Modeling rebound bone loss following denosumab discontinuation and sequential zoledronate therapy in TgRANKL osteoporotic mice

Vagelis Rinotas¹Eleftheria-Dimitra Ntouskou¹Melina Dragolia²Vasileios Ntafis²Maria P. Yavropoulou³Athanasios D Anastasilakis⁴Polyzois Makras⁵Eleni Douni^1,6*

• ¹Institute for Bioinnovation, Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece
• ²Institute for Fundamental Biomedical Research, Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece
• ³Endocrinology Unit, 1st Department of Propaedeutic and Internal Medicine, Medical School, National and Kapodistrian University of Athens, Athens, Greece
• ⁴Department of Endocrinology, Diabetes and Metabolism, 424 General Military Hospital, Thessaloniki, Greece
• ⁵Department of Endocrinology and Diabetes and Department of Medical Research, 251 Hellenic Air Force; VA General Hospital, Athens, Greece
• ⁶Laboratory of Genetics, Department of Biotechnology, Agricultural University of Athens, Athens, Greece

Abstract Denosumab, a monoclonal antibody against RANKL, is a highly effective antiresorptive agent for postmenopausal osteoporosis, while its discontinuation causes rapid bone loss and increased fracture risk. Using TgRANKL mice, which express human RANKL and respond to denosumab, we replicated this rebound effect and investigated sequential therapies. Administration of zoledronate after denosumab treatment effectively prevented rebound bone loss, highlighting its promise as a transition strategy. However, its discontinuation led to a delayed rebound bone loss. Our findings establish TgRANKL mice as a unique osteoporotic model for investigating the mechanisms driving denosumab rebound and testing sequential antiresorptive strategies. The rebound phenomenon that occurs after discontinuation of RANKL inhibition with denosumab represents a significant challenge in osteoporosis management. A major limitation of preclinical models investigating this effect is the lack of denosumab activity against murine RANKL. This study overcomes that limitation by exploring the rebound phenomenon in a transgenic mouse model expressing human RANKL (TgRANKL) and evaluating the impact of sequential zoledronate therapy. TgRANKL mice were divided into four experimental groups: vehicle control, continuous denosumab treatment, denosumab withdrawal, and sequential denosumab followed by zoledronate, including an additional follow-up phase after zoledronate discontinuation. Skeletal alterations were characterized using microCT, histomorphometric assessments, serum bone turnover markers (BTMs), and bone gene expression analyses. Denosumab therapy increased bone mass relative to untreated TgRANKL mice, whereas its discontinuation resulted in a rebound bone loss accompanied by elevated and overshooting bone turnover markers. Sequential administration of zoledronate attenuated the rebound response, although bone loss reappeared after zoledronate withdrawal. Denosumab also inhibited bone marrow adipose tissue formation in TgRANKL mice, while its discontinuation led to moderate reformation of marrow adiposity. The TgRANKL mouse model recapitulates the clinical rebound effects observed after denosumab withdrawal and provides a valuable preclinical platform for mechanistic studies and for evaluating future therapeutic interventions.