Differential Action of the Bisphosphonates (3-Amino-1-Hydroxypropylidene)-1,1-Bisphosphonate (APD) and Disodium Dichloromethylidene Bisphosphonate (Cl2MDP) on Rat Macrophage-mediated Bone Resorption In Vitro

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Abstract
The bisphosphonates (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) and disodium dichloromethylidene bisphosphonate (Cl2MDP) effectively inhibit the accelerated bone resorption associated with some skeletal disorders, e.g., Paget's disease. However, it has not been established whether these compounds exert their inhibitory effect by rendering the bone mineral more resistant to degradation, by diminishing the activity of resorbing cells, or through some combination of both activities. In this study, we have tested these possibilities using an in vitro resorption assay system consisting of elicited rat peritoneal macrophages co-cultured with particles of 45Ca-labeled, devitalized rat bone. This assay system permits the quantitative assessment of the action of APD and Cl2MDP on the two major phases of bone resorption (cell-substrate attachment and osteolysis) under circumstances where the drugs are present continuously or, most importantly for the issues in question, after the separate pretreatment of the particles or the resorbing cells. Our data indicate that (a) Both APD and Cl2MDP at concentrations ≥5 × 10-6 M diminish macrophage-mediated 45Ca release (i.e., bone resorption) in a log dose-dependent fashion. (b) A 10-min pretreatment of bone particles with either bisphosphonate (P-C-P) similarly inhibits resorptive activity, but is most pronounced with Cl2MDP. However, only APD is effective in reducing resorption when cells are preincubated (for 24 h) with P-C-P. (c) In cultures containing both labeled and unlabeled bone, significant inhibition occurs only when the labeled particles are coated with P-C-P (indicating that the action of P-C-P-treated bone is highly localized). (d) P-C-P does not diminish cell-bone particle attachment, an essential step in the resorptive process. On the other hand, delaying the addition of P-C-P until after cell-bone attachment is completed significantly reduces the resorption-inhibiting effect of these compounds. (e) Cl2MDP reduces culture DNA content in proportion to its inhibitory effect on resorption, and both the inhibitory and cytotoxic actions of this P-C-P are dependent upon the presence of bone. On the other hand, APD is cytotoxic only at very high concentrations (10-4 M), acts independently of the presence of bone, and inhibits resorption without killing cells. We conclude that the mechanisms of action of APD and Cl2MDP are markedly different. Cl2MDP is a potent cytotoxin in the presence of bone and apparently exerts its inhibitory effect in this manner. APD is noncytotoxic at levels adequate to suppress resorption and, therefore, must inhibit macrophage activity by some other mechanism. Neither P-C-P appears to limit resorption by decreasing the solubility of mineralized bone matrix.