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2016 Drug Design and Delivery Symposium
http://bit.ly/2016ddds
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Thursday, September 22, 2016
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www.acs.org/acswebinars Slides available now! Recordings are an exclusive ACS member benefit.
2016 Drug Design and Delivery Symposium
“Crystallography as a Drug Design and Delivery Tool”
The 2016 DDDS is co-produced with ACS Division of Medicinal Chemistry and the AAPS
Vincent Stoll Research Fellow and Associate
Director of Structural Biology, AbbVie
Robert Wenslow Vice President, Business
Development, Crystal Pharmatech
Andrew Brunskill Associate Principal Scientist,
Merck
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Drug Design Using Structural Biology
Vincent S. Stoll, Ph.D. Associate Director Structural Biology R&D, AbbVie
No Pro Approval Code A4360572
Protein Biochemistry Molecular Biology
The Drug Design Cycle
Iterative structure-based drug design cycle
3D Structure Determination Crystallography/NMR Homology Modeling
Structure-based Drug Design Cycle Analog Design
Lead Generation
Chemical Synthesis
Biological Assays
Medicinal Chemistry-based Cycle
Preclinical Studies
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Protein Production – Protein Biochemistry Protein production pathway – an iterative process
Project team Chemists Biologists
Purification Characterization
Fermentation Cloning and expression
Crystallography NMR
Production Customers Beneficiaries .
Cycle to improve expression, solubility, activity, stability
Cycle to improve crystallizability,
spectral quality, etc.
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How Do We Determine the Crystal Structure?
Solution of target protein
Crystal of protein in
drop
Add precipitant
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How Do We Determine the Crystal Structure?
Solution of target protein
Crystal of protein in
drop
Add precipitant
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Soak ligand into crystal
How Do We Determine the Crystal Structure?
Solution of target protein
Crystal of protein in
drop
Add precipitant
Ligand soaks in over 2-24 hours
X-ray Generator
Crystal mounted at liquid nitrogen temperature
(–196 °C)
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Protein Crystal X-ray Diffraction
mounted crystal
X-ray beam
Intensities transformed to electron density
Scatters X-rays in discrete directions (image 0.2° wedge of 180° crystal rotation scan)
3D atomic model
Integrate with AbbVie Projects
Single wavelength (~0.1 nm) Single crystal (10~100 µm)
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22 SBBD| Drew Medicinal Chemistry Course| 6.8.16
Global X-ray Crystallography
Using State of the Art Technologies • Highly automated crystallization process
– UV Florescence and Absorbance crystal viewing
– Lipidic cubic phase dispenser – Micro crystal mounting system
– Currently manual, automation in development
– Image viewing software – Novel approach to compound solubility
measurement (Critical Aggregation Concentration) – Helps characterize and prioritize
compounds for crystallization
• Commercially purchased automation – Mosquito for nanoliter solution dispensing
• Protein supplied by centralized Protein Biochemistry
Driving FBDD & SBDD on small Molecules and Biologics
Crystallization Screening
Crystallization Optimization
Crystal Plate Viewing Tower
Crystal Identification
Microcrystal Scooping
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Enabling Challenging Experiments
• Diffraction Rastering sample centering • Collect-Along-Vector
• Mini-Beam 50, 20, 10, 5 m
• Stable Beam auto positional feedback
• High-Precision Alio Goniometer reliable sample positioning
• High Flux >1013 ph/s @ 12.4 keV
• Micro-Focused Beam 30 m(v) x 70 m(h) FWHM
• Fast Detector Pilatus 6M, no readout noise
Weakly Diffracting
Crystals
Small Crystals
Lipidic Cubic Phase
Crystals
Radiation SensitiveC
rystals
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Crystallography Experiments @ IMCA-CAT
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20,000+ structures annually
INDUSTRY DISCOVER CAPABILITIES PRODUCTIVITY EXPERIMENT
IMCA-CAT Subscribers
New subscriptions available
IMCA Members
www.imca-cat.org
• proprietary
• rapid & frequent access
