SYNTHESIS AND VALIDATION OF DM-22, A NOVEL H2S-RELEASING AMINO-BISPHOSPHONATE WHICH STIMULATES OSTEOBLAST DIFFERENTIATION

Objective: Hydrogen sulfide (H₂S) is a gasotransmitter recently shown to regulate bone metabolism by inhibiting osteoclast development in vitro and inducing the differentiation of osteoprogenitor cells both in vitro and in vivo. Moreover, endogenous levels of serum H₂S are significantly decreased in estrogen-deficient mice, suggesting that H₂S-replacement therapy could be a novel therapeutic approach for osteoporosis. Based on the dual action exerted by H₂S, we developed a H₂S-releasing bisphosphonate compound, named DM-22, prototype of a novel family of hybrid molecules aiming at treating bone loss. Methods: DM-22 was derived from alendronate (AL), hybridized with an aryl-sothiocyanate-based H₂S-releasing moiety; H₂S release was assessed by amperometry and the effects of DM-22 or AL (1-33μM) were investigated in vitro on human osteoclasts (OCs) and mesenchymal stromal cells (MSC) differentiation and function. Gene expression of osteogenic markers was analyzed by RT-PCR and LDH assay was used to assess cytotoxicity. Results: The incubation of DM22 at 1 mM in aqueous solution lead to along-lasting release of H₂S (peak concentration: 42μM of H₂S). LDH assay revealed that, contrary to AL, DM-22 is devoid of cytotoxicity on OCs and MSC even at the high dose of 33μM. DM-22 dose-dependently inhibited OCs differentiation reaching up to 40% relative inhibition in the total number of TRAP+ OC at 33μM. DM-22 dose-dependently increased mineral apposition, as revealed by Alizarin Red staining, compared to unstimulated MSC; moreover, DM-22 at the high concentration of 33μM lead to a 3-fold increase in the mRNA expression of Collagen Iand BSP. Conclusions: This work describes the synthesis of a new H₂S-hybrid N-BPs able to induce osteogenic differentiation of h-MSCs and, while retaining an anti-osteoclastogenic activity in vitro