Rheumatoid arthritis (RA) is a chronic, systemic disease characterized by persistent inflammatory synovitis that typically involves peripheral joints in a symmetric distribution 1. The synovial inflammation can cause cartilage destruction and bone erosions that are irreversible. To minimize the radiographic damage, it has been recognized that initiation of therapy with disease-modifying antirheumatic drugs (DMARDs) within 3 months after disease diagnosis is critical 2. The folic acid antagonist methotrexate (MTX) is the DMARD most commonly selected for initial therapy 2 and whose mechanism of action has been attributed, at least in part, to its ability to function as an antimetabolite. As such, the compound inhibits cell proliferation in the inflamed synovium but can affect other proliferating tissues, including gut and bone marrow, producing associated side effects. The use of biological response modifiers, such as tumor necrosis factor (TNF) antagonists, has grown due to efficacy observed in many patients and reasonable safety profile 3. However, the incomplete efficacy and/or toxicities observed with agents such as these create a need for additional therapies with novel mechanisms of action.

The key role that T cells appear to play in the pathogenesis of the disease has supported evaluation of calcineurin inhibitors such as cyclosporin A and tacrolimus in RA patients 4. Clinical efficacy for both calcineurin inhibitors has been reported, particularly in combination with other DMARDs such as methotrexate. However, the use of cyclosporine and tacrolimus in this patient population may be limited based upon the multiplicity and severity of associated adverse reactions. CP-690550 is a novel immunosuppressant that has not exhibited the safety liabilities associated with calcineurin inhibition, yet has demonstrated efficacy in a number of animal models including delayed-type hypersensitivity and cardiac allograft rejection 56. CP-690550 is a small molecule inhibitor of the tyrosine kinase Janus kinase 3 (JAK3), an enzyme that is associated with the common gamma chain (γc) of various cytokine receptors and is critical for signal transduction by interleukin (IL)-2, -7, -15 and -21 7. Interestingly, JAK3 expression has been shown to decrease in the synovial tissue biopsies from active rheumatoid arthritics receiving and responding to DMARD therapy 8.

Since multiple cytokines whose receptors signal through pathways involving JAK3 have been associated with progression of arthritis, experiments were designed to evaluate the effects of CP-690550 in rodent models of the disease. Neither murine collagen-induced arthritis (CIA) nor adjuvant-induced arthritis (AA) in rats are identical to RA, but both share the common features of inflammation of the synovial membrane, erosion of bone, and cartilage degradation. In both models of RA, we observed dose-dependent inhibition of disease endpoints that correlated with reduction in histological changes. These data support JAK3 inhibition as a new target for the treatment of RA.

CP-690550 produced significant dose-dependent attenuation of inflammatory swelling, cell influx and cartilage damage in two well characterized rodent models. A T cell contribution to disease has been demonstrated in both models 151617. In murine CIA, the magnitude of effects observed at the highest dose of the JAK3 inhibitor tested (15 mg/kg/d) were greater than those following administration of anti-TNF antibody (TN-1912) when assessing clinical scores and histology. The magnitude of effect of anti-TNF that we observed on the clinical arthritis score is consistent with that reported previously 101112 when animals were dosed with the same anti-TNF mAb. Anti-TNF treatment is efficacious in murine CIA when dosed before or immediately after the onset of CIA (see review of the role of TNF and IL-1 in CIA; 18). Even though we did begin treating the mice immediately after disease induction, the fact that anti-TNF treatment was not as efficacious as treatment with CP-690550 in murine CIA could be due to the role of IL-1 or other inflammatory mediators in this animal model.

CP-690550 doses/exposures that produced effects in this model are consistent with those demonstrating immune suppression in other murine models including delayed-type hypersensitivity and cardiac allograft transplantation 56. Interestingly, both CP-690550 (78% reduction vs control) and the anti-TNF mAb (68% reduction vs control) significantly reduced serum IL-6 levels. IL-6 has been proposed to play an important role in the development of CIA based upon delay in onset and reduction in disease magnitude observed in mice genetically deficient in this cytokine 19. The effects of anti-TNF on IL-6 are consistent with other reports in which inhibition of TNF action, either via genetic ablation of its receptor 20 or via anti-TNF mAb 2122 were found to down-modulate levels of IL-6. However, in our studies, anti-TNF mAb treatment reduced serum IL-6 by a similar magnitude as CP-690550 but did not demonstrate the same degree of efficacy, which suggests the JAK3 inhibitor, affected other inflammatory mediators important for expression of disease in this model. A role for IL-6 in rheumatoid arthritis has been proposed based upon the ability of the cytokine to activate inflammatory responses and osteoclastogenesis and is supported by positive clinical data obtained with the anti-IL-6 mAb tocilizumab in this patient population 23.

The efficacy produced by CP-690550 in the rodent models of arthritis may result from its ability to affect signaling of a number of cytokines including IL-2, -7, -15 and -21 as a consequence of JAK3 inhibition 5. IL-2 mRNA was found to be markedly increased in arthritic paws from mice with CIA during the early phases of disease 24. This may explain the efficacy observed following prophylactic administration of an anti-IL2R antibody in this model 25. When mice with established disease were treated with cyclosporine 50 or 75 mg/kg/day, disease was also attenuated 26. Tacrolimus is another, albeit more potent, calcineurin inhibitor that has also demonstrated efficacy in experimental models of rheumatoid arthritis 27. In rat arthritis models, tacrolimus suppressed paw inflammation, type II collagen antibody formation and delayed-type hypersensitivity to type II collagen 2728. While clinical trials of tacrolimus in rheumatoid arthritis have been conducted, it appears that the compound has a narrow therapeutic window which limits its utility 29.