• on-site, remote, mail-in • focused, intense beam
• mini beam 5-50 m
• pucks: Uni, ACTOR, ALS
• high-throughput
• fast, encrypted data transfer
• real-time integration to company pipelines
Beamline 17-ID @ APS
• micro crystals
• membrane proteins
• MAD / SAD
• in situ
• diffraction rastering
• collect-along-vector
• auto collect & process
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Structure-based Drug Design
Bcl Inhibitors, Fragment-based Drug Design, and Anti-targets
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Bcl-2 Family Inhibitor – a Protein-Protein Interaction
• Potential as a therapeutic option in a range of cancers
106 Business Week | December 12, 2005
SBBD| Drew Medicinal Chemistry Course| 6.8.16
(ABT-737is an investigational Compound. Safety and Efficacy have not been established)
27
Bcl-2 Family Inhibitor Helps Turns on Death Switch
Bcl-xL
Overproduction of Bcl-2 protein binds to death proteins and shuts off the death switch allowing
cancers to stay alive
Death Protein
In cancer cells
Muchmore et al., Nature 381, 335 (1996) Sattler et al., Science, 275, 983 (1997)
How Bcl-xL binds to death proteins
Bcl-2
like
BH3- only +
Cancer cells live
Bcl-2
like
BH3- only
Bcl-2 family inhibitor
binds to Bcl-2 and helps blocks death protein interaction, allowing death protein to kill cancer
Cancer cells die
BH3- only
Bcl-2
like +
Bcl-2
like
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Bcl-xL Structure of Binding Site
• Goal is to design a compound to mimic Bak peptide binding
• Hydrophobic residues interact with Bcl-xL:
– Leu 78, Ile 85
• Hydrophilic residues point out into solvent:
– Arg 76, Asp 83, Asp 84
• Take advantage of groups to design compound
R139
L78
D83
I85
Bcl-xL complexed to Bak peptide
R76
29
O
O Screen for fragment #2
OH
Screen for fragment #1
1H (ppm)
13C
(p
pm
)
Fragment Screening and Linking – SAR by NMR
NH
N
O
HN
NHN
N
N
N
O
Cl
HN O NH2
CH3
H3CO
CH3
H3C
COOH
10,000 fragment
library
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16
• Perform 2nd site screen
• Determine ternary structure
• Design linkers and synthesize
• Confirm design with structure
O
O Screen for fragment #2
OH
Screen for fragment #1
Fragment Screening and Linking – SAR by NMR
NH
N
O
HN
NHN
N
N
N
O
Cl
HN O NH2
CH3
H3CO
CH3
H3C
COOH
10,000 fragment
library
Link fragments
O
O
O
SBBD| Drew Medicinal Chemistry Course| 6.8.16 31
Fragment Collection Fragment Screening Fragment Optimization
NMR SPR
Triage
Lead
Ro3 and Ro3.5 Collections
Overall Fragment Screening Paradigm
BP Collection
BP Similars
HTS
BioPhys (BP)
BPS
SBDD
Screening and confirmation
Biochemical assays XRC
Synthesis Screening
Design (XRC and Props)
Purification and sample
logistics
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Screen for First-Site Ligands using NMR
• 10,000 compound library
• <MW> ~ 215
• [Compound] = 1 mM
9.0 8.5 8.0
111
.011
0.0
109
.01
08
.01
07
.01
06
.01
05
.0
1H ppm
15N
ppm
OH
OF
Kd = 300 M
Monitor Binding with 15N-HSQC spectrum
G94 G196
G138
SBBD| Drew Medicinal Chemistry Course| 6.8.16 33
R139
NMR Structure of Bound Fragment
Binds to peptide “hot spot”
• Two key interactions maintained (Leu78 and Asp83)
Second site accessible • Ile85 pocket of Bak peptide
SBBD| Drew Medicinal Chemistry Course| 6.8.16 34
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Screen for Second-Site Ligand
• Screen in excess of biaryl acid
• 3,500 compound library
• <MW> ~ 150
• [Compound] = 5 mM
OH
Kd = 2000 M
• Binds to second “hot spot” ─ Ile of Bak peptide
R139
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Linking Strategy
6.1 Å
F97 F
OHO
OH
Kd = 2000 – 6000 M
Kd = 300 M
linker
O
O
O
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Linking Strategy
• Accesses hydrophobic second site
• 200-fold gain in potency
– Expected >150-fold
• Still room for improvement
FPA IC50 = 1.4 µM
F
O OH
SBBD| Drew Medicinal Chemistry Course| 6.8.16 37
Acylsulfonamide Linking Strategy
New trajectory: avoids F97
Maintains acidic nature
Acidic Hydrogen
Kd = 300 µM Kd = 320 µM
R = Me
F
OHO
F
NH
OS
R
O O
O
O
O
F97
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Diversity Approach to 2nd Site Binders
Parallel synthesis
F
O OH
React with 120 diverse
sulfonamides
R-SO2NH2
F
OHN
S
HN
O O
NS
NO2
Kd = 245 nM
React with 125 diverse amines
HNRR'
F
OHN
S
O O
NO2HN
S
Kd = 36 nM
Kd = 300,000 nM
Kd = >10 µM in 10% serum!
