Spleen tyrosine kinase (Syk) is involved in regulation of B-cell receptor (BCR) and Fc receptor downstream signal pathways. Syk plays an essential role in production of inflammatory mediators and differentiation in various immune cells and is therefore an attractive target for treating inflammatory conditions, such as autoimmune and allergic diseases. We identified TAS05567 as a highly selective Syk inhibitor and evaluated its therapeutic potential in animal models. In vitro biochemical assays were performed with available kinase assay panels. Inhibitory effects of TAS05567 on immune cells were analyzed by assessing the Syk downstream signaling pathway and production of inflammatory factors. In vivo effects of TAS05567 were evaluated in animal models of autoimmune diseases and antigen-specific IgE transgenic mice. TAS05567 inhibited only 4 of 191 kinases tested but inhibited Syk enzymatic activity with high potency. TAS05567 inhibited BCR-dependent signal transduction in Ramos cells, FcR-mediated tumor necrosis factor- production in THP-1 cells, and FcR-mediated histamine release from RBL-2H3 cells. In rheumatoid arthritis models, TAS05567 suppressed hind-paw swelling in a dose-dependent manner compared with vehicle. Moreover, TAS05667 markedly reduced histopathologic scores in an established rat arthritis model. In a mouse immune thrombocytopenic purpura model, platelet counts were reduced with injection of anti-platelet antibody. TAS05567 prevented the platelet count decrease in a dose-dependent manner. Finally, TAS05567 treatment suppressed IgE-mediated ear swelling in vivo. Collectively, our data indicate TAS05567 is a selective Syk inhibitor and potential therapeutic candidate for treating humoral immune-mediated inflammatory conditions such as autoimmune and allergic diseases.

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BackgroundThe family of Janus kinases (JAKs) plays important roles in signalling pathway mediated by various cytokine receptors. An aberrant activation of JAK-STAT signalling has been reported to be involved in the pathogenesis of autoimmune diseases1. Pan-JAK inhibitors have shown a good efficacy in patient with rheumatoid arthritis (RA)2. However, their use is limited due to safety concerns, including severe herpes zoster infection, by inhibiting JAK1-mediated interferon signaling3. Therefore, a selective JAK3 inhibitor would provide a better balance between efficacy and safety than pan-JAK inhibitors.ObjectivesWe identified the characteristics of TAS8274, a novel highly selective inhibitor of JAK3, using in vitro assays, a mouse model of collagen-induced arthritis (CIA), and a mouse model of herpes simplex virus (HSV)−1 infection.MethodsIn vitro biochemical assay was performed using available kinase assay panels. The effects on anti-inflammatory responses were assessed by examining cytokine productions. IL-2, IL-3, and IFN-α–induced phosphorylation of STAT proteins in peripheral blood mononuclear cells (PBMCs) were analysed by a flow cytometry method. NK cell cytotoxicity in the presence of IFN-α was evaluated by Cr51 release assay. In a mouse skin HSV-1 infection model, TAS8274 and tacrolimus were administered for 7 days before inoculation of the virus on the back skin, and then were administered for another ten consecutive days. At the end of this experiment, the number of papules on the back was counted. To evaluate the therapeutic efficacy using mouse CIA model, TAS8274 was orally administered to CIA mice after the disease onset. Disease severity was evaluated by clinical score of paw swelling, and the scores of inflammation, pannus, cartilage, and bone damage were performed using a modified Mankin score system.ResultsTAS8274 inhibited the enzymatic activity of JAK3 (IC50=0.16 nM), and showed more than 1000-fold selectivity against other JAK kinases. In the cell-based assays, TAS8274 strongly inhibited IL-17 production from differentiated Th17 cells. TAS8274 also suppressed the IL-2-induced STAT5 phosphorylation in PBMCs, but had much lower inhibitory effects on the IFN-α-induced STAT1 phosphorylation. In contrast, Tofacitinib and Baricitinib had robust inhibitory effects on the IFN-α-induced STAT1 phosphorylation. Furthermore, Tofacitinib and Baricitinib dose-dependently reduced the NK cell cytotoxicity, while TAS8274 had little effect on that. Tacrolimus-treated group significantly increased the number of papules compared with vehicle-treated group in a mouse HSV-1 infection model, but TAS8274-treated group did not increase the number of papules. In an established mouse CIA model, TAS8274 dose-dependently reduced the severity of arthritis and histopathological scores compared with vehicle-treated mice.ConclusionsTAS8274 did not inhibit the JAK3-independent STAT signalling pathway in vitro and showed potent efficacy at dose range without exacerbation of the risk of HSV-1 infection. Our study demonstrates t...