Inhibition of Phosphate Transporters Ameliorates the Inflammatory and Necrotic Side Effects of the Nitrogen-Containing Bisphosphonate Zoledronate in Mice

Abstract

Bisphosphonates (BPs) are pyrophosphate analogs. They are widely used against enhanced boneresorption in various diseases. Nitrogen-containing BPs (N-BPs) exhibit strong anti-bone-resorptive effects but have inflammatory 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 inflammatory/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 inflammation/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 inflammatory/necrotic effects of zoledronate, but not those of pyrophosphate or of non-N-BPs. Conversely, pyrophosphate, at non-inflammatory/necrotic concentrations, reduced the inflammatory/necrotic effects of non-N-BPs, but not those of zoledronate. The efficacies of the protective effects against the inflammatory/ necrotic effects of zoledronate were clodronate > etidronate > phosphonoformate. These findings suggest that (i) the N-BP zoledronate may enter soft-tissue cells via phosphonoformate-inhibitable phosphatetransporters, (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 phosphatetransporters.

Inflammatory Effects of Nitrogen-Containing Bisphosphonates (N-BPs): Modulation by Non-N-BPs

“…10) Here, we compared the effects of 50 mM PFA, Eti, and Clo on the inflammatory effects of other N-BPs. As shown in Fig.…”

“…8,9) Concerning the mechanism underlying such interesting effects of Eti and Clo, our pharmacological studies have suggested that they can competitively inhibit the entry of N-BPs into soft-tissue cells, and that this possibly occurs via SLC20 and/or SLC34 phosphate transporters. 10) Tiludronate (Til, not used in Japan) is a cyclic non-N-BP. Thus, it might be expected that tiludronate would be an effective inhibitor of the entry of cyclic N-BPs into softtissue cells, thereby reducing or preventing their inflammatory side effects.…”

“…1), all of which are used in Japan. In this study, unlike in previous studies, [8][9][10] inflammatory effects were evaluated by measuring the swelling induced in hind-paw-pads by subcutaneous injection of test reagents into them. Such swelling is more precisely and objectively measurable than the inflammation/necrosis in ear-pinnas, on which the previous method depended.…”

Phosphonocarboxylates Can Protect Mice against the Inflammatory and Necrotic Side Effects of Nitrogen-Containing Bisphosphonates by Inhibiting Their Entry into Cells <i>via</i> Phosphate Transporters

“…Among these phosphate transporter families, SLC20 and/or SLC34 have been suggested to be involved in the transport of N-BPs. 16) Among the antibodies that would be required to detect them by immunostaining, antibodies against only SLC20a1 and SLC20a2 are commercially available. Figure 9 shows immunostaining results obtained using those two antibodies.…”

A Strategy against the Osteonecrosis of the Jaw Associated with Nitrogen-Containing Bisphosphonates (N-BPs): Attempts to Replace N-BPs with the Non-N-BP Etidronate

“…It should be noted that such cytotoxic effects of N-BPs are initiated by their entry into soft-tissue cells, 12) and that there are few reports of inflammatory/necrotic side effects in patients treated with non-N-BPs. 10) Indeed, the non-N-BPs etidronate and clodronate almost completely lack inflammatory and necrotic effects in mice, 7,16) and they are hardly retained at all within soft-tissues in mice after intravenous injection.…”