QsarDB: chemo-informatics solution for preserving and efficient use of in silico physicochemical, toxicological, biochemical and human health endpoint models

Uko Maran, Sulev Sild, Villu Ruusmann, Geven Piir, Alfonso T. García-Sosa, Kalev Takkis, Mare Oja, Elmar Laigna*, Priit Ahte*, Iiris Kahn*, Andre Lomaka*

Institute of Chemistry, University of Tartu, Tartu, Estonia, *Institute of Chemistry, Tallinn University of Technology, Tallinn, Estonia

Since the seminal work by Hanch and Fujita (J Am Chem Soc, 1964, 86, 1616) QSAR got boost particularly in the area of medicinal chemistry. (Q)SARs have been at the forefront of explaining obscure physical, chemical, biological phenomena and playing important role in drug discovery and development scenarios when utilizing in silico model for ADMET properties. Many (Q)SARs have been developed, most of them have found way to scientific publications that are unfortunately very difficult to reuse. Very few of proposed models have found their way to the practical applications in commercial or public software solutions. The current presentation gives an overview of perspective reuse of published models from physic-chemical properties to human health endpoints. Presentation also looks into web repositories and integrated web modelling environments available for (Q)SAR/(Q)SPR models and discusses the lifecycle of models and how models reach the intended audience from the perspective of model developers and users and how web repositories provide help via reducing the time to decision. In more detail the QSAR DataBank approach for the digital organization and archiving of QSAR model information [1] and QsarDB repository [2] (http://qsardb.org/) for archiving, uniquelly citing and interactively accessing models is discussed. The repository stores models in an open QsarDB data format that is a generic solution for the electronic organization and archiving of (Q)SAR/(Q)SPR model information according to open standards. At the time of writing this abstract, the QsarDB repository contains 346 unique models for 58 different endpoints and about 17 species in the following mathematical representations: regression models, classification models, decision trees, neural networks, random forests, support vector machines and ensemble (consensus) models. Many of those models are for describing and predicting ADMET and human health endpoints, for example membrane permability [3]. Acknowledgement: European Union, Regional Development Fund (3.2.1201.13-0021) [1] V. Ruusmann, S. Sild, U. Maran, QSAR DataBank - an approach for the digital organization and

archiving of QSAR model information. J. Cheminf. 6 (2014) 25 [2] V. Ruusmann, S. Sild, U. Maran, QSAR DataBank Repository: open and linked qualitative and

quantitative structure-activity relationship models. J. Cheminf. 7 (2015) 32. [3] M. Oja, U. Maran, The permeability of an artificial membrane for wide range of pH in human

gastrointestinal tract: experimental measurements and quantitative structure-activity relationship. Mol. Inf. 34 (2015) 493–506.

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Simulation of in vitro Caco-2 Papp from molecular structure (Kubinyi vs. QSAR) and estimation of intracellular Km for efflux transporters

Michael B. Bolger, Viera Lukacova, James M. Mullin, Ke Xu-Szeto

Simulations Plus, Inc., Lancaster, CA 939534, USA

Purpose To build in silico models based on molecular structure that estimate the rate of passive diffusion into and out of the cell membrane and to combine those estimates with a cellular simulation of Caco-2 apparent permeability to determine the intracellular unbound Km for efflux transporters.

Methods A cellular simulation program (MembranePlus

TM, Simulations Plus, Inc. Lancaster CA) of

Caco-2 apparent permeability was used to calibrate a simple QSAR model of the passive rate into and out of the cellular membrane bilayer for 22 diverse compounds. This was compared to the drug partitioning of forward and reverse rate constant method of Kubinyi (Kubinyi 1978). A separate cellular simulation for 8 different concentrations of digoxin with and without inhibition of P-glycoprotein (P-gp) (Troutman and Thakker 2003) was used to fit the value of the intracellular unbound Km for P-gp. Finally, the Km value for P-gp determined from fitting in the cellular simulation was used to build a mechanistic oral absorption and physiologically-based pharmacokinetic (PBPK) model of digoxin using GastroPlus

TM . To account for the in vivo distribution and clearance of digoxin, the PBPK

model incorporated permeability-limited liver, kidney, and muscle tissues with basolateral influx and apical efflux transporters. The PBPK model simulation results were compared to literature plasma concentration time data for escalating doses of digoxin by intravenous and oral administration routes (Ochs, Greenblatt et al. 1978; Greiner, Eichelbaum et al. 1999).

Results The cellular simulation for 22 diverse drug molecules using the QSAR model was more accurate than a model based on Kubinyi's drug partitioning theory. The fitted intracellular

unbound Km value for digoxin (95.3 M) was significantly lower than Km values measured in vitro by Troutman et al. in either the absorptive (1150 mM) or secretory (177 mM) directions (Troutman and Thakker 2003). The PBPK simulations of Cp vs. time compared well to the observed clinical data for digoxin.

Conclusions A combination of cellular simulation of in vitro experiments and PBPK modeling of in vivo plasma concentration vs. time profiles can provide a good estimation of the significance of efflux transporters on dose linearity of absorption, distribution, and excretion.

References: Greiner, B., et al. (1999). J Clin Invest 104(2): 147-53. Kubinyi, H. (1978). J Pharm Sci 67(2): 262-3. Ochs, H. R., et al. (1978). Am Heart J 96(4): 507-11.

Troutman, M. D. and D. R. Thakker (2003). Pharm Res 20(8): 1200-9.

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Structure-activity relationship analysis of new antipseudomonal gemini-imidazolium chlorides

Łukasz Pałkowski, Jerzy Błaszczyński*, Andrzej Skrzypczak**, Jan Błaszczak**, Alicja Nowaczyk***, Roman Słowiński*, Jerzy Krysiński

Nicolaus Copernicus University, Department of Pharmaceutical Technology, Jurasza 2, 85-094 Bydgoszcz, Poland

*Poznań University of Technology, Institute of Computing Science, Piotrowo 2, 60-965, Poznań, Poland

**Poznań University of Technology, Institute of Chemical Technology, Skłodowskiej-Curie 2, 60-965 Poznań, Poland

***Nicolaus Copernicus University, Department of Organic Chemistry, Jurasza 2, 85-094 Bydgoszcz, Poland

This paper presents the results of studies of the structure activity relationship (SAR) of 140 3,3'-(α,ω-dioxaalkan)bis(1-alkylimidazolium) chlorides [1]. For those compounds minimum inhibitory concentration (MIC) against strains of Pseudomonas aeruginosa, opportunistic pathogen with ability to rapidly develop resistance to multiple classes of antimicrobial agents, was determined [2]. In order to perform the SAR analysis, tabular information system was formed, in which tested compounds were described by means of condition attributes characterizing: structure (spacer length n, the type of R substituent), surface properties (critical micelle concentration, surface tension at point of CMC, surface excess, area occupied by one molecule of the surfactant and the free energy of adsorption of molecules), and molecular properties (Moriguchi octanol-water partition coefficient, Balaban index, Narumi topological index, total structure connectivity index, Wiener index, electric dipole moment, radius of gyration, molecular weight). Decision attribute, classify-ing compounds, corresponded to MIC value against Pseudomonas aeruginosa ATCC 27853 strain. In SAR analysis a new tool, namely, Dominance-based Rough Set Approach (DRSA), was used. The parameter on the basis of which the ranking of condition attributes was constructed (attributes influence the correctness of the classification of objects) is the Bayesian confirmation factor s. Decision rules, presenting the most important relation-ships between the descripttion of chemical compounds, their surface properties, and mo-lecular descriptors and MIC value, were generated. Analyzed compounds show a good antimicrobial activity ranging from 0.01 to 33.87 mM/L. It was shown that number of carbon atoms in R-substituent, Narumi topological index, molecular weight influence the most on the increase of antipseudomonal activity of gemini-imidazolium chlorides. Obtained decision rules and condition attributes with high confirmation coefficients are a valuable guidance in the planning of the synthesis of new chemical compounds with potentially high antimicrobial activity. Decision rules represent ranges of parameters of compounds to be included in the synthesis of new antimicrobial active gemini-imidazolium chlorides.

[1] Ł. Pałkowski, J. Błaszczynski, A. Skrzypczak et al. Antimicrobial Activity and SAR Study of New Gemini Imidazolium-based Chlorides, Chem. Biol. Drug. Des. 83 (2014) 278-288.

[2] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biofilms 358 (2001) Lancet, 135-138.

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Antibacterial drug release from a biphasic polymeric system: mathematical modelling

Mario Grassi, Samuel Golob*, Fabio Pontelli, Michela Abrami**, Gianluca Chiarappa, Gabriele Grassi**, Beatrice Perissutti*, Dario Voinovich*

Dept of Eng and Architecture, Trieste University, Via A. Valerio 8, I-34127, Trieste, Italy *Dept of Chem and Pharm Sci, Trieste University, Via L. Giorgeri 1, I-34127, Trieste, Italy

**Dept of Life Sciences, Trieste University, Strada di Fiume 447, I-34149 Trieste, Italy

Bacterial infections represent an important problem in the orthopaedic field as they can develop either immediately after the chirurgical intervention or after some years [1]. In particular, they can be very problematic in the case of implants as, often, their elimination requires the surgical removal of the infected implant. Accordingly, a possible solution strategy is to act locally by coating the implant by an antibacterial system that has to be easily applicable, biocompatible (it must not hinder implant osseointegration) and able to provide the desired release kinetics of the selected antibacterial drug. In this frame, we focussed the attention on a biphasic polymeric system made up by a thermoreversible gel matrix, constituted by Poloxamer 407/water system, hosting a dispersed phase represented by PLGA microparticles loaded by the antibacterial drug (vancomycin hydrochloride). While below room temperature, the Poloxamer 407/water system behaves as a solution and it is easily spreadable on the implant surface, upon temperature rise to physiological values, the Poloxamer 407/water solution undergoes gelation. Accordingly, the gel phase ensures that the PLGA microparticels remain in situ, between the implant surface and the growing bone. The prolonged release of vancomycin hydrochloride is due to the PLGA microparticles, acting as reservoir systems. In principle, the mathematical modelling of drug release from such a biphasic system is not simple, as we have to account for the drug radial diffusion in the microparticles, the drug axial diffusion in the gel matrix and the spatial distribution of microparticles inside the gel matrix. Thus, a three-dimenional diffusive model should be considered. In this work, we propose a simplified approach assuming that microparticles have the same diameter and that they are uniformly distributed in the gel matrix. In addition, we assume that drug release can be considered one-dimensional both in the gel matrix and in the microparticles. The connecting equation between the two diffusive phenomena is represented by a proper boundary condition at the microparticles-gel matrix interface. This condition simply ensures that the drug flux exiting from the particle surface is equal to that going in the gel matrix. As the drug concentration in the gel matrix depends on the axial position, the exiting flux will depend on the position of the microparticle. In order to account for the presence of microparticles, the differential equation describing drug diffusion in the gel matrix will contain a term depending on the microparticle volume fraction. The two differential equations describing drug diffusion in the gel matrix and in the microparticles will be solved by means of the control volume method [2]. [1] N. M. Bernthal et al. PLoS ONE 5(9) (2010). PLoS ONE 5(9): e12580. [2] S. Patankar, Numerical Heat Transfer and Fluid Flow, McGraw-Hill, New York, 1990

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The role of mathematics in mass spectrometry driven species identification

Ena Melvan, Antonio Starcevic, Jurica Zucko, Mario Cindric*, Ivo Ugrina**, Daslav Hranueli

Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia *Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia

**Faculty of Science, Mathematics Department, Zagreb, Croatia Although the application of mathematics to biology has already been included within the discipline of applied mathematics, only recently there has been an explosion of interest in the field. Since the enormous expansion of data-rich information sets, due to genomics revolution, and recent development of mathematical tools such as chaos theory a rapid synergy between biology and mathematics is significantly extending both fields in the coming decades. Progressively, biologists are using mathematical skills to create simulations of real life examples that can help us understand complex, non-linear mechanisms in biology. Other important aspect of this synergy is increasing interest in in silico experimentation due to ethical consideration, unreliability, risk and other complications involved in human and animal research. Applying mathematical models one can display otherwise invisible worlds hidden within forests of data generated by various „-omics“, such as mass spectrometry based proteomics. Now days, Mass-spectrometry based proteomic assays are becoming golden standards in many clinical microbiology laboratories. There are numerous examples of these methods being used to identify bacteria at the genus and species level. In our lab, we are developing a solution for identifying microorganisms by replacing numerous unrelated single protein searches with something we call a “suspect species fingerprint”. By diving each protein into n-grams and making a vector of n-gram frequencies we can represent species proteome using term vector model. The similarity between species can then be calculated by comparing the deviation of angles between each species vector and the query vector. Unfortunately, the accuracy is seriously affected by the problem of data sparsity and scalability. Therefore, we propose a proficient dimensionality reduction-based algorithm. Singular Value Decomposition (SVD) often used by Latent Semantic Indexing (LSI) is utilized to obtain a reduced data representation, therefore solving the sparsity and scalability issues. As a result, significant improvements in computational time are observed together with increased quality of returned results when matching species. In addition, this approach decreases the memory requirements.

This research is funded by HRZZ (Croatian Science Foundation) project “Clinical proteomics of microorganisms”

References 1.Ferguson, P.L., Smith, R.D., 2003. Proteome analysis by mass spectrometry. Biomol. Struct. 32,

399–424. 2.P. W. FOLTZ, Using Latent Semantic Indexing for information filtering, in Proceedings of the ACM

Conference on Office Information Systems (COIS), 1990, pp. 40–47.

