Inhibition of Syk Activity Is Sufficient for Inhibitory Effect in Animal Models of Autoimmune Diseases

Spleen tyrosine kinase (Syk) serves as a key mediator of Fc receptor-mediated signaling in immunologic signaling cascades. Human Phase II trials have reported that dual inhibition of Syk and Janus kinases (JAK) by agents such as R788 (tamatinib fosdium) result in clinical benefit in rheumatoid arthritis (RA) and idiopathic thrombocytopenia purpura (ITP) patients. In order to delineate if inhibition of Syk alone was sufficient to produce a treatment benefit, we utilized compounds from two chemically diverse series of oral Syk inhibitors with differing kinase specificity in mouse models of RA and ITP. Compounds from the first series (P142–76, P459–72) were potent inhibitors of purified Syk (IC₅₀ = 4 to 43nM) and when tested at a ten fold higher concentration (300–500nM), lacked JAK inhibitory activity. Even at a concentration of 5μM, P142–76 did not inhibit JAK activity. Compounds from the second series (P420–1, P420–89) were potent Syk inhibitors (IC₅₀ = 15 to 31nM) and also exhibited comparable inhibitory potency against purified JAK (IC₅₀ JAK1 = 6–7nM, JAK2 = 2–3nM, JAK3 = 0.6–0.8nM). The compounds inhibited B cell receptor-induced activation of primary mouse splenocytes; P420–89 (IC₅₀ ~ 50nM) being somewhat more potent than P459–72 (IC₅₀ = 125–250nM). Importantly, phorbol 12-myristate 13-acetate (PMA)-induced B cell activation was not inhibited by any of the compounds at the tested concentration (1μM). Further, oral dosing of these compounds inhibited B cell receptor-induced B cell activation in mice. The RA model (n=12/group) investigated collagen antibody induced arthritis over a seven to ten day time course. The ITP model (n=10/group) utilized rapid clearance and destruction of antibody-coated platelets by phagocytic cells such as spleen macrophages. In this model, mice injected with a rat anti-mouse CD41 antibody became thrombocytopenic (average of 49% reduction in number of circulating platelets) over the 8h time course of experimentation.

In the RA studies, in addition to macroscopic signs of arthritis and assessment of clinical score, positive results were also verified histologically by reductions in peri-articular edema and neutrophil count and by scoring of intra-articular neutrophils and fibrin. In the ITP studies, mice pre-treated with P142–76, P420–1 or P420–89 were protected from loss of platelets, while mice pre-treated with vehicle alone displayed no protection. Our results highlight the role of Syk inhibition by small molecule oral inhibitors and suggest that inhibition of Syk activity, without dual inhibition of Syk/JAK, may be a promising strategy for therapeutic intervention in human autoimmune and inflammatory diseases.