BackgroundBruton's tyrosine kinase (BTK) is a member of the Tec family kinases, and is expressed in B cells, monocytes/macrophages, mast cells, basophils and osteoclast1,2. BTK is a critical effector molecule that activates B cell receptor signaling pathway in B cells and Fc-γ receptor signaling pathway in macrophages3. In osteoclast, RANKL (receptor activator of NF-kB ligand) binds to its receptor RANK, and induces differentiation and bone resorption of osteoclast through BTK-PLCγ signaling pathway. These various effector cells are reported to be associated with disease progression of rheumatoid arthritis4,5. Therefore, BTK would be an attractive target for a therapeutic agent in autoimmune disease such as rheumatoid arthritis (RA). We discovered a novel synthesized compound which inhibits BTK with a highly selectivity.ObjectivesWe report characteristics of a highly selective and potent BTK inhibitor, TAS5315, in vitro and in vivo models for RA.MethodsThe kinase selectivity of TAS5315 was evaluated in a series of biochemical assays. The expression of CD69, a lymphocyte activation marker, stimulated by anti-IgM in the mouse splenic B cells was measured using flow cytometry. The bone resorption activity of mouse osteoclast was evaluated using osteoclast culture kit. Male DBA/1 mice were injected on day-27 and-6 with an emulsion of complete Freund's adjuvant and bovine type II collagen. TAS5315 (0.05, 0.1, 0.2 and 0.4 mg/kg) was administrated orally once daily for 15 consecutive days.ResultsTAS5315 showed a potent inhibitory activity against anti-IgM-induced phosphorylation of BTK with an IC50 in sub nmol/L range. TAS5315 also inhibited only 4 kinases (TEC, BMX, TXK and ITK) out of 276 off-target kinases with high inhibitory activity (over 80% at 100 nmol/L). The up-regulation of CD69 on stimulated mouse splenic B cells was suppressed by TAS5315 in a dose-dependent manner (IC50 =0.2 nmol/L). In pit formation assay, TAS5315 also inhibited bone resorption activity of osteoclast in a dose-dependent manner (IC50 =2.2 nmol/L). In a mouse collagen-induced arthritis model, TAS5315 dose-dependently and significantly decreased the clinical score in arthritic mice compared with that in vehicle-treated mice, with an ED50 value of 0.12 mg/kg. The treatment group with TAS5315 (0.4 mg/kg) completely ameliorated arthritic symptoms on day 14. All four paws of CIA mice were then examined by histopathological analysis. TAS5315-treated mice had a marked reduction in the severity of inflammation, pannus, cartilage destruction and bone destruction in a dose-dependent manner. In the micro CT analysis of hind paws of CIA mice, TAS5315 (1mg/kg)-treated mice showed a remarkable recovery of bone mineral density compared with vehicle-treated mice.ConclusionsOur study demonstrates that TAS5315 is a highly selective BTK inhibitor and has significant efficacy in the mouse CIA model. These data suggests that TAS5315 could be a promising new therapeutic agent for RA.ReferencesCurr Opin Immunol. 2000;12, 282-288,Int Arch Allergy Immunol. 2004; 134,...

