AB0124 Iguratimod, a novel DMARD, inhibits osteoclastogenesis and bone resorption in vitro

Background Iguratimod (T-614), a novel DMARD, was originally discovered by Toyama Chemical. The drug has been reported to have good efficacy in the ACR 20 response rate in the clinical trials on Japanese patients with rheumatoid arthritis (RA) (1). Using animal models of arthritis or autoimmune disease, we reported that iguratimod exhibited anti-inflammatory and immunomodulating effects (2). Notably, it was found to show a significant improvement in bone and cartilage destruction in rat type II collagen-induced arthritis (3). Objectives To elucidate the mechanism for improvement effect of iguratimod on the progression of articular destruction, we investigated its effect on osteoclastogenesis in vitro, and compared with other nonbiologic DMARDs. Methods Murine RAW264.7 cells and primary osteoclast precursor monocytes derived from mouse bone marrow (BMMs) were used for experiments. To differentiate into osteoclast-like cells, RAW264.7 cells and BMMs were cultured with receptor activator of nuclear factor kappa B ligand (RANKL) and RANKL + M-CSF, respectively. Tartrate-resistant acid phosphatase (TRACP)-positive multinucleated (>3 nuclei) cells (MNCs) were counted as osteoclast-like cells, and TRACP activity in the cell lysate was measured using para-nitrophenylphosphate as a substrate. Bone resorption activity was measured using fluoresceinated calcium phosphate-coated plate. To examine the effect of iguratimod on nuclear factor of activated T cells c1 (NFATc1), expression of NFATc1 was determined by western blot analysis using anti-NFATc1 monoclonal antibody. DNA binding was assessed using TransAM NFATc1 transcriptional factor assay kit. Results Iguratimod remarkably inhibited the increase of TRACP activity in a dose dependent manner (IC50 =0.7 μM) without inhibiting cell proliferation, and decreased the number of TRACP-positive MNCs at 1 μM in RANKL-stimulated RAW264.7 cells. Teriflunomide (active metabolite, leflunomide) also showed inhibitory effect on TRACP activity (IC50 =1.3 μM). On the other hand, reductions of TRACP activity by methotrexate were coincident with inhibition of the cell proliferation. Salazosulfapyridine did not show any clear effects. Similar results were also obtained with assay of TRACP activity in BMMs. Furthermore, iguratimod inhibited the bone resorbing activity of mature osteoclasts. Western blot analysis revealed that iguratimod significantly suppressed the NFATc1 expression induced by RANKL-stimulation, but not the DNA binding