Presentation by E. Berg at the CHI Conference on Drug Discovery Chemistry: Anti-inflammatory Small Molecule Approaches, April 17, 2012, San Diego, CA

1. Selection of safer and more effective anti-inflammatory
kinase inhibitors using a platform of primary human cell
based disease models (BioMAP® systems)
Ellen L. Berg
17 April 2012

2. Challenges of Kinase Drug Discovery
 Defining optimal target selectivity
• Kinase gene family members (> 500 members) are highly related
• Biochemical ≠ cellular efficacy
 Limited knowledge about target biology
• Individual kinases can play a role in multiple pathways - complex signaling
• Is there target biology that we are missing?
 Unclear significance of secondary activities
• There will be secondary targets at a high enough concentration
• Are secondary targets affecting safety and/or efficacy?
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3. Solution – Better Biology Tools
 BioMAP Systems
• Primary human cell-based disease & tissue models
• Link in vivo complexity with in vitro reproducibility
 Simultaneous evaluation of compounds across a broad
range of human biology
• Reveal unexpected activities
• Test compounds in more “physiological” settings
• Build confidence in therapeutic hypotheses, prioritized leads
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4. BioMAP® Technology Platform
BioMAP Reference Predictive
Assays Profile Database Informatics Tools
Compound Validation
Lead Optimization
LPS Efficacy Biomarkers
Safety Pharmacology
BF4T
Adverse Effects
SM3C
Human primary cells Biomarker responses to drugs Specialized informatics tools are
Disease-models are stored in the database used to predict clinical outcomes
30+ systems
Human Biology Integrated into a Robust, Scalable Platform
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5. BioMAP® Systems
Human primary cell-based assays
Engineered to model complex human disease biology
 Physiologically relevant assay conditions
• Mixtures of stimulation factors, co-cultures of cells
• Complex culture conditions selected to achieve stable
signaling networks that reflect in vivo tissue & disease states
LPS
 Quantitative and reproducible
• Robust readouts (proteins, mediators); standardized assays
BF4T
• Assay formats manage disease variations among donors
 Validated with known drugs
HSM3C
• Large reference database
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6. Example Compounds in Development
 Tofacitinib (CP-690,550)
• Jak 3 kinase inhibitor – FDA review for rheumatoid arthritis
 Fostamatinib (R788)
• Syk kinase inhibitor – Phase III for rheumatoid arthritis
 CAL-101 (GS-1101)
• PI3K- inhibitor – Phase III oncology
 RN786
• BTK inhibitor - Preclinical
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7. Tofacitinib
Tofacitinib
CP-690,550
 Jak 3 kinase selective inhibitor
 Kinase activity: EC50s: Jak3 (1 nM); Jak2 (20 nM); Jak1 (112 nM); Rock-II (3.4 M) and Lck
(3.8 M) (Changelian, Science 2003, 302:875). Binding: JAK3 (2.2 nM) and Jak2 (5 nM)
(Karaman, Nat Biotech 2008, 26:127).
 Safe and effective in rheumatoid arthritis
 Kremer, Arthr & Rheum 2012, 60:1895; Fleischmann, Arth & Rheum 2012, 64:619
 In review at the FDA for rheumatoid arthritis (Pfizer)
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8. BioMAP Profile of Tofacitinib
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9. BioMAP® Systems
BioMAP Systems
 10 primary human cell types
 12 assay systems
• Different cell types, co-culture combinations
• Different activation conditions – disease models
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10. BioMAP® Systems – Cell Types & Co-cultures
BioMAP Systems
Endothelial Cells
Peripheral Blood Mononuclear Cells
B cells
Bronchial Epithelial Cells
Dermal Fibroblasts
Smooth Muscle Cells
Keratinocytes
Lung Fibroblasts
Macrophages
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11. BioMAP® Systems – Biology Covered
Vascular Biology
Immune Biology
Lung Biology
Skin Biology
Wound Healing
Th1 Inflammation
Th2 Responses
Th17 Biology
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12. BioMAP Profile of Tofacitinib
BioMAP Systems
95% significance envelope
Control (no drug)
Log expression ratio Dose
(Drug/DMSO control) Response
Cytotoxicity Readouts
Readout Parameters (Protein Biomarkers)
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13. BioMAP Profile of Tofacitinib (≤ 370 nM)
IL-2
IL-6 Lin, 2010
Eotaxin-3 TNF De Paz, 2010
IgG Kutukculer, 1998
 Key activities of Tofacitinib (CP-690,550 ) ≤ 370 nM
• Inhibition of IL-4 dependent signaling in endothelial cells (4H system)
• Selective inhibition of T-cell-dependent B cell activation (BT system)
• Several activities consistent with clinical efficacy biomarkers (in red)
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14. BioMAP Profile of Tofacitinib (≥ 370 nM)
IL-2
CD69
IL-6 ICAM VCAM MIG
VCAM CD38 HLA-DR IP-10
CD40 TNF IP-10
Eotaxin-3 IP-10 HLA-DR
MIG IL-17A, F MIG
MIG MIG
HLA-DR IgG
 Many additional activities at higher doses (≥ 370 nM):
• Most are clinical efficacy biomarkers for RA (Kuan, 2010; Klimiuk, 2002; Kutukcular,
1998; Dolhain, 1998; Metawi, 2011) (in red)
 Activities are consistent with clinical effects and dosing
• Van Gurp, Transpl. 2009, 87:79
• Cmax in one clinical study was ~1 M (Cohen, BJCP 2010, 69:143)
 Higher dose (less selective) profile is more “efficacious” in BioMAP
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and in the clinic

