Paul S. Changelian

Discovery of a Novel, Potent, and Src Family-selective Tyrosine Kinase Inhibitor STUDY OF Lck- AND FynT-DEPENDENT T CELL ACTIVATION

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

A new modality for immunosuppression: targeting the JAK/STAT pathway

Thousands of organs are transplanted each year and millions of people suffer from autoimmune diseases, which creates a need for an armamentarium of immunosuppressive drugs. Unfortunately, immunosuppressants have unwanted side effects owing, in part, to the fact that they have ubiquitous molecular targets. Cytokines have emerged as important controllers of the immune response, and work during the past decade has identified Janus kinases (JAKs) and signal transducers, and activators of transcription (STATs), as crucial intracellular elements in cytokine signalling. Here, we discuss the potential of the JAK/STAT pathway as a target for new immunosuppressants. In particular, the inhibition of JAK3 seems to be an excellent strategy, because of the selective expression and precise functions of this kinase.

Discovery of CP-690,550: A Potent and Selective Janus Kinase (JAK) Inhibitor for the Treatment of Autoimmune Diseases and Organ Transplant Rejection

There is a critical need for safer and more convenient treatments for organ transplant rejection and autoimmune disorders such as rheumatoid arthritis. Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are involved in the signaling of multiple cytokines important for various T cell functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was anticipated to provide therapeutic immunosuppression/immunomodulation. The Pfizer compound library was screened against the catalytic domain of JAK3 resulting in the identification of a pyrrolopyrimidine-based series of inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast proliferation assay. Select compounds were evaluated in rodent efficacy models of allograft rejection and destructive inflammatory arthritis. Optimization within this chemical series led to identification of CP-690,550 1, a potential first-in-class JAK inhibitor for treatment of autoimmune diseases and organ transplant rejection.

Cartilage preservation by inhibition of Janus kinase 3 in two rodent models of rheumatoid arthritis

IntroductionCP-690550 is a small molecule inhibitor of Janus kinase 3 (JAK3), a critical enzyme in the signaling pathway of multiple cytokines (interleukin (IL)-2, -7, -15 and -21) that are important in various T cell functions including development, activation and homeostasis. The purpose of this study was to evaluate CP-690550 in murine collagen-induced (CIA) and rat adjuvant-induced (AA) models of rheumatoid arthritis (RA).MethodsCIA and AA were induced using standard protocols and animals received the JAK3 inhibitor via osmotic mini-pump infusion at doses ranging from 1.5–15 mg/kg/day following disease induction. Arthritis was assessed by clinical scores in the CIA models and paw swelling monitored using a plethysmometer in the AA model until study conclusion, at which time animals were killed and evaluated histologically.ResultsCP-690550 dose-dependently decreased endpoints of disease in both RA models with greater than 90% reduction observed at the highest administered dose. An approximate ED50 of approximately 1.5 mg/kg/day was determined for the compound based upon disease endpoints in both RA models examined and corresponds to CP-690550 serum levels of 5.8 ng/ml in mice (day 28) and 24 ng/ml in rats (day 24). The compound also reduced inflammatory cell influx and joint damage as measured histologically. Animals receiving a CP-690550 dose of 15 mg/k/d showed no histological evidence of disease.ConclusionThe efficacy observed with CP-690550 in CIA and AA suggests JAK3 inhibition may represent a novel therapeutic target for the treatment of RA.

The Novel JAK-3 Inhibitor CP-690550 Is a Potent Immunosuppressive Agent in Various Murine Models

JAK‐3 has been shown to play a key role in cytokine signaling via γc, e.g. IL‐2, 4, 7, 9, 15, 21. The current study describes the immunosuppressive effects of CP‐690550, a novel, small molecule inhibitor of JAK‐3, in various murine models. In vitro, CP‐690550 effectively inhibited a murine mixed lymphocyte reaction (MLR) (IC50= 91 nm). Mice chronically dosed with CP‐690550 (1.5–15 mg/kg/day) demonstrated dose‐ and time‐dependent alterations in lymphocyte subsets when examined by flow cytometry. The most dramatic change observed was a 96% reduction in splenic NK1.1 + TCRβ– cell numbers following 21 days of treatment. Delayed‐type hypersensitivity (DTH) responses in sensitized mice were reduced in a dose‐dependent manner following treatment with the JAK‐3 inhibitor (1.87–30 mg/kg, s.c.). Extended survival of neonatal Balb/c hearts implanted into the ear pinna of MHC mismatched C3H/HEN mice was observed with CP‐690550 monotherapy (10–30 mg/kg/day), but improved upon combination with cyclosporin (10 mg/kg/day). These data support the participation of JAK‐3 in various lymphocyte homeostatic functions in mature mice. Furthermore, the ability of CP‐690550 to extend cardiac allograft survival in murine models suggests it may afford a new treatment for prevention of transplant rejection.

