Takefumi Oizumi

Inhibition of necrotic actions of nitrogen-containing bisphosphonates (NBPs) and their elimination from bone by etidronate (a non-NBP): a proposal for possible utilization of etidronate as a substitution drug for NBPs.

PURPOSE Nitrogen-containing bisphosphonates (NBPs) have powerful anti-bone-resorptive effects (ABREs). However, recent clinical applications have disclosed an unexpected side effect, osteonecrosis of the jaw. We previously found in mice that etidronate (a non-NBP), when coadministered with alendronate (an NBP), inhibited the latters inflammatory effects. However, etidronate also reduced the ABRE of alendronate. The present study examined in mice the modulating effects of etidronate on the inflammatory and necrotic actions of zoledronate (the NBP with the strongest anti-bone-resorptive activity and the highest incidence of osteonecrosis of the jaw) and on ABREs of various NBPs including zoledronate. MATERIALS AND METHODS NBPs were subcutaneously injected into ear pinnas of mice and ensuing inflammation and necrosis at the site of the injection were evaluated. ABREs of NBPs were evaluated by analyzing sclerotic bands induced in mouse tibias. RESULTS Coinjection of etidronate reduced inflammatory and necrotic reactions induced by zoledronate, and also reduced the amount of zoledronate retained within the ear tissue. When both agents were intraperitoneally injected, etidronate reduced the ABRE of zoledronate and those of other NBPs. Notably, etidronate reduced the ABRE of zoledronate even when this non-NBP was injected 16 hours after the injection of zoledronate. Bone scintigram indicated that etidronate reduced the amount of zoledronate that had already bound to bone. CONCLUSIONS These results suggest that etidronate may 1) inhibit the entry of NBPs into cells related to inflammation and/or necrosis, 2) inhibit the binding of NBPs to bone hydroxyapatite, 3) at least partly eliminate (or substitute for) NBPs that have already accumulated within bones, and thus 4) if used as a substitution drug for NBPs, be effective at treating or preventing NBP-associated osteonecrosis of the jaw.

Necrotic actions of nitrogen-containing bisphosphonates and their inhibition by clodronate, a non-nitrogen-containing bisphosphonate in mice: potential for utilization of clodronate as a combination drug with a nitrogen-containing bisphosphonate.

Nitrogen-containing bisphosphonates (NBPs) exhibit powerful anti-bone-resorptive effects (ABREs) via inhibition of farnesyl pyrophosphate synthase during cholesterol biosynthesis. Clinical applications have disclosed an unexpected side effect, namely osteonecrosis of jaw bones, and although thousands of cases have been documented in the last few years the mechanism remains unclear. Since NBPs accumulate in bone-hydroxyapatite, more jaw bone osteonecrosis cases may come to light if NBPs continue to be used as they are being used now. We have previously reported that in mice, systemic (intraperitoneal) injection of clodronate (a non-NBP) prevents the inflammatory effects of NBPs. Here, we examined in mice the local necrotic actions of various NBPs and the anti-necrotic effects of clodronate. A single subcutaneous injection of an NBP into the ear pinna induced necrosis at the injection site (relative potencies of necrotic actions of NBPs: zoledronate >> pamidronate > or = alendronate > risedronate), while non-NBPs lacked this effect. Clodronate, when injected together with an NBP, reduced or prevented the necrosis induced by that NBP, but not its ABRE. Clodronate reduced the amount of each NBP retained within tissues. These results, together with those of previous studies, suggest that (i) clodronate inhibits the inflammatory and necrotic actions of NBPs by inhibiting their incorporation into cells related to inflammation and/or necrosis, (ii) clodronate could be useful as a combination drug with NBPs for preventing their necrotic actions while retaining their ABREs and (iii) clodronate could also be useful as a substitution drug for NBPs in patients at risk of osteonecrosis of jaw bones.

In vitro cytotoxicity of zoledronate (nitrogen-containing bisphosphonate: NBP) and/or etidronate (non-NBP) in tumour cells and periodontal cells

