Uptake of a fluorinated bisphosphonate by cultured bones

Abstract

The uptake of bisphosphonates into bone was studied using 19-day-old fetal rat bones cultured with a new fluorinated bisphosphonate, difluoromethylidene bisphosphonate (F 2MBP). F 2MBP uptake was assessed by determining the weight percent of fluoride using electron probe microanalysis. By 30 min the weight percent of fluoride was significantly greater in the F 2MBP-treated bones than in controls and continually increased throughout the duration of the experiment to reach a fluoride concentration 6-fold greater than controls after 120 h of incubation. When the peripheral cortical bone was analyzed separately from the interior trabecular bone in the F 2MBP-treated bones, the fluoride concentration in the periphery increased until 24 h and then remained somewhat constant, while the interior, which is more actively remodeling, showed a continual increase. The uptake of F 2MBP during the 1 to 6 h time intervals demonstrated no differences between vital and devitalized bone and, thus, is not cell-mediated. Because analysis of free fluoride in F 2MBP media incubated with bones showed that the concentration of fluoride was less than 1% of the total amount of fluoride, the fluoride detected by the probe was most likely that of the intact molecule and not free fluoride. The rapid uptake of the F 2MBP molecule was supported by assessing the effects of short-term F 2MBP treatment on subsequent bone resorption, as determined by the release of 45Ca from prelabeled bones. Bones treated with F 2MBP for only 5 min exhibited reductions in the percentage of 45Ca released during the remainder of the 120 h incubation period similar to that when F 2MBP was continuously in the medium. These results indicate that this bisphosphonate compound rapidly enters the bone, localizes at sites which are actively remodeling, and exerts a prolonged and irreversible inhibitory effect on bone resorption. Furthermore, the findings that bone resorption was not inhibited with a significantly weaker chelating agent, tetrafluoroethylene bisphosphonate, and when bones were devitalized, support the concept that both binding of F 2MBP to the calcium of bone mineral and cellular effects are essential for F 2MBP-induced inhibition of bone resorption.