INHIBITION OF BONE-RESORPTION BY BISPHOSPHONATES - INTERACTIONS BETWEEN BISPHOSPHONATES, OSTEOCLASTS, AND BONE

Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl2MBP) inhibits bone resorption through injury to the cells that resorb Cl2MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorption in vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl2MBP. The greater sensitivity of resorption to AHPrBP in vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormones in vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl2MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generation in vivo in AHPrBP-treated rats. These results suggest that the comparisons of potency in vitro do not include all the factors responsible for determining bisphosphonate potency in vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10(-3) M AHPrBP. Inhibition was associated with degenerative changes in osteoclasts and a more rapid decrease in the number remaining on the bone surface than occurred with Cl2MBP. The effect was specific for osteoclasts, could be prevented if bone resorption was suppressed by calcitonin, and was not seen in osteoclasts incubated in AHPrBP on plastic coverslips. These observations suggest that AHPrBP inhibits bone resorption through injury to osteoclasts when they solubilize bisphosphonate-contaminated bone. We found that the concentration of AHPrBP used in the preincubation phase could be reduced by an order of magnitude if the volume of the AHPrBP solution was correspondingly increased. This implies that the concentration of bisphosphonate is less relevant to potency comparisons than the density of bisphosphonate on the bone surface. The latter will be strongly influenced in vivo not only by affinity for bone but by the pharmacokinetic and other properties of the compound.