selection of safer and more effective anti-inflammatory kinase inhibitors using a platform of...
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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
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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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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BioMAP® Technology Platform
BioMAPAssays
Human primary cells Disease-models
30+ systems
LPS
BF4T
SM3C
ReferenceProfile Database
Predictive Informatics Tools
Biomarker responses to drugs are stored in the
database
Specialized informatics tools are used to predict
clinical outcomes
Human Biology Integrated into a Robust, Scalable Platform
Compound Validation
Lead Optimization
Efficacy Biomarkers
Safety Pharmacology
Adverse Effects
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BioMAP® Systems
Human primary cell-based assays Engineered to model complex human disease biology
LPS
BF4T
HSM3C
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
Quantitative and reproducible • Robust readouts (proteins, mediators); standardized assays
• Assay formats manage disease variations among donors
Validated with known drugs• Large reference database
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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-d inhibitor – Phase III oncology
RN786• BTK inhibitor - Preclinical
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Tofacitinib
TofacitinibCP-690,550
Jak 3 kinase selective inhibitor Kinase activity: EC50s: Jak3 (1 nM); Jak2 (20 nM); Jak1 (112 nM); Rock-II (3.4 mM) and Lck
(3.8 mM) (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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BioMAP Profile of Tofacitinib
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BioMAP® SystemsBioMAP Systems
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10 primary human cell types 12 assay systems• Different cell types, co-culture combinations• Different activation conditions – disease models
BioMAP® Systems – Cell Types & Co-cultures
B cells
Endothelial Cells
Bronchial Epithelial Cells
Keratinocytes
Smooth Muscle Cells
Dermal Fibroblasts
Lung Fibroblasts
Peripheral Blood Mononuclear Cells
Macrophages
BioMAP Systems
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BioMAP® Systems – Biology Covered
Vascular Biology
Wound Healing
Th1 Inflammation
Skin Biology
Th2 Responses
Immune Biology
Th17 Biology
Lung Biology
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BioMAP Profile of Tofacitinib
95% significance envelope
Control (no drug)
DoseResponse
Readout Parameters (Protein Biomarkers)
Cytotoxicity Readouts
BioMAP Systems
Log expression ratio (Drug/DMSO control)
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BioMAP Profile of Tofacitinib (≤ 370 nM)
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)
Eotaxin-3
IgG
TNFa
IL-2
IL-6
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IgG
TNFaIL-6
De Paz, 2010Lin, 2010
Kutukculer, 1998
BioMAP Profile of Tofacitinib (≥ 370 nM)
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 mM (Cohen, BJCP 2010, 69:143)
Higher dose (less selective) profile is more “efficacious” in BioMAP and in the clinic
Eotaxin-3
IgG
TNFa
IL-2
MIGHLA-DR
VCAM CD38
MIG
CD40IL-17A, F
MIG
HLA-DRMIG
HLA-DRICAM
IP-10
MIGIP-10IP-10
VCAMIL-6
CD69
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IgG
TNFa
MIG
VCAM CD38
MIG IL-17A, FMIG
MIGIP-10IP-10
VCAMIL-6 ICAM
IP-10MIG
Fostamatinib (R788)
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)
R788Fostamatinib
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BioMAP Profile of Fostamatinib
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 mM) reported in clinical studies (Podolanczuk, Blood 2009, 113:154)
Eotaxin-3 TNFa
TF uPAR
MIG
uPAR
IL-6
IL1auPAR
MCP-1
MIP1auPAIL-8IL-1a
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MMP3
TNFa
uPAR
MIG
uPAR
IL-6
uPAR
MCP-1
IL-8 MMP3
Expression of Syk Kinase in BioMAP Systems
Expression of Syk mRNA in BioMAP Systems (AU units)
Fostamatinib is active in systems where syk kinase mRNA is not
expressed
Fostamatinib is not a selective syk inhibitor at clinically relevant doses
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
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PI3-Kd and BTK Inhibitors
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
PI3Kd and BTK inhibitors are highly selective • Is this sufficient for efficacy in rheumatoid arthritis??18
IgG
IL-6IL-2
B cellProliferation
TNFa
Clinical Standards of Care
E-sel
IgG
TNFaIL-8
E-sel
IL-17
IL-2
TNFa
IP-10IL-8
MMP-9IL-6
MCP-1 VCAM
IL-8
MCP-1VCAM
E-sel
IL-8
SAA
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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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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Acknowledgements
BioSeek• Alison O’Mahony• Mark A. Polokoff• Dat Nguyen
Roche• Daigen Xu• Dinesh Srinivasan• Jay Fine• Julie DeMartino
Cellzome• Oliver Rausch
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