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LC-SPE-(cryo)NMR/MS as routine analysis technique

C. Daolio

Bruker BioSpin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany [email protected]

The latest development in the field of hyphenated techniques is the usage of a Solid Phase Extraction (SPE) unit as an interface between liquid chromatography (LC) and NMR. This relatively new technique goes especially well together with mass spectroscopy. The SPE allows the usage of completely non-deuterated solvent environment during the chromatographic separation, what simplifies the acquired MS spectra. The most recent coupling of a time-of-flight mass spectrometer provides the exact mass of the analyzed peak, and therefore, access to the sum formula. Also, the sample enrichment through repeated chromatographic runs can be accomplished much more broadly through the use of highly sensitive mass chromatogram. The molecular formula, from high resolution MS, and a complete set of 1D- and 2D-NMR data acquired in reasonable working time due to the CryoProbe™ Prodigy technology, which boosts NMR sensitivity, can be bounded together in a newly developed software tool. Structures which are in agreement with the experimental data are generated and ranked based on

13C chemical shift prediction providing, finally, a good starting point for

the completely unbiased structure validation. Examples for the successful application of the methods described above will be presented

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Exploration of tetrahelical DNA structures as dynamic drug targets by NMR

J. Plavec

Slovenian NMR Center, National Institute of Chemistry, Ljubljana, Slovenia; University of Ljubljana Faculty of Chemistry and Chemical Technology, Slovenia and

EN-FIST Center of Excellence, Ljubljana, Slovenia [email protected]

In addition to double helical structure, DNA can fold into a wide range of structures that are associated with its unique biological roles and functions. A wide distribution of G-rich repetitive sequences throughout genome and transcriptome has raised a high interest. G-quadruplexes are four-stranded DNA structures that have become increasingly popular within the last 20 years when alternative DNA structures were shown to represent a novel level of control in DNA replication, transcription and replication processes. A large repertoire of possible G-quadruplex topologies differing in orientation of DNA strands and types of connecting loops that a G-rich oligonucleotide can adopt has made the prediction of G-quadruplex topology very challenging. Folding into a given G-quadruplex topology is a consequence of several not well understood intertwined factors, such as sequence details, type and concentration of stabilizing cations, concentration of oligonucleotide and interplay of interactions between loop and G-quadruplex core forming residues. Nuclear magnetic resonance (NMR) spectroscopy is currently the most versatile spectroscopic techniques for the characterization of molecular structure and dynamics of G-quadruplexes in solution. LNA modified VEGF aptamer RNV66 adopts a single unprecedented parallel-stranded monomeric G-quadruplex with three G-quartets.

1-3

Assessment of hydrodynamic properties on d(G4T4G4) originating from 1.5 telomeric repeats of Oxytricha nova has shown that unfolded oligonucleotides do not adopt an extended, but rather some type of random coiled structures. The pre-organized structure held together by transient hydrogen bonds with distinctive fingerprint features represents an intermediate on pathway to G-quadruplex formation. An oligonucleotide with repetitive G-rich segments found in the regulatory region of the PLEKHG3 gene in the 14

th human chromosome that represents a potential candidate

contributing to the risk of autism does fold into a G-quadruplex.Nevertheless, it adopts a well-defined tetrahelical structure. No G-quartets or Hoogsteen-type H-bonded guanine residues are present and the overall topology is conserved in the presence of Li

+, Na

+, K

+

and NH4+ ions. Its four-stranded architecture is stabilized by four G-C base pairs in Watson-

Crick geometry, four G∙A base pairs in N7-N1 amino carbonyl and six G∙G base pairs in N1-carbonyl symmetric geometry. References 1. S.L. Edwards, V. Poongavanam, J. R. Kanwar, K. Roy, K. M. Hillman, N. Prasad, R. Leth-Larsen, M.

Petersen, M. Marusic, J. Plavec, J. Wengel, R. N. Veedu, Chem. Commun. 51 (2015) 9499-9502. 2. M. Trajkovski, E. Morel, F. Hamon, S. Bombard, M.-P. Teulade-Fichou, J. Plavec, Chem. Eur. J. 21

(2015) 7798-7807. 3. M. Marusic, H. Tateishi-Karimata, N. Sugimoto, J. Plavec, Biochimie 108 (2015) 169-177.

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The Application of NMR within Drug Discovery: Overview and Case Studies

K. Embrey

AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom

[email protected]

An overview of the role of the UK biomolecular NMR group within drug discovery at AstraZeneca will explained. Furthermore, the role of biophysics within the PHGDH project will be presented as a case study of an early lead generation project. Studies have linked the enzyme 3-phosphohyrdoxyglycerate dehydrogenase (PHGDH) to in vivo tumourgenesis in aggressive breast tumours. PHGDH utilises the cofactor NAD+ to convert 3-phosphohydroxyglycerate to 3-phoshohydroxypyruvate, the rate limiting step in the synthesis of serine. A number of tumours have an increased metabolic flux of glucose to lactate, with a large proportion of glycolytic carbon being diverted into the serine and glycine metabolism through PHGDH. Given the role of PHGDH in potentially driving tumour proliferation and tumourgenesis, we explored the druggability of this unprecedented target. Our hit finding strategies included an X-ray screen of an AstraZeneca fragment library. Multiple ligand efficient binders were identified, and the rich structural output enabled structure-based design strategies to greatly improve binding affinity in both biophysical and biochemical assays. The pivotal role of biophysics and structure in the optimization of an indole fragment hit into a series of nM inhibitors will be presented.

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Fluorine NMR in pharma development: from drug substance to drug product

T. Biljan

PLIVA CROATIA Ltd., TAPI R&D Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia [email protected]

Among the numerous marketed pharmaceuticals more than 150 drugs are fluorinated compounds

1,2. The occurrences of a fluorine substituent in commercial drugs

continuously increase and today it can be estimated that globally 20-25% of drugs contain at least one fluorine atom. This is a huge frequency when comparing with other halogen containing drugs and especially when one considers the fact that there are virtually no natural products that contain fluorine. Incorporation of fluorine atoms in drugs are driven by the special properties of fluorine atom such as strong electronegativity, small size and the low polarisability of the C-F bond that can significantly impact the behavior of drug molecule in biological environment

3.

NMR spectroscopy is widely used technique in pharmaceutical industry in all phases of drug development. In the context of fluorine containing drugs

19F NMR is very powerful

method for studying such molecules. Fluorine-19 nucleus is very attractive for NMR studies due to the 100% natural abundance, high intrinsic sensitivity and a magnetogyric ratio only slightly smaller than that of proton. The fluorine-19 chemical shifts are also highly sensitive to even subtle changes in the magnetic environment, the property that can be used as a probe for distinguishing closely related molecules. The analysis of complex reaction mixtures, formulated drug products and drug metabolites play an important role in pharmaceutical research. However, the analysis of such samples is often very difficult by severe resonance overlap in proton spectrum. Fluorine-19 NMR is a good alternative in such cases if the molecule contains fluorine atoms. Normally excipients, solvents and biological matrices do not contain fluorine atoms and identification of target molecule is much easier. Solid state fluorine-19 NMR is also very useful for identification and quantification of fluorine containing drug molecules and its polymorphs. The use of NMR based screening in drug discovery is very well known. In recent years it has been demonstrated that ligand and substrate based fluorine-19 NMR screening is a powerful tool for identification of novel active compounds

4.

The overview of fluorine-19 NMR spectroscopy use in pharmaceutical research from drug substance to drug product will be presented. References 1. S. Purser, P. R. Moore, S. Swallow, V. Gouverneur, Chem. Soc. Rev. 37 (2008) 237-237. 2. C. Isanbor, D. Hagan, J. Fluorine Chem. 127 (2006) 303-319. 3. J. P. Bégué, D. Bonnet-Delpon, J. Fluorine Chem. 127 (2006) 992-1012. 4. C. Dalvit, Prog. NMR. Spectrosc., 2007, 51, 243-271.

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Poster Presentations


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Small-molecule inhibitors of influenza A virus that act by disrupting PB2 cap-binding of the viral polymerase

Shuofeng Yuan, Jie Zhou, Richard Y. Kao, Bo-Jian Zheng

Department of Microbiology, University of Hong Kong, Hong Kong SAR, China

Objectives The conserved residues 318−483 in PB2 subunit (PB2cap) of influenza A polymerase is an independently folded domain that exhibits a distinct binding mode from other cap-binding host proteins. This study aims to identify a new class of anti-influenza inhibitors that specifically disrupts PB2 cap-binding. Methods A novel fluorescence polarization (FP) assay was established for primary screening, followed by antiviral potency and cytotoxicity analyses. Selected compounds were characterized by multi-cycle virus growth assays, minireplicon assays, time-of-addition assays, cross-protection tests, synergy evaluations, docking simulations and mouse study. Results Several PB2 cap-binding inhibitors were identified. Notably, 4-hydroxy-3-[(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-2H-chromen-2-one, designated as ANA-1, was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus in Madin-Darby canine kidney (MDCK) cell cultures, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2. The docking analyses predicted that ANA-1 occupied the cap-binding pocket of PB2 in a similar mode with m

7GTP, suggesting its role as a viral cap-

binding competitor. Combinational treatment of zanamivir and ANA-1 exerted synergistic anti-influenza effect in vitro. The intranasal administration of ANA-1 could enhance survival rate and reduce lung viral loads in H1N1 virus infected BALB/c mice. Conclusion: The identified inhibitor shows potential to be optimized as an anti-influenza drug. The established platform provides new insights for the development of a new category of influenza therapeutic agents that directly targets on the PB2 cap-binding domain.

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Mast cell inhibitors with potential benefits in tumor angiogenesis

Stefana Avram, Anca M. Cimpean*, Marius Raica*

Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România *Department of Histology, Faculty of Medicine, University of Medicine and Pharmacy

“Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România Melanoma, a highly malignant form of skin cancer, though intensively investigated is still confronted with high mortality. Tumor microenvironment through mast cells that are known to secrete proangiogenic factors is involved in tumor invasiveness. Inhibitors of mast cell degranulation could represent a therapeutic approach for the limitation of cancer progression. We evaluated two known drugs, sodium cromoglycate and ketotifen on a cancer cell model implemented in vivo using the chick embryo chorioallantoic membrane (CAM). The CAM model was performed on fertilized eggs, by inoculating human melanoma A375 cells. Stereomicroscopy and histologic evaluation were performed. Results showed the formation of highly vascularized, unpigmented tumors on the chorionic epithelium for the control samples. The mast cell inhibitors, acting differently, induced tumor cell death and the impairment of the intratumoral vessel architecture. Our data indicates that mast cell stabilizers have potential antiangiogenic effects in tumor microenvironment. Acknowledgement: This study was published under the frame of European Social Found, Human Resources Development Operational Programme 2007-2013, project no. POSDRU/159/1.5/S/136893 Reference [1] Rabenhorst, A., Hartmann, K., http://www.embrn.eu [2] Dehelean, C. A., Feflea, S., Gheorgheosu, D., Ganta, S., Cimpean, A. M., Muntean, D., Amiji, M.

M., Anti-Angiogenic and Anti-Cancer Evaluation of Betulin Nanoemulsion in Chicken Chorioallantoic Membrane and Skin Carcinoma in Balb/c Mice, J. Biomed. Nanotechnol. 9 (2013) 577–589.

[3] Ribatti, D, The Chick Embryo Chorioallantoic Membrane as an In Vivo Assay to Study Antiangiogenesis, Pharmaceuticals 3 (2010) 482–513.

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Triterpene derivatives effects on angiogenesis and melanoma

Stefana Avram, Anca M. Cimpean*, Camelia Oprean**, Ionut Ledeti***, Adriana Fulias****, Corina Danciu, Ioana Z. Pavel, Sorin I. Avram*****,

Cristina A. Dehelean******

Department of Pharmacognosy , Faculty of Pharmacy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România

*Department of Histology, Faculty of Medicine, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România

**Department of Pharmaceutical Chemistry , Faculty of Pharmacy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România ***Department of Pharmaceutical Physics , Faculty of Pharmacy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România ****Department of Analytical Chemistry , Faculty of Pharmacy, University of Medicine

and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România *****Department of Computational Chemistry, Institute of Chemistry of Romanian

Academy Timisoara, Mihai Viteazul Avenue 24, Timisoara 300223, Romania ******Department of Toxicology, Faculty of Pharmacy, University of Medicine and Pharmacy “Victor Babeş“, Eftimie Murgu Square, No. 2, 300041 Timişoara, România

Melanoma is the most aggressive form of malignant skin cancer that arises from unregulated growth of melanocytes. Despite the latest therapeutic advances, resistance and still very low survival rates are signaled [1]. Natural compounds are investigated for their chemopreventive and anti-invasion potential preventing the angiogenesis process. Pentacyclic triterpenes, as betulin, betulinic acid, and some derivatives are known for anticancer, antiviral, anti-inflammatory and antiangiogenic effects [2]. In this study the triterpenes were evaluated on a melanoma assay using the chick embryo chorioallantoic membrane (CAM). The in vivo CAM model was performed in ovo on fertilized eggs, by inoculating human melanoma A375 cells. Compared to the control specimens, the tested compounds induced a low angiogenic index around the primary tumor and limited the invasion from secondary formed tumor sites, showing a low vascular density. Having a good tolerability, the triterpenes reduced angiogenesis and tumor invasiveness in the A375 CAM melanoma model. Acknowledgements: This work was supported by Internal grant at UMFT Victor Babes, PROGRAME III - C1 – PCFI - 2014/2015, UMFT Victor Babes, SYNTANTITUM, Project Director Fulias Adriana Reference [1] Finn, L., Markovic, S.N., Joseph, R.W., Therapy for metastatic melanoma: the past, present, and

future, BMC Med 10 (2012) 23 [2] Laszczyk, M.N., Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in

cancer therapy, Planta Med 75 (2009) 1549–1560.

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Pharmacokinetics, in vitro and in silico assessment of anti-inflammatory alkaloids from Isatis tinctoria

Evelyn A. Jähne, Mouhssin Oufir, Veronika Butterweck*,**, Daniela E. Eigenmann,

Maxime Culot***, Roméo Cecchelli***, Fruzsina Walter****, Mária A. Deli****, Martin Smiesko, Matthias Hamburger

Department of Pharmaceutical Sciences, University of Basel, Switzerland *Department of Pharmaceutics, University of Florida, Gainesville, USA **Pharma Technology, School of Life Sciences, Muttenz, Switzerland

***Université Lille Nord, Lens, France ****Biophysics, Hungarian Academy of Sciences, Szeged, Hungary

We previously identified the alkaloids tryptanthrin (1), indirubin (2) and indolinone (3) as pharmacologically active compounds in woad (Isatis tinctoria L.). They inhibit COX-2, 5-LOX catalyzed leukotriene synthesis, mast cell degranulation at low μM to nM concentrations [1–3], and possess drug-like physico-chemical properties. In a pilot pharmacokinetic study in rats (2 mg/kg i.v. b.w.), 1 and 2 showed a t1/2 of 30-40 min, whereas 3 was rapidly eliminated (t1/2 ~ 4min). In silico predictions for 1-3 indicated high oral absorption and high blood-brain barrier (BBB) permeation. Data obtained with animal and human in vitro BBB models showed a high BBB permeation for 1 and 3 (Papp values > 19x10

-6 cm/s). In the Caco-2 intestinal absorption model, 1 was well absorbed across Caco-

2 monolayers (Papp > 20x10-6

cm/s). Due to low recovery of 3 in the Caco-2 assay, further investigations on its metabolism are needed.

