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Mark E Flanagan,
Todd A Blumenkopf,
William H Brissette,
Matthew F Brown,
Jeffrey M Casavant,
Chang Shang-Poa,
Jonathan L Doty,
Eileen A Elliott,
Michael B Fisher,
Michael Hines, [......],
Kelly S Magnuson,
Sandra P McCurdy,
Michael J Munchhof,
Bret D Perry,
Perry S Sawyer,
Timothy J Strelevitz,
Chakrapani Subramanyam,
Jianmin Sun,
David A Whipple, Paul S Changelian
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ABSTRACT: 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.
Journal of Medicinal Chemistry 12/2010; 53(24):8468-84. · 4.80 Impact Factor
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Journal of Investigative Dermatology 03/2009; 129(9):2299-302. · 6.31 Impact Factor
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Paul S Changelian,
Deborah Moshinsky,
Cyrille F Kuhn,
Mark E Flanagan,
Michael J Munchhof,
Thomas M Harris,
David A Whipple,
Jonathan L Doty,
Jianmin Sun,
Craig R Kent,
Kelly S Magnuson,
David G Perregaux,
Perry S Sawyer,
Elizabeth M Kudlacz
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ABSTRACT: PF-956980 is a selective inhibitor of JAK3, related in structure to CP-690550, a compound being evaluated in clinical trials for rheumatoid arthritis and prevention of allograft rejection. PF-956980 has been evaluated against a panel of 30 kinases, and found to have nanomolar potency against only JAK3. Cellular and whole blood activity of this compound parallels its potency and selectivity in enzyme assays. It was effective in vivo at inhibiting the delayed type hypersensivity reaction in mice. We compared 2 commercially available JAK3 inhibitors (WHI-P131 and WHI-P154) in the same panel of biochemical and cellular assays and found them to be neither potent nor selective for JAK3. Both were found to be nanomolar inhibitors of the EGF receptor family of kinases. As these compounds have been used in numerous publications in the transplant and autoimmune disease literature, their specificity should be considered when interpreting these results.
Blood 03/2008; 111(4):2155-7. · 9.90 Impact Factor
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ABSTRACT: Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common gamma chain. The rationally designed inhibitor of JAK3, CP-690,550, prevents acute allograft rejection in rodents and in nonhuman primates. Here we investigated the ability of CP-690,550, to prevent allograft vasculopathy in a rodent model of aorta transplantation. Aortas from AxC Irish (RT1(a)) or Lewis (RT1(l)) rats were heterotopically transplanted into the infra-renal aorta of Lewis recipients and harvested at 28 or 56 days. Treated recipients received CP-690,550 by osmotic pumps (mean drug exposure of 110 +/- 38 ng/ml). Significant intimal hyperplasia was demonstrated in untreated allografts when compared with isografts at 28 days (2.08 +/- 0.85% vs. 0.43 +/- 0.2% luminal obliteration, respectively, P = 0.001) and 56 days (5.3 +/- 2.4% vs. 0.38 +/- 0.3%, P = 0.002). Treatment caused a 51% reduction in intimal hyperplasia at day 56. CP-690,550-treated animals also had a significant reduction of donor-specific IgG production and of the gene expression for suppressor of cytokine signaling-3 and with unchanged levels of expression of RANTES, IP-10 and transforming growth factor-beta1. These results are the first to show that JAK3 blockade by CP-690,550 effectively prevents allograft vasculopathy in this rat model of aorta transplantation.
Transplant International 01/2007; 19(12):1014-21. · 2.92 Impact Factor
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Dominic C Borie,
Michael J Larson,
Mona G Flores,
Andrew Campbell,
Geraldine Rousvoal,
Sally Zhang,
John P Higgins,
Douglas J Ball,
Elizabeth M Kudlacz,
William H Brissette,
Eileen A Elliott,
Bruce A Reitz, Paul S Changelian
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ABSTRACT: 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).
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.
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.
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.
Transplantation 01/2006; 80(12):1756-64. · 4.00 Impact Factor
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ABSTRACT: Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates.
Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-gamma production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry.
In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-gamma production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 microM) and CD71 (IC50; 1.6 microM), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 microM). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8 effector memory T-cell populations were unaffected.
Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.
Transplantation 12/2005; 80(9):1283-92. · 4.00 Impact Factor
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ABSTRACT: Purpose of review: Most current immunosuppressive drugs have side effects. It is recognized that the mechanisms for such side effects are similar to that responsible for efficacy and reflects undesirable effects of the drug outside of the immune system. In this review, we will discuss recent developments that led to the introduction of a compound selectively targeting immune cells and that appears devoid of classical side effects observed with current immunosuppressive drugs.
Recent findings: A large number of cytokines exert their effect by binding to receptors that activate the Janus kinase/signal transducer and activator of transcription pathway, so targeting intracellular signaling pathways is a logical strategy. A selective inhibitor of Janus kinase 3 has been generated and is effective for the prevention of transplant rejection in nonhuman primates and other models. Consistent with predictions from murine studies, drug treatment results in significant reductions in numbers of natural killercells and modest reductions in effector memory CD8+ cells. The side-effect profile appears limited to anemia observed only at high exposure. None of the dose-limiting side effects observed with other immunosuppressants that have been linked with early graft rejection and patient demise have been observed with the inhibition of Janus kinase 3.
Summary: A selective inhibitor of Janus kinase 3 has now been generated. It most likely represents a new class of effective immunosuppressants. Because of its particular mechanism of action - targeting a wide array of immune cells (T, B, and natural killer) - the compound may offer solutions to acute problems as well as chronic allograft rejection.
