Kinase inhibitors in rheumatoid arthritis: JAK/ STAT signaling pathway

Kinase inhibitors in rheumatoid arthritis: JAK/ STAT signaling pathway Professor Peter C. Taylor MA, PhD, FRCP Tyrosine Kinase Inhibitors in Health and Rheumatic Diseases Ankara, Turkey. 12 March 211

Cytokines Regulate Many Biological Processes Hematopoietic SCF, IL-3, TPO, EPO, GM-CSF, G-CSF, M-CSF Growth/Differentiation PDGF, EGF, FGF, IGF, TGFβ, VEGF Immunoregulatory TGFβ, IFNγ, IL-2, IL-4, IL-5, IL-7, IL-9, IL-1, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-23 2 Pro-inflammatory IL-1α, IL-1β, TNFα, LT, IL-6, LIF, IL-17, IL-18 2, IL-33 3 Anti-inflammatory IL-1RA, IL-4, IL-1, IL-13 Chemotactic IL-8, MIP-1α, MIP-1β, MCP-1, RANTES *The definition of cytokine function is ever-changing, with many cytokines playing multiple roles. Some cytokines, such as IL-4 and IL-13, can be either pro -or anti-inflammatory, depending on the environment and circumstances. 1. Arend WP. Arthritis Rheum. 21;45:11-16; 2. Murphy K, et al. Janeway s Immunobiology. 7 th ed. NY: Garland; 28; 3. Hsu C-L, et al. PLOS One. 21;5:E1944. 2

Cytokines Are Implicated in Each Phase of RA Pathogenesis Cellular recruitment 1 Immunologic activation and organization 1 Cellular retention and survival 1 Tissue Response 1,2 BMPs=bone morphogenetic proteins, RANKL=receptor activator of nuclear factor Kappa B ligand. 1. McInnes IB, Liew FY. Nat Clin Pract Rheumatol. Nov 25;1:31-39. 2. Schett G, et al. Arthritis Rheum. 28;58:2936-2948. Figure adapted from McInnes IB, Liew FY. Nat Clin Pract Rheumatol. Nov 25;1:31-39. 3

Cytokines and Intracellular Signaling Pathways in RA

Cytokine Receptor Superfamilies Type I/II Cytokine TNF TGFβ Toll/IL-1 Chemokine Receptor tyrosine kinase Heterodimeric or homodimeric receptors Trimeric proteins usually associated with cell surface Receptor serine/ threonine kinase Single-pass membrane receptors with Toll-IL-1 receptor domains G-proteincoupled receptor family Enzyme-linked receptors specific for tyrosine residues Adapted from Baker SJ, et al. Oncogene. 27;26:6724-6737 and Murphy K, et al. Janeway s Immunobiology. 7 th ed. NY: Garland ; 28. 5

Type I/II Cytokine Receptor Families Growth hormone, erythropoietin, prolactin, thrombopoietin IL-2, IL-4 IL-7, IL-9 IL-15, IL-21 1 IL-3, IL-5 GM-CSF IL-6 IL-11, IL-31 2, (IL-12,23,27,35) 3 OSM,CNTF,CT-1 IFN α/β IFN γ IL-19,2,22, 25,28,29 α β γ c α β c α gp13 α β CNTF=ciliary neurotrophic factor, OSM=oncostatin M. 1. Leonard WJ. Nat Rev Immunol. 21;1:2-28; 2. Zhang Q, et al. Cytokine Growth Factor Rev. 28;19(5-6):347 356.3; 3. Collison LW, et al. Immunol Rev. 28;226:248 262. Figure adapted from Baker SJ, et al. Oncogene. 27;26:6724-6737. 6

JAK Pathways 1 Cytokine binding to its cell surface receptor leads to receptor polymerization and autophosphorylation of associated JAKs JAK JAK 2 Activated JAKs phosphorylate the receptors that dock STATs STAT P STAT STAT P 3 Activated JAKs phosphorylate STATs, which dimerize and move to the nucleus to activate new gene transcription STAT P STAT Gene transcription P 7 Figure adapted from Shuai K, et al. Nat Rev Immunol. 23;3:9-911. 7

Cytokines Signal Through Different JAK Combinations JAK 1 JAK 3 JAK 1 JAK 1 JAK 1 TYK 2 JAKs work in pairs of 2 different JAKs or pairs of identical JAKs 1,2 IL-2 IL-4 IL-7 IL-9 IL-15 IL-21 IL-1 IL-6 IL-11 OSM LIF CTNF IL-22 IFN-αβ JAK1 and JAK3 are required for γ-chain cytokine receptor signaling 1,2 JAK 1 JAK 2 JAK 2 JAK 2 JAK 2 TYK 2 IFN-γ G-CSF EPO TPO IL-3 IL-5 Leptin GM-CSF Prolactin GH IL-12 IL-23 GH=growth hormone. 1. Murray P. J Immunol. 27;178:2623-2629; 2. Ghoreschi K, et al. Immunol Rev. 29;228:273-287. 8