1 2 3 [1] H. Danz, S. Stoyanova, P. Wippich, A. Brattström, M. Hamburger, Identification and isolation of

the cyclooxygenase-2 inhibitory principle in Isatis tinctoria, Planta Med. 67 (2001) 411–416. [2] T. Ishihara, K. Kohno, S. Ushio, K. Iwaki, M. Ikeda, M. Kurimoto, Tryptanthrin inhibits nitric oxide

and prostaglandin E2 synthesis by murine macrophages, Eur. J. Pharmacol. 407 (2000) 197–204. [3] S. Kiefer, A.C. Mertz, A. Koryakina, M. Hamburger, P. Küenzi, (E,Z)-3-(3′,5′-Dimethoxy-4′-

hydroxybenzylidene)-2-indolinone blocks mast cell degranulation, Eur. J. Pharm. Sci. 40 (2010) 143–147.

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Antifungal styrylquinolines probably act as a cellular razor blade

Jacek Mularski, Wioleta Cieślik, Joanna Szczepaniak*, Anna Krasowska*, Robert Musioł

University of Silesia, Institute of Chemistry, Department of Organic Chemistry, Katowice *University of Wroclaw, Faculty of Biotechnology, Wroclaw

Ergosterol, a fungal specific cellular building block, is most abundand in the plasma membrane, but has been found within membranes of many organelles as well. Its ubiquity allows to design ergosterol specific drug therapies. Azoles are the most widely deployed antifungals in clinics and were found to inhibit the biosynthesis of ergosterol. Nonetheless, multi drug resistance has arisen from their excessive use. On the other hand polyenes such as amphotericin (AmB) primarily kill yeast by simply binding plasma membrane ergosterol [1]. Such agregates form ion channels what leads to cell damage. Unfortunatelly, some AmB affinity for cholesterol has been observed and intrinsic high toxicity correlates with exceptional activity profile, limiting its use only to fight systemic fungal infections as the last line of defense. Despite azoles are in treatment more than two decades, our understanding of the specific cellular processes disrupted by ergosterol deprivation remains insufficient.

O

OH

OH

O

O

OO

OH

NH2

OH

O OHOH

OH

OHOH

OH

OH

N

Cl

Cl

OH

O

amphotericin B [AmB]

styrylquinoline [SQ]

Recently, we have been evaluating styrylquinolines (SQ) bioactivity profile [2]. It appears that these small molecules reveal excellent antifungal activity. The ongoing research has already exposed some mechanistic diversity. For instance, resembling some AmB structural relationships, such as conjugated molecular π orbitals, which are known for creating a rigid molecular surface. Therefore, it can be assumed that SQ may acts as ergosterol-selective cellular razor blade. There is also evidence of intracellular ergosterol complexation. As an assistance to those observations and development of further lead optimization we've designed NMR – based experiment.

1H NMR has been chosen for

preliminary studies. This sensitive technique enables maintenance of solutes at marginal desired level. Chemical shifts as well through-space coupling constants of SQ – ergosterol supramolecular complexes should be a measure of their antifungal activity and SQ – cholesterol as a toxicity coefficient. The experiment can also illustrate qualitatively how the drug interacts with its particular sterol ligand, and quantitatively, determining its complexation ratio at particular concentration.

Acknowledgement: National science centre grant no 2013/09/B/NZ7/00423

Reference [1] Proc. Natl. Acad. Sci. USA 109 (2012) 11156-11159 [2] Bioorg. Med. Chem. (2012) 6960-6968

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Controlled suspensions as a method for small scale dissolution profiling of poorly soluble compounds

Sara Andersson, Caroline Alvebratt, Johan Gråsjö, Christel Bergström

Department of Pharmacy, Uppsala University, Sweden The purpose of this study was to identify a method that more rapidly determines dissolution rate and solubility of highly lipophilic, poorly soluble compounds than the powder and disc methods currently in use. Controlled suspensions [1, 2] were prepared by milling the compounds at 600 rpm for 4 x 20 minutes. The compounds were milled in a phosphate buffer containing 1% (w/w) PVP and 0.2% (w/w) SDS. The IDR was measured from controlled suspensions, powder and discs in FaSSIF for a series of highly lipophilic compounds using the µDISS profiler (pION INC, MA). The added amount of material (powder or suspension) was calculated depending on the apparent solubility (Sapp) of the compound. To be able to measure the size of the particles the highly concentrated, milled suspensions were filtered through a 5 µm pore size filter. The particle size was measured using a Zetasizer (Malvern Instruments, UK) from which the particle area was calculated taking use of the density of the compound. The raw data of the dissolution profiles together with the particle area was used to calculate an IDR from controlled suspensions. The crystalline form of the drug after being subjected to micronisation was analysed using differential scanning calorimetry (DSC). Milling original material can be used to produce controlled suspensions that remain stable for several days. The particle size measured for the controlled suspensions was smaller than that calculated by the µDISS software. This indicates that there is a need to modify the methods to measure IDR from powder. Further, dissolution profiling from discs are time-consuming for class II and IV compounds (15-83 hours) [3] compared to controlled suspensions. For the latter, a time scale of 1-2 hours was sufficient to obtain the IDR. The results obtained herein indicate that the IDR measurement may be more accurate when taking use of the particle surface area of controlled suspensions. Controlled suspensions significantly decrease the time needed for IDR measurements suggesting it to be a promising technique. The experimental results can be used to calculate IDR corrected for particle size. Acknowledgements: This work has received support from the Innovative Medicines Initiative Joint Undertaking (http://www.imi.europa.eu) under Grant agreement no. 115369.

References [1] L. Lindfors, P. Skantze, U. Skantze, J. Westergren, U. Olsson, Amorphous drug nanosuspensions.

3. Particle dissolution and crystal growth, Langmuir. 23 (2007) 9866-74. [2] M. Mosharraf, C. Nyström, The effect of particle size and shape on the surface specific

dissolution rate of microsized practically insoluble drugs, Int. J. Pharm. 122 (1995) 35-47. [3] A. Avdeef, O. Tsinman, Miniaturized rotating disk intrinsic dissolution rate measurement: effects

of buffer capacity in comparisons to traditional wood's apparatus, Pharm. Res. 25 (2008) 2613-27.

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High throughput lipophilicity determination of novel amidino substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives

V. Kelava, M. Ilijaš, S. Dragojević, S. Koštrun, M. Hranjec*, V. Gabelica Marković

Fidelta Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia *Faculty of Chemical Engineering and Technology, Department of Organic Chemistry,

Marulićev trg 19, 10000 Zagreb, Croatia

The role of lipophilicity in drug discovery and design is a critical one. Lipophilicity is a key physicochemical property that plays a crucial role in determining ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties and predicting druglikeness of a molecule. Recently, a set of novel amidino substituted benzimidazole and benzimidazo [1,2-a]quinoline derivatives were synthesized and their antitumor activity was investigat-

ed1. In order to determine the lipophilicity range of these compounds, Chromatographic Hydrophobicity Index was measured for a set of selected quinoline derivatives. Chromatographic Hydrophobicity Index by gradient reverse-phase HPLC was used at different pH values. The advantage of high-throughput PhysChem determination is the small amount of sample needed, which is favourable for the early stage of drug

discovery2. Analysis of measured chromatographic parameters, their comparison with calculated data as well as structural influence will be presented and discussed. References: 1 M. Hranjec, M. Kralj, I.Piantanida, M. Sedić, L. Šuman, K. Pavelić, G. Karminski-Zamola, J. Med.

Chem. 50 (2007) 5696; M. Hranjec, B. Lučić, I. Ratkaj, S. Kraljević Pavelić, I. Piantanida, K. Pavelić, G. Karminski-Zamola, Eur. J. Med. Chem. 46 (2011) 2748; M. Hranjec, I. Piantanida, M. Kralj, L. Šuman, K. Pavelić, G. Karminski-Zamola, J. Med. Chem. 51 (2008) 4899.

2 K. Valko, in: R.T. Borchardt, E.H. Kerns, C.A. Lipinski, D.R. Thakker, B. Wang(Eds.), Biotechnology: Pharmaceutical Aspects, vol. 1, AAPS Press, 2004, pp. 127-182

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Lipophilicity determination of zwitterionic compounds: a comparative study

Clara Ràfols, Xavier Subirats, Martí Rosés, Elisabeth Bosch

Departament de Química Analítica – Institut de Biomedicina, Universitat de Barcelona Martí i Franquès 1-11, 08028 Barcelona

Lipophilicity is a fundamental physicochemical property of molecules of pharmaceutical interest, because it measures the capacity of a specific drug to penetrate a lipid membrane and reach a proposed target. The present work deals with the lipophilicity determination of ordinary and zwitterionic ampholytes. For the former, the lipophilicity profile shows a log D maxium at pH values in the in the range between the acidic and basic pKas, corresponding to the neutral form of the compound. On the contrary, in the case of zwitterionic ampholytes two different lipophilicity profiles are possible: log D can adopt around the isoelectric point a maximum (e.g. some antibiotics and anti-hypertensives), or a minimum value (e.g. aminoacids). Therefore, intramolecular effects have to play a key role [1]. In this work the lipophilicity profiles of several ampholytic drugs have been determined using the classical methods based on potentiometry and shake-flask with chromatographic UV detection in a wide range of pH values, and the results have been compared with two different kinds of chromatographic profiles: conventional reversed-phase liquid chromatographic in isocratic elution mode, and the chromatographic hydrophobicity index (CHI) [2]. In addition, the obtained retention factors around the isoelectric point have been used to estimate the log Po/w of neutral zwitterionic compounds by means of the polarity model previously developed in the research group [3]. Finally, the lipophilicity profiles computed by the ACD/Labs Percepta platform [4] have been compared with the obtained experimental data. References: [1] A. Pagliara, P.A. Carrupt, G. Caron, P. Gaillard, B. Testa, Lipophilicity Profiles of Ampholytes,

Chem. Rev. 97 (1997) 3385-3400. [2] M. Rosés, E. Bosch, C. Ràfols, E. Fuguet, Chromatographic hydrophobicity index (CHI), Advances

in Chromatography, CRC Press, Boca Raton, FL, US, 2012. [3] J.M. Pallicer, C. Calvet, A. Port, M. Rosés, C. Ràfols, E. Bosch, Extension of the liquid

chromatography/quantitative structure-property relationship method to assess the lipophilicity of neutral, acidic, basic and amphotheric drugs, J. Chromatogr. A 1240 (2012) 113-122.

[4] Advanced Chemistry Development, Toronto Canada, www.acdlabs.com

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Microemulsion Electrokinetic Chromatography as a very suitable tool for lipophilicity determination of acidic and basic compounds

Xavier Subirats, Alejandro Carrillo, Martí Rosés

Departament de Química Analítica – Institut de Biomedicina, Universitat de Barcelona Martí i Franquès 1-11, 08028 Barcelona

Lipophilicity is a fundamental physicochemical property in the drug discovery process provided that the capacity of a specific compound to penetrate a lipid membrane and reach a proposed target is a key factor of a potential drug candidate. The partition ratio between the immiscible phases 1-octanol and water, log Po/w, is the most widely used lipophilicity index, but reference methods are excessively time consuming for screening purposes and require a high purity sample. Alternatively, traditional chromatographic methods solve these overcomes, but they present a limited pH range of applicability due to the degradation of the column at extreme acidic and basic conditions. In addition, conversion between the particular chromatographic lipophilicity index and log Po/w very often requires the inclusion of molecular descriptors [1]. These drawbacks are avoided by Microemulsion Electrokinetic Chromatography (MEEKC), which is based in the different partition of the solutes between the aqueous (or hydroorganic) phase of the microemulsion and its oil droplets (pseudo-stationary phase) [2]. In the present work a methodology for the determination of lipophilicity of acids and bases is presented, allowing the log Po/w determination of neutral solutes in the range comprised between -0.8 and 5.1, choosing the appropriate microemulsion of pH 2.0, 7.4, 10.0, or 12.0, and using a conventional capillary electrophoresis instrument. Acidic pH is very convenient for acidic analytes, typically carboxylic acids of low pKa values, and the most basic one is suitable for the study of compounds containing aliphatic amines and phenolic groups. The addition of acetonitrile (up to 15%) increases the resolution of the technique for the most lipophilic compounds. Under these conditions, the retention factors of the compounds can be converted into log Po/w values by simple linear correlations. The microemulsion solution is very stable (at least 4 months, stored at room temperature, light protected), and once filtered and introduced in the CE vial allows not less than thirty consecutive analysis (about 12 hours of continuous running). When the acetonitrile content is increased from 10% to 15%, the stability of the microemulsion is reduced about to half. References: [1] B. Sethi, M. Soni, S. Kumar, G.D. Gupta, S. Mishra, R. Singh, Lipophilicity Measurement Through

Newer Techniques, J. Pharm. Res. 3 (2010) 345-351. [2] W.W. Buchberger, Microemulsion Electrokinetic Chromatography, in P. Schmitt-Kopplin (Ed.),

Methods in Molecular Biology, vol. 384, Humana Press Inc, Totowa, NJ, 2008.

