Identifying therapeutic biomarkers of zoledronic acid by metabolomics

Zoledronic acid (ZOL) is a potent antiresorptive agent that increases bone mineral density (BMD) and reduces fracture risk in postmenopausal osteoporosis (PMOP). The anti-osteoporotic effect of ZOL is determined by annual BMD measurement. In most cases, bone turnover markers function as early indicators of therapeutic response, but they fail to reflect long-term effects. We used untargeted metabolomics to characterize time-dependent metabolic shifts in response to ZOL and to screen potential therapeutic markers. In addition, bone marrow RNA-seq was performed to support plasma metabolic profiling. Sixty rats were assigned to sham-operated group (SHAM, n = 21) and ovariectomy group (OVX, n = 39) and received sham operation or bilateral ovariectomy, respectively. After modeling and verification, rats in the OVX group were further divided into normal saline group (NS, n = 15) and ZOL group (ZA, n = 18). Three doses of 100 μg/kg ZOL were administrated to the ZA group every 2 weeks to simulate 3-year ZOL therapy in PMOP. An equal volume of saline was administered to the SHAM and NS groups. Plasma samples were collected at five time points for metabolic profiling. At the end of the study, selected rats were euthanatized for bone marrow RNA-seq. A total number of 163 compound were identified as differential metabolites between the ZA and NS groups, including mevalonate, a critical molecule in target pathway of ZOL. In addition, prolyl hydroxyproline (PHP), leucyl hydroxyproline (LHP), 4-vinylphenol sulfate (4-VPS) were identified as differential metabolites throughout the study. Moreover, 4-VPS negatively correlated with increased vertebral BMD after ZOL administration as time-series analysis revealed. Bone marrow RNA-seq showed that the PI3K-AKT signaling pathway was significantly associated with ZOL-mediated changes in expression (adjusted-p = 0.018). In conclusion, mevalonate, PHP, LHP, and 4-VPS are candidate therapeutic markers of ZOL. The pharmacological effect of ZOL likely occurs through inhibition of the PI3K-AKT signaling pathway.