SBBD| Drew Medicinal Chemistry Course| 6.8.16 39
Structure-Based Optimization of Bcl-xL Inhibitors
Kd = 36 nM
Structure-based reduction in
protein-binding
Polar isostere for phenyl ring
Accessing a “third” pocket In the groove
Nature 435, 677-681 (2005)
Tail collapses increases potency
F
NH
OS
O O
NH
S
NO2
N
N
NH
OS
O O
NH
S
NO2
H
N
Cl
ABT-737 KD < 1 nM
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ABT-737 – The Clinical Candidate
ABT-737 Clinical candidate
Kd < 1 nM
F
NH
OS
O O
NH
S
NO2
N
N
NH
OS
O O
NH
S
NO2
H
N
Cl
Fits the structure-based design rubric
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42
Audience Survey Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
• They identify and bind key hotspots in a binding site.
• Linking two fragments together is easy.
• They often lead to more efficient drugs with better physical properties.
• Fragments are easy to optimize in chemistry without protein structural information.
What are some of the advantages of using fragments in drug discovery?
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ABT-263: Making an Oral Drug
Main liability of ABT-737 is its poor oral bioavailability
• Must be dosed IV
This is due to limited absorption
• Very basic dimethyl amine
• Rigid chloro-biphenyl
Nitro group potentially toxic
N
OS
HN
OO
HN
NO2
S
N
N
Cl
ABT-737 Oral Bioavailability ~ 5 %
SBBD| Drew Medicinal Chemistry Course| 6.8.16 43
N
OS
HN
OO
HN
NO2
S
N
N
Cl
ABT-263: Making an Oral Drug
N
OS
HN
OO
HN
NO2
S
N
N
Cl
ABT-737 Oral Bioavailability ~ 5 %
N
OS
HN
OO
HN
S
S
N
N
Cl
OO
F3C
Remove potential toxicity
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ABT-263: Making an Oral Drug
N
OS
HN
OO
HN
NO2
S
N
N
Cl
N
OS
HN
OO
HN
S
S
N
N
Cl
OOO
F3C
Decrease basicity by one log unit
Remove potential toxicity
ABT-737 Oral Bioavailability ~ 5 %
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ABT-263: Making an Oral Drug
N
OS
HN
OO
HN
NO2
S
N
N
Cl
N
OS
HN
OO
HN
S
S
N
N
Cl
OOO
F3C
Decrease basicity by one log unit
Remove potential toxicity
Decrease rigidity, improve absorption
ABT-737 Oral Bioavailability ~ 5 %
SBBD| Drew Medicinal Chemistry Course| 6.8.16 46
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ABT-263: Making an Oral Drug
N
OS
HN
OO
HN
NO2
S
N
N
Cl
N
OS
HN
OO
HN
S
S
N
N
Cl
OOO
F3C
Decrease basicity by one log unit
Remove potential toxicity
Decrease rigidity, improve absorption
ABT-263 Oral Bioavailability ~ 30 %
ABT-737 Oral Bioavailability ~ 5 %
SBBD| Drew Medicinal Chemistry Course| 6.8.16 47
ABT-263 – The Current Clinical Candidate
Also fits the structure-based design rubric
ABT-263 Clinical candidate
Kd < 1 nM
N
OS
HN
OO
HN
S
S
N
N
Cl
OOO
F3C
48
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Navitoclax Induces Thrombocytopenia Bcl-xL driven toxicity
• Platelet survival is dependent on Bcl-xL
– Mason, et. al., Cell 128, 1173, (2007).