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Modelling tadpole narcosis by means of chromatographic systems

M. Rosés*, E. Fuguet*,**, S. Amézqueta*, A. Fernandez-Pumarega*



Toxicity has been emulated in tadpole species through chromatographic systems. The parameter studied to evaluate the non-specific toxicity of a compound is the narcosis concentration (Cnar), which is defined as the concentration needed for the immobilization of the organism. Because experimental investigation with animals is lengthy, costly, technically difficult, and ethically questionable, there is a great interest in developing surrogate physicochemical systems able to emulate biological systems to obtain the same information in a faster, more economic, and easier manner. In order to see which chromatographic systems would be able to emulate tadpole narcosis, both, tadpole narcosis data and chromatographic and electrophoretic data, were fitted to a common QSPR model [1]. Thus, by comparison of the models it was possible to see which of the chromatographic systems were more similar to the biological one. With this aim, four comparison tools were used and, according to these tools, the physicochemical systems that could best emulate the tadpole narcosis were an HPLC system based on an immobilized artificial membrane (IAM) column, and two micellar electrokinetic chromatography systems based on sodium taurocholate (STC) and a mixture of sodium dodecylsulphate (SDS) and Brij 35 as surfactants. A system based on a RP18 HPLC column also was selected because it is a common column in most analytical laboratories. To establish the models, a set of compounds with known Cnar values [2-3] were analyzed in the chromatographic, and electrophoretic selected systems and, then, the retention factor (k) was correlated to the concentration of narcosis. For each physicochemical system, three models were developed, one for the species R. Japonica, another for the species R. Temporaria and a global model (which includes values from both tadpole’s species). Statistics show that the system based on STC micelles is the best to emulate toxicity in tadpoles, and also for the species R. Temporaria. However, for the species R. Japonica the best model corresponds to an HPLC system with an IAM column. Finally, the robustness and predictive ability of the models that best emulate narcosis concentration were validated. [1] M. H. Abraham, Chem. Soc. Rev. 22 (1993) 73-83. [2] K. R. Bowen, K. B. Flanagan, W. E. Acree Jr., M. H: Abraham, C. Ràfols, Sci. Total Environ. 371

(2006) 99-109. [3] E. Overton, Studies on Narcosis, Chapman and Hall, London, 1991.

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Performance of chromatographic systems to model aquatic toxicity

Elisabet Fuguet*,**, Martí Rosés*, Susana Amézqueta*, Alejandro Fernández-Pumarega*,

Sandra Farré*, Laura Muñoz-Pascual*



In this work, the ability of chromatographic systems to model aquatic toxicity has been studied. On one hand, eight animal species usually used in in vivo studies (Tetrahymena Pyriformis (protozoa), Chilomonas Paramecium (protozoa), Streptococcus Sobrinus (), Pseudomonas Putida (bacterium), Daphnia Magna (water flea), Rana tadpoles (tadpoles), Fathead Minnow (fish), and Golden Orfe (fish)) have been considered. On the other hand, five chromatographic systems based on HPLC and MECK techniques were selected after considering the results obtained using four mathematical tools to compare the similarity between chromatographic and biological (toxicological) systems, both characterized through the Abraham solvation parameter model [1]. HPLC systems are based on separation using C18 or immobilized artificial membrane columns; and MECK systems use as surfactants sodium taurocholate, tetradecyltrimethylammonium bromide and a mixture of sodium dodecyl sulphate and polyoxyethylene(23) dodecyl ether (Brij 35). Chemical substances with known aquatic toxicity values to these species have been analysed by the five diferent chromatographic methods. Then the biological property (different non-specific toxicological parameters depending on the aquatic system evaluated) logarithm [2-5] has been correlated to the chromatographic property (retention factor) logarithm. It has been possible to emulate toxicity in the aquatic species Tetrahymena Pyriformis (protozoa), Streptococcus Sobrinus (bacteria), Daphnia Magna (water flea), Rana tadpoles (tadpoles) and Fathead Minnow (fish), through some of the selected chromatographic systems. Those ones showing the best correlation parameters have been successfully validated proving their robustness and prediction ability. Further efforts have to be done to find suitable chromatographic systems to model the toxicity to Chilomonas Paramecium (protozoa), Pseudomonas Putida (bacterium) and Golden Orfe (fish). [1] M. H. Abraham, Chem. Soc. Rev. 22 (1993) 73-83. [2] K. R. Bowen, K. B. Flanagan, W. E. Acree, M. H. Abraham, Sci. Total Environ. 369 (2006) 109–118. [3] K. R. Bowen, K. B. Flanagan, W. E. Acree, M. H. Abraham, C. Ràfols, Sci. Total Environ. 371 (2006)

99–109. [4] K. R. Hoover, K. B. Flanagan, W. E. Acree, M. H. Abraham, Toxicology 6 (2007) 165–174. [5] C. Mintz, W. E. Acree, M. H. Abraham, QSAR Combin. Sci. 25 (2006) 912–920.

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Critical comparison of several methods for lipophilicity determination

Adriana Port, Magda Bordas, Raquel Enrech, Martí Rosés*, Clara Ràfols*, Elisabeth Bosch*, Xavier Subirats*

ESTEVE, Parc Científic de Barcelona, Baldiri Reixac, 4-8, 08028 Barcelona *Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de

Barcelona, Martí i Franquès 1-11, 08028 Barcelona.

Optimal lipophilicity (logPo/w) in pharmaceutical compounds should be addressed in the early phases of drug discovery in order to assure the quality of development candidate compounds. Several methodologies have been considered for lipophilicity determination but their widespread application to all type of compounds is far from reality. In this study, the applicability of fully experimental methods like shake flask (with UV, MS or NMR

1 detection) or potentiometry together with chromatographic measurements

2

have been carefully examinated. The logPo/w of a wide set of representative drugs of different classes (neutral, acidic, basic, amphoteric and zwitterionic compounds) have been evaluated using the mentioned approaches. No significant differences are observed between shake-flask, potentiometric and chromatographic methodologies for compounds with a range of logPo/w from 0.5 to 5. However, careful selection of the methodology is required in case of extreme logPo/w or for amphoteric and zwitterionic compounds, because their peculiar nature can lead to variation among methods. Additionally, it is important to take into account other physicochemical properties, such as solubility, since they can affect in a large extent the experimental lipophilicity values. 1 Mo H, Balko KM, Colby DA, A practical deuterium-free NMR method for the rapid

determination of 1-octanol/water partition coefficients of pharmaceutical agents, Bioorg.Med.Chem. Lett. 20 (2010) 6712-6715.

2 Pallicer JM, Calvet C, Port A, Rosés M, Ràfols C, Bosch E, Extension of the liquid chromatogra-phy/quantitative structure-property relationship method to assess the lipophilicity of neutral, acidic, basic and amphoteric drugs, J. Chromatogr. A 124 (2012) 113-122.

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Medium-throughput determination of lipophilicity and dissociation constant by liquid chromatography–mass spectrometry

Paweł Wiczling, Łukasz Kubik, Wiktoria Struck-Lewicka, Danuta Siluk, Michał Jan Markuszewski, Roman Kaliszan

Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416 Gdańsk, Polanda

Convenient methods for testing drug candidates’ lipophilicity and acidity are highly requested in modern pharmaceutical research and drug development strategies. Reversed-phase high-performance liquid chromatography (RP HPLC) might be particularly useful for the determination of both dissociation constant and the (pH-dependent) partition coefficient related parameters, applicable in high-throughput analysis of multi-component mixtures. The general theory of combined pH/organic modifier gradient has recently provided equations relating gradient retention time and pH of the mobile phase. The purpose of this work was to facilitate the identification of analytes in this technique by its transfer to RP HPLC coupled with time-of-flight mass spectrometry with electrospray ionization source (ESI-TOF-MS). The accuracy of the proposed methodology was assessed by analyzing a set of known drugs. The ammonium formate, ammonium acetate or ammonium bicarbonate buffers were used to control pH during chromatographic analysis. In result, the pKa and lipophilicity parameters were determined and the accuracy of the estimated values was assessed by comparing them with literature data. The gradient RP HPLC coupled with ESI-TOF-MS methods allowed for the rapid determination of dissociation constant and lipophilicity and was shown to be especially applicable for complex mixtures. The use of ESI-TOF-MS detection allowed us to achieve the medium-throughput screening rate (100 compounds/day) and provided a simple approach to assess pharmacokinetically important physicochemical properties of drugs [1] P. Wiczling, W. Struck-Lewicka, L. Kubik, D. Siluk, M.J. Markuszewski, R. Kaliszan, J Pharm Biomed

Anal. 94 (2014)180-7.

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Implementation of Analytical Quality by Design (AQbD) approach to RP-HPLC method for dissolution testing

Maja Bartolec, Josip Pranjić, Senka Radošević, Maja Lusina Kregar

PLIVA Croatia Ltd., Research and Development, Prilaz baruna Filipovića 29, Zagreb, Croatia

The primary objective of our work was to implement Quality by Design (QbD) principles to RP-HPLC method development for the analysis of dissolution samples of immediate release film-coated tablets. Film-coated tablets of interest contain one active pharmaceutical ingredient which is practically insoluble in water, slightly soluble in alcohols and soluble in DMSO and DMF. It can be classified as a BCS class II molecule. Firstly, generic fishbone with Critical Method Variables and Critical Method Attributes was prepared. Mobile phase composition (% ACN in the mobile phase), Column type/brand, Column temperature, Injection volume and Flow rate were identified as Critical Method Variables. Retention time, Tailing Factor, Theoretical Plates Number, SST Recovery, Peak height and %RSD were defined as Critical Method Attributes. The requirements for Tailing Factor, Theoretical Plates Number, SST Recovery, Peak height and %RSD were in line with common method validation specifications. The requirement for Retention time was set to a range between 0.5 - 0.8 minutes in order to have a run time of 1 minute. For Initial Risk Assessment, the estimation of Method Variable risk was made through a semi-quantitative method, using traffic lights color table. In order to evaluate influence of the Critical Method Variables on Critical Method Attributes, Design of Experiment (DoE) study was done. Commercialy available statistical software JMP® was used for selecting DoE, establishing the model, sequence generation and for statistical analysis. Experimentally obtained data was statistically analyzed using least squares fitting method and significant cause and effect relationship was identified. Factors with p-values <0.05 were considered statistically significant. Prediction Profiler platform was used for the simulations based on fitted models. Method Attributes Tailing Factor, SST Recovery and %RSD were not shown to be statistically significant factors. Method Variables Mobile phase composition (% ACN in the mobile phase), Flow rate, Column temperature and Column Type/Brand show a significant effect on Critical Method Attributes Retention time and Theoretical Plates Number. Method Variables Injection volume, Mobile phase composition (% ACN in the mobile phase), Flow rate and Column Type/Brand have statistically significant effect on Critical Method Attribute Height. Based on the DoE study results, updated Risk assessment was prepared.

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Filtration as phase separation technique in saturation shake-flask solubility determination method

Gergely Völgyi, Krisztina Takács-Novák

Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary

Knowledge of the physico-chemical parameters (ionization, solubility, lipophilicity and permeability) of drug molecules is essential in the estimation of their pharmacokinetic properties. Among these parameters, solubility is used to predict the absorption of orally administered drugs from the gastrointestinal tract. Equilibrium solubility of drugs and drug-like molecules is measured by different methods, among them new potentiometric methods. However, the “gold standard” method has remained the classical saturation shake-flask method. The phase separation technique is a key part of the saturation shake-flask method. Various methods (sedimentation, centrifugation or filtration) are used for separation of solid and liquid phases. Sedimentation is considered to be the safest technique for separation of phases. However, solutions sometimes fail to clarify, for example with compounds that form different aggregates or micelles and produce opalescent solutions. For such samples, the separation of phases can only be carried out by centrifugation or filtration. The aim of the present study was to examine the effect of the filtration on solubility. The equilibrium solubility of hydrochlorthiazide, diclofenac sodium and papaverine hydrochloride was determined at different pH values using saturation shake-flask method. Hydrophilic polyvinylidene fluoride and polyether sulfone filters and hydrophobic nylon filters as well as analytical filter papers were chosen for this study. The concentration was measured by UV spectrophotometry. In this poster, a comprehensive, systematic investigation of the filtration, and its influence on equilibrium solubility will be presented.

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Chitosan/xanthan multilayer films as a prospective vehicle for buccal administration

Bissera Pilicheva, Yordanka Uzunova, Ivan Bodurov*, Ivanka Vlaeva**, Asya Viraneva*, Ginka Exner*, Sotir Sotirov**, Tsenka Grancharova*, Maria Marudova*,

Temenuzhka Yovcheva*

Medical University Plovdiv, Faculty of Pharmacy, 15A Vassil Aprilov blvd., 4002 Plovdiv, Bulgaria, [email protected]

* Plovdiv University “Paisii Hilendarski”, Faculty of Physics, Department of Experimental Physics, 24 Tsar Asen str., 4000 Plovdiv, Bulgaria

** University of Food Technologies, Department of Mathematics and Physics, 26 Maritsa blvd., 4002 Plovdiv, Bulgaria

The aim of the present study was to evaluate chitosan/xanthan multilayer films as a potential drug delivery system for buccal administation. The multilayer films were prepared by alternative dipping of corona pretreated poly-ε-caprolactone substrate into 0.1% chitosan solution and 0.05% xanthan solution in acetate buffer for 15 min. After each dipping process the samples were rinsed in the same acetate buffer for 5 min. The procedure was carried out until 8, 14 or 20 xanthan/chitosan or chitosan/xanthan layers were deposited. After that the multilayer structures were dried at room temperature for 48 h and stored in desiccator at RH 54%. The films were evaluated for chitosan/xanthan interactions, surface pH, percentage moisture absorption and moisture loss, swelling behaviour and mucin adsorption capability. FTIR-ATR spectra of the fims did not show specific peaks

corresponding to asymmetric and symmetric vibrations of NH3+ (at 1632 and 1522 cm

-1) of

chitosan and ionized carboxyl group (COO-) of xanthan at 1620 cm

-1 which proved the

formation of complex between two polymers.

The film surface pH was measured to determine the risk of buccal mucosa irritancy due to acidity or alkalinity. The surface pH of all prepared films was found in the neutral pH range indicating high tolerance. The percentage swelling of the films ranged between 38.5 % to 113.3 % which confirmed big relevance for buccal administration forshadowing high drug diffusion rate and preventing excessive polymer erosion. The adsorption of mucin on film surface was used as a method to assess mucoadhesion by the Bradford colorimetric assay. According to the results obtained the multilayer films showed good mucoadhesive properties and could therefore be used as a promising drug delivery system for buccal administration.

Acknowledgements: This study was funded by Bulgarian National Scientific Fund, Project No DFNI B-02/7.