Signaling Through JAK Pathways Is Critical for Immune Cells Lethal JAK1 Perinatal lethality 1,2 JAK2 Embryonic lethality 1,2 Viable JAK3 SCID 1 TYK2 Pathogen hypersensitivity 1 SCID: Severe Combined Immunodeficiency Disease. 1. Shuai K, Liu B. Nat Rev Immunol. 23;3:9-911; 2. Leonard WJ. Nat Rev Immunol. 21;1:2-28. 9

JAK Pathway Signaling Is Important for Immune Cell Development, Survival, Proliferation, and Differentiation IL-2Rα IL-15Rα IL-2 IL-4 IL-7 IL-9 IL-15 IL-21 γ c γ c γ c γ c γ c IL-21R γ c IL-2Rβ IL-4R IL-7R IL-9R IL-2Rβ T-cell proliferation Cell death B-cell proliferation Th2 cell development T-cell and B-cell development Mucus production Mast-cell proliferation NK-cell development T-cell homeostasis Actions on T cells, NK cells, and B cells Figure adapted from Leonard WJ. Nat Rev Immunol. 21;1:2-28. 1

An Important Subset of Pro-inflammatory Cytokines Utilize JAK Pathways Key cytokines in the pathogenesis of RA 1 IFNα and IFNβ IL-6 IL-7 IL-1 IL-12 IL-15 IL-23 IL-1 IL-17 IL-18 TGF-β TNF Key cytokines in RA that utilize JAK 2,3 IFNα and IFNβ IL-6 IL-7 IL-1 IL-12 IL-15 IL-23 1. McInnes IB, et al. Nat Rev Immunol. 27;7:429-442; 2. Leonard WJ. Nat Rev Immunol. 21;1:2-28. 3. Riese RJ, et al. Best Pract Clin Res Rheumatol. 21;24:513-526. 11

Cytokine Expression and JAK3-specific Localization Indicate JAK Expression In the Synovium Normal synovium STAT1 STAT6 STAT4 JAK3 RA synovium Walker JG, et al. Ann Rheum Dis. 26;65:149-156. 12

Signaling Pathways in the Cycle of Chronic Inflammation 1 Pro-inflammatory cytokines recruit cells Cytokines 2 Cytokines activate cells through several signaling pathways 3 Signaling induces production of further proinflammatory signals Activated immune cells 4 Further recruitment and activation of cells and effector function occurs McInnes IB, Liew FY. Nat Clin Pract Rheumatol. 25;1(1):31-39. 13

Selected protein kinase inhibitors in clinical development Compound (developer) Target(s) Selected indications (Phase) INCB285 (Eli Lilly, Incyte) JAK1/2 Rheumatoid arthritis (Phase II) Tofacitinib (CP-69,55) (Pfizer) JAK1/3 Rheumatoid arthritis (Phase III), psoriasis (Phase II), inflammatory bowel disease (Phase II) VX-59 (Vertex) JAK3 Rheumatoid arthritis (Phase II) VX-72 (Vertex) p38 MAPK Rheumatoid arthritis (Phase II) BMS-582949 (Bristol-Myers Squibb) Fostamatinib/R-788 (AstraZeneca, Rigel Pharmaceuticals) p38 MAPK SYK Rheumatoid arthritis, atherosclerosis (Phase II), psoriasis (Phase I-II) Rheumatoid arthritis, B-cell lymphoma, immune thrombocytopaenic purpura, peripheral T-cell lymphoma, solid tumours (Phase II) JAK = janus kinase; p38 MAPK = mitogen-activated protein kinase; SYK = spleen tyrosine kinase Opar A. Nat Rev Drug Discov 21;9:257-8

