Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.

https://asbmr.onlinelibrary.wiley.com/doi/pdf/10.1359/jbmr.2005.20.7.1265

Nitrogen-Containing Bisphosphonates Inhibit the Mevalonate Pathway and Prevent Post-Translational Prenylation of GTP-Binding Proteins, Including Ras

BACKGROUND
Bisphosphonates currently are the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia, Paget's disease, and osteoporosis. These compounds have high affinity for calcium and therefore target to bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function.

METHODS
This article reviews the pharmacology of bisphosphonates and the relation between the chemical structure of bisphosphonates and antiresorptive potency, and describes recent new discoveries of their molecular mechanisms of action in osteoclasts.

RESULTS
Bisphosphonates can be grouped into two pharmacologic classes with distinct molecular mechanisms of action. Nitrogen-containing bisphosphonates (the most potent class) act by inhibiting the mevalonate pathway in osteoclasts, thereby preventing prenylation of small GTPase signaling proteins required for osteoclast function. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that may involve the formation of cytotoxic metabolites in osteoclasts or inhibition of protein tyrosine phosphatases.

CONCLUSIONS
Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts. After more than 30 years of clinical use, their molecular mechanisms of action are only just becoming clear. Cancer 2000;88:2961–78. © 2000 American Cancer Society.

Cellular and molecular mechanisms of action of bisphosphonates

Bisphosphonates: The first 40 years

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo, although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently, several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase, thereby preventing protein prenylation in osteoclasts. In this study, we examined the potency of a wider range of nitrogen-containing bisphosphonates, including the highly potent, heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro, to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro, and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations > or = 1 nM zoledronic acid or minodronate, the order of potency (zoledronic acid approximately equal to minodronate > risedronate > ibandronate > incadronate > alendronate > pamidronate) closely matching the order of antiresorptive potency. Furthermore, minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates, giving rise to less potent inhibitors of bone resorption in vivo, also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo, and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase.

Structure-Activity Relationships for Inhibition of Farnesyl Diphosphate Synthase in Vitro and Inhibition of Bone Resorption in Vivo by Nitrogen-Containing Bisphosphonates

Bisphosphonates: from the laboratory to the clinic and back again.

Bisphosphonates (BPs), such as clodronate and pamidronate, are inhibitors of bone resorption and are used on a widespread basis in the treatment of hyper-resorptive bone diseases. At the cellular level, BPs inhibit osteoclasts, but the precise molecular mechanisms are unclear. BPs have also been shown to affect the survival of macrophages, cells ontogenetically related to osteoclasts. We show that both clodronate and pamidronate induce apoptosis in isolated osteoclasts. Clodronate, when administered in liposomes, also induced apoptosis in rat peritoneal macrophages in vitro and in liver macrophages of mice in vivo but not in murine macrophage-like RAW-264 cells. The subcellular localization and staining intensity of Bcl-2, an anti-apoptotic protein known to protect several cell types against drug-induced apoptosis, were similar in RAW-264 and peritoneal macrophage cells, as revealed by immunofluorescence. The clodronate-induced apoptotic pathway was further characterized in isolated osteoclasts cultured on glass coverslips through the use of clodronate-containing liposomes and several inhibitors of the apoptotic cascade. None of the agents tested could totally prevent clodronate-induced osteoclast death. Partial protection was, however, obtained by the addition of staurosporine or homocysteine. The results suggest that primarily cytoplasmic, protein kinase C-activated mechanisms are involved in the execution of clodronate-induced apoptosis of osteoclasts.

Characteristics of Clodronate-Induced Apoptosis in Osteoclasts and Macrophages

Calcitonin promotes osteoclast survival in vitro

Clinicians have been concerned that fractures do not heal properly in individuals exposed to bisphosphonate treatment, a treatment that strongly affects bone metabolism. The current study attempted to clarify the long-term effects of clodronate (dichloromethylene bisphosphonate) treatment on fracture healing in growing rats. Clodronate was administered subcutaneously twice a week in a dose of 2 mg/kg or 10 mg/kg. Physiologic saline served as a control. After 24 weeks of treatment, the tibiae were fractured, and the treatment was continued for another 4 weeks and 8 weeks. At both end points the cross-sectional areas of the callus, measured by peripheral quantitative computed tomography, were greater in the clodronate-treated rats than in controls, but there were no significant differences in bone mineral density. There were no significant differences between treatments in radiologic healing, histomorphometry, or in mechanical failure load of the callus with the exception of increased tensile stiffness at a dose of 2 mg/kg at 4 weeks. Clodronate treatment does not seem to prolong the fracture healing process, even when administered on a long-term basis before the fracture. Clodronate increases the size of the callus, but has only a minor effect on its biomechanical properties. The current results suggest that long-term clodronate treatment does not inhibit fracture healing.

Long-term administration of clodronate does not prevent fracture healing in rats.

Fracture healing and callus formation have traditionally been evaluated by using X-ray radiography Here we