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Permeation of betamethasone esters through excised pig skin

Tina Jarc, Katja Berginc*, Mateja Erdani Kreft, Katja Kristan*

Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia

*Sandoz Development Center, Lek Pharmaceuticals, d.d, Verovškova 57, 1526 Ljubljana, Slovenia

Since their introduction to dermatology, topical corticosteroids are used for treatment of various dermatoses, mainly due to their immunosuppressive, anti-inflammatory and antiproliferative properties. Although their use is limited by the occurrence of undesirable side effects; glucocorticoids depress the local defence mechanisms and induce immunodeficiency [1,2]. The in vitro study of skin permeability plays an essential role in the development of transdermal dosage forms. Prior permeability assays, skin integrity was checked in fresh pig ear skin samples and samples after transportation, after dermatomization and after longer storage at -20°C. Betamethasone (BM) and four betamethasone esters (BM-21-acetate, BM-17-valerate, BM-21-valerate, BM-17,21-dipropionate) with different lipophilic proprieties were used to perform the in vitro experiments by using static Franz diffusion cells and excised pig ear skin as a membrane. Franz cells were filled with 30% (v/v) isopropanol in water as acceptor medium and equilibrated to 32°C. Betamethasone and different esters, dissolved in oleyl alcohol (0.1 %), were applied on the skin surface in the donor compartment. The medium in acceptor compartment was stirred at 710 rpm. Sample volumes (0.2 ml) were taken for UPLC analysis up to 4 days and fresh preheated medium replacements of the same volume were reintroduced into the acceptor compartment. The flux through skin was calculated for all tested compounds. The results revealed no morphological damage during skin sample removal. The skin ultrastructure remained the same and with conserved desmosome junctions between the cells before and after transportation. Dermatomization influenced the detachment of stratum corneum on some areas. Freezing and thawing did not damage stratum corneum and stratum granulosum, however the differences were observed in stratum spinosum and stratum basale. Drug permeability tests have shown that out of five different compounds, only BM-17,21-dipropionate permeated pig skin with flux value of 2.2 × 10

4

mg/(cm2 × h

1/2). BM-17,21-dipropionate has the highest logP value (4.07) and is the most

lipophilic compound. Thus, the lipophilicity of a drug is an important factor for prediction of the skin permeability. Reference [1] L. Uva, D. Miguel, C. Pinheiro, J. Antunes, D. Cruz, J. Ferreira, P. Filipe, Mechanisms of Action of

Topical Corticosteroids in Psoriasis, Int. J. Endocrinol. 2012 (2012) 1-16. [2] W. Raab, Effects of local corticosteroids in skin infections, Dermatologica 152 (1976) 67-79.

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Introduction of Golem v2, a new model for biorelevant dissolution studies

Ivan Stupák, Lucie Gruberová*, Jakub Vysloužil, Jiří Dohnal, Martin Čulen

Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1-3, Brno 612 42, Czech Republic

*Faculty of Chemical Technology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague, Czech Republic

Generic drug products represent a dominant portion of the pharmaceutical market. This results in a considerable interest in the topics of generic formulation development and bioequivalence studies. In this respect, our team focuses on innovative in vitro dissolution studies employing a dynamic biorelevant dissolution instrument – Golem; developed for physiologically relevant simulation of drug dissolution process occurring in human stomach and small intestine [1,2]. Recently, we have introduced new improvements to the instrumental design of Golem, developed in the course of our continuous research. Here, we present the modifications incorporated in Golem v2 and the initial optimization assays. The in vitro performance of new compartment design and peristaltics simulation was assessed using an immediate release drug formulation and compared with USP 2 dissolution. Reference: [1] Culen M., Rezacova A., Jampilek J., Dohnal J. Designing a dynamic dissolution method: a review of

instrumental options and corresponding physiology of stomach and small intestine. Journal of Pharmaceutical Sciences. 2013;102(9):2995–3017. doi: 10.1002/jps.23494.

[2] Culen M, Tuszyński PK, Polak S, Jachowicz R, Mendyk A, Dohnal J. Development of In Vitro-In Vivo Correlation/Relationship Modeling Approaches for Immediate Release Formulations Using Compartmental Dynamic Dissolution Data from “Golem”: A Novel Apparatus. BioMed Research International. 2015;2015:328628. doi:10.1155/2015/328628.

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Furosemide solvates: can they serve as precursors to different polymorphs?

A. A. Beloborodova*,**; V. S. Minkov*

,**, V. A. Drebushcak*

,***, E. V. Boldyreva*

,**

*Novosibirsk State University, Russia **Institute of Solid State Chemistry and Mechanochemistry SB RAS, Russia

***Institute of Geology and Mineralogy SB RAS, Russia

The importance of polymorphism of molecular crystals is hard to overestimate, especially when dealing with compounds used as materials or drugs. Different polymorphs of a drug substance may have different properties related to their manufacturing, therapeutic usage, or storage (density, hygroscopicity, melting point, thermal stability, solubility, dissolution rate, surface free energy, toxicity, bioavailability, tableting, etc.). Different polymorphs, solvates, and co-crystals can be patented, and this opens the way for a competition with brand drugs. Since the energies of different polymorphs are sometimes very close, producing desirable crystalline forms is challenge and can also be complicated by the phenomena of concomitant polymorphism (when several polymorphs crystallize simultaneously from the same batch), or erratic and poor reproducibility (when crystallization gives different polymorphs even at seemingly identical experimental conditions). The aim of the present study was to crystallize various solvates of furosemide, to check whether these solvates can be used as precursors for producing different polymorphs of pure furosemide on their subsequent decomposition upon heating, and to search any correlation between the crystal structure of the solvates and on the furosemide polymorphs produced by desolvation. In this work several solvates of furosemide with tetrahydrofuran, dioxane, dimethylformamide, and dimethylsulfoxide were crystallised. The detailed structural analysis of furosemide-containing crystal structures showed that the molecule of furosemide had a high conformational lability because of the rotations of the sulfamoyl and furanylmethylamino fragments. Some of the furosemide conformations were shown to be stabilized by the intermolecular N-H…Cl hydrogen bond. Desolvation of the solvates was studied by TG and X-ray diffraction and was shown to give different products depending on the precursor and particle size.

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Mannitol as co-milling agent for the preparation of theophyline inhalable nanocrystal agglomerates

Maria Malamatari, S. Somavarapu, M. Bloxham*, G. Buckton

UCL School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UK *GSK Medicines Research Centre, Gunnels Wood Road, Stevenage,

Hertfordshire, SG1 2NY, UK

Purpose: To produce inhalable nanocrystal agglomerates of theophylline which is a drug with needle-like morphology and forms hydrates in water, by applying wet bead milling with mannitol in isopropanol followed by spray drying. Methods: Wet bead milling of theophylline in isopropanol was applied with increasing amount of mannitol and the obtained nanosuspensions were solidified by spray drying. The effects of mannitol on micromeritic properties, solid state, dissolution, redispersibility, flow and aerosolisation performance of the engineered agglomerates were investigated. Particle size was determined by laser diffraction and morphology by scanning electron microscopy (SEM) with further analysis of SEM images by the ImageJ software. Specific surface area was measured using a BET surface area analyser with nitrogen as the adsorbate. Solid state was assessed by X-ray powder diffraction (XRPD) and flowability was determined using density measurements under tapping and uniaxial compression. In vitro aerosolisation performance was investigated using the next generation impactor (NGI) and Aeroliser

® inhaler device, at a volumetric flow rate of 60 L min

-1.

Results Agglomerates of 4.64 ± 0.87 μm and 2.18 ± 0.30 μm mean particle size of theophylline alone and theophylline: mannitol 25:75 mass ratio mixtures, respectively, were produced. SEM images revealed that spray dried suspension of theophylline alone consisted of elongated micron-sized crystals while those of theophylline and mannitol mixtures consisted of theophylline nanocrystals embedded in porous microcomposites. XRPD showed that theophylline retained the crystalline state of the raw material (anhydrous form I) in all formulations. The inclusion of mannitol at 25:75 mass ratio resulted in formulations which exhibited a 2.18-fold increase in the fine particle fraction indicating enhanced aerosolisation performance comparatively to the formulation without mannitol, which can be attributed to their smaller size, more spherical shape and increased specific surface area and porosity. Conclusion: Wet bead milling in organic solvents and in the presence of mannitol followed by spray drying can be used as a platform for the production of respirable particles of (moderately) water-soluble drugs. Maintenance of the crystalline state makes this approach preferable to micronisation by dry milling techniques, which is associated with limitations as drug amorphisation and performance variability.

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The composites of betulin and its diacyles with improved biological activity

Tatyana P. Shakhtshneider, Mikhail A. Mikhailenko, Ilia V. Eltsov, Vladimir V. Boldyrev, Elena V. Boldyreva, Svetlana A. Kuznetsova*, Yury N. Malyar*, Anna S. Zamay*,

Alexander S. Kozlov**

Institute of Solid State Chemistry and Mechanochemistry SB RAS, Novosibirsk, Russia, Novosibirsk State University, Russia

*Institute of Chemistry and Chemical Technologies SB RAS, Krasnoyarsk, Russia, Siberian

Federal University, Krasnoyarsk, Russia **Institute of Chemical Kinetics and Combustion SB RAS, Novosibirsk, Russia

The purpose of this work was to obtain the composites of betulin and its esters, betulin diacetate (BDA) and betulin dipropionate (BDP), with improved dissolution properties and study their structure and pharmacological activity. Betulin and its esters were obtained directly from birch bark according to the original methods [1, 2]. Arabinogalactan was isolated from Siberian larch wood [3]. The composites of betulin, BDA, and BDP with water-soluble polymers, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and arabinogalactan (AG), were prepared by ball-milling the mixtures of the components in a SPEX 8000 mixer mill (CertiPrep Corp., USA). In the case of the mechanocomposites, the solubility of betulin and its derivatives increased as compared to the initial substances. The obtained composites were non-toxic and exhibited antitumor properties against the ascites adenocarcinoma Ehrlich cells. The composites of betulin esters with AG were prepared also as amorphous thin films readily soluble in water. In vitro studies revealed that these composites exhibited the highest antitumor activity against lung adenocarcinoma in comparison with the initial substances and their mixtures with AG. For better understanding the structure of the complexes in solution, supramolecular ensembles of AG and its complexes with BDA in water were studied using ablation induced by submillimeter radiation from the Novosibirsk Free Electron Laser. It was shown that the ensembles of molecules were larger in the solution of the AG – BDA complex than in pure AG aqueous solution, and the distribution was narrow. This was likely due to BDA incorporation into AG side chains and the consequent decrease in their mobility. The role of side chain interactions in the formation of AG-BDA complexes in aqueous solutions was confirmed by NMR. The work was partly supported by the Russian Foundation for Basic Research (project No. 14-03-31900) and by the Council for Grants of the President of RF for Support of Leading Scientific Schools (project no. NSh-279.2014.3). References [1] Kuznetsova S.A., Kuznetsov B.N., et al., R.F. Patent 2324700, May 20, 2008. [2] Kuznetsova S.A., Skvortsova G.P., et al., R.F. Patent 2469043, December 10, 2012 [3] Kuznetsova S.A., Kuznetsov B.N., et al., R.F. Patent 2273646, April 6, 2006.

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Novel synthons in sulfamethizole cocrystals: Structure−property relations and solubility

Kuthuru Suresh, Vasily S. Minkov*, Kranthi Kumar Namila, Elizaveta Derevyannikova*, Evgeniy Losev*, Ashwini Nangia, Elena V. Boldyreva*

School of Chemistry, University of Hyderabad, India *Novosibirsk State University, Russia and

Institute of Solid State Chemistry and Mechanochemistry SB RAS, Russia

In the pharmaceutical industry, it is the poor biopharmaceutical properties rather than toxicity or lack of efficacy that are the main reasons why less than 1% of active pharmaceutical compounds eventually appear into the marketplace. Improving the solubility of drugs is currently one of the main challenges for the pharmaceutical industry. Many approaches have been adopted for improving the aqueous solubility of drugs including micronisation, salt formation, emulsification, solubilisations using co-solvents, and the use of polymer drug vehicles for delivery of poorly soluble drugs. Although these techniques have been shown to be effective at enhancing oral bioavailability, success of these approaches is dependent on the specific physicochemical nature of the molecules being studied. Over the last decade, there has been growing interests in the design of pharma-ceutical cocrystals, which emerges as a potential method for enhancing the bioavailability of drugs with low aqueous solubility. Apart from offering potential improvements in solubility, dissolution rate, bioavailability and physical stability, pharmaceutical cocrystals can enhance other essential properties of the APIs such as flowability, chemical stability, compressability and hygroscopicity. Sulfamethizole (SMT) is a sulfonamide class antibiotic that acts through the competitive inhibition of folate synthesis in microorganisms. Most sulfonamide class drugs exhibit moderate to high solubility, but bioavailability is limited due to fast elimination half-life. As a result the dose strength of sulfonamide drugs has to be higher. SMT has good solubility (1.05 g/L at 37 °C in water), but it has a short half-life (2.1 h) due to rapid metabolism and systematic elimination. Our goal in this study was to optimize pharmaceutical cocrystals of SMT and control solubility of the drug. The SMT has rich hydrogen bond functionalities (donors: amine NH2 and imine NH; acceptors: sulfonyl O, thiazolidine N and S, and imidine N), which makes it a functionally diverse molecule to form cocrystals. A cocrystal screen of SMT with COOH, NH2, pyridine, and CONH2 functional group containing coformers, e.g., p-aminobenzoic acid (PABA), vanillic acid (VLA), p-aminobenzamide (ABA), 4,4-bipyridine (BIP), suberic acid (SBA), oxalic acid (OA), and adipic acid (ADP), resulted in six cocrystals and one salt, namely, SMT−ADP (1:0.5), SMT−PABA (1:1), SMT−VLA (1:1), SMT−ABA (1:1), SMT−BIP (1:1), SMT−SBA (1:0.5), and SMT−OA (1:1). The novel crystalline adducts were synthesized by liquid-assisted cogrinding and isothermal solvent crystallization. In addition to single-crystal X-ray diffraction, the phase composition of the powder samples was confirmed by powder X-ray diffraction and DSC. Hydrogen bonding interactions between the coformers and SMT are analyzed as six different synthons. In addition to strong N−H∙∙∙O and O−H∙∙∙N hydrogen bonds, the cocrystal structures are sustained by weak C−H∙∙∙O hydrogen bonds. The not so common chalcogen−cha-lcogen (S∙∙∙O) type II intermolecular interaction in SMT−ADP cocrystal and chalcogen−nicogen (S∙∙∙N) type II interaction in SMT−BIP cocrystal were observed. The products were characterized by vibrati-onal spectroscopy to obtain information on the strengths of the intermolecular interactions. Solubi-lity and dissolution experiments on SMT−ADP, SMT−SBA, and SMT−OA showed a lower intrinsic dis-solution rate (IDR) and equilibrium solubility compared to SMT in 0.1 N HCl medium, which is ascri-bed to stronger N−H∙∙∙O, N−H∙∙∙N, and O−H∙∙∙O hydrogen bonds and better crystal packing. The de-creased IDR could be useful in controlled/extended release of SMT to improve therapeutic activity of the drug by minimizing its fast systemic elimination in vivo. Furthermore, we observed that SMT−OA salt is formed spontaneously when the components were mixed in acidic medium (0.1 N HCl), whereas in neutral medium (phosphate buffer) no SMT−OA salt formation was observed.

