Inhibition of phosphate transporters ameliorates the inflammatory and necrotic side effects of the nitrogen-containing bisphosphonate zoledronate in mice

Bisphosphonates (BPs) are pyrophosphate analogs. They are widely used against enhanced bone-resorption in various diseases. Nitrogen-containing BPs (N-BPs) exhibit strong anti-bone-resorptive effects but have inflammaatory and necrotic side effects. The non-nitrogen-containing BPs (non-N-BPs) etidronate and clodronate lack such side effects, but their anti-bone-resorptive effects are weak. In mice, etidronate and clodronate reduce the inflammaatory/necrotic effects of N-BPs, even those of zoledronate, the N-BP with the strongest anti-bone-resorptive effect yet reported and the highest risk of inflammaation/necrosis. Here, to explore the mechanisms underlying this protection, we used a mouse model in which a single reagent or a mixture of two reagents was injected subcutaneously into ear-pinnas. These reagents included zoledronate, four non-N-BPs, pyrophosphate, and inhibitors of various organic-anion-transporters. Pyrophosphate and two of the four non-N-BPs (not etidronate or clodronate) had inflammatory/necrotic effects. These effects were reduced by etidronate and clodronate, but not by phosphonoformate, an inhibitor of two of the three known phosphate-transporter families. Phosphonoformate reduced the inflammaatory/necrotic effects of zoledronate, but not those of pyrophosphate or of non-N-BPs. Conversely, pyrophosphate, at non-inflammaatory/necrotic concentrations, reduced the inflammaatory/necrotic effects of non-N-BPs, but not those of zoledronate. The effcacies of the protective effects against the inflammaatory/ necrotic effects of zoledronate were clodronate > etidronate > phosphonoformate. These fndings suggest that (i) the N-BP zoledronate may enter soft-tissue cells via phosphonoformate-inhibitable phosphate-transporters, (ii) other phosphate-transporters may carry pyrophosphate and inflammatory/necrotic non-N-BPs into such cells, and (iii) etidronate and clodronate inhibit all these transporters, and they ameliorate the side effects of zoledronate by inhibiting phosphonoformate-inhibitable phosphate-transporters.