Tofacitinib inhibits the JAK/Signal Transducer of Activated Transcription (STAT) Pathway Orally available, small molecule, highly selective inhibitor of the JAK family of kinases Displays nanomolar potency against JAK3; exhibits functional selectivity for JAK1 and JAK 3 over JAK2 Inhibits gamma cytokine signalling Some attenuation of signalling through additional cytokines, such as IFN-γ and IL-6 Tofacitinib Demonstrated efficacy in patients with rheumatoid arthritis (RA), 1,2 psoriasis, 3 and the prevention of acute renal allograph rejection 4 Also in Phase I/II trials in patients with ulcerative colitis, Crohn s disease, and dry eye 5 1 Kremer et al. Arthritis and Rheumatism 29; 6 (7): 1895-195; 2 Kremer et al. Arthritis and Rheumatism 28; 58 (12): 43-431 3 Boy et al. J Invest Dermatol 29; 129: 2299-232; 4 Busque et al. Am J Transplant 29; 9: 1936-1945 5 West. Curr Opin Investig Drugs 29; 1(5): 491-54 JAK, Janus kinase; IFN, interferon; IL, interleukin

Selectivity of Tofacitinib for the JAK Family of Kinases Small-molecule kinase interaction map depicts selectivity for the JAK family of kinases 1 Displays ~1-fold selectivity over 82 other kinases tested in a selectivity panel compared with the potency against JAK3 (1 nm) 2 Tyk2 (62nM) Jak2 (5nM) Jak3 (2.2nM) 1. Karaman MW, et al. Nature Biotech. 28;26(1):127-132; 2. Investigator s brochure, June 29. 2. Investigator s brochure and data on file.

CP-69,55 Inhibits JAK1 & JAK 3 with Functional Selectivity Over JAK2 JAK Enzyme JAK 3 JAK 1 JAK 2 TYK2 IC 5 nm (SEM) (Ki) 1.6 (.2). 2 3.2 (1.4) (.7) 4.1 (.4) (1.) 34 ( 6) (4) Cell IC 5 (nm) (JAK 1/3) (JAK1/2) (JAK 2) (TYK2/JAk2 HU. WB (SEM) 56 (6) 287 (83) 1377 (185) Mouse WB (SEM) 42 (12) 186 (73) 4379 (655) 3 ND % Control JAK inhibition in vitro (Human blood) 125 1 75 5 25-25 FACS analysis of Phospho-STAT 1 1 1 [CP-69,55] (nm) IL-15 CD8 T cells IL-6 Monocytes IL-6 CD8 T cells GM-CSF Monocytes JAK inhibition in vitro (Mouse blood) % of Control 125 1 75 5 25 FACS analysis of Phospho-STAT IL15 (JAk1/3) IL6 (JAk1/2)) -25 1 1 1 [CP-69,55] (nm) GMCSF (JAk 2)

CP-6955 Shows a Prolonged PD Effect Mouse CIA vehicle.5 mg/kg JAK mediated STAT phosphorylation assay 5 mg/kg dose 5 mg/kg 5 mg/kg Efficacy achieved in mouse CIA model at exposures that only inhibit JAK 1/3 & 1/2 signaling for 4-6 hrs per 12 hr dosing interval; little to no inhibition at minimum plasma concentrations Comparable efficacy is achieved in rat AIA model with BID, QD, and every-other-day dosing Ghosh, Lee, Jesson, Warner, Funckes-Shippy

CP-69,55 Normalizes Plasma Cytokines and Chemokines in the CIA Model 8 p =.8 4 p =.5 8 p =.1 6 5 p=.5 gm/ml plasma 6 4 2 pg/ml plasma 3 2 1 pg/ml plasma 6 4 2 pg/ml 4 3 2 1 SAA (CRP) IL-6 G-CSF CXCL9/MIG 12 p =.25 24 p =.1 6 p =.16 24 p =.44 pg/ml plasma 9 6 3 pg/ml plasma 18 12 6 pg/ml plasma 45 3 15 pg/ml plasma 18 12 6 KC/CXCL1 (IL8) IP-1/CXCL1 MCP-1/CCL2 MIP-1α /CCL3 Normal Vehicle CP-69,55

Rat Adjuvant-Induced Arthritis (AIA)- Model for Robust Bone Absorption Immunize M Butyricum in Squalene 5. 4.5 Therapeutic dosing design Sort into matched groups by edema QD dosing, ED8 @ 6.2 mg/kg Day 16 4hr 2 23 Assess for arthritis Rat AIA model T-cell mediated Primary arthritic lesion is synovitis, followed by pannus formation, lymphocyte infiltration, & robust bone destruction Increased level of inflammatory cytokines and acute phase proteins Paw volume (ml) 4. 3.5 3. 2.5 * * vehicle tasocitinib normal * p=.2 Monitor effects of Tasocitinib therapeutic dosing at 4h, 4 days and 7 days 2. 4 h 4 days Time after onset of therapy 7 days