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Amorphous protic ionic systems as promising active pharmaceutical ingredients:

The case of sumatriptan drug

Z. Wojnarowska, J. Cielecka-Piontek*, M. Paluch

Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland and SMCEBI, 75 Pulku Piechoty 1A, 41-500 Chorzow, Poland

*Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland

In this presentation we highlight the benefits coming from the application of amorphous protic ionic systems as active pharmaceutical ingredients (APIs). Using the case of sumatriptan (STR) drug we show that the conversion of non-ionic API to partially ionized amorphous succinate salt (STR SUCC) brings a substantial improvement in apparent solubility. Since in general the disordered systems reveal a tendency to self-arrangement during storage the dominant part of this paper is dedicated to the physical stability issue of sumatriptan and its ionic counterpart. To recognize the crystallization tendency of the studied systems the molecular/ions mobility, usually considered as the most critical factor governing the spontaneous nucleation process from the amorphous and supercooled state, is discussed. Analysis of dielectric response of ionic and non-ionic sumatriptan complemented by calorimetric measurements reveals many similarities in relaxation dynamics and thermal properties of these APIs as well as distinct differences in their resistance against crystallization in the supercooled liquid state. To determine the long-term physical stability of these materials at room temperature conditions the time scale of structural relaxation below their glass transition temperatures are estimated. We show that in contrast to non-ionic materials τα predictions of STR SUCC are much more complex and require aging experiments.

Acknowledgements:The authors Z.W. and M.P. are deeply grateful for the financial support by the National Science Centre within the framework of the Opus3 project (Grant No. DEC-2012/05/B/NZ7/03233).

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Measuring and modelling pH-dependence of passive transport in artificial membrane for wide range of pH

Mare Oja, Uko Maran

Institute of Chemistry, University of Tartu, Estonia Human intestinal absorption is one of the vital properties for orally administrated drugs. Human gastrointestinal tract is very diverse system with wide range of pH. Transport through intestinal epithelium can be active or/and passive; it is estimated that around 90% of drugs are transported passively. Passive transport can be described using parallel artificial membrane permeability assay (PAMPA). PAMPA method is robust and can be easily modified, which makes this method useful for describing regions corresponding to different pH-s in human gastrointestinal tract. In the early stage of drug discovery process, very often interesting active compound is not yet available (not synthesised), which means that compound should be described without experimental measurements. For this in silico prediction models serve good alternative. So far all prediction models for membrane permeability are developed only for neutral or near to neutral pH, while pH in gastrointestinal tract ranges approximately from 3 to 9. The purpose of current research is to consider influence of pH to the membrane permeability and take this into account in QSAR models. For this PAMPA experiments were performed for four pH-s (pH 3, 5, 7.4 and 9) over 48 hour. PAMPA values were measured for ~170 compounds, in total around 680 datapoints. Measured compounds were structurally diverse and belong to different chemical classes. The experimental values were analysed and used for in silico modelling resulting in two different types of QSAR models: (i) models corresponding to one pH; (ii) model for highest membrane permeability values over four pH. Two-parameters QSAR models were developed using stepwise forward selection of molecular descriptors. The results shows that membrane permeability is highly depend on molecule ionization state. Obtained QSAR models for certain pH are statistically significant and able to predict membrane permeability at that pH. But more precise and universal model was developed using only highest membrane permeability values over four pH. Additionally, also intrinsic membrane permeability values [1] were used to develop prediction model for membrane permeability of uncharged species and the results were compared. In conclusion, considering different pH-s in modelling of biochemical properties gives valuable information on behaviour of chemicals and improves the prediction quality over uncharged species. Acknowledgement: Estonian Ministry of Education and Research (grant IUT34-14) [1] A. Avdeef, Absorption and Drug Development: Solubility, Permeability, and Charge State, second

ed., John Wiley & Sons, Inc., Hoboken, New Jersey, 2012.

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Exploring and predicting in QsarDB repository: membrane permeability QSAR model over gastrointestinal tract pH-range

Mare Oja, Sulev Sild, Uko Maran

Institute of Chemistry, University of Tartu, Estonia Computational pharmacology tools on the Internet are gaining momentum. These tools relay on consolidated and abstracted information in the form of computational models. This presentation gives an example of use of quantitative structure-activity relationship (QSAR) in open internet environment where user can access, explore and use models and data for the model development and validation. As concrete example maximum membrane permeability QSAR model mimicking human intestinal absorption uploaded to QsarDB repository (qsardb.org) is used. Human intestinal absorption is important property for the orally administrated drugs. Direct measurements methods for the human intestinal absorption are expensive and complicated. Therefore several indirect methods are developed for estimating human absorption, like the parallel artificial membrane permeability assay (PAMPA). 90% of drugs are transported passively making PAMPA measurements important source of information in the early stage of drug discovery. Experimental measurements however expect that the new drug candidates are already synthesised or one needs an estimate in order to select correct conditions for the experimental measurements. For this in silico methods, like QSAR, serve as good alternative. The current example is based on the original PAMPA experimental values describing the full pH-range (3, 5, 7.4 and 9) in gastrointestinal tract [1]. For the modelling only the highest PAMPA values for each compound were selected. Developed model includes two descriptors, the logarithmic octanol-water partition coefficient and the hydrogen bonding surface area, and both have concrete mechanistic explanation for processes occurring during the transport through membrane. Model is published [1] and uploaded to the QsarDB repository [2] for citing (archive DOI) and interactive access. QsarDB includes several applications for property, descriptor and applicability domain analyses. Additionally, QsarDB repository includes prediction capabilities. In summary, QsarDB gives excellent opportunity to integrate derived knowledge and computational pharmacology tools with internet resources via making developed models accessible for drug discovery tasks. Acknowledgement: European Union, Regional Development Fund (3.2.1201.13-0021) [1] M. Oja, U. Maran, The permeability of an artificial membrane for wide range of pH in human

gastrointestinal tract: experimental measurements and quantitative structure-activity relationship, Mol. Inf. 34 (2015) 493-506.

[2] M. Oja, U. Maran, QDB archive #137. QsarDB repository, 2015. http://dx.doi.org/10.15152/QDB.137

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Exponential repulsion for molecular docking

Václav Bazgier, Karel Berka*, Michal Otyepka*, Pavel Banáš*

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc, 17. listopadu 12, 771

46 Olomouc, Czech Republic *Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc, 17. listopadu 12, 771 46

Olomouc, Czech Republic

Molecular docking is a powerful tool for theoretical prediction of the preferred conformation and orientation of small molecules within protein active sites. The obtained poses can be used for estimation of binding energies, which indicate the inhibition effect of designed inhibitors, and therefore might be used for in silico drug design. However, the evaluation of ligand binding affinity critically depends on successful prediction of the native binding mode of the ligand in the target site. Contemporary docking methods are often based on scoring functions derived from molecular mechanical potentials. In such potentials, non-bonded interactions are typically represented by electrostatic interactions between atom-centered partial charges and a standard 6-12 Lennard-Jones potential. Here, we present implementation and testing of a scoring function in DOCK 6.5 based on more physically justified exponential repulsion with damped dispersion instead of the Lennard-Jones potentials in either standard 6-12, scaled 6-12 or 6-9 forms. In addition, we applied different mixing rules of van der Waals pair parameters and tested their impact on the predictability of the docking algorithm. Overall, we found that the scoring function based on exponential repulsion coupled with more appropriate arithmetic mixing rules significantly improved prediction of the native binding modes of 15 CDK2 inhibitors as well as in other 1058 protein-ligand complexes from Rizzo’s SB2010 dataset [1]. Acknowledgement: This work was supported by projects CR NPUI LO1204 (V.B.) and LO1305 (P.B., K.B., M.O.) of the Ministry of Education, Youth and Sports, and by student project IGA_PrF_2015_027 and IGA_PrF_2015_021 of Palacký University. Reference [1] Mukherjee, Sudipto, Trent E. Balius, and Robert C. Rizzo. "Docking validation resources: protein

family and ligand flexibility experiments." Journal of chemical information and modeling 50.11 (2010): 1986-2000.

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Human serum albumin binding of certain antimalarials

Olivera S. Marković, Ilija N. Cvijetić*, Mario V. Zlatović, Igor M. Opsenica, Nataša V. Terzić-Jovanović**, Bogdan A. Šolaja, Tatjana Ž. Verbić

Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Serbia *Innovation Center of the Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Serbia

Department of Chemistry-IChTM, University of Belgrade, Njegoševa 12,Serbia. [email protected]

Tested compounds, previously synthesized, are derivatives of chloroquine, drug commonly used in the treatment and prevention of malaria. Human serum albumin (HSA) has the role in transport of endogenous (fatty acids, hormones, bile acids, amino acids) and exogenous compounds (drug molecules and nutrients). Interaction between tested compounds and HSA has been studied by fluorescence spectroscopy in phosphate buffered saline (1× PBS, pH 7.4) [1]. Results show that among tested compounds, all positively charged at pH 7.4, derivatives with thiophene substructure bind to HSA. Molecular docking studies were used to determine HSA–compound binding mode.

Figure 1. Stern-Volmer plots for binding of a) 1 (1-16 mol. eq.), b) 2 (1-15 mol. eq.), and

c) 3 (1-15 mol. eq.) to HSA (c=5×10-7

M); 1× PBS, pH = 7.4

Fluorescence quenching data were processed using Stern-Volmer (S-V) equation [2]. Almost linear S-V plot for binding of 1 to HSA (Fig. 1a) indicates single type of quenching mechanism.

Results show that Ksv decreases (20C: (2.60±0.07)×105 M-1; 25C: (2.33±0.07)×105 M-1 and

37C: (2.18±0.08)×105 M-1) as temperature increases indicating static quenching mechanism. Downward curvature in S-V plots of 2 and 3 (Fig. 1b and 1c) indicates that tryptophan residues are not fully accessible to the drug and that dynamic quenching dominates over static. Fraction of tryptophan residues that are buried and inaccessible to the quencher and effective quenching constants can be determined by modified S-V equation. The effective quenching constant for 2 and 3 increases as temperature increases, this is another indication that dynamic quenching process is dominant in binding of 2 and 3 to HSA. Effective quenching constants of all three compounds are in the order of 105 M-1, meaning that these compounds can be effectively carried and stored by HSA in the human body.

Acknowledgement: Ministry of Education, Science, and Technological Development of Serbia (Grant No. 172008) and FP7 RegPot project FCUB ERA GA (No. 256716) supported this work.

References: [1] R. Punith, A. Hegde, S. Jaldappagari, J. Fluoresc. 21 (2011) 487-495. [2] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3

rd ed., Springer Science Business Media,

New York, USA, 2006.

0 1 2 3 4 5 6 7 8 9

1.2

1.6

2.0

2.4

2.8 293K

298K

310K

F0/F

[Q]10-6 (M)

a)

0 1 2 3 4 5 6 7 8

1.2

1.6

2.0

2.4

2.8 293K

298K

310K

F0/F

[Q]10-6 (M)

b)

0 1 2 3 4 5 6 7 8

1.2

1.4

1.6

1.8

2.0

2.2 293K

298K

310KF

0/F

[Q]10-6 (M)

c)

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Physico-chemical analysis of new synthetic derivatives from triterpenoid class

Adriana Fuliaş, Ionuţ Ledeţi, Gabriela Vlase*, Lenuţa-Maria Şuta, Titus Vlase*

University of Medicine and Pharmacy “Victor Babeş”, Faculty of Pharmacy, Eftimie Murgu Square 2, Timişoara, RO-300041, Romania

*West University of Timisoara, Research Centre for Thermal Analysis in Environmental Problems, Pestalozzi Street 16, Timişoara, RO-300115, Romania

Two new derivatives, C1 and C2 were obtained and characterized. The spectra of these triterpenic substances are very similar. For all compounds, the ϑas(COO) vibration is identified as the strong band in the range 1712–1690 cm

-1. The band assigned to the

vibration of ϑs(COO) is observed in the range 1460–1385 cm-1

. In the case of C1 and C2, the symmetrical vibrations for carboxyl group are split into two peaks. In the IR spectra of these compounds, there are also vibrations of betulonic acid.

C1, where R=-N(H)-C(NH2)=NH C2, where R=-OH

The characteristic band attributed to C=N stretching vibration are found at 1642 cm

-1 for

all three compounds in the region mentioned in literature (between 1610 and 1665 cm-1

) [1]. For the C2 compound, the functionalized group of oxime (C=N-OH) are represented by the vibrations placed at wavenumber: ϑ= 3300 cm

-1 for O-H stretch; 1642 cm

-1 for C=N

stretch and 945 cm-1

for N-O, respectively. [2] Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babeş” Timişoara (Grant PIII-C1-CFI-2014/2015-03 to A.F., I.L. and L.-M.S.). 1. L. D. Frederickson, An Infrared Study of the C=N Stretching Vibration in Azine Derivatives of

Aldehydes and Ketones. Anal. Chem. 36 (1964) 1349–1355. 2. Z. A. Saveleva, G. V. Romanenko, L. A. Shelud-yakova, Stanislav V, Larionov. Synthesis and

structure of a complex with the coordinated triaminoguanidinium(+2) ion,[Cu(TAGH2)Cl3]Cl∙H2O. Polyhedron. 17 (2000) 1737–40.

