STUDIES ON 2 NEW VITAMIN-D ANALOGS, EB-1089 AND KH-1060 - EFFECTS ON BONE-RESORPTION AND OSTEOCLAST RECRUITMENT IN-VITRO

We have investigated the effects on bone resorption of two new potent antiproliferative vitamin D-3 analogs, EB 1089 and KH 1060, by studying recruitment of osteoclasts in murine bone marrow cultures and Ca-45 release from prelabeled neonatal mouse calvarial bones. Binding studies to vitamin D receptor protein, from human osteosarcoma MG-63 cells, demonstrated k(d) values of 8.5 x 10(-11) for 1 alpha,25(OH)(2)D-3, 6/5 x 10(-11) for KH 1060, and 2.7 x 10(-10) for EB 1089, 1 alpha,25(OH)(2)D-3 and EB 1089 were equipotent stimulators of osteoclast recruitment in murine bone marrow cultures, with EC(50) at 10(-10) mol/L, whereas KH 1060 was about tenfold more potent with an EC(50) at 10(-11) mol/L. In serum-free media, 1 alpha,25(OH)(2)D-3 enhanced Ca-45 release from neonatal mouse calvarial bones with EC(50) at 10(-11) mol/L, but in the presence of 10% fetal calf serum (FCS) the stimulatory effect was significantly diminished, with a threshold value at 10(-10) mol/L. EB 1089 stimulated bone resorption with an estimated EC(50) at 3 x 10(-11) mol/L, whereas KH 1060 was about tenfold more potent than 1 alpha,25(OH)(2)D-3, and stimulated bone resorption with an EC(50) at 10(-12) mol/L. The effects of EB 1089 and KH 1060 on Ca-45 release were not significantly affected by the addition of 10% FCS, Addition of vitamin D binding protein to serum-free incubations of neonatal mouse calvarial bones significantly inhibited the bone resorbing effect of 1 alpha,25(OH)(2)D-3, but did not affect EB 1089 and KH 1060 induced Ca-45 release, These data show that the k(d) values for the binding to Vitamin D receptors are similar to the EC(50) values for stimulation of bone resorption and osteoclast differentiation, in vitro, for 1 alpha,25(OH)(2)D-3, EB 1089, and KH 1060. The discrepancy in the effect of FCS on Ca-45 release between 1 alpha,25(OH)(2)D-3 and the new analogs suggests that EB 1089 and KH 1060 do not bind to vitamin D binding protein, indicating that pharmacokinetic differences may partially explain their less calcemic effects in vivo.