Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor

Abstract

Spleen tyrosine kinase (Syk), a key mediator of immunoreceptor signaling in inflammatory cells, is essential for immune complex-mediated signal transduction initiated by activated receptors for immunoglobulin G. In collagen-induced arthritis, R788/R406, a novel and potent small molecule Syk inhibitor suppressed clinical arthritis, bone erosions, pannus formation, and synovitis. Serum anti-collagen type II antibody levels were unaltered, while the half-life of exogenous antibody was extended when co-administered with R406. Expression of the targeted kinase (Syk) in synovial tissue correlated with the joint level of inflammatory cell infiltrates and was virtually undetectable in treated rats. Syk inhibition suppressed synovial cytokines and cartilage oligomeric matrix protein (COMP) in serum, suggesting a sensitive and reliable biomarker for R406 activity. These results highlight the role of activating Fcgamma receptors in inflammatory synovitis and suggest that interruption of the signaling cascade with a novel Syk inhibitor may be a useful addition to immunosuppressive disease-modifying anti-rheumatic drugs currently used in the treatment of human autoimmune diseases such as rheumatoid arthritis.

Introduction

Spleen tyrosine kinase (Syk) is an intracellular protein tyrosine kinase that serves as a key mediator of Fc receptor and B cell receptor signaling in inflammatory cells, including mast cells, macrophages, dendritic cells (DC), NK cells, and neutrophils. Through its interaction with the phosphorylated gamma chains of the heterodimeric Fcγ receptors, Syk is essential for the signal transduction initiated by activated receptors for immunoglobulin G. Aggregation of the Fc receptors, induced by antibody–antigen complexes, can induce many cellular functions including phagocytosis, degranulation, NK cell antibody-dependent cellular cytotoxicity, and cytokine secretion, depending on the cell type. The initiation of inflammatory and destructive responses by these effector cells is believed to be crucial for the development and pathogenesis of autoimmunity [1]. Immune complex (IC)-mediated acute inflammatory tissue injury has been implicated not only in rheumatoid arthritis (RA), but in a variety of human autoimmune diseases, including systemic lupus erythematosus and certain types of glomerulonephritis [2], [3].

Several recent studies in Fc receptor (FcR)-deficient mouse strains suggest that signaling through this receptor is the critical step in the progression of tissue destruction in murine models of inflammation [4], [5], [6], [7]. In KRN TCR transgenic mice, in which the arthritis is chronic and symmetric with pannus formation and destructive lesions of joints and bones, FcγRIII-deficient mice have markedly diminished paw swelling following treatment with K/BxN serum, and mast cell-deficient mice have markedly attenuated synovitis [5]. Ji et al. [8] confirmed that the arthritogenic immunoglobulins act through Fc receptors (FcγRIII and complement). Furthermore, in mice immunized with collagen, on an arthritis susceptible DBA background, mice lacking FcRγ chains were protected relative to wild-type controls, although both groups produced similar levels of anti-collagen antibody [6].

Despite extensive efforts, the etiology and pathogenesis of RA remain poorly understood, and numerous cell populations have been implicated including T cells, B cells, monocytes/macrophages, mast cells, dendritic cells and fibroblasts [9]. Chemokines, matrix metalloproteinases, adhesion molecules, angiogenic growth factors, and dysregulated intra-articular expression of proinflammatory cytokines (in particular IL-1β and TNFα) play key roles in the pathogenesis of RA [10]. It is known that complement is activated in RA synovial fluids, and complement components are even locally produced [11]. Moreover, immune complexes are deposited at the cartilage level [12]. Humoral mediators may play a role as well; rheumatoid factors, found in 90% of patients, are associated with a poor prognosis. Genetic linkage studies implicate FcR polymorphisms as a potentially important disease susceptibility factor. In humans, there are three classes of FcγR (I–III); the level of expression and localization of FcγR (activating and inhibitory) restricts specific biological properties to certain cell types. Functional polymorphic variants within FCRG2A or FCRG2B, or within FCGR3A or FCGR3B, may additionally contribute to disease susceptibility and pathogenesis [13], [14], [15], [16], [17]. FcγR polymorphisms appear to be important predisposing factors in systemic lupus erythematosus (SLE) and multiple sclerosis (MS) [15], [18]. In addition, IgG receptor polymorphisms are also reported to influence the efficacy of intravenous immunoglobulin therapy for patients with autoimmune diseases [19].

The effects of a novel small molecule, R406, have been examined in several cell systems, and its activity is consistent with Syk inhibition in isolated mast cells, macrophages, DC, and synoviocytes. R406 potently inhibits IgG and IgE-induced mast cell degranulation in cell-based assays with an EC₅₀ of approximately 50 nM [20], [21]. Selectivity towards Syk inhibition was demonstrated in mast cells activated by FcεR1 cross-linking, in which R406 inhibited the phosphorylation of linker for activation of T cell (LAT) tyrosine Y191 (a Syk substrate) about 50-fold more potently than the phosphorylation of Syk itself, which is phosphorylated by Lyn kinase. Subsequent signaling events in that pathway, including JNK and ERK1/2, but not p38, were also inhibited [21]. IC stimulation of DC cytokine secretion is also inhibited by R406. R406 prevented cytokine release (IL-13) from DC isolated from the bone marrow of ovalbumin (OVA)-sensitized mice and cultured in the presence of R406 and OVA-IgG immune complexes [22]. Recent data implicate Syk signaling in TNFα-induced inflammatory cytokine and matrix metalloproteinase (MMP) production by fibroblast-like synoviocytes (FLS) isolated from joint tissues of RA patients [23], and treatment of cultured FLS with R406 markedly suppressed TNFα-induced IL-6 and MMP-3 production in RA FLS. Syk is an attractive target for immune and inflammatory diseases because inhibition of Syk can suppress numerous overlapping pathways simultaneously [1], [23], [24].

Given that Syk has a pivotal role in mediating FcR activation in hematopoietic cells, we investigated R406 and R788 (prodrug of R406) for their capacity to suppress the reverse passive Arthus (RPA) reaction and collagen-induced arthritis (CIA).