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Comparative thermal stability of statins

Ionuţ Ledeţi, Gabriela Vlase, Titus Vlase, Lenuţa-Maria Şuta*, Ionel Ciucanu, Adriana Fuliaş*

West University of Timisoara, Research Centre for Thermal Analysis in Environmental Problems, Pestalozzi Street 16, Timişoara, RO-300115, Romania

*University of Medicine and Pharmacy “Victor Babeş”, Faculty of Pharmacy, Eftimie Murgu Square 2, Timişoara, RO-300041, Romania

This study describes the results obtained for investigation by instrumental hyphenated techniques the thermal stability of three similar-structured antihyperlypidemic drugs – namely lovastatin (LOVA), pravastatin (PRAV) and simvastatin (SIMV), by employment of methods based on classical-Arrhenius kinetic study of degradation in solid state.

a) b) c) Fig.1. Structures of analysed compounds: a) LOVA; b) PRAV and c) SIMV

The obtained results obatiend by Kissinger, Coats-Redfern and Li-Tang were compared with the ones obtained by the employment of kinetic methods recommended by ICTAC 2000 protocol. Comments over the differences observed between classical kinetic methods and isoconversional were carried out, suggesting that the decomposition mechanism consist in parallel steps. Acknowledgement: This work was performed at West University of Timişoara and was supported by the strategic grant POSDRU/159/1.5/S/137750, Project “Doctoral and Postdoctoral programs support for increased competitiveness in Exact Sciences research” co-financed by the European Social Fund within the Sectoral Operational Programme Human Resources Development 2007-2013” to Ionuţ Ledeţi.

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Employment of hyphenated techniques for analysis of betulin-type compounds

Ionuţ Ledeţi, Gabriela Vlase*, Lenuţa-Maria Şuta, Titus Vlase*, Adriana Fuliaş

University of Medicine and Pharmacy “Victor Babeş”, Faculty of Pharmacy, Eftimie Murgu Square 2, Timişoara, RO-300041, Romania

*West University of Timisoara, Research Centre for Thermal Analysis in Environmental Problems, Pestalozzi Street 16, Timişoara, RO-300115, Romania

The thermal degradation of two triterpenic substances in air atmosphere was monitored by the analysis of evolving gaseous species from the triterpenoid derivatives using coupled FTIR spectrometric gas cell as detector connected to the furnace of thermal balance. The components of released gaseous mixtures have been monitored and identified on the basis of their FTIR spectra using Gram-Schmidt profile and thermogravimetric curves recorded at a heating rate β=20°C∙min

-1. As studied compounds, natural occurring

triterpenes and semi-synthetic compounds with betulin-type skeleton were used. In each case, a discussion regarding the probabilistic mechanism of decomposition was realised. The EGA (evolved gas analysis) study was a fast and reproducible tool which allowed a clear interpretation over the thermal behaviour of samples – according to this, the compounds were separated into two main classes: compound which undergo sublimation and compounds that undergo thermal oxidation. Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babeş” Timişoara (Grant PIII-C1-CFI-2014/2015-03 to I.L., L.-M.S. and A.F.).

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Spectroscopic characterisation of newly synthesised bile acid derivatives

Srđan Bjedov, Dušan Škorić, Marija Sakač, Mihalj Poša*, Janoš Čanadi

Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg D. Obradovića 3, Novi Sad, Serbia,

*Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Hajduk Veljka 3, 21000 Novi Sad, Serbia

Besides the bile acids already known capability of transporting drugs by acting as micellization and liposolubilization agents [1], recently it was discovered that they can also act as hormones [2]. Therefore, chemical transformations of bile acids are very important for modern pharmacochemistry.

Bile acid salts are amphiphilic steroid molecules, able to form micelles that can bind and transport lipophilic drugs. Their ability to bind drugs depends on bile acid hydrophobicity. Above critical micellar concentrations bile acid salts damage the cell membrane. Oxo derivatives of bile acids have decreased membranotoxicity, but their solubilisation capacity is also lowered. Our approach to the modification of hydrophobicity is introducing different side chains at the bile acid skeleton. However, it can be challenging to determine the structure of newly synthesized bile acid derivatives, since they frequently won’t produce crystals suitable for crystallographic methods. Additionally, NMR spectra are crowded with the signals of diastereotopic CH2 hydrogens. Herein, we want to report synthesis of compounds 1 and 2 starting from cholic acid and detailly present the analysis of their FTIR and NMR spectra in structure elucidation. Acknowledgments: Financial support by the AP of Vojvodina Republic of Serbia 114-451-1023/2015 for conducting the research is gratefully acknowledged. [1] I. Kuhajda, M. Poša, V. Jakovljević, V. Ivetić, M. Mikov, Effect of 12-monoketocholic acid on

modulation of analgesic action of morphine and tramadol, Eur. J. Drug. Metab. Pharmacokinet., 34 (2009), 73-8.

[2] S. Fiorucci, A. Mencarelli, G. Palladino, S. Cipriani, Bile-acid-activated receptors: targeting TGR5 and farnesoid-X-receptor in lipid and glucose disorders, Trends Pharmacol. Sci. 30 (2009) 570–80.

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A Quality by Design (QbD) based automated analytical method development for determination of impurities in new pharmaceutical drug product

Ana Georgieva, Irena Brašnarska, Sonja Ugarkovič

Institute for Research and Development, Alkaloid AD-Skopje, Blv. Aleksandar Makedonski 12, 1000 Skopje, Macedonia

Analytical methods are commonly developed using a one-factor-at-a-time (OFAT) approach in which one parameter alone is optimized for the expected response whilst others remained constant. Because of this common practice analytical method development can be a time-consuming process that often provides a limited understanding of method capabilities and method robustness. On the other hand, regulatory authorities FDA and ICH are encouraging and promoting implementation of Quality by Design (QbD) principles in the pharmaceutical industry environment. A Quality by Design (QbD) approach to analytical method development refers to a software-driven method development. These softwares are using statistical design of experiments (DoE) to develop a robust method ‘design space’. The design space defines an experimental region in which changes to method parameters will not significantly affect the results. A key benefit of defining a design space is that working within this space is not considered as a change, and therefore would not initiate a regulatory post approval change process. QbD approach/concept for development of a high pressure reversed phase liquid chromatography (HPLC) method for determination of impurities in a drug product that contains two active ingredients was performed. Fusion AE Method Development software that is QbD based LC method development software with built-in robustness metrics was used. The study was carried out on an Agilent 1290 Infinity Automated Method Development system equipped with a column and solvent selection valves that allow automated exploration and screening of a wide range of conditions that are common critical parameters in HPLC (different columns, pH ranges, and organic modifiers). In conjunction with ChemStation, Fusion AE manages complex statistics, builds experiments, analyzes data and presents results as visual and numeric method predictions.

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Rapid and green chemistry catalysis in synthesis of hexahydroquinazolinones and cyclopenta, cyclohepta[d]pyrimidinones with prediction of biological

activity via PASS INET

Ahmed M. M. El-Saghier, Eman A. Ahmed, Ahmed M. A. Mahmoud

Chemistry Department, Faculty of Science, Sohag University, Sohag, 82524, Egypt, [email protected]

Ceric Ammonium Nitrate (CAN), turns out to be commercially available, efficient and mild catalyst for synthesis of polyhydro-2H-cycloalka[d]pyrimidinones and hexahydroquin-azolinones in few minutes. Accompanied with Predictions of the activity spectra of some selected compounds using PASS INET. at Pa > 50%, showing high probability of Protein kinase (CK1) inhibitor and Alpha7 nicotinic receptor agonist. However Cycloheptene systems showed some important targets including, Insulin growth factor agonist, and Acetylcholine nicotinic agonist and Tumour necrosis factor agonist.

O X

NH2

H2N

CHO

+

x = S, On = 1, 2, 3

R

NH

NH

n

R

X

R

CAN (10 % mol)

Solvent Free

n

about 30 derivatives

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Identification, structural caracterization and qualification of unidentified impurity in Bisoprolol film-coated tablets

Ivana Mitrevska, Ema Kikovska, Gjorgji Petruševski, Ana Georgieva, Sonja Ugarkovič, Aneta Dimitrovska*

Institute for Research and Development, Alkaloid AD-Skopje, Blv. Aleksandar Makedonski 12, 1000 Skopje, Macedonia

*Institute of Applied Chemistry and Pharmaceutical Analyses, Faculty of Pharmacy, University Ss. Cyril and Methodius, Majka Tereza 47, 1000 Skopje,Macedonia

The focus of this study is identification, structural characterization and qualification of a degradation impurity of Bisoprolol with RRt 0.95. This degradation product is considered as principal thermal-degradation impurity identified in Bisoprolol film-coated tablets. The impurity has been observed in the stress thermal degradation study as well as the general stability study of the drug product. Using HPLC / DAD / ESI-MS method tentative structure was obtained, followed by detailed structural characterization using 1D

1H-NMR and

13C-NMR spectroscopy, 2D HSQC

1H-

13C

spectroscopy, mass spectroscopy and 31

P spectroscopy. The structure of the impurity RRt 0.95 was elucidated as phospho monoester of Bisoprolol, having relative molecular mass 406 (positive ionization mode). The structural characterization was followed by qualification of the impurity RRt 0.95 using several different in silico methodologies: Toxtree, TEST, and Toolbox. From the results obtained it can be concluded that no new structural alerts have been generated for the Impurity RRt 0.95 relative to the parent compound Bisoprolol. The current study presents in depth- analysis on the full characterization and qualification of unidentified impurity in a drug product with the purpose in properly defining the quality specification of the product.

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Chemical stability and in vitro and clinical efficacy of bis-retinamido methylpentane, a novel hybrid retinoid derivative

Ki Hyun Kim, Nam Ah Kim, So-Hyun Park*, Jin Hun Cho*, Seong Hoon Jeong

College of Pharmacy, Dongguk University, Gyeonggi 410-820, Republic of Korea *Coway Cosmetics R&D Center, Seoul 153-803, Republic of Korea

The anti-aging agent, retinol, has fewer side effects [1] and similar biological activity compared to retinoic acid [2]. However, retinol becomes unstable when exposed to light and heat [3]. A novel hybrid retinoid derivative, bis-retinamido methylpentane (RS-2A), was newly developed to overcome the limitations. This study evaluated the chemical stability of RS-2A under thermal and light conditions by examining degradation profiles, and assessed the in vitro biological activity, cytotoxicity, and clinical efficacy. Chemical stability and degradation profiles were investigated with HPLC and LC-MS. Especially, photo-stability of RS-2A was analyzed under various conditions, such as change of physical state and concentration, different solvents, and various excipients. For analyses of cellular activity and cytotoxicity, human dermal fibroblasts were cultured with RS-2A. To evaluate the safety and efficacy of the compound with the cellular results, RS-2A was applied to women who had moderate to severe wrinkles at the periorbital region. All of the experiments were conducted with retinol as a reference. RS-2A was more stable than retinol to thermal conditions, especially in solution. Both RS-2A and retinol were unstable to light, but RS-2A showed enhanced photo-stability with regard to concentration, more polar solvent, and addition of proper excipients. RS-2A exhibited decreased cytotoxicity and enhanced effects on collagen synthesis compared with retinol. In a clinical study, a 4-week treatment with RS-2A significantly improved the appearance of periorbital wrinkles without any side effects. The results indicate that RS-2A might have potential as an anti-aging agent for cosmeceutical preparations because of its enhanced chemical stability, biological activity, safety, and clinical efficacy. Reference [1] J. Fluhr, M. Vienne, C. Lauze, P. Dupuy, W. Gehring, M. Gloor, Tolerance profile of retinol,

retinaldehyde and retinoic acid under maximized and long-term clinical conditions, Dermatology (Basel). 199 (1998) 57-60.

[2] G.J. Fisher, S.C. Datta, H.S. Talwar, Z.Q. Wang, J. Varani, S. Kang, J.J. Voorhees, Molecular basis of sun-induced premature skin ageing and retinoid antagonism, Nature 379 (1996) 335-339.

[3] K. Yoshida, T. Sekine, F. Matsuzaki, T. Yanaki, M. Yamaguchi, Stability of vitamin A in oil-in-water-in-oil-type multiple emulsions, J. Am. Oil Chem. Soc. 76 (1999) 1-6.

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Synthesis of PLGA /nano-ZnO composite particles for biomedical application

A. Stanković, M. Lukić, M. Jović, M. Sezen*, M. Milenković**, M. Stevanović

Institute of Technical Sciences of the Serbian Academy of Science and Arts, Belgrade, Serbia

*Sabanci University Nanotechnology Research and Application Center (SUNUM) Orhanli, Istanbul, Turky

**Departments of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, Serbia

Copolymer poly (DL-lactide-co-glycolide) (PLGA), due of its biodegradable and biocompatible nature, is widely used in various medical applications; controlled release of delivering drugs, carriers in the tissue engineering, etc. On the other hand, zinc oxide (ZnO) is extensively used in medicine and pharmacy for personal care products, as well as in biomedical materials like dental composites, as a material for treatment of a variety of skin irritations, to enhance the antibacterial activity of different medicaments, etc. In this research we have dealt with a procedure to prepare particles of poly (lactide-co-glycolide) and nano zinc oxide (PLGA/nano-ZnO). Nano-ZnO has been synthesized using a microwave synthesis method and additionally immobilized within PLGA by physicochemical solvent/non-solvent method. Firstly, ZnO has been dispersed in acetone and then additionally added dropwise in the PLGA/ethyl acetate (PLGA/nano-ZnO(EtAc) or PLGA/acetone (PLGA/nano-ZnO(Ac)) solutions, respectively. The as-prepared dispersions were dry in air atmosphere for 24 h. The characterization of the prepared samples was performed using X-ray powder diffraction (XRPD) method for the structure properties, field emission scanning electron microscopy (FE SEM) for the investigation of particles morphology, as well as Malvern’s Mastersizer instrument for particle size distribution.

DTA-TG measurements were performed in order to investigate the samples thermal stability and mass loss percentage. The antimicrobial behavior of the synthesized

PLGA/nano-ZnO particles was tested against gram-negative and gram-positive bacteria

cultures and also against Candida Albicans, diploid fungus.