15. Fostamatinib (R788)
R788
Fostamatinib
 Syk kinase inhibitor
 Kinase activity: IC50= 41 nM (active form). Braselmann, JPET 2006, 319:998
 Well tolerated in Phase II studies for rheumatoid arthritis
 Genovese, Arth & Rheum 2011, 63:337
 Currently in Phase III clinical trials for RA (Rigel/AstraZeneca)
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16. BioMAP Profile of Fostamatinib
TF IL-8 MMP3uPAR
uPAR uPA uPAR MIP1
IL-1 IL1
Eotaxin-3 MCP-1 IL-6
TNF
MIG
 Key activities of Fostamatinib (R788)
• Broadly active in endothelial cells, leukocytes, epithelial cells and SMC
• Inhibition of monocyte activation, T cell activation, and T-dependent B cell activation
• Many activities (in red) consistent with clinical efficacy biomarkers (Szekanecz, 1997;
Klimiuk, 2002, 2005; Kutukculer, 1998; Kaun, 2010)
 Consistent with Clinical effects
• Peak serum concentrations of > 800 ng/ml (1.4 M) reported in clinical studies
(Podolanczuk, Blood 2009, 113:154)
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17. Expression of Syk Kinase in BioMAP Systems
Expression of Syk mRNA in BioMAP Systems (AU units)
3C 4H LPS SAg BT BF4T BE3C CASM3C HDF3CGF KF3CT MyoF /Mphg
100.4 98.5 133 137.3 550 251.3 165.3 179.1 108.3 87 108.1 733.7
 Fostamatinib is active in systems where syk kinase mRNA is not expressed
 Fostamatinib is not a selective syk inhibitor at clinically relevant doses
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18. PI3-K and BTK Inhibitors
B cell IL-6
Proliferation IL-2
IgG TNF
 Cal-101 (GS-1101) – PI3-K delta inhibitor
• Lannutti, Blood 2011, 117:591
• In clinical trials for CLL (Phase 3) and NHL (Phase 2)
 RN486 – BTK inhibitor
• Active in CIA and AIA models of RA
• Xu, JPET 2012, 341:90
 PI3K and BTK inhibitors are highly selective
18 • Is this sufficient for efficacy in rheumatoid arthritis??

19. Clinical Standards of Care
SAA
E-sel IL-8 IP-10 MMP-9 IL-8
TNF IL-8 IL-6
E-sel MCP-1 VCAM MCP-1
IL-2
IL-8 VCAM
IL-17 E-sel
TNF IgG
 Rheumatoid Arthritis - Clinical Standards of Care
• Methotrexate – inhibitor of DHFR
• Selective inhibitor of T cell dependent B cell activation
• Prednisolone – corticosteroid
• Inhibition of macrophage > monocyte activation; T cell activation
• Remicade – TNF antagonist
• Inhibition of monocyte > macrophage activation; myofibroblast activation
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20. Summary
 Clinically effective kinase inhibitors for rheumatoid
arthritis are not selective
• Tofacitinib and fostamatinib are broadly acting agents
• Selective PI-3K delta inhibitors are progressing in oncology
indications, but not arthritis
• Selective BTK inhibitors remain to be tested in patients
 BioMAP systems of primary human cell based assays
can be useful for characterizing novel kinase inhibitors
• Comparison to standards of care
• Testing combination therapies
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21. Acknowledgements
 BioSeek  Roche
• Alison O’Mahony • Daigen Xu
• Mark A. Polokoff • Dinesh Srinivasan
• Dat Nguyen • Jay Fine
• Julie DeMartino
 Cellzome
• Oliver Rausch
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