Elizabeth M. Kudlacz

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.

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.

The JAK3 inhibitor CP-690550 selectively reduces NK and CD8+ cell numbers in cynomolgus monkey blood following chronic oral dosing

Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase associated with the common γ chain, an integral component of cytokine receptors of the interleukin (IL)‐2 family, including IL‐4, ‐7, ‐9, ‐15, and ‐21. CP‐690550 is a JAK3 inhibitor with immunosuppressive properties under development for transplantation. We evaluated alterations in circulating lymphocyte subsets in cynomolgus monkey blood following chronic (3‐week), oral CP‐690550 administration. Natural killer (NK) and CD8+ T cell numbers were reduced in a dose‐ and time‐dependent manner; the latter was a primary effect on memory subsets. CD4+ T and B cell numbers were unaffected or slightly increased, respectively. NK cell numbers were reduced ∼80% (vs. 35% in vehicle‐treated animals) and returned to baseline levels within 3 weeks following treatment cessation. CD8+ T cells declined by a maximum 43% (vs. 25% for vehicle‐treated animals) but rebounded significantly (300%) within 2 weeks after the last dose. Although CP‐690550 did not result in reduction of CD4+ T cell number, these cells also increased (225%) within 2 weeks of treatment cessation. IL‐15 is important for maintaining homeostasis of these cell types, and CP‐690550 inhibited IL‐15‐induced CD69 expression in NK cells [inhibitory concentration 50% (IC50)=48.0±8.4 nM] and CD8+ T cells (IC50=16.2±1.5 nM).

Randomized Phase 2b Trial of Tofacitinib (CP‐690,550) in De Novo Kidney Transplant Patients: Efficacy, Renal Function and Safety at 1 Year

In this Phase 2b study, 331 low‐to‐moderate risk de novo kidney transplant patients (approximately 60% deceased donors) were randomized to a more intensive (MI) or less intensive (LI) regimen of tofacitinib (CP‐690, 550), an oral Janus kinase inhibitor or cyclosporine (CsA). All patients received basiliximab induction, mycophenolic acid and corticosteroids. Primary endpoints were: incidence of biopsy‐proven acute rejection (BPAR) with a serum creatinine increase of ≥0.3 mg/dL and ≥20% (clinical BPAR) at Month 6 and measured GFR at Month 12. Similar 6‐month incidences of clinical BPAR (11%, 7% and 9%) were observed for MI, LI and CsA. Measured GFRs were higher (p < 0.01) at Month 12 for MI and LI versus CsA (65 mL/min, 65 mL/min vs. 54 mL/min). Fewer (p < 0.05) patients in MI or LI developed chronic allograft nephropathy at Month 12 compared with CsA (25%, 24% vs. 48%). Serious infections developed in 45%, 37% and 25% of patients in MI, LI and CsA, respectively. Anemia, neutropenia and posttransplant lymphoproliferative disorder occurred more frequently in MI and LI compared with CsA. Tofacitinib was equivalent to CsA in preventing acute rejection, was associated with improved renal function and less chronic allograft histological injury, but had side‐effects at the doses evaluated.

Combined use of the JAK3 inhibitor CP-690,550 with mycophenolate mofetil to prevent kidney allograft rejection in nonhuman primates

Background. Immunosuppression via Janus kinase (JAK) 3 inhibition affords significant prolongation of allograft survival. We investigated the effects of an immunosuppressive regimen combining the JAK3 inhibitor CP-690,550 with mycophenolate mofetil (MMF) in nonhuman primates (NHPs). Methods. Life-supporting kidney transplantations were performed between ABO-compatible, MLR-mismatched NHPs. Animals were treated orally twice a day with CP-690,550 and MMF (n=8) or MMF alone (n=2) and were euthanized at day 90 or earlier due to allograft rejection. Results. Mean survival time (±SEM) in animals treated with MMF alone (23±1 days) was significantly extended in animals that concurrently received CP-690,550 (59.5±9.8 days, P=0.02). Combination animals exposed to higher levels of CP-690,550 had a significantly better survival (75.2±8.7 days) than animals that received less CP-690,550 (33.3±12.6 days, P=0.02). Three combination therapy animals were euthanized at day 90 with a subnormal renal function and early-stage acute graft rejection. Rejection, delayed by treatment, ultimately developed in other animals. Anemia and gastrointestinal intolerance was seen in combination therapy animals that otherwise did not show evidence of viral or bacterial infection besides signs consistent with subclinical pyelonephritis (n=3). One incidental lymphosarcoma was noted. Conclusions. Addition of CP-690,550 to MMF significantly improved allograft survival. The observed side effects appear amenable to improvements upon alteration of dosing strategies. Efficacy of this combination regimen suggests that it could become the backbone of calcineurin inhibitor-free regimens.

Identification and chemical synthesis of MDL 105,212, a non-peptide tachykinin antagonist with high affinity for NK1 and NK2 receptors

We have synthesized and identified MDL 105,212, a non-peptide tachykinin receptor antagonist that has high affinity for human NK1 (IC50=3.11 nM) and NK2 (IC50=8.40 nM) receptors. The chemical synthesis of MDL 105,212 and the SAR of a series of racemic amide analogs are described.

Synthesis and structure-activity relationships for a series of substituted pyrrolidine NK1/NK2 receptor antagonists

Abstract We recently described the synthesis and characterization of MDL 105,212, a non peptide tachykinin antagonist with high affinity for NK1 and NK2 receptors.1 Here we report the synthesis and structure-activity relationships for a series of analogs of MDL 105,212 with regards to: NK1 and NK2 receptor binding affinity, physical-chemical characterization; in vitro absorption potential; in vitro metabolic stability; and efficacy in a capsaicin-challenge conscious guinea pig model after oral administration.