• Bcl-xL inhibition with navitoclax results in concentration and dose dependent thrombocytopenia
• Thrombocytopenia is the clinical dose limiting toxicity of navitoclax Roberts et al., J Clin Oncol 27:15s, abstr 3505 (2009)
Wilson et al., Lancet Oncol 11, 1149, (2010)
R2 = 0.9466
0
10
20
30
40
50
60
70
80
90
1 10 100 1000
Dose (mg)
% o
f B
as
elin
e P
late
lets
Platelet count vs. drug exposure Platelet count vs. dose %
of
Ba
se
lin
e P
late
lets
AUC0-inf (ug·h/mL)
(Navitoclax is an investigational Compound. Safety and Efficacy have not been established)
49
Bcl-xL
navitoclax
Bcl-2
Dose-limiting
thrombocytopenia
Navitoclax Inhibition Profile Dictates Efficacy/Toxicity
• Bcl-2 is an important survival factor in lymphoid malignancies
– CLL, NHL
• Bcl-xL is required for survival of circulating platelets
– Bcl-xL inhibition leads to dose-limiting thrombocytopenia
SBBD| Drew Medicinal Chemistry Course| 6.8.16
(Navitoclax is an investigational Compound. Safety and Efficacy have not been established)
Target efficacy in leukemia and lymphoma
50
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26
Bcl-xL
navitoclax
Bcl-2
Dose-limiting thrombocytopenia
Target efficacy in leukemia and lymphoma
Bcl-2 selective
Bcl-2 Selective Inhibitors: Key Hypotheses
• Bcl-2 selective inhibitors will maintain efficacy in lymphoid malignancies
• Bcl-2 selective (Bcl-xL sparing) inhibitors will not induce thrombocytopenia
• Will result in improved therapeutic window, allowing higher exposures and greater efficacy in Bcl-2 dependent malignancies
SBBD| Drew Medicinal Chemistry Course| 6.8.16
(Navitoclax is an investigational Compound. Safety and Efficacy have not been established)
51
Bcl-2 & Bcl-xL Binding Grooves Show High Similarity
• No naturally occurring, Bcl-2 selective BH3-only protein
• Only 4 residues differ within binding groove
R126 S122 L108 M112
A104 D108
E96 D100
Bcl-2 Bcl-xL
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N
N
OHN
Cl
SO O
HN
N+O-O
N
S
O
Reverse Engineering Leads to Bcl-2 Selectivity
• Modification of P4 binding region • Loss of Bcl-xL affinity • Reduced Bcl-2 affinity • No cellular potency
N
N
OHN
Cl
SO O
HN
N+O-O
N
O
TR FRET, Ki, [nM]
Bcl-2 Bcl-xL
1 0.20 1.3
2 29 >660
3 18 >660
1 2 3
-P4 binder
-amine N
N
OHN
Cl
SO O
HN
N+O-O
O
SBBD| Drew Medicinal Chemistry Course| 6.8.16 53
