Diane E. Gingrich

A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis

IntroductionJanus kinase 2 (JAK2) is involved in the downstream activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 and is responsible for transducing signals for several proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), including interleukin (IL)-6, interferon γ (IFNγ) and IL-12. In this paper, we describe the efficacy profile of CEP-33779, a highly selective, orally active, small-molecule inhibitor of JAK2 evaluated in two mouse models of RA.MethodsCollagen antibody-induced arthritis (CAIA) and collagen type II (CII)-induced arthritis (CIA) were established before the oral administration of a small-molecule JAK2 inhibitor, CEP-33779, twice daily at 10 mg/kg, 30 mg/kg, 55 mg/kg or 100 mg/kg over a period of 4 to 8 weeks.ResultsPharmacodynamic inhibition of JAK2 reduced mean paw edema and clinical scores in both CIA and CAIA models of arthritis. Reduction in paw cytokines (IL-12, IFNγ and tumor necrosis factor α) and serum cytokines (IL-12 and IL-2) correlated with reduced spleen CII-specific T helper 1 cell frequencies as measured by ex vivo IFNγ enzyme-linked immunosorbent spot assay. Both models demonstrated histological evidence of disease amelioration upon treatment (for example, reduced matrix erosion, subchondral osteolysis, pannus formation and synovial inflammation) and reduced paw phosphorylated STAT3 levels. No changes in body weight or serum anti-CII autoantibody titers were observed in either RA model.ConclusionsThis study demonstrates the utility of using a potent and highly selective, orally bioavailable JAK2 inhibitor for the treatment of RA. Using a selective inhibitor of JAK2 rather than pan-JAK inhibitors avoids the potential complication of immunosuppression while targeting critical signaling pathways involved in autoimmune disease progression.

CEP-28122, a Highly Potent and Selective Orally Active Inhibitor of Anaplastic Lymphoma Kinase with Antitumor Activity in Experimental Models of Human Cancers

Anaplastic lymphoma kinase (ALK) is constitutively activated in a number of human cancer types due to chromosomal translocations, point mutations, and gene amplification and has emerged as an excellent molecular target for cancer therapy. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity and cellular ALK tyrosine phosphorylation. It induced concentration-dependent growth inhibition/cytotoxicity of ALK-positive anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cells, and displayed dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xenografts in mice, with substantial target inhibition (>90%) for more than 12 hours following single oral dosing at 30 mg/kg. Dose-dependent antitumor activity was observed in ALK-positive ALCL, NSCLC, and neuroblastoma tumor xenografts in mice administered CEP-28122 orally, with complete/near complete tumor regressions observed following treatment at doses of 30 mg/kg twice daily or higher. Treatment of mice bearing Sup-M2 tumor xenografts for 4 weeks and primary human ALCL tumor grafts for 2 weeks at 55 or 100 mg/kg twice daily led to sustained tumor regression in all mice, with no tumor reemergence for more than 60 days postcessation of treatment. Conversely, CEP-28122 displayed marginal antitumor activity against ALK-negative human tumor xenografts under the same dosing regimens. Administration of CEP-28122 was well tolerated in mice and rats. In summary, CEP-28122 is a highly potent and selective orally active ALK inhibitor with a favorable pharmaceutical and pharmacokinetic profile and robust and selective pharmacologic efficacy against ALK-positive human cancer cells and tumor xenograft models in mice. Mol Cancer Ther; 11(3); 670–9. ©2011 AACR.

Depletion of Autoreactive Plasma Cells and Treatment of Lupus Nephritis in Mice Using CEP-33779, a Novel, Orally Active, Selective Inhibitor of JAK2

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC50 = 1.3 nM; JAK3 enzyme IC50/JAK2 enzyme IC50 = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1β, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138+ plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.

2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazines: new variant of an old template and application to the discovery of anaplastic lymphoma kinase (ALK) inhibitors with in vivo antitumor activity.

A novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine scaffold has been designed as a new kinase inhibitor platform mimicking the bioactive conformation of the well-known diaminopyrimidine motif. The design, synthesis, and validation of this new pyrrolo[2,1-f][1,2,4]triazine scaffold will be described for inhibitors of anaplastic lymphoma kinase (ALK). Importantly, incorporation of appropriate potency and selectivity determinants has led to the discovery of several advanced leads that were orally efficacious in animal models of anaplastic large cell lymphoma (ALCL). A lead inhibitor (30) displaying superior efficacy was identified and in depth in vitro/in vivo characterization will be presented.

A Selective, Orally Bioavailable 1,2,4-Triazolo[1,5-A]Pyridine-Based Inhibitor of Janus Kinase 2 for Use in Anticancer Therapy: Discovery of Cep-33779.

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

Discovery of an Orally Efficacious Inhibitor of Anaplastic Lymphoma Kinase

Anaplastic lymphoma kinase (ALK) is a promising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and culminating in the recent FDA approval of the ALK inhibitor crizotinib. Through a series of targeted modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase selectivity, and overall pharmaceutical properties. Optimization of this scaffold has led to the identification of a potent and efficacious inhibitor of ALK, 25b. A striking feature of 25b over previously described ALK inhibitors is its >600-fold selectivity over insulin receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment in advanced preclinical models.

2,7-Pyrrolo[2,1-f][1,2,4]triazines as JAK2 inhibitors: Modification of target structure to minimize reactive metabolite formation

The JAK2/STAT pathway has important roles in hematopoiesis. With the discovery of the JAK2 V617F mutation and its presence in many patients with myeloproliferative neoplasms, research in the JAK2 inhibitor arena has dramatically increased. We report a novel series of potent JAK2 inhibitors containing a 2,7-pyrrolotriazine core. To minimize potential drug-induced toxicity, targets were analyzed for the ability to form a glutathione adduct. Glutathione adduct formation was decreased by modification of the aniline substituent at C2.

Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK)

Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

Optimization of a novel kinase inhibitor scaffold for the dual inhibition of JAK2 and FAK kinases.

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.

2,7-Disubstituted-pyrrolotriazine kinase inhibitors with an unusually high degree of reactive metabolite formation.

There are numerous published studies establishing a link between reactive metabolite formation and toxicity of various drugs. Although the correlation between idiosyncratic reactions and reactive metabolite formation is not 1:1, the association between the two is such that many pharmaceutical companies now monitor for reactive metabolites as a standard part of drug candidate testing and selection. The most common method involves in vitro human microsomal incubations in the presence of a thiol trapping agent, such as glutathione (GSH), followed by LC/MS analysis. In this study, we describe several 2,7-disubstituted-pyrrolotriazine analogues that are extremely potent reactive metabolite precursors. Utilizing a UPLC/UV/MS method, unprecedented levels of GSH adducts were measured that are 5-10 times higher than previously reported for high reactive metabolite-forming compounds such as clozapine and troglitazone.