Background RANK/RANKL signaling is the pathway of osteoclast differentiation and activation. In many bone diseases, the balance of bone formation and bone resorption is impaired. In osteoporosis, bone resorption is predominant with accelerated osteoclast differentiation by RANKL over-expression, and in rheumatoid arthritis, the osteoclasts activated by over-expressed RANKL on activated T cells induce bone erosion. Denosumab, a human anti-RANKL antibody, has been shown to be clinically effective for osteoporosis, and in a phase II study on rheumatoid arthritis patients, denosumab attenuated the progression of bone erosion. Whilst anti-RANKL antibodies have been developed, the potent and small molecule RANK/RANKL signal inhibitors have not been described yet.We carried out the high-throughput screening to discover the RANK/RANKL signal inhibitor. We optimized the lead molecule from high-throughput screening and found AS2690168.
Objectives To characterize in vitro and in vivo anti-osteoclastogenic effects of AS2690168.
Methods Osteoclastogenesis was evaluated by tartrate-resistance acid phosphatase (TRAP) staining in RAW 264 cells. The bone resorption was determined by Ca2+ concentration in the media of mouse calvaria stimulated with parathyroid hormone (PTH). Quantitative gene expression analysis for NFATc1 was performed by Real time quantitative PCR. A rat ovariectomy (OVX) was performed as an osteoporosis model. AS2690168 was daily administered from the day after OVX treatment. Femur total bone mineral density (BMD) was measured by peripheral quantitative computed tomography, and osteocalcin and TRAP-5b level in plasma were measured by ELISA. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc.
Results AS2690168 inhibited (1) RANKL-induced differentiation of octeoclast progenitors RAW 264 to osteoclasts, dose-dependently, (2) the expression of NFATc1 of RAW 264 cells stimulated with RANKL, and (3) the bone resorption of mouse calvaria stimulated with PTH. In a rat OVX-induced osteoporosis model, AS2690168 (0.3-3 mg/kg) suppressed loss of femur total BMD dose-dependently, with similar potency to risedronate. AS2690168 inhibited the increase of TRAP-5b which is the bone resorption marker in serum, but did not affect osteocalcin level which is the osteogenic marker.
Conclusions These results indicate that AS2690168 is selective inhibitor of RANKL- induced bone resorption and has a potential therapeutic benefit in treating bone diseases.
Disclosure of Interest Y. Kato Employee of: Astellas Pharma Inc., N. Morikawa Employee of: Astellas Pharma Inc., E. Hamachi Employee of: Astellas Pharma Inc., H. Nakayama Employee of: Astellas Pharma Inc., Y. Takata Employee of: Astellas Pharma Inc., K. Tamaki Employee of: Astellas Pharma Inc., H. Mizuhara Employee of: Astellas Pharma Inc., K. Nishimura Employee of: Wakunaga Pharmaceutical Co., LTD., H. Akamatsu Employee of: Wakunaga Pharmaceutical Co., LTD., Y. Taguchi Employee of: Wakunaga Pharmaceutical Co., LTD., T. Yamaguchi Employee of: Wakunaga Pharmaceutical Co., LTD., J. Miyata Employee of: Astellas Pharma Inc., Y. Higashi Employee of: Astellas Pharma Inc.
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