Double-Blind, Placebo-Controlled, Dose-Escalation Study to Evaluate the Pharmacologic Effect of CP-690,550 in Patients With Psoriasis

CONFLICT OF INTEREST The authors state no conflict of interest. Penkanok Sriwiriyanont, Akira Hachiya, William L. Pickens, Shigeru Moriwaki, Atsushi Ohuchi, Takashi Kitahara, Yoshinori Takema, William J. Kitzmiller, Marty O. Visscher, Alexander Bello, Ryoji Tsuboi and Gary P. Kobinger Skin Sciences Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Kao Biological Science Laboratories, Haga, Tochigi, Japan; Department of Dermatology, Tokyo Medical University, Tokyo, Japan; Department of Surgery, University of Cincinnati, Cincinnati, OH, USA; Special Pathogens Program, Department of Medical Microbiology, National Microbiology Laboratory, Public Health Agency of Canada, University of Manitoba, Winnipeg, Canada E-mail: gary_kobinger@phac-aspc.gc.ca

Unexpected Effects of FERM Domain Mutations on Catalytic Activity of Jak3: Structural Implication for Janus Kinases

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.

The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in a murine model of pulmonary eosinophilia.

Janus kinase 3 (JAK-3) is a tyrosine kinase that has been shown to participate in the signaling of several cytokines that are believed to play a role in allergic airway disease, e.g. IL-2, 4 and 9. The current study describes the immunosuppressive effects of CP-690550, a novel, small molecule inhibitor of JAK-3, in a murine model of allergic pulmonary inflammation. In vitro, CP-690550 potently inhibited IL-4 induced upregulation of CD23 (IC(50)=57 nM) and class II major histocompatibility complex (MHCII) expression (IC(50)=71 nM) on murine B cells. Repeat aerosol exposure to ovalbumin in wild-type mice sensitized to the antigen resulted in preferential recruitment of Th2-like cells (IL-4+ and IL-5+) into bronchoalveolar lavage fluid (BAL). The importance of IL-4 in the development of pulmonary eosinophilia was supported by a marked (90%) reduction in the influx of these cells in IL-4KO mice similarly sensitized and ovalbumin exposed. Animals dosed with CP-690550 (15 mg/kg/d) during the period of antigen sensitization and boost demonstrated marked reductions in BAL eosinophils and levels of IL-13 and eotaxin following ovalbumin aerosol exposure. The JAK-3 inhibitor (1.5-15 mg/kg/d) also effectively reduced the same parameters when administered during the period of antigen challenge. In contrast, the calcineurin inhibitor tacrolimus (10 mg/kg) was effective only when administered during the period of ovalbumin aerosol exposure. These data support the participation of JAK-3 in processes that contribute to pulmonary eosinophilia in the allergic mouse model. CP-690550 represents an intriguing novel therapy for treatment of allergic conditions associated with airway eosinophilia including asthma and rhinitis.

Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (&ggr;c). Because mutations in genes encoding &ggr;c or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. Methods. Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n=18) or its vehicle (controls, n=3) and were euthanized at day 90 or earlier if there was allograft rejection. Results. Mean survival time (± standard error of mean) in animals treated with CP-690,550 (53±7 days) was significantly longer than in control animals (7±1 days, P=0.0003) and was positively correlated with exposure to the drug (r=0.79, P<0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46±7 days from transplantation vs. 7±1 days in controls, P=0.0003). Persistent anemia, polyoma virus-like nephritis (n=2), and urinary calcium carbonate accretions (n=3) were seen in animals with high exposure. Natural killer cell and CD4+ and CD8+ T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. Conclusions. CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

Jakinibs: a new class of kinase inhibitors in cancer and autoimmune disease

Cytokines are critical for normal cell growth and immunoregulation but also contribute to growth of malignant cells and drive immune-mediated disease. A large subset of immunoregulatory cytokines uses the type I and type II cytokine receptors and pharmacological targeting of these cytokines/cytokines receptors has proven to be efficacious in treating immune and inflammatory diseases. These receptors rely on Janus family of kinases (Jaks) for signal transduction. Recently the first Jak inhibitor (jakinib) has been approved by the FDA and a second has been recommended for approval. Many other Jakinibs are likely to follow and in this brief review, we will discuss the state-of-the art of this new class of pharmacological agents.