OBJECTIVE Nitrogen-containing bisphosphonates (NBPs), the first-choice drugs for diseases that cause enhanced bone resorption, may injure jawbones and gastrointestinal tissues. In rodents, NBPs cause necrosis at injection sites. Bisphosphonates accumulate within bones, especially where there is inflammation. We hypothesized that if jawbone-accumulated NBPs are released, they may directly injure cells around the jawbones. To examine this hypothesis, we compared the direct effects of zoledronate (NBP) and/or etidronate (non-NBP) on various cells, including periodontal cells. DESIGN Various human tumour cells (such as squamous carcinoma cells and prostate adenocarcinoma cells) and periodontal cells (such as gingival fibroblasts and periodontal ligament cells) were incubated with or without zoledronate and/or etidronate. Cell viability and cytotoxicity were determined by tetrazolium dye assay and by FITC-Annexin V/propidium iodide assay, respectively. RESULTS Zoledronate, at 100μM, was toxic to all types of cells tested, while its toxicity varied among cells at both 1 and 10μM. There was no clear difference between tumour cells and non-tumour cells in sensitivity to the cytotoxicity of zoledronate. In contrast, etidronate was not toxic at 1-100μM in any of the cells tested. Interestingly, etidronate reduced the cytotoxicity of zoledronate in many cell-types, including gingival fibroblasts. CONCLUSIONS These results, together with those reported by others and those from our previous in vivo experiments, suggest that NBPs, upon release from jawbones (e.g., during dental surgery or bone infection), may directly injure various cells located around the jawbones, and that etidronate may be protective against the cytotoxicity of NBPs in periodontal tissues.

Pro-IL-1β accumulation in macrophages by alendronate and its prevention by clodronate.

Nitrogen-containing bisphosphonates (NBPs), anti-bone-resorptive drugs, exhibit inflammatory side effects (fever, jaw osteomyelitis or osteonecrosis, etc.). We previously reported that in mice: (i) a single intraperitoneal injection of alendronate (an NBP, 40 μmol/kg or less) induces various inflammatory reactions, (ii) these effects, which are minimal in IL-1-deficient mice, can be prevented by co-administration of clodronate (a non-NBP, 40 μmol/kg or less), and (iii) alendronate increases IL-1β in tissues (liver, spleen, and lung), but strangely not in blood. Here, we found the following in mice. (a) The IL-1β in tissues is pro-IL-1β. (b) Unlike LPS, alendronate induces minimal activation of caspase-1 (pro-IL-1β-processing enzyme). (c) The tissue pro-IL-1β elevations are largely absent in macrophage-depleted mice. (d) In vitro, 100 μM alendronate directly stimulates RAW 264 cells (murine macrophage-like cells) to produce pro-IL-1β, and 1 μM clodronate inhibits this effect. These results suggest that in mice: (i) the major pro-IL-1β-producing cells in response to alendronate are macrophages, (ii) alendronate directly stimulates them to produce pro-IL-1β, but the release of mature IL-1β is below detectable levels due to insufficient activation of caspase-1, and (iii) clodronate inhibits the pro-IL-1β production by acting directly on macrophages, although the in vivo mechanism may differ from the in vitro one.

Promotion of arthritis and allergy in mice by aminoglycoglycerophospholipid, a membrane antigen specific to Mycoplasma fermentans

Mycoplasmas, which lack a cell wall and are the smallest self-replicating bacteria, have been linked to some chronic diseases, such as AIDS, rheumatoid arthritis (RA), and oncogenic transformation of cells. Their membrane components (lipoproteins and glycolipids) have been identified as possible causative factors in such diseases. Glycoglycerophospholipid (GGPL)-III, a unique phosphocholine-containing aminoglycoglycerophospholipid, is a major specific antigen of Mycoplasma fermentans, and has been detected in 38% of RA patients. Unlike those of lipoproteins, which induce inflammation via Toll-like receptor 2 (TLR2), the pathologic effects of GGPL-III are poorly understood. RA and metal allergies are chronic inflammatory diseases in which autoantigens have been implicated. Here, we examined the effects of chemically synthesized GGPL-III in murine arthritis and allergy models. GGPL-III alone exhibited little inflammatory effect, but promoted both collagen-induced arthritis and nickel (Ni) allergy, although less powerfully than Escherichia coli lipopolysaccharide. The augmenting effect of GGPL-III on Ni allergy was present in mice deficient in either T cells or active TLR4, but it was markedly weaker in mice deficient in macrophages, interleukin-1, or the histamine-forming enzyme histidine decarboxylase than in their control strains. These results suggest that GGPL-III may play roles in some types of chronic diseases via the innate immune system.

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

Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) can occur when enhanced bone-resorptive diseases are treated with nitrogen-containing BPs (N-BPs). Having previously found, in mice, that the non-N-BP etidronate can (i) reduce the inflammatory/necrotic effects of N-BPs by inhibiting their intracellular entry and (ii) antagonize the binding of N-BPs to bone hydroxyapatite, we hypothesized that etidronate-replacement therapy (Eti-RT) might be useful for patients with, or at risk of, BRONJ. In the present study we examined this hypothesis. In each of 25 patients receiving N-BP treatment, the N-BP was discontinued when BRONJ was suspected and/or diagnosed. After consultation with the physician-in-charge and with the patients informed consent, Eti-RT was instituted in one group according to its standard oral prescription. We retrospectively compared this Eti-RT group (11 patients) with a non-Eti-RT group (14 patients). The Eti-RT group (6 oral N-BP patients and 5 intravenous N-BP patients) and the non-Eti-RT group (5 oral N-BP patients and 9 intravenous N-BP patients) were all stage 2-3 BRONJ. Both in oral and intravenous N-BP patients (particularly in the former patients), Eti-RT promoted or tended to promote the separation and removal of sequestra and thereby promoted the recovery of soft-tissues, allowing them to cover the exposed jawbone. These results suggest that Eti-RT may be an effective choice for BRONJ caused by either oral or intravenous N-BPs and for BRONJ prevention, while retaining a level of anti-bone-resorption. Eti-RT may also be effective at preventing BRONJ in N-BP-treated patients at risk of BRONJ. However, prospective trials are still required.