Inflammation and Osteoclast Mediated Bone Resorption is Reversed by Tofacitinib Inflamm. Severity score Bone Severity score 5 4 3 2 1 5 4 3 2 1 vehicle tasocitinib normal 4 h 4 d 7 d Time after onset of therapy D vehicle tasocitinib normal * * vehicle 7 days # E vehicle 7 days * * tasocitinib 7 days F tasocitinib 7 days * 4 h 4 d 7 d Time after onset of therapy LaBranche, Shevlin Osteoclast Osteoblast

Tofacitinib Development Plan in RA 26 27 28 29 21 211 212 213 POC Mono 119 + MTX 125 Mono DMARD IR MTX IR Mono 135 MTX IR Japan Mono Japan Vs. TNFi (+ MTX) TNFi IR (+ MTX) DMARD naïve Monotherapy Structure Open Label Extension Studies Follow-up Study

Clinical Responses; tofacitinib studies % Response 6 wk MTX IR 12 wk MTX IR 12 wk mono 12 wk MTX IR 2 yr 12 wk mono 119 125 135 139 124 145 5mg 15mg 5mg 1mg 5mg 1mg 5mg 1mg 5/1 5mg 1mg ACR2 7 81 61 6 59 71 96 81 8 6 66 ACR5 33 54 37 31 37 44 82 58 6 31 37 ACR7 13 22 18 13 12 25 33 35 4 15 2 HAQ 57% 71% -.49 -.39 -.48 -.64 NR NR -.7 -.5 -.57 DAS28-2. -2.3 NR NR -2.1-2.5-2.8-3. -2.86-2.4-2.26 DAS28<2.6 15 35 18 29.6 1.8 16.1 35 45 NR 6 9.6 Phase II studies LTE Phase III

Safety; tofacitinib studies 6 wk MTX IR 12 wk MTX IR 12 wk mono 12 wk MTX IR 2 yr 12 wk mono % 119 125 135 139 124 145 Dose 5mg 15mg 5mg 1mg 5mg 1mg 5mg 1mg 5/1 5mg 1mg AE 59 75.4 66.2 67.6 55.1 59 7.4 76.9 86.8 51 56.7 SAE 1.4 5.6 9.5 1.6 3.7 7.7 17.5 <1 2. SIE 1.4 1.4 1.4 NR NR 3.2 <1 OI NR- Deaths NR D/Cs 2 AE 1.6 8.6 4.2 6.8 2 1.6 14.8 15.4 6.5 <1 2.4 AST (% pts ) NR NR 22 21 15 16 14.8 3.8.3 9.5 11.8 AL T (% pts ) NR NR 24 27 11 11 22.2 7.7.8 9.5 11.4 ANC ( ) 94 162 5 5 85 155 13 164? 83 135 LDL (% pts ) 16.4 28.4 17 2 18 26 3.7 3.8 NR 13.6 19.1 Phase II studies LTE Phase III

Tofaocitinib vs PBO in DMARD-IR First Phase 3 Trial Report 5 mg BID (n=243) 5 mg BID (n=243) 1 mg BID (n=245) 1 mg BID (n=245) Placebo (n=61) 5 mg BID (n=61) Placebo (n=61) 1 mg BID (n=61) Day 1 Randomization Month 3 Primary Endpoint Month 6 Study End Fleischmann, et al. ACR 21, Atlanta, #L8

Tofacitinib vs PBO in DMARD-IR Primary end-point at Month 3: ACR2, HAQ-DI improvement, % DAS28 remission PBO Tofa 5 mg BID Tofa 1 mg BID Baseline characteristics n=122 n=243 n=245 TJC, mean 28.9 29 29.1 SJC, mean 17.3 16.3 17. HAQ-DI, mean 1.53 1.53 1.5 DAS28(ESR), mean 6.63 6.71 6.67 ESR, mm/h 5.9 53.1 52.1 CRP, mg/l 17.8 22.9 19.1 RF and/or anti-ccp + (%) 72.9 83.1 8.4 Prior TNF inhibitor, % 19.7 14. 16.7 Prior MTX (%) 83.6 86. 84.5 Fleischmann, et al. ACR 21, Atlanta, #L8 26

Tasocitinib vs Placebo in DMARD IR: Efficacy ACR2 ACR5 ACR7 Patients (%) Patients (%) Patients (%) 1 6 2 1 2 3 4 5 6 6 4 2 4 3 2 * 1 2 3 4 5 6 PBO Tofa 5 mg BID Tofa 1 mg BID PBO 5 mg BID PBO 1 mg BID * p<.5 ** p<.1 p<.1 vs PBO 1 ** * ** * * 1 2 3 4 5 6 Month Fleischmann, et al. ACR 21, Atlanta, #L8