Current Opinion in Organ Transplantation 11/2005; 10(4):273-278. · 2.97 Impact Factor
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ABSTRACT: A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.
Therapeutic Drug Monitoring 11/2005; 27(5):608-16. · 2.49 Impact Factor
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Dominic C Borie, Paul S Changelian,
Michael J Larson,
Ming-Sing Si,
Ricardo Paniagua,
John P Higgins,
Bari Holm,
Andrew Campbell,
Macy Lau,
Sally Zhang,
Mona G Flores,
Geraldine Rousvoal,
Jennifer Hawkins,
Douglas A Ball,
Elizabeth M Kudlacz,
William H Brissette,
Eileen A Elliott,
Bruce A Reitz,
Randall E Morris
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ABSTRACT: Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (gammac). Because mutations in genes encoding gammac 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.
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.
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.
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.
Transplantation 05/2005; 79(7):791-801. · 4.00 Impact Factor
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ABSTRACT: The field of organ transplantation has had tremendous success because of the availability of immunosuppressive drugs that efficiently prevent acute organ rejection. Numerous and severe side effects are, however, associated with all current immunosuppressive therapies and justify a search for drugs with better efficacy and safety profiles. Janus kinase (JAK) 3, a tyrosine kinase that is crucial for mediating signals from the common gamma-chain of cytokine receptors, is peculiar in that its expression, contrarily to the targets of most current immunosuppressive drugs, is limited to cells that actively participate to the immune response to allografts. The recent demonstration in stringent preclinical models that JAK3 inhibition results in efficacy for the prevention of allograft rejection with a narrow side-effect profile might lead to a new era in the field of immunosuppression.
Trends in Molecular Medicine 12/2004; 10(11):532-41. · 10.35 Impact Factor
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dressNature Reviews Drug Discovery 08/2004; 3(7):555-64. · 29.01 Impact Factor
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ABSTRACT: Although current immunosuppressive drugs are effective, they have numerous severe side effects that mandate the search for new agents. Mutations in the gene for janus kinase (JAK)3 result in severe combined immune deficiency with severely impaired humoral and cellular immunity, an observation that has prompted the development of JAK3 inhibitors. Due to its central role in lymphocyte activation, proliferation and homeostasis, targeting the JAK/signal transducer and activator of transcription (STAT) pathway may provide the required efficacy, without the toxicities associated with current therapies. Several studies conducted in rodents have validated the proof-of-concept, with a variety of JAK3 inhibitors demonstrating efficacy for immune suppression. In addition, the selective JAK3 inhibitor CP-690550 (Pfizer Inc) significantly improved allograft survival in a stringent preclinical model in primates and exhibited a good safety profile in non-human primates. This, along with studies of protein kinase inhibitors for cancer treatment, could demonstrate that development of effective, safe and selective kinase inhibitors for immunosuppression is possible.
Current opinion in investigational drugs (London, England: 2000) 12/2003; 4(11):1297-303. · 3.31 Impact Factor
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Paul S Changelian,
Mark E Flanagan,
Douglas J Ball,
Craig R Kent,
Kelly S Magnuson,
William H Martin,
Bonnie J Rizzuti,
Perry S Sawyer,
Bret D Perry,
William H Brissette, [......],
Michael Larson,
Ming-Sing Si,
Ricardo Paniagua,
John Higgins,
Bari Holm,
Bruce Reitz,
Yong-Jie Zhou,
Randall E Morris,
John J O'Shea,
Dominic C Borie
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ABSTRACT: Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.
Science 11/2003; 302(5646):875-8. · 31.20 Impact Factor
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ABSTRACT: Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with
cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic
activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating
its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain.
Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3
is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant
Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on
Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, γc and STAT5A. In contrast,
mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These
results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity.
These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations
of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important
to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance
of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these
and other tyrosine residues in the control of kinase activity and hence cytokine signaling.
Proceedings of the National Academy of Sciences 12/1997; 94(25):13850-13855. · 9.68 Impact Factor
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ABSTRACT: JAK3 is a protein tyrosine kinase that specifically associates with the common γ chain (γc), a shared subunit of receptors for interleukin (IL) 2, 4, 7, 9, and 15. Patients deficient in either JAK3 or γc presented with virtually identical forms of severe combined immunodeficiency (SCID), underscoring the importance of the JAK3–γc interaction. Despite the key roles of JAK3 and γc in lymphocytic development and function, the molecular basis of this interaction remains poorly understood. In this study,
we have characterized the regions of JAK3 involved in γc association. By developing a number of chimeric JAK3–JAK2 constructs, we show that the binding specificity to γc can be conferred to JAK2 by transferring the N-terminal domains of JAK3. Moreover, those JAK3–JAK2 chimeras capable of binding
γc were also capable of reconstituting IL-2 signaling as measured by inducible phosphorylation of the chimeric JAK3–JAK2 protein,
JAK1, the IL-2 receptor β chain, and signal transducer and activator of transcription 5A. Subsequent deletion analyses of
JAK3 have identified the N-terminal JH7-6 domains as a minimal region sufficient for γc association. Furthermore, expression of the mutant containing only the JH7-6 domains effectively competed with full-length
JAK3 for binding to γc. We conclude that the JH7-6 domains of JAK3 are necessary and sufficient for γc association. These studies offer clues toward a broader understanding of JAK-mediated cytokine signaling and may provide
a target for the development of novel therapeutic modalities in immunologically mediated diseases.
Proceedings of the National Academy of Sciences 06/1997; 94(13):6910-6915. · 9.68 Impact Factor
