A Novel Inhibitor of I-KappaB Kinase Beta Ameliorates Experimental Arthritis through Downregulation of Proinflammatory Cytokines in Arthritic Joints

Abstract

Inhibitor of kappaB (IκB) kinase beta (IKKβ) plays a critical role in nuclear factor-kappaB (NF-κB) activation and production of proinflammatory cytokines in various inflammatory diseases including rheumatoid arthritis. We previously reported a novel IKKβ inhibitor Compound D, 4-[6-(cyclobutylamino)imidazo[1,2-b]pyridazin-3-yl]-2-fluoro-N-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methyl}benzamide, which is efficacious in experimental arthritis models. In the present study, we characterized the pharmacological properties of Compound D and investigated the mechanisms of the anti-arthritic effect. Compound D inhibited IKKβ kinase activity with 160-fold selectivity against IKKα. The cellular analyses revealed that Compound D selectively blocked NF-κB promoter activity among major cellular signaling pathways, such as the activator protein-1 pathway, consistent with inhibition of the NF-κB signaling pathway including phosphorylation of IκBα. In addition, Compound D inhibited NF-κB-driven production of tumor necrosis factor alpha (TNFα) and interleukin-6 comparably. The correlation between inhibitory effect on TNFα production and plasma concentration of the compound was observed in vivo. Consecutive administration of Compound D decreased gene expression of proinflammatory cytokines and inflammatory mediators in the paws of arthritic mice with attenuation of paw swelling. Notably, Compound D was rapidly distributed to the arthritic paws, rather than healthy paws, and where it decreased the gene expression of proinflammatory cytokines by a single oral administration. Furthermore, Compound D completely inhibited arthritis progression even when treatment occurred after disease development. These data suggest that the downregulation of proinflammatory cytokines in local inflamed joints is one of the mechanisms underlying the anti-arthritic effect of the IKKβ inhibitor, Compound D.