Effects of two novel bisphosphonates on bone cells in vitro

Abstract

Bisphosphonates are now widely used in the treatment of bone diseases, particularly where there is uncontrolled bone resorption, as they are known to be potent inhibitors of osteoclasis. It is still unclear whether the bisphosphonates act by inhibiting osteoclast maturation or by blocking the mechanism of bone resorption, and little is known of their effects on osteoblasts. Recent studies with 3-amino-1, hydroxypropylidene-1,1-bisphosphonic acid (APD) in the treatment of osteolytic metastases in breast cancer have suggested that APD may affect osteoblasts directly. We have now investigated the effects of two novel bisphosphonates, CGP 47072 and CGP 42446A on osteoclastogenesis in fetal rat calvariae cultured on collagen gels and on human osteoblasts (hOB) cultured as expiants from bone taken from patients at surgery. We also compared the action of these new bisphosphonates with that of APD, which at concentrations of 2.5 × 10⁻⁶ M to 2.5 × 10⁻¹⁰ M inhibited osteoclast recruitment, even when this was stimulated by conditioned medium from MCF7 breast cancer cells. This bisphosphonate was particularly potent if cultured with calvaria taken at 19 days gestation, when more immature osteoclast precursors are present. If calvariae from 20 days gestation were used, which contain more mature cells, it produced less inhibition. In contrast, CGP 42446A (2.5 × 10⁻⁶ M to 2.5 × 10⁻⁸ M) was more effective in inhibiting osteoclast maturation in calvariae from 20 days gestation than in those from 19 days. CGP 47072 had a similar pattern of effects but was less potent than either of the other two compounds. APD or CGP 42446A at 10⁻⁵ M significantly inhibited hOB numbers and DNA synthesis, but lower concentrations had little effect. CGP 47072 did not inhibit human osteoblast replication. It is unlikely that these effects are due to calcium chelation, as none of these compounds mimicked results obtained with EDTA, which was effective only at 2.5 × 10⁻⁶ M in reducing osteoclast size and 10⁻⁴ M in human osteoblast cultures. These results demonstrate that all three bisphosphonates are able to inhibit osteoclast formation at low concentrations. APD may be able to influence less mature osteoclast precursors and CGP 42446A and CGP47072 may exert their effects on the fusion of more mature precursor cells on the bone surface. At these concentrations, however, there is little or no effect on osteoblasts.