Abstract The PIM family of serine/threonine kinases has roles in proliferation, differentiation, and cell survival. In lymphocytes, their activity is regulated by inducible expression following cytokine or antigen receptor signaling. Using small molecule inhibitors, we have examined the role of PIM1/3 kinases in B cells in vitro and in vivo and in the MRL/LPR model of lupus. PIM kinases were expressed 1-3 hours after B cell activation with anti-IgM or anti-CD40 but not BAFF. Inhibition of PIM-1/3 kinase activity decreased anti-IgM- and anti-CD40-induced B cell proliferation without inducing apoptosis. AR-254 also prevented IL-4-, and IFNy-induced B cell survival, but had no affect on BAFF-induced B cell survival consistent with the inability of BAFF to upregulate PIM expression. AR-770 administration in the HEL immunization model resulted in a 90% reduction of HEL-specific IgG2a and IgG3, but had no effect on IgG1 suggesting a role for PIM kinases in IFNy-mediated class switching. In the MRL/MpJ-Fas lupus model, mice treated with AR-770 had >90% reduction in proteinuria AUC, normalized anti-dsDNA antibody titers and ~80% decrease in kidney glomerulo- and interstitial nephritis as well as vessel and protein cast formation. Under specific conditions, Taken together, the data suggests that inhibition of PIM kinases may be a useful strategy for patients with B cell-mediated diseases such as lupus.

RATIONALE: Prostaglandin D2 (PGD2), is a potent prostanoid released during allergic/asthmatic responses. CRTH2 (aka DP2), a PDG2 receptor expressed on cells involved in allergic inflammation, mediates chemotaxis and mast cell-dependent activation of basophils, eosinophils and Th2 lymphocytes. Thus, CRTH2 antagonists may be useful in allergic diseases. METHODS: ARRY-005 and ARRY-006 are potent, selective, orally bioavailable, competitive antagonists of CRTH2 (IC50 <10 nM). Human blood or basophils from healthy and allergic subjects were used for flow cytometry and chemotaxis assays. Balb/c mice were sensitized to OVA then challenged days on 28-30 with inhaled OVA in the asthma studies monitoring airway hyperresponsiveness (AHR) and inflammation or challenged with intranasal OVA on days 28-33 in the AR studies monitoring respiratory frequency (RF) or nasal resistance (Rna). Inhibitors were administered orally 1hr before each challenge. NC/Nga mice were used in the AD studies. RESULTS: ARRY-005 inhibited PGD2-mediated chemotaxis of isolated basophils (IC50 1 nM) and in human blood inhibited PGD2-induced eosinophil shape change (IC50 180 nM) and CRTH2 receptor internalization (IC50 ∼200 nM). ARRY-005 (30 mg/kg QD) decreased AHR and reduced BAL eosinophil and lymphocyte accumulation. In the AR model, ARRY-005 (10 mg/kg QD) normalized early and late phase decreases in RF and prevented the increases in Rna in the late phase. In the AD model, ARRY-006 (30 mg/kg, QD) inhibited ear thickening, erythema, and pruritis. CONCLUSIONS: Selective CRTH2 antagonists, ARRY-005 and -006, are potent inhibitors of basophils and eosinophils in vitro and exhibited significant protective activities in models of three allergic diseases, supporting their advancement to clinical development.

RATIONALE: Pim kinases are a family of 3 serine/threonine kinases whose activity is regulated by expression and can be induced by cytokines involved in allergy and asthma. Pim kinases play a role in the survival of eosinophils and basophils and their expression is increased in the BAL of asthmatic patients after allergen provocation. We investigated effects of Pim kinase inhibition on Th2 cells and following allergen sensitization and challenge. METHODS: ARRY-770 is an orally available Pim inhibitor with cellular IC50 = 110 nM. Th2 cells were generated in vitro using DO11.10 CD4+ T cells. Proliferation and cytokines were measured using FACS and ELISAs. BALB/c mice were sensitized by intraperitoneal injection of OVA (with alum) on days 0 and 14, followed by three nebulized OVA challenges on days 28-30. On each challenge day, 100mg/kg ARRY-770 was administered orally BID. AHR and BAL assessments were performed 48hrs after final airway challenge. RESULTS: ARRY-770 prevented development of Th2 effector cells and inhibited established Th2 effector cells from proliferating and producing IL-4. Following treatment with ARRY-770, development of AHR and eosinophilic airway inflammation were significantly reduced. In BAL fluid, levels of IL-4, IL-5, IL-13 and IFNγ were significantly lower than in control groups. CONCLUSIONS: ARRY-770, a novel Pim kinase inhibitor, decreases Th2 responses and was effective in reducing AHR, airway inflammation and cytokine production in sensitized and challenged mice. These data identify an important role for this kinase in the development of allergen-induced lung allergic responses.