IL-15 is a cytokine with close homology to IL-2 whose receptor shares signaling through the common gamma chain. Previous studies from our lab have demonstrated that CP-690550 inhibits IL-15-mediated up-regulation of activation markers on CD8+ T cells and NK cells 30. Upon chronic treatment with CP-690550, there is a preferential loss of these cells from the circulation, which is consistent with a role for IL-15 in their survival 630. Evidence is emerging for the importance of IL-15 in the pathogenesis of rheumatoid arthritis. Elevated serum levels of the cytokine have been reported in arthritic patients, the primary source of which may be macrophages residing in the synovial lining layer of inflamed joints 31. IL-15 produces a number of effects which may be relevant to the pathogenesis of arthritis including recruitment and activation of T lymphocytes into the synovial membrane and induction of TNFα production 3233. A soluble fragment of the murine IL-15Rα chain inhibited development of collagen-induced arthritis in DBA/1 mice 34. Administration of an IL-15 mutant/Fcγ2c fusion protein in established murine CIA blocked disease progression and reduced long term articular inflammation and destruction 33. The therapeutic benefit achieved by inhibiting IL-15 is supported by evidence that HuMax-IL-15, a fully human anti-IL-15 mAb, produced encouraging signs of efficacy in rheumatoid arthritis patients 35.

IL-21 is a cytokine produced by activated CD4+ T cells that also signals through JAK3. It enhances T cell activation, proliferation and secretion of pro-inflammatory cytokines such as TNFα and IL-21R has been shown to be over-expressed in inflamed synovial membrane and peripheral blood or synovial fluid leukocytes of rheumatoid arthritis patients 36. A recent publication reported that blockade of IL-21 effects with a murine IL-21 receptor Fc fusion protein attenuated disease in both mouse and rat models of arthritis 22. Effects in a 'semi-therapeutic' murine CIA model (compound administration begun when 10% of mice began to exhibit clinical signs of disease) included reduction in disease severity scores (including histology) and serum IL-6 levels. Effects produced by IL-21RFc were even more profound in a rat adjuvant-induced arthritis model in which full amelioration of clinical signs was achieved in conjunction with significant reduction in histological damage 22. Recent evidence demonstrates that IL-21 is a key cytokine involved in the generation of Th17 cells which have been shown to mediate tissue inflammation via production of IL-17 3738. Thus it is possible that CP-690550, through inhibition of IL-21R signaling, may also be efficacious in the CIA model by reducing IL-17 producing Th17 cells which have been proposed to play an important role in the pathogenesis of autoimmune diseases.

IL-7 represents another member of the IL-2 family that signals through the common gamma chain. It plays a key role in T cell homeostasis supporting growth, proliferation and survival of developing and mature T cells. In mice, unlike humans, its absence or blockade results in a diminution of B cell numbers as was evident in our own studies that examined the effects of chronic CP-690550 administration on circulating lymphocytes 6. IL-7 has also been suggested to play a role in rheumatoid arthritis based upon the observation of increased levels of the cytokine in this patient population, its ability to induce TNFα and induction of bone loss by stimulation of RANKL-dependent osteoclastogenesis 39.

The potential for CP-690550 to attenuate multiple cytokines associated with rheumatoid arthritis by virtue of its ability to inhibit JAK3 may provide improved efficacy vs a single agent. For example, TNF antagonists rarely induce complete disease remission and not all patients respond to TNF-blocking therapies 3. IL-1 antagonism also demonstrates some effectiveness albeit to a lesser extent than TNF blockers 40. However, combined inhibition of these two cytokines has been shown to provide increased benefit relative to inhibition of either alone 41. Hence, JAK3 inhibition provides a potentially beneficial target for the treatment of RA based upon its ability to inhibit multiple cytokines known to be involved in the pathogenesis of the disease.

CP-690550, a potent inhibitor of JAK3, reduced the clinical and histological manifestations of joint inflammation, including bone and cartilage damage, when administered therapeutically in murine CIA and rat AA. The effects of CP-690550 were dose dependent and higher doses were required for suppression of CIA histopathology than clinical manifestations. These data support the evaluation of CP-690550 for DMARD activity in RA patients.

AA = adjuvant-induced arthritis; CIA = collagen-induced arthritis; DMARD = disease-modifying antirheumatic drug; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; JAK3 = Janus kinase 3; LLOQ = lower limit of quantification; RA = rheumatoid arthritis; TNF = tumor necrosis factor.

All authors were, or currently are employed by Pfizer Global Research and Development. Pfizer is financing the publication of this manuscript.

AJM was responsible for histology, data analysis and manuscript writing, EK for data analysis and manuscript writing, LA for all Pfizer in vivo animal work, SZ for assistance with manuscript writing, and PC for concept and assistance with manuscript writing.