Design Cues From X-ray Crystal Artifact
• Overall ligand binding conformation maintained
• P4 pocket occupied by Trp28 in N-terminal loop of neighboring protein in crystal
• Trp side chain forms H-bond with Asp100 of Bcl-2
– Corresponds to Glu96 of Bcl-xL
• Suggests alternate approach to Bcl-2 P4 hot spot
Bcl-2 X-ray
P4
Bcl-2 X-ray
Asp 100
Intercalating Trp
Asp 100
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ASP 100
• ~100-fold increase in Bcl-2 affinity • Selectivity vs. Bcl-xL maintained
• Affinity & cellular efficacy still ~10-fold lower than navitoclax • Target second hydrogen bond and improve cellular activity
Indole Substituent Fills P4 & Captures H-Bond
O
HN
N
N
Cl
NO2
ONH
SOO
ASP 100
TR FRET, Ki, [nM] Bcl-2 Bcl-xL
navitoclax 0.04 0.05
3 18 >660
4 0.30 >660
3 4
2.8
R104
D100
O
O
HN
N
N
Cl
NO2
O
NH
NH
SOO
+ P4 indole
SBBD| Drew Medicinal Chemistry Course| 6.8.16 55
ABT-199: a Selective Bcl-2 Inhibitor
• High Bcl-2 affinity
• Lower affinity for Bcl-xL, Mcl-1
• Ineffective in Bcl-xL-dependent human tumor cell lines (H146)
Affinity Cellular Efficacy, EC50, nM
TR FRET, Ki, nM Human Tumor Cell Lines, 10%HS
Agents Bcl-2 Bcl-xL Bcl-w Mcl-1 RS4;11 (Bcl-2) H146 (Bcl-xL)
navitoclax 0.04 0.05 7 >440 110 75
4 0.30 >660 NT >440 1180 >10,000
ABT-199 < 0.01 48 21 >440 8 3600
4 ABT-199
O
O
HN
N
N
Cl
NO2
O
NH
NH
SOO
O
O
HN
N
N
Cl
NO2
O
N NH
NH
SOO
Souers et al, Nature Medicine 2013 (ABBV-199 is an investigational Compound. Safety
and Efficacy have not been established)
56
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29
Physicochemical Properties
Property ABT-199
Mol Wt (g/mol) 868.44
cLogP 8.1
PSA 183
Experimental logD 5.4
Aqueous solubility pH 1
25 °C, (g/mL) 2.3 ± 0.2
Aqueous solubility pH 7.4 ,
25 °C, (g/mL), <0.004
• High molecular weight • Hydrophobic
• Low aqueous solubility
O NH
O
HN
N
N
Cl
N NH
N+O–O
SO
O
O
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ABT-199 is Orally Bioavailable
• Modest oral bioavailability across species
– Low clearance
– Good plasma half-life
• Amorphous solid dispersion allows high exposures and good dose linearity
PEG400/DMSO i.v. t1/2
(h)
i.v. V
(L/kg)
i.v. CLP
(L/h/kg)
Oral t1/2
(h)
Oral F
(%)
Mouse 2.5 0.38 0.10 2.7 28
Rat* 5.8 2.0 0.24 5.3 29
Dog 12.0 0.3 0.02 13.8 28
Cyno 2.2 0.9 0.27 2.0 9
* PEG400:Cremaphor EL:Oleic Acid
Dog 2.5 mg/kg i.v. or p.o.
