James D. Rodgers

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM), and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) > 400nM). In a mouse model of JAK2V617F(+) MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

Selective Inhibition of JAK1 and JAK2 Is Efficacious in Rodent Models of Arthritis: Preclinical Characterization of INCB028050

Inhibiting signal transduction induced by inflammatory cytokines offers a new approach for the treatment of autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown promising oral disease-modifying antirheumatic drug potential with efficacy similar to anti-TNF biologics. Direct and indirect inhibition of the JAKs, with small molecule inhibitors like CP-690,550 and INCB018424 or neutralizing Abs, such as the anti-IL6 receptor Ab tocilizumab, have demonstrated rapid and sustained improvement in clinical measures of disease, consistent with their respective preclinical experiments. Therefore, it is of interest to identify optimized JAK inhibitors with unique profiles to maximize therapeutic opportunities. INCB028050 is a selective orally bioavailable JAK1/JAK2 inhibitor with nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple proinflammatory cytokines including IL-6 and IL-23 at concentrations <50 nM. Significant efficacy, as assessed by improvements in clinical, histologic and radiographic signs of disease, was achieved in the rat adjuvant arthritis model with doses of INCB028050 providing partial and/or periodic inhibition of JAK1/JAK2 and no inhibition of JAK3. Diminution of inflammatory Th1 and Th17 associated cytokine mRNA levels was observed in the draining lymph nodes of treated rats. INCB028050 was also effective in multiple murine models of arthritis, with no evidence of suppression of humoral immunity or adverse hematologic effects. These data suggest that fractional inhibition of JAK1 and JAK2 is sufficient for significant activity in autoimmune disease models. Clinical evaluation of INCB028050 in RA is ongoing.

Enantioselective Synthesis of Janus Kinase Inhibitor INCB018424 via an Organocatalytic Aza-Michael Reaction

An enantioselective synthesis of INCB018424 via organocatalytic asymmetric aza-Michael addition of pyrazoles (16 or 20) to (E)-3-cyclopentylacrylaldehyde (23) using diarylprolinol silyl ether as the catalyst was developed. Michael adducts (R)-24 and (R)-27 were isolated in good yield and high ee and were readily converted to INCB018424.

Preclinical Evaluation of Local JAK1 and JAK2 Inhibition in Cutaneous Inflammation

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100  nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

Downmodulation of key inflammatory cell markers with a topical Janus kinase 1/2 inhibitor.

INCB018424 is a novel, potent Janus kinase (JAK)1/JAK2 inhibitor that blocks signal transduction of multiple proinflammatory cytokines.

Abstract B266: Systemic inflammation and tissue atrophy in cancer cachexia is mediated by cytokine‐dependent protease activation: Physiological and functional improvements through JAK1/2 inhibition

Cancer‐associated cachexia (CAC) is a complex syndrome characterized by a detrimental loss of fat and muscle. Approximately 50% of patients suffer from CAC which accounts for a significant proportion of cancer deaths in addition to debilitating constitutional symptoms and poor quality of life. Recently, the Cancer Cachexia Study Group has proposed that systemic inflammation is one of the 3 factors that defines CAC. Consistent with this, we previously demonstrated that inflammatory cytokines are elevated in cachectic mice bearing subcutaneous tumor xenografts. Because many inflammatory cytokines utilize JAK1 and/or JAK2 kinases (JAK1/2) for intracellular signaling, we hypothesized and demonstrated that selective inhibition of JAK1/2 improved tissue preservation and physical performance. Here we extend these studies beyond subcutaneous xenografts and present biochemical data in an effort to better understand the role of JAKs muscle atrophy. Subcutaneous xenograft models are useful tools but may lack host‐tumor interactions relevant to CAC. Therefore, we utilized two immune competent models of CAC. Mice inoculated with BaF/3 B‐cells transformed with a constitutively active form of JAK2 (JAK2V617F) common in patients with myeloproliferative neoplasms (MPNs) suffer from an aggressive disseminated disease characterized by splenomegaly and weight loss, similar to MPN patients. Compared to normal mice, those inoculated with JAK2V617F expressing cells lost 80% of their fat stores and 15% of skeletal muscle and had severely enlarged spleens (450%). Treatment with an oral selective JAK1/2 inhibitor prevented tissue loss and dramatically reduced spleen size. Similar findings were observed in the Apc Min/+ mouse model of spontaneous colon cancer and CAC. Here, treatment of male mice with a JAK1/2 inhibitor resulted in 120% and 268% increases in fat and muscle stores, respectively. Splenomegaly was also completely eliminated. Importantly, treatment with a JAK1/2 inhibitor markedly improved physical performance as assessed by grip strength and exercise wheel activity (#x003E; 200% of controls). Because JAK activating cytokines are elevated in our models (e.g. IL‐6) and have been implicated as causal factors in CAC, we wanted to better understand the impact of aberrant JAK activation on muscle tissue. C2C12 murine myotubes treated with recombinant IL‐6 demonstrated increased JAK signaling as measured by elevated levels of pSTAT3 and pSTAT5, canonical substrates of JAK kinases. IL‐6 treatment also increased the activity of two intracellular proteases previously linked to muscle catabolism in CAC ‐ cathepsins B and L. Inhibition of JAK1/2 reduced aberrant JAK/STAT signaling in IL‐6 treated C2C12 myotubes and in vivo . Moreover, cathepsin activity was decreased explaining, at least in part, the muscle sparing effects of JAK1/2 inhibition observed in vivo . In summary, the data suggest that JAK‐activating cytokines are elevated in CAC and contribute to physiological and functional deficits. JAK1/2 inhibition improves signs of inflammation and prevents loss of fat stores and skeletal muscle, the latter, through suppression of cathepsin activity. Clinical exploration of selective JAK inhibition is therefore warranted for the prevention or alleviation of CAC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B266.