The effects of NO-NSAID and their novel analogues on bone metabolism in vitro and in vivo
Accelerated osteoclastic bone resorption plays an important role in the pathogenesis of osteoporosis and other common bone diseases such as Paget's disease of bone, rheumatoid arthritis and cancer-associated bone disease. The prevention and treatment of these diseases is based on agents that inhibit osteoclast (OC) formation and resorption. Nitrated non-steroidal anti-flammatory drugs (NO-NSAID) are a recently developed group of compounds that contain a conventional NSAID linked to a nitric oxide (NO)-donor group. As prostaglandins and NO can both stimulate and inhibit OC formation and bone resorption, I investigated whether NO-NSAD may affect the bone loss observed in bone diseases. Both Flurbiprofen and its nitrated derivative NO-Flurbiprofen (HCT1026) inhibit IL-1-stimulated OC formation and resorption in mouse co-cultures. However, HCT1026 was much more potent than the parent compound Flurbiprofen. As resorption was completely blocked by HCT1026 even though there were still OC present, I studied the effect of this drug on actin ring formation as an indicator of OC activity HCT1026 significantly decreased the percentage of active OC within 4 hours and all actin rings had disappeared after 24 hours, whereas Flurbiprofen had no effects. HCT1027, a HCT1026 derivative that lacks the NO-donor properties, was a potent inhibitor of OC formation and resorption in both murine and human cultures. The fact that HCT1027 and HCT1026 were equipotent clearly demonstrates that the anti-resorptive effects of these compounds are not dependent on NO-donor properties. Both HCT1026 and HCT1027 were potent inducers of apoptosis in OC and J774 cells as evidenced by numbers of apoptotic nuclei and caspase-3 activation. Treatment of primary osteoblasts (OB) with HCT1026 for up to 72 hours failed to induce OB apoptosis, suggesting that this compound acts only on cells of the monocyte-macrophage lineage such as macrophages and OC. Intraperitoneal injection with ABD56 (10 mg/kg/day) completely prevented ovariectomy-induced bone loss in mice and maintained BMD at level comparable to those of non-ovariectomised Sham controls. Histomorphometrical analysis of the proximal tibial metaphysis indicated that ABD56 totally recovered ovariectomy-induced trabecular bone loss and significantly decreased OC number in both Sham- and ovariectomy-operated mice, whereas it had not significant effects on OB numbers. In conclusion, we successfully developed a novel class of anti-resorptive drugs using structure-based drug design. The non-nitrated biphenyl carboxylic acid derivatives ABD56 and ABD68 are presented here as anti-resorptive agents that inhibit osteoclast formation, survival and resorption by a mechanism that is independent of NO production, COX inhibition and formation of metabolites. ABD56 prevents ovariectomy-induced bone loss and induces apoptosis in mature OC in vitro by inhibiting NFkB and p42/44 activity. Although its exact mechanism of action is yet unclear, ABD56 is a promising therapeutic candidate for the treatment of bone diseases associated with accelerated bone loss due to osteoclast activation such as osteoporosis, RA and cancer associated bone disease.