ASD formulation Dog AUC vs. p.o. Dose
Dose (mg/kg)
AU
C (
ug
*hr/
mL
)
R 2
= 0.9746
0
500
1000
1500
2000
2500
0 50 100 150 200
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ABT-199 Has Platelet Sparing Potential
• Navitoclax 5 mg/kg po qdx1 results in ~95% platelet loss in dogs within 6 hours
• ABT-199 has minimal platelet effect at ~20-fold higher dose and exposure
• ABT-199 is significantly less potent than navitoclax against human platelets ex vivo (n=9)
dogs in vivo Single oral dose
0.083 M 5.483 M
Human platelets treated ex vivo
100 90 80 70 60 50 40 30 20 10 00 500 1,000 1,500 2,000 2,500
AUC (µgh/ml) Max platelet reduction (%)
ABT-199
navitoclax
vehicle
150 mg/kg
50 mg/kg
25 mg/kg
5 mg/kg
Souers et al, Nature Medicine 2013
SBBD| Drew Medicinal Chemistry Course| 6.8.16
(ABBV-199 is an investigational Compound. Safety and Efficacy have not been established)
59
ALL Cells are ‘Primed for Death’ ABT-199 helps induces tumor regressions in preclinical models
0
500
1000
1500
2000
2500
3000
20 25 30 35 40 45 50 55
ABT-199 100 mg/kg PO, qdx7
Vehicle
Tu
mo
r vo
lum
e (
mm
3)
RS4;11 (ALL) flank xenograft In female C.B-17 SCID Mice
Days post-inoculation
• High Bcl-2 & Bim expression
• High levels of Bcl-2:Bim complex
• ABT-199 induces complete regression of established tumors
Tumor Cell Lines
Del Gaizo Moore, et.al. Blood. 111, 2300, (2008)
SBBD| Drew Medicinal Chemistry Course| 6.8.16
(ABBV-199 is an investigational Compound. Safety and Efficacy have not been established)
60
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Summation
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Conclusions
• Protein-protein interactions can be suitable drug targets
• Structural understanding of the PPI is key
- Know your target: protein hot spots
- Learn from nature: endogenous ligand binding
• Fragment based screening identifies multiple hot spot binders
• Chemical tools are key to understanding biological processes
• Targeting apoptotic machinery may be an effective approach to treat certain cancer types
SBBD| Drew Medicinal Chemistry Course| 6.8.16 62
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Structure-based Drug Design Concepts
• Initial lead-protein complex structures usually provide greatest benefit – Identify ligand binding site and binding mode
– Show critical ligand-protein interactions
– Explain structure-activity relationships
– Suggest positions and groups for modification
• Achieve a consistent binding mode – Allows for reliable prediction
• Develop strategy for modification – Add and combine substituents
– Link fragments
• Focus on all drug properties – Potency, metabolism, solubility, protein binding, pharmacokinetics,
bioavailability
SBBD| Drew Medicinal Chemistry Course| 6.8.16 63
Bcl Project Team
High Throughput Screening
and Fragment Screening
Team
Structural Biology
Acknowledgments
+ many, many others
SBBD| Drew Medicinal Chemistry Course| 6.8.16 64
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33
Never Give Up!
SBBD| Drew Medicinal Chemistry Course| 6.8.16 65
Crystal Pharmatech
– Your Partner for Pharmaceutical Solid State
Research and Development Solutions
Crystallography as a Drug Design and Delivery Tool:
Robert Wenslow, Ph. D. [email protected]
Crystal Pharmatech
66
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34
67
• Importance of API Crystals
• Types of Crystals
• Choosing the right Crystal (or lack thereof)
– Salts
– Amorphous
– Co-Crystal
Outline
68
•The most significant challenge facing discovery organizations is understanding reasons why drugs are failing in development.
•A review of Phase II failures (2008-2011) in Nature Reviews Drug Discovery showed that efficacy was the major cause (51%) followed by strategic reasons (29%) and safety (19%).
Why do Compounds Fail
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69
Active Pharmaceutical
Ingredient
API Crystals Chemical purity PSD, SA, morphology, density, etc. Stability (chemical and/or physical) Hygroscopisity, color, caking, etc.
Drug Product Bioavailability Efficacy Safety
Crystallization
Critical Role in Ensuring Drug Product Quality
Cryst. Growth Des., 2015, 15 (12), pp 5645–5647
M3
Molecules, Materials, Medicines
70
Late Stage Polymorph
Change
Hydrate impacts dissolution
Crystal Form leads to new
indication
Extensive
Intellectual Property battles
Salt for consistent
bioavailability and shelf life
Crystal Form Impact on Products
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71
Solid forms include • Polymorphs, hydrates, solvates,
amorphous
• Salts and cocrystals
– Polymorphs, hydrates, solvates and amorphous can exist for each salt/cocrystal
• Amorphous dispersions
– Multiple polymers
and concentrations
Harmon et al. AAPS Newsmagazine, 2009, Sept, 14-20.