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https://en.wikipedia.org/wiki/Diploid

https://en.wikipedia.org/wiki/Fungus


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Effect of expression system on assay performance of human ABC transporters of pharmacological importance in vesicular transport assays

Balázs Vaskó, Beáta Tóth, Viktória Juhász, Ildikó Nagy, Anita Kurunczi, Krisztina Herédi-Szabó, Joseph K. Zolnerciks, Emese Kis, Rémi Magnan,

Kent K. Grindstaff, Peter Krajcsi, László Szilágyi

Solvo Biotechnology, 52 Középfasor str., Szeged H-6726, Hungary Purpose: ABC (ATP binding cassette) transporters are expressed at important pharmacological barriers and have an effect on ADME (absorption–distribution–metabolism–excretion) properties of drugs as well as on drug toxicity / adverse reactions. Current guidelines /draft guidance enlist several efflux transporters to be tested or considered for testing in inhibition and substrate assays. In vitro testing of ABC transporters in both inhibition and substrate assays whole cells or membrane preparations are used. Expression system and assay selection depends on the passive permeability of the compound and on the question addressed. For inhibition assays a membrane-based assay, the vesicular transport assay is applicable for compounds in any segment of the permeability space. Methods: Membrane vesicles were prepared from selected or stably transfected mammalian cell lines and insect cells infected with baculoviruses harboring the cDNA of the respective transporter. Vesicular transport assays were carried out to characterize the membranes. Results: The MDR1 (ABCB1/P-gp), BCRP (ABCG2/MXR), BSEP (ABCB11) MRP2 (ABCC2) and the MRP3 (ABCC3) transporters were expressed in insect cells such as Sf9 and Hi5 or in mammalian cells such as HEK293. Our aim was to study the effect of the expression system, including the lipid environment on transport. Signal-to-noise ratios and IC50 values will be presented. The mammalian expression systems were superior to the insect expression systems with an increase of upto 16-fold in the signal-to-noise ratios. IC50 data will also be presented. Conlusions: Our data suggest that mammalian cell-based vesicles yield a more robust assay system to measure drug transport as well as transporter mediated drug-drug interactions in a vesicular transport assay format. Most transporters seem to work more efficiently in a physiologically more relevant membrane environment. Grant support: XTTPSRT1, OM-00230/2005

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Selenium nanoparticles as a potential candidate in cancer treatment

Nenad Filipović, Jana Nunić*, Metka Filipič*, Milos Filipović**, Magdalena Stevanović

Institute of technical sciences of the Serbian Academy of Science and Arts, 11000 Belgrade, Serbia

*Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia

**Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, 91058 Erlangen, Germany

The broad spectrum of selenium applications in pharmacy and medicine has been known for a while and strongly depends on its chemical form, size and shape. However, the use of Se often requires consumption over the long period, so the toxicity of Se is always a crucial concern. Majority of available pharmaceutical products contain organic forms of selenium or its salts, but recently, when it comes to cancer treatment, elemental selenium nanoparticles (SeNPs) have emerged as a novel selenium source with the advantage of reduced risk of selenium toxicity, but with same bioavailability and efficacy in increasing the activities of selenoenzimes. In this work we are presenting the fast, reproducible method for producing stable colloidal suspension of amorphous SeNPs (<80 nm). These SeNPS were successful incorporated within PCL microspheres by combining the high speed homogenization and the precipitation in a solvent/non-solvent system. The obtained PCL/SeNPs were charac-terized by Fourier transform infrared spectroscopy (FTIR), electron microscopy (SEM and TEM), X-ray diffraction (XRD) and thermal analysis methods (TGA-DTA). The cytotoxicity and the formation of intracellular reactive oxygen species of SeNPs as well as of PCL/SeNPs were investigated employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay and using a fluorescent probe (DCFDA test) respectively. Both systems have shown good biocompatibility. The anticancer activity of SeNPs was examined on the HeLa cell line and it was demonstrated that SeNPs exhibits strong, a dose dependent, anticancer activity by preventing further HeLa cells growth and division. Bearing in mind that PCL is well known biodegradable polymer with low degradation rate, it is our opinion that PCL/SeNPs possess a great potential for cancer treatment.

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Author Index

A Abrami, M ................................................... 33 Adrjanowicz, K ............................................ 28 Ahmed, EA .................................................. 73 Ahte, P ........................................................ 30 Alvebratt, C ................................................. 46 Amézqueta, S ........................................ 50, 51 Andersson, S ............................................... 46 Avdeef, A .............................................. 12, 13 Avram, S ................................................ 42, 43 Avram, SI ..................................................... 43

B Bálint, M ..................................................... 19 Banáš, P ...................................................... 66 Bartolec, M ................................................. 54 Bazgier, V .................................................... 66 Beloborodova, AA ....................................... 59 Berginc, K .................................................... 57 Bergström, C ............................................... 46 Berka, K ....................................................... 66 Biljan, T ....................................................... 38 Bjedov, S ..................................................... 71 Błaszczak, J .................................................. 32 Błaszczyński, J ............................................. 32 Bloxham, M ................................................. 60 Bobal, P ....................................................... 10 Bodurov, I ................................................... 56 Boldyrev, VV ............................................... 61 Boldyreva, EV ............................ 24, 59, 61, 62 Bolger, MB .................................................. 31 Bordas, M ................................................... 52 Bosch, E .......................................... 15, 48, 52 Brašnarska, I ............................................... 72 Buckton, G .................................................. 60 Butterweck, V ............................................. 44

C Cabot, JM ...................................................... 7 Carrillo, A .................................................... 49 Cecchelli, R .................................................. 44 Cernikova, A ................................................ 10 Chang, T-C ................................................... 18 Chiarappa, G ............................................... 33 Cho, JH ........................................................ 75 Cielecka-Piontek, J ...................................... 63 Cieślik, W ..................................................... 45 Cimpean, AM ........................................ 42, 43 Cindric, M .................................................... 34 Ciucanu, I .................................................... 69 Comer, J ........................................................ 6 Culot, M ...................................................... 44 Cvijetić, IN ............................................. 12, 67

Č Čanadi, J ...................................................... 71 Čulen, Č ....................................................... 58

D Danciu, C ..................................................... 43 Daolio, C ...................................................... 35 Dehelean, CA ............................................... 43 Deli, MA ...................................................... 44 Derevyannikova, E....................................... 62 Dimitrovska, A ............................................. 74 Ding, Z ......................................................... 22 Dohnal, J...................................................... 58 Dragojević, S................................................ 47 Drebushcak, VA ........................................... 59 Dulski, M ..................................................... 25


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E Eigenmann, DE ............................................ 44 El-Saghier, AMM ......................................... 73 Eltsov, IV ..................................................... 61 Embrey, K .................................................... 37 Enrech, R ..................................................... 52 Erdani Kreft, M ........................................... 57 Exner, G ...................................................... 56

F Farré, S ........................................................ 51 Fernandez-Pumarega, A ............................. 50 Fernández-Pumarega, A ............................. 51 Filipič, M ..................................................... 78 Filipović, M ................................................. 78 Filipović, N .................................................. 78 Fuguet, E ..................................... 7, 15, 50, 51 Fuliaş, A .................................... 43, 68, 69, 70

G Gabelica Marković, V .................................. 47 García-Sosa, AT ........................................... 30 Georgieva, A ......................................... 72, 74 Golob, S ...................................................... 33 Grancharova, Ts .......................................... 56 Gråsjö, J ...................................................... 46 Grassi, G ...................................................... 33 Grassi, M ..................................................... 33 Grindstaff, KK .............................................. 77 Gruberová, L ............................................... 58 Grzybowska, K ............................................ 25

H Herédi-Szabó, K .......................................... 77 Hetényi, Cs .................................................. 19 Hilfiker, R .................................................... 26 Honeywell, RJ.............................................. 20 Horváth, I .................................................... 19 Horváth, V ................................................... 12 Hranjec, M .................................................. 47 Hranueli, D .................................................. 34

I Ilijaš, M ....................................................... 47

J Jähne, EA ..................................................... 44 Jampilek, J ................................................... 10 Jan Markuszewski, M .................................. 53 Janković, B ................................................... 27 Jarc, T .......................................................... 57 Jeong, SH ..................................................... 75 Jeszenői, N .................................................. 19 Jović, M ....................................................... 76 Juhász, V ...................................................... 77

K Kahn, I ......................................................... 30 Kaliszan, R ................................................... 53 Kao, RY ........................................................ 41 Kelava, V...................................................... 47 Kikovska, E .................................................. 74 Kim, KH ........................................................ 75 Kim, NA ....................................................... 75 Kis, E ............................................................ 77 Knapik, J ...................................................... 25 Kónya, Z ...................................................... 29 Koštrun, S .................................................... 47 Kozlov, AS .................................................... 61 Krajcsi, P .................................................. 3, 77 Krasowska, A ............................................... 45 Kregar, ML ................................................... 54 Kristan, K ..................................................... 57 Krysiński, J ................................................... 32 Kubik, Ł ........................................................ 53 Kumar Namila, K ......................................... 62 Kurunczi, A .................................................. 77 Kuznetsova, SA ............................................ 61

L Laigna, E ...................................................... 30 Ledeti, I ....................................................... 43 Ledeţi, I ........................................... 68, 69, 70 Li, HW .......................................................... 21 Llinàs, A ................................................... 6, 16 Lomaka, A ................................................... 30 Losev, E ....................................................... 62 Lovrić, J ....................................................... 11 Lukacova, V ................................................. 31 Lukić, M ....................................................... 76


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M Madarász, J ................................................. 12 Magnan, R ................................................... 77 Mahmoud, AMA ......................................... 73 Malamatari, M ............................................ 60 Malyar, YN .................................................. 61 Maran, U ......................................... 30, 64, 65 Marković, OS ............................................... 67 Marudova, M .............................................. 56 Melvan, E .................................................... 34 Meštrović, E ................................................ 23 Mikhailenko, MA ........................................ 61 Milenković, M ............................................. 76 Minkov, VS ............................................ 59, 62 Mitrevska, I ................................................. 74 Mularski, J ................................................... 45 Mullin JM .................................................... 31 Muñoz-Pascual, L ........................................ 51 Musioł, R ..................................................... 45

N Nagy, I ......................................................... 77 Nangia, A .................................................... 62 Nowaczyk, A ............................................... 32 Nunić, J ...................................................... 78

O Oja, M ............................................. 30, 64, 65 Oprean, C .................................................... 43 Opsenica, IM ............................................... 67 Otyepka, M ................................................. 66 Oufir, M ...................................................... 44

P Pałkowski, Ł ................................................ 32 Paluch, M ........................................ 25, 28, 63 Park, S-H ..................................................... 75 Pavel, IZ ...................................................... 43 Perissutti, B ................................................. 33 Pešić, MP .................................................... 12 Peters, GJ .................................................... 20 Petruševski, G ............................................. 74 Piir, G .......................................................... 30 Pilicheva, B .................................................. 56 Pintye-Hódi, K ............................................. 29 Plavec, J ...................................................... 36

Pontelli, F .................................................... 33 Popović, GV ................................................. 12 Port, A ......................................................... 52 Poša, M ....................................................... 71 Pranjić, J ...................................................... 54

R Radošević, S ................................................ 54 Ràfols, C .......................................... 15, 48, 52 Raica, M ...................................................... 42 Rams-Baron, M ........................................... 25 Regdon, G, jr. .............................................. 29 Reppas, R ...................................................... 9 Rosés, M .......................... 7, 48, 49, 50, 51, 52 Ruiz, R ........................................................... 6 Ruusmann, V ............................................... 30

S Sakač, M ...................................................... 71 Serajuddin, A ................................................. 8 Sezen, M ..................................................... 76 Shakhtshneider, TP ..................................... 61 Shoghi, E ..................................................... 15 Sild, S ..................................................... 30, 65 Siluk, D ........................................................ 53 Sipos, B ........................................................ 29 Skrzypczak, A ............................................... 32 Słowiński, R ................................................. 32 Somavarapu, S ............................................ 60 Sotirov, S ..................................................... 56 Sovány, T ..................................................... 29 Srčič, S ......................................................... 27 Stanković, A ................................................. 76 Starcevic, A.................................................. 34 Stevanović, M ........................................ 76, 78 Struck-Lewicka, W ....................................... 53 Stupák, I ...................................................... 58 Subirats, X ....................................... 48, 49, 52 Suresh, K ..................................................... 62 Şuta, L-M ......................................... 68, 69, 70 Szczepaniak, J .............................................. 45 Szilágyi, L ..................................................... 77

Š Škorić, D ...................................................... 71 Šolaja, BA .................................................... 67


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T Tabiova, K ................................................... 10 Takács-Novák, K .................................... 14, 55 Takkis, K ...................................................... 30 Tam, K ......................................................... 21 Terzić-Jovanović, NV ................................... 67 Tian, Y ......................................................... 22 Tóth, B ........................................................ 77 Tsinman, K .................................................... 5

U Ugarkovič, S .......................................... 72, 74 Ugrina, I ...................................................... 34 Uzunova, Y .................................................. 56

V Valko, K ......................................................... 4 van der Spoel, D .......................................... 19 Vaskó, B ...................................................... 77 Verbić, TŽ .............................................. 12, 67 Viraneva, A.................................................. 56 Vlaeva, I ...................................................... 56 Vlase, G ........................................... 68, 69, 70 Vlase, T ........................................... 68, 69, 70 Voinovich, D ................................................ 33 Völgyi, G ................................................ 14, 55 Vysloužil, J ................................................... 58

W Walter, F ..................................................... 44 Wan, H ........................................................ 17 Wang, Z ....................................................... 22 Wiczling, P ................................................... 53 Wojnarowska, Z .................................... 25, 63 Wong, R ....................................................... 21

X Xu-Szeto, K .................................................. 31

Y Yovcheva, T ................................................. 56 Yuan, S ........................................................ 41 Yung, K ........................................................ 21

Z Zamay, AS .................................................... 61 Zhang, H ...................................................... 22 Zheng, B-J .................................................... 41 Zhou, J ......................................................... 41 Zhou, W ....................................................... 21 Zlatović, MV ................................................ 67 Zolnerciks, JK ............................................... 77 Zucko, J........................................................ 34

Ž Živanović, VS ............................................... 12

3rd World Conference on Physico-Chemical Methods in Drug Discovery and Development

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fourth world conference on physico-chemical methods in ... · 4th world conference on...

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