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[Preprint]. 2024 May 3:rs.3.rs-4237258.
doi: 10.21203/rs.3.rs-4237258/v1.

Hydroxychloroquine and a low activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects

Zhenqiang Yao  1 , Akram Ayoub  1 , Venkatesan Srinivasan  2 , Jun Wu  1 , Churou Tang  1 , Rong Duan  3 , Aleksa Milosavljevic  2 , Frank Ebetino  2 , Alison Frontier  2 , Brendan Boyce  1
Affiliations

Hydroxychloroquine and a low activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects

Zhenqiang Yao et al. Res Sq. .

Update in

Abstract

Osteoporosis is incurable because there are no dual antiresorptive and anabolic therapeutic agents that can be administered long-term. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation and thus have limited effect in preventing osteoporotic fracture. 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 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.

Keywords: TRAF3; bisphosphonate; hydroxychloroquine; osteoblast; osteoclast; osteoporosis.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
HABP-HCQ stimulates OB and inhibits OC differentiation. (A) Synthesis of HABP – Hydrolysis of tetraethyl HABP with bromotrimethylsilane. (B) C57Bl6 mouse BM cells were cultured with M-CSF for 2 d followed by treatment with RANKL plus the indicated doses of HCQ, HABP-HCQ, or HABP for 3 d. OC number and surface were evaluated after TRAP staining. (C) BM cells were cultured for 5 d in 24-well plates to expand stromal cells, which were induced for OB differentiation in the presence of the indicated doses of HCQ or HABP-HCQ intermittently, 6 h each d, for 5 d. ALP staining was performed to evaluate OB differentiation. (D) Effects of 1 mM HABP vs. 1 mM HABP-HCQ and HCQ on OB differentiation, tested in 12-well plates, as above in (B). *p<0.05 & **p<0.01 vs. vehicle, or #p<0.01; one-way ANOVA+Dunnett’s test.
Figure 2
Figure 2
HABP-HCQ stabilizes TRAF3 in OC and OB progenitor cells. (A) C57Bl6 mouse BM cells were cultured with M-CSF for 3 d to expand macrophages, which were treated with vehicle or RANKL plus 3 uM of the indicated compounds for 24 hr. Protein levels of TRAF3 and its related level normalized to actin were analyzed by WB. (B) BdMPCs were treated with TGFb1 plus 3 uM of the indicated compounds for 24 hr, and levels of TRAF3 and actin protein were analyzed by WB. *p<0.05 & **p<0.01 vs. vehicle, #p<0.01 between the two groups, one-way ANOVA+/Dunnett’s test.
Figure 3
Figure 3
Low dose of HABP-HCQ effectively prevents PTH-induced OC formation and BM fibrosis in mice. (A) Diagram showing the treatment schedule of HABP-HCQ vs HCQ and PTH in mice. (B) TRAP-stained calvarial bones were used to quantify OC number (upper panel) and H&E-stained tibial bones were used to quantify fibrosis around trabeculae (lower panel). (C) An independent experiment was performed to evaluate the effect of HABP on PTH-induced OC formation in calvarial bones (upper panel) and BM fibrosis in tibial bones (lower panel). 5 mice per group for all experiments. *p<0.05, **p<0.01 vs. Vehicle or the two groups with line, one-way ANOVA+Dunnett’s test.
Figure 4
Figure 4
HABP-HCQ prevents OVX-induced bone loss independent of TRAF3. 10-wk-old female TRAF3f/f and TRAF3f/f LysMcre (cKO) mice on a C57Bl6 background had sham or OVX surgery. From the 3rd d, OVX mice for each phenotype were randomly assigned to 5 treatment groups, which were given 5 doses per wk of vehicle, HCQ (31.25 mM/kg), HABP-HCQ (6.25 mM/kg) or PTH (80 ng/mouse) or 1 weekly dose of zoledronate acid (0.15 mg/kg) for 5 wk. (A) The spines were micro-CT scanned to quantify the bone structural parameters in L1 vertebrae. BP-H = HABP-HCQ, Zol = zoledronate acid. (B) TRAP-stained L4 vertebrae was used to quantify OC number and surface on eroded surfaces. (C) OB surfaces on trabeculae were quantified on H&E-stained sections from L4 vertebrae. 8–9 mice per group for all experiments. *p<0.05, **p<0.01, one-way ANOVA+Dunnett’s test.
Figure 5
Figure 5
Low-dose HABP-HCQ restores the lost bone caused by OVX. 10-wk-old female C57Bl6 mice had sham (1 group) or OVX surgery (8 groups; 8–9 mice per group. 4 wk post-surgery, the sham and one group of ovariectomized mice were micro-CT scanned alive to confirm bone loss in ovariectomized mice (which then were euthanized). From the 5th wk, the 8 groups of ovariectomized mice were randomly assigned to 5 doses per wk of the indicated treatments for 5 wk. (A) The spines were micro-CT scanned to quantify the bone structural parameters in L1 vertebrae. (B) TRAP-stained sections from L4 vertebrae were used to quantify OC numbers and surfaces on eroded surfaces. (C) OB surfaces on trabeculae were quantified on H&E-stained sections of L4 vertebrae. (D) Dynamic bone formation parameters were measured in plastic-embedded undecalcified sections from L1 vertebrae. *p<0.05, **p<0.01, one-way ANOVA+Dunnett’s test.
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References

    1. Looker A. C., Melton L. J. 3rd, Harris T. B., Borrud L. G. & Shepherd J. A. Prevalence and trends in low femur bone density among older US adults: NHANES 2005–2006 compared with NHANES III. J Bone Miner Res 25, 64–71 (2010). 10.1359/jbmr.090706 - DOI - PMC - PubMed
    1. Baghdadi S. et al. Mortality following proximal femoral fractures in elderly patients: a large retrospective cohort study of incidence and risk factors. BMC Musculoskelet Disord 24, 693 (2023). 10.1186/s12891-023-06825-9 - DOI - PMC - PubMed
    1. Mashiba T. et al. Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib. J Bone Miner Res 15, 613–620 (2000). 10.1359/jbmr.2000.15.4.613 - DOI - PubMed
    1. Eastell R. et al. Effects of denosumab on bone turnover markers in postmenopausal osteoporosis. J Bone Miner Res 26, 530–537 (2011). 10.1002/jbmr.251 - DOI - PubMed
    1. Wasnich R. D. & Miller P. D. Antifracture efficacy of agents are related to changes in bone density. J Clin Endocrinol Metab 85, 231–236 (2000). 10.1210/jcem.85.1.6267 - DOI - PubMed

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