Tofacitinib vs PBO in DMARD-IR: Efficacy 1 2 3 4 5 6 Month HAQ-DI DAS28 <2.6 LS mean change from BL Patients (%) -.3 -.6 -.9 3 2 1 * ** Red dotted line indicates MCID HAQ (-.22) PBO Tofa 5 mg BID Tofa 1 mg BID PBO 5 mg BID PBO 1 mg BID DAS28(ESR) Improvement LS mean change from BL -1-2 -3 1 2 3 4 5 6 1 2 3 4 5 6 * p<.5 ** p<.1 p<.1 vs PBO Fleischmann, et al. ACR 21, Atlanta, #L8

Tofacitinib vs PBO in DMARD-IR: Safety Months 3 Months 3 6 PBO Tofa 5 Tofa 1 PBO Tofa 5 Tofa 5 PBO Tofa 1 Tofa 1 n=122 n=243 n=245 n=61 n=243 n=61 n=245 AE 127 279 295 4 17 54 27 Pts with AE (%) 54.9 51. 56.7 36.1 39.9 39.3 41.2 SAE (%) 4.9 <1. 2. 1.6 2.1 2.9 SIE (%) <1. 1.6 <1. 1.2 Confirmed Op Inf Deaths (%) <1. D/Cs 2 AE (%) 4.1 <1. 2.4 <1. 2. Fleischmann, et al. ACR 21, Atlanta, #L8

Tofacitinib vs PBO in DMARD-IR: Toxicity Month 3 Month 6 PBO Tofa 5 Tofa 1 Tofa Tofa 5 n=243 n=245 n=61 n=243 PBO Tofa 1 n=61 PBO n=122 Tofa 1 n=245 LS mean change from BL ANC, 1 3 /mm 3 -.6 -.83* -1.35** -.9 -.78-1.18-1.15 Hgb, g/dl -.12.28.3.21.25 -.22.15 % LDL change 3.5 13.6* 19.1** 16.9 12.8 16.8 19.1 Serum.4.5.6.6.8.8 creatinine Patients (%) n=122 n=243 n=245 n=57 n=239 n=52 n=232 ALT >3x ULN <1. <1. 1.75 <1. <1. AST >3x ULN <1. 1.7 1.8 <1. No significant differences between groups in AST/ALT 1 3x ULN; * P<.1;**P<.1 vs PBO Tofacitinib monotherapy efficacy and safety similar to Phase II Fleischmann, et al. ACR 21, Atlanta, #L8

Tofacitinib : Long-term extension study 1,7 pts from double-blind studies of Tofacitinib 5 mg or 1 mg BID 1 Allowed rescue drugs, background therapy, adjustments in dosages Efficacy maintained over 24 months 5 4 3 2 1 Incidence (%) per 1 pt-yrs Tofa Tofa + MTX 4,2 2,8 2,3 1,5 1,7 1,9,2 Anemia AST/ALT Cytopenia SIE SAE 2 : 66 Tofa vs 57 Tofa + MTX 18.4% infections 32 SIE (2.62/1 pt-yrs) 7 discontinued due to AEs All with SIE discontinued Clinical responses maintained in LTE 1. Connell, et al. ACR 21, Atlanta, #1129; 2. Connell, et al. Ibid, Atlanta, #2171

JAK 1/2 Inhibitor from Incyte / Lilly Phase II in 127 DMARD-IR pts Responders (%) 1 EP: Week 12 ACR and LDAS 4, 7 or 1 mg QD vs PBO 1 EP at 12 weeks *P<.5 (one sided) Disease duration: 7 9 yrs; BL DAS scores 6.1 6.3 * * * Failed biologics: 13, 38, 6 vs 2% PBO Frequent TEAEs: HA, URI, diarrhoea H. zoster: 2.1% active vs PBO HDL, LDL, and HDL:LDL ratios 1.6% vs PBO Positive proof of concept at Week 12 Greenwald, et al. ACR 21, Atlanta, #2172 *TEAE: Treatment emergent adverse emergent

Conclusions JAK pathways operate as one of several hubs in the inflammatory cytokine network. JAK pathways modulate the incoming signal of an important subset of proinflammatory cytokines. Tofacitinib is an orally available, small molecule, highly selective inhibitor of the JAK family of kinases with nanomolar potency against JAK3; exhibits functional selectivity for JAK1 and JAK 3 over JAK2 Phase 2 trials demonstrate impressive efficacy in patients with an incomplete response to MTX in combination with MTX and as monotherapy in DMARD failures Stable safety profile: Decreased neutrophils (not associated with infection) Elevation of lipids Elevation of transaminases (especially in combination with MTX)