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

Bisphosphonates (BPs) are used against diseases involving increased bone-resorption. Among BPs, nitrogen-containing BPs (N-BPs) have much stronger anti-bone-resorptive effects than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs carry the risk of inflammatory/necrotic effects, including osteonecrosis of jawbones. When injected into mouse ear-pinnas, N-BPs induce inflammatory/necrotic effects within the ear-pinna. We previously found that (a) the non-N-BPs clodronate and etidronate can reduce such side effects of N-BPs, and (b) phosphonoformate (an inhibitor of the phosphate transporters SLC20 and SLC34) can reduce the inflammatory/necrotic effects of zoledronate (the N-BP with the highest reported risk of side effects). However, it is not clear (i) whether phosphonoformate can reduce the side effects of other N-BPs, too, and (ii) whether other phosphonocarboxylates have such inhibitory effects. Here, using the mouse ear-pinna model, we compared the effects of etidronate, clodronate, and four phosphonocarboxylates on the inflammatory/necrotic effects of N-BPs of the alkyl type (alendronate) or cyclic type (zoledronate and minodronate). Like phosphonoformate, the other three phosphonocarboxylates protected against the inflammatory/necrotic effects of all the N-BPs. The protective potencies were clodronate>etidronate>phosphonoacetate>phosphonoformate>phosphonopropionate>phosphonobutyrate. With a similar order of potencies, these agents reduced the amount of (3)H-alendronate retained within the ear-pinna after its injection therein. The mRNAs of SLC20 and SLC34 were detected in untreated ear-pinnas. These findings suggest that the inhibition of phosphate transporters by phosphonocarboxylates, as well as by etidronate and clodronate, might be a useful preventive strategy against the side effects of both alkyl- and cyclic-type N-BPs.

Antagonism Between Bisphosphonates (BPs) and Structurally Related Substances in the Effects of BPs in Mice

The anti-bone-resorptive effects (ABREs) of nitrogen-containing BPs (NBPs) are much more powerful than those of non-nitrogen-containing BPs (non-NBPs). However, NBPs have inflammatory and necrotic side effects (INSEs) including osteonecrosis of jaw bones. Here, we examined antagonism between BPs and structurally related substances in terms of ABREs and INSEs. Our findings suggest that a certain type(s) of transporters or related mechanisms may be involved in the intracellular uptake of NBPs, leading to INSEs.

Prevention of necrotic actions of nitrogen-containing bisphosphonates (NBPs) in mice by non-NBPs (clodronate and etidronate)

Nitrogen-containing bisphosphonates (NBPs) have powerful antibone-resorptive effects (ABREs), but they induce unexpected side effect, osteonecrosis of jaw-bones (ONJ). The mechanism underlying NBP-associated ONJ is unclear. Zoledronate, the strongest NBP, exhibits the highest incidence of ONJ. We previously found that clodronate and etidronate (non-NBPs) inhibit NBP-induced inflammation. Here, we found the following. (a) Subcutaneous injection of zoledronate into ear-pinnas induced inflammation and then necrosis at these sites. These effects of zoledronate were strongest among NBPs tested, while non-NBPs lacked these effects. (b) Coinjection of clodronate or etidronate reduced the amount of zoledronate retained within the ear tissue and reduced the inflammatory and necrotic effects of zoledronate. (c) When zoledronate and clodronate were intraperitoneally injected, clodronate little affected the ABRE of zoledronate, as well as those of other NBPs. In contrast, etidronate markedly reduced the ABRE of zoledronate. Notably, etidronate reduced the ABRE of zoledronate even when it was injected 16 h after the injection of zoledronate. These results suggest that (a) clodronate and etidronate may inhibit the entry of NBPs into cells related to inflammation and/or necrosis and prevent NBPs’ side effects, (b) clodronate could be useful as a combination drug with NBPs for preventing their side effects while retaining their ABREs, (c) etidronate (but not clodronate) may competitively inhibit the binding of NBPs to bone hydroxyapatite (BHA), and this reagent may reduce NBPs that have already accumulated within bones, and (d) etidronate, if used as a substitution drug for NBPs, may be effective at treating or preventing NBP-associated ONJ.