Osteoporosis remains incurable. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation, while the anabolic agent, teriparatide, does not inhibit bone resorption, and thus they have limited efficacy in preventing osteoporotic fractures and cause some side effects. Thus, there is an unmet need to develop dual antiresorptive and anabolic agents to prevent and treat osteoporosis. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited marrow fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

骨质疏松症仍然无法治愈。使用最广泛的抗骨吸收剂双膦酸盐 （BPs） 也抑制骨形成，而合成代谢剂特立帕肽不抑制骨吸收，因此它们在预防骨质疏松性骨折方面的疗效有限，并引起一些副作用。因此，开发双重抗骨吸收剂和合成代谢剂来预防和治疗骨质疏松症的需求尚未得到满足。用于治疗类风湿性关节炎的羟氯喹 （HCQ） 可防止 TNF 受体相关因子 3 （TRAF3） 的溶酶体降解，TRAF3 是一种 NF-κB 衔接蛋白，可限制骨吸收并维持骨形成。我们试图将 HCQ 与预期抗吸收活性低的羟基烷基 BP （HABP） 共价连接，以靶向将 HCQ 递送到骨骼，以测试这种靶向是否增加了其防止骨微环境中 TRAF3 降解的功效，从而减少骨吸收和增加骨形成，同时减少其全身副作用。出乎意料的是，发现 HABP-HCQ 以盐的形式存在于水溶液中，由质子化的 HCQ 阳离子和去质子化的 HABP 阴离子组成。然而，它在体外抑制破骨细胞生成，刺激成骨细胞分化，并增加 TRAF3 蛋白水平。HABP-HCQ 显著抑制每日多次 PTH 注射小鼠的破骨细胞形成和骨髓纤维化。相比之下，HCQ 抑制骨髓纤维化，但不抑制破骨细胞形成，而单独 HABP 抑制小鼠破骨细胞形成，但不抑制纤维化。 HABP-HCQ 而不是 HCQ 阻止了小鼠卵巢切除术后的骨小梁丢失，重要的是，增加了已确定骨丢失的卵巢切除小鼠的骨量，因为 HABP-HCQ 同时增加了骨形成和骨吸收参数降低。相比之下，HCQ 增加了骨形成，但没有降低骨吸收参数，而 HABP 也恢复了卵巢切除小鼠的骨损失，但它抑制了骨吸收和形成的参数。我们的研究结果表明，HABP 和 HCQ 的组合可能具有双重抗吸收和合成代谢作用，以预防和治疗骨质疏松症。