BackgroundThe erosions of bone and cartilage are a cardinal feature of rheumatoid arthritis (RA) and associated with disease severity and poor functional outcome1. Although several anti-inflammatory drugs improve symptoms of articular inflammation, they are less effective against bone erosion. The bone erosions in RA are associated with aberrant activations of osteoclasts induced by pro-inflammatory cytokines and receptor activator of nuclear factor κB ligand (RANKL)2. Bruton's tyrosine kinase (BTK), which is expressed in immune cells and mature osteoclast, is reported to be a key molecule in inflammatory response and bone resorption3,4. Thus, targeting BTK may be efficacious against not only inflammation but also bone erosion through direct regulation of activation of effector cells such as B cells, macrophages and osteoclasts in RA.ObjectivesIn this study, we evaluated the effect of TAS5315, a novel BTK inhibitor, on in vitro osteoclasts activation and bone erosion in mouse collagen-induced arthritis.MethodsKinase selectivity of TAS5315 was assessed by available kinase assay panels. The effects of TAS5315 on macrophages and osteoclasts were assessed by examining phosphorylation of BTK, cytokine productions, osteoclast differentiation and bone resorptions. The effects of TAS5315 were investigated in mouse collagen-induced arthritis (CIA). Disease severity was evaluated by clinical score of paw swelling. Changes in bone mineral density (BMD) and bone erosion were assessed using microCT. TNF blocker was used as a control drug.ResultsTAS5315 selectively inhibited the enzyme activity of BTK and had less off target inhibition against other kinases. TAS5315 dose-dependently inhibited cytokine productions by macrophages, phosphorylation of BTK, osteoclastogenesis and bone resorbing activity in osteoclasts. In established mouse CIA, TAS5315 significantly ameliorated paw swelling in a dose dependent manner and the anti-inflammatory effect of TAS5315 (0.3 mg/kg, once daily) was comparable to that of TNF blocker. Most importantly, improvement of BMD and bone erosion were observed in TAS5315 treated mice at a doses of higher than 0.1 mg/kg within 13 days from treatment initiation, but not in TNF blocker-treated mice. The onset of action of TAS5315 on BMD and bone erosion was earlier and stronger compared with that of TNF blocker. These data suggest that TAS5315 had direct effect against osteoclasts function and led to improvement of bone erosion in murine model for RA.ConclusionsOur study demonstrates that TAS5315, a novel BTK inhibitor, would be an ideal RA therapeutic agent that could inhibit bone destruction as well as inflammation.References Nat Rev Rheumatol. 2012;8,656–64.Nat Rev Rheumatol. 2015;11,189–94.Cell. 2008;132,794–806.Drug Discov Today. 2014;19,1200–4. Disclosure of InterestNone declared

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.

BackgroundBone erosions and cartilage damages are a pathological hallmark of rheumatoid arthritis (RA) and are associated with poor functional outcome1. Aberrant activations of osteoclasts induced by receptor activator of nuclear factor κB ligand (RANKL)2 are involved in the bone erosions of RA. It has also been recently shown that under chronic inflammatory conditions such as RA, inflammatory cytokines in joints induce pathological osteoclast differentiation and cause excessive bone resorption independent of RANKL-RANK signaling3. The Bruton’s tyrosine kinase (BTK) signaling pathway plays a pivotal role in inflammatory response and bone resorption4. Thus, targeting BTK may be efficacious against not only inflammation but also bone erosion by regulating activation of effector cells such as B cells, macrophages and osteoclasts in RA. We have already shown the inhibitory effects of TAS5315 on RANKL-dependent osteoclast activation. However, it remained uncertain whether TAS5315 inhibits osteoclast activation induced by inflammatory cytokines.ObjectivesIn this study, we evaluated the effects of TAS5315 on osteoclast activation induced by inflammatory cytokines (in vitro) and the infiltration of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the joint of a mouse CIA model.MethodsIn vitro biochemical assay was performed using available kinase assay panels. The BioMAP® Diversity PLUS panels were used to determine the profile of TAS5315 in primary human cell systems. The effects of TAS5315 on osteoclasts were assessed by examining osteoclast-mediated bone resorption. TAS5315 was orally administrated once a day in an established mouse CIA model. TRAP-positive osteoclasts were counted manually. Bone mineral density (BMD) and bone erosion were assessed using micro-CT analysis. The mechanical properties of the tibia were evaluated by a compression test of proximal metaphysis using a material-testing machine.ResultsTAS5315 selectively inhibited the enzyme activity of BTK and had less off target inhibition against other kinases. In BioMAP® systems, TAS5315 decreased the production of IgG and the expression of cytokines (TNF-α, IL-6, IL-17A). TAS5315 also inhibited osteoclast-mediated bone resorption induced by inflammatory cytokines. On the other hand, anti-RANKL antibody did not inhibit bone resorption induced by inflammatory cytokines. Futhermore, in the mouse CIA model, TAS5315 significantly ameliorated paw swelling and pathological changes. TAS5315 significantly decreased the infiltration of TRAP-positive osteoclasts in the joint and also showed improvement of BMD and bone erosion by time-dependent micro-CT analysis. In vehicle-treated mice, the mechanical strength of tibia was decreased compared with normal mice. TAS5315-treated mice recovered the decreased parameters of the mechanical strength compared with vehicle-treated mice.These data suggests that TAS5315 improves bone erosion in murine model for RA through direct inhibition of osteoclast activation induced by inflammatory cytokines as well as RAN...

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