Solid Form Diversity
72
Audience Survey Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
• About one third
• About half
• About two thirds
• Almost all
In a 2007 study of over 245 compounds, what percentage had multiple crystal forms (Polymorphs, hydrates, solvates)
Stahly. Crystal Growth & Design. 2007, 7, 1007-1026
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73
BCS Classification
74
Current Reality
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75
Ku, Amer. Pharm. Rev. 2010, 13, 22-30
Items Sulfate Form A
p-Tosylate Form A
Stiochiometry Molar
Ratio(API/Acid) 1:1 (IC) 1:1 (NMR)
Counter ion Safety
Safety Class I II
Solid Characterization
Crystal Form Anhydrate Anhydrate
Crystallinity Crystalline Crystalline
Hygroscopicity Slightly
hygroscopic Slightly
hygroscopic
Purity (% Area) 99.1% 98.4%
Solubility
FaSSIF (4 hr) (mg/mL)
2.9 2.0
SGF (1 hr) (mg/mL)
25.5 21.8
Stability Bulk, 80oC, 39h
Physically stable
Chemically stable
Physically unstable
Chemically stable
Recommendation
Sulfate Form A is recommended for further
evaluation
Salts
When Salts Properties
76
Audience Survey Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
• About one third
• About half
• About two thirds
• Almost all
What percentage of drugs are marketed as salts?
P H Stahl and C G Wermuth, Handbook of Pharmaceutical Salts, Wiley VCH, 2002 L. Kumar, A. Amin & A.K. Bansal; Pharm. Technol., March 2008
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• HIV protease inhibitor (MK-693, L-735,524)
• Marketed as CrixivanTM
– Approved 1996
• Low aqueous solubility (< 0.02 mg/mL), pH dependant
• Free base monohydrate first form investigated
Lin et al. The Story of Crixivan Chapter 11 in Integration of Pharmaceutical Discovery and Development: Case Studies, edited by Borchardt et al, Plenum Press, New York, 1998, p 233-255
77
Crixivan
• Initial animal studies used 0.5% methocel and citric acid solutions
– Showed relatively low and variable oral bioavailability with methocel
– Higher and more reproducible results with acid solutions
– Acid solutions were not stable; same degradation seen with solid free form
• Needed a soluble salt
– pKa 3.68, 6.00
Lin et al. The Story of Crixivan Chapter 11 in Integration of Pharmaceutical Discovery and Development: Case Studies, edited by 3- Borchardt et al, Plenum Press, New York, 1998, p 233-255
78
Crixivan
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• Crystalline sulfate salt found
– Ethanolate
– Highly soluble (500 mg/mL)
– Excellent absorption in rats and dogs
– Very hygroscopic
• Sulfate ethanolate selected for development
Lin et al. The Story of Crixivan Chapter 11 in Integration of Pharmaceutical Discovery and Development: Case Studies, edited by 3- Borchardt et al, Plenum Press, New York, 1998, p 233-255
79
Crixivan
• Stability issues investigated
– Crystalline sulfate ethanolate compared to amorphous
– Salt exhibited minimal degradation; estimated shelf life of 2 yrs at 25C/33% RH
• Satisfactory chemical stability at low RH
Lin et al. The Story of Crixivan Chapter 11 in Integration of Pharmaceutical Discovery and Development: Case Studies, edited by 3- Borchardt et al, Plenum Press, New York, 1998, p 233-255
• Handling of bulk and formulated drug product below 30% RH and dessication of final product recommended
80
Crixivan
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Yeh et al. Antimicrob Agents and Chemother 1998, 42, 332-338
• Free base and sulfate salt used in human studies
• Sulfate salt in fasted state or with low fat meal gave highest AUC and Cmax
81
Crixivan
• Summary
– Early salt/form selection simplified development
– Problems with early free base properties solved with sulfate salt
– Early animal and clinical studies showed utility of salt
– Sulfate salt physical properties not ideal, but additional studies provided needed solutions for handling, formulation, packaging
82
Crixivan
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• disintegrants / solubilizers • lipid-based delivery systems
83
• prodrugs
Chemical Modification
• micronized
• nano-particles
Particle-size Reduction
• Polymorphs, amorphous
• co-crystals Form Change
Formulation Systems
Not Just Salts
Amorphous “Structure”
84
Local domains with short-range order (20-25 Å)
Microstructure
Microvoids (defects)
Higher-state energy than crystalline
84
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85 Therapeutic Delivery, February 2015 ,Vol. 6, No. 2 , Pages 247-261
Marketed Amorphous Drugs
10+ marketed amorphous drug products
> 80% with improved bioavailability (Newman A. et al J Pharm Sci. 2012)
Overcome Solubility of late stage polymorph
Dispersion - co-crystal also
works
Amorphous coated pellets
– Co-crystal also works
86
Law et al., J. Pharm. Sci. 93 (2004) 563
Douslin et al. JACS 1946, 68, 173
A higher-energy form that offers enhanced solubility, dissolution rate and oral bioavailability
Kaletra
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87
Solid ccx
Exposure to water/SGF
Meta-stable
Free base
free base
acid
Extraction of co- former from lattice of ccx
time
con
cen
trat
ion
co
nce
ntr
atio
n
time
BA BA
No BA change No BA change
ccx = cocrystal
+
= “parachute” prolongs supersaturation (excipient(s) or crystal form by itself)
Co-crystals – potential mechanism
88
Audience Survey Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
• 1776
• 1942
• 1840
• 1996
What year was the first recorded co-crystal discovered?
Kobell FV, Prakt JF. D-glucose: Sodium chloride monohydrate. Chemie. 1843;28:489
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• Weak base, extremely low aqueous solubility
– ~nanograms / mL RT solubility in water at pH 3 – 8
– 0.6 mg/mL at pH 1.2
• High dose drug, significant first-pass metabolism
• Inconsistent oral bioavailability was a major challenge
• Salt forms were intrinsically acidic and hygroscopic
89
N N ONN
N
O
H3C
CH3
O
O
N
N
N
Cl ClH
Itraconazole
• Co-crystals display improved dissolution compared to the crystalline free base
• Comparable dissolution to the amorphous drug coated on beads – SPORANOX® capsule formulation
90
8x10-4
6
4
2
0
[1] (M
)
4003002001000
Time (min)
Sporanox® beads l-malic acid co-crystal
l-tartaric acid co-crystal succinic acid co-crystal
cis-itraconazole
Remenar et al. JACS, 125, 8456 (2003)
Co-crystal vs. marketed product
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91 ICH Q6A, Federal Register, 2000, 65(251), 83041-83063
Regulatory
IDENTIFY all relevant Crystals (or amorphous) for your compound
92 ICH Q6A, Federal Register, 2000, 65(251), 83041-83063
Regulatory
Understand the IMPACT of all relevant Crystals (or amorphous) for your product
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93
Lessons Learned
Crystals (or lack thereof) have significant impact on product quality and performance
Choosing the right Crystal is not a unit operation Staged decision points
Constant Vigilance
Crystals make your “molecule” a “medicine”
94
Special Thanks!
Ann Newman – Seventh Street Development Group and Crystal Pharmatech
Jun Huang – Crystal Pharmatech
Elizabeth Vadas – InSciTech Inc.
Örn Almarsson – Moderna Therapeutics
Carlos Sanrame – Crystal Pharmatech
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2016 Drug Design and Delivery Symposium
“Crystallography as a Drug Design and Delivery Tool”
The 2016 DDDS is co-produced with ACS Division of Medicinal Chemistry and the AAPS
Vincent Stoll Research Fellow and Associate
Director of Structural Biology, AbbVie
Robert Wenslow Vice President, Business
Development, Crystal Pharmatech
Andrew Brunskill Associate Principal Scientist,
Merck
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2016 Drug Design and Delivery Symposium
“Crystallography as a Drug Design and Delivery Tool”
The 2016 DDDS is co-produced with ACS Division of Medicinal Chemistry and the AAPS
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Director of Structural Biology, AbbVie
Robert Wenslow Vice President, Business
Development, Crystal Pharmatech
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Merck
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Future Protective Materials for First Responders, Football Players, and Astronauts: Shear Thickening Fluids
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