New 19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 analogs strongly stimulate osteoclast formation both in vivo and in vitro

2-Methylene-19-nor-(20 S )-1α,25-dihydroxyvitamin D 3 (2MD), an analog of 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ], has been shown to strongly induce bone formation both in vitro and in vivo . We have synthesized four substituents at carbon 2 of 2MD (2MD analogs), four stereoisomers at carbon 20 of the respective 2MD analogs (2MD analog-C20 isomers) and four 2MD analogs with an oxygen atom at carbon 22 (2MD-22-oxa analogs) and examined their ability to stimulate osteoclastogenesis and induce hypercalcemia. 2MD analogs were 100 times as potent as 1α,25(OH) 2 D 3 in stimulating the formation of osteoclasts in vitro and in inducing the expression of receptor activator of NF-κB ligand (RANKL) and 25-hydroxyvitamin D 3 -24 hydroxylase mRNAs in osteoblasts. The osteoclast-inducing activities of 2MD analog-C20 isomers and 2MD 22-oxa analogs were much weaker than those of 2MD analogs. In addition, the activity of a 2MD analog in inducing dentine resorption was much stronger than that of 1α,25(OH) 2 D 3 in the pit formation assay. Affinities to the vitamin D receptor and transcriptional activities of these compounds did not always correlate with their osteoclastogenic activities. Osteoprotegerin-deficient (OPG −/− ) mice provide a suitable model for investigating in vivo effects of 2MD analogs because they exhibit extremely high concentrations of serum RANKL. The same amounts of 2MD analogs and 1α,25(OH) 2 D 3 were administered daily to OPG −/− mice for 2 days. The elevation in serum concentrations of RANKL and calcium was much greater in 2MD analog-treated OPG −/− mice than in 1α,25(OH) 2 D 3 -treated ones. A 2MD analog was much more potent than 1α,25(OH) 2 D 3 in causing hypercalcemia and in increasing soluble RANKL with enhanced osteoclastogenesis even in wild-type mice. In contrast, the administration of the 2MD analog to c- fos -deficient mice failed to induce osteoclastogenesis and hypercalcemia. These results suggest that new substituents at carbon 2 of 2MD strongly stimulate osteoclast formation in vitro and in vivo , and that osteoclastic bone resorption is indispensable for their hypercalcemic action of 2MD analogs in vivo .