RN486, a Selective Bruton's Tyrosine Kinase Inhibitor, Abrogates Immune Hypersensitivity Responses and Arthritis in Rodents

Abstract

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopro-, in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fc receptor cross-linking-induced degranulation in mast cells (IC 50 ϭ 2.9 nM), Fc␥ receptor engagement-mediated tumor necrosis factor ␣ production in monocytes (IC 50 ϭ 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC 50 ϭ 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and boneprotective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function

“…Btk inhibition results in reduced Fc␥R-mediated cytokine production in monocytes and macrophages (19,27,28). However, the effect of reduced Btk function on phagocytosis is less clear, with studies showing significant (29 -31) or only marginal (32) effects.…”

Selective Inhibition of BTK Prevents Murine Lupus and Antibody-Mediated Glomerulonephritis

“…PF-06250112 profoundly reduced both of these populations, whereas RN486 had more modest effects. In this regard, it is noteworthy that PF-06250112 and RN486 exhibit distinct selectivity windows for TEC kinase (23 and 2063, respectively) (41). TEC is a nonreceptor tyrosine kinase that is expressed by both B and T cells.…”

Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune System

“…Indeed, in preclinical models, BTK inhibitors result in therapeutic efficacy in oncologic and autoimmune settings, including models of rheumatoid arthritis and systemic lupus erythematosus (SLE) (18)(19)(20)(21)(22)(23)(24). In the clinic, promising results with the BTK inhibitor ibrutinib have been observed in relapsed chronic lymphocytic leukemia and relapsed or refractory mantle cell lymphoma (25,26).…”

“…All mice were used between the ages of 8 and 12 wk. The following immunogens were used: NP-LPS (25 mg), NP 23 -CGG (25 mg), NP 3 -CGG (25 mg), and NP-Ficoll (100 mg) (all from Biosearch Technologies); PE (10 mg; Chromaprobe); alum (100 ml; Thermo Fisher Scientific), and ODN1826 (25 mg; InvivoGen). For in vivo B cell activation, 200 ml anti-IgD antisera (eBioscience) or 100 mg anti-CD40 (clone 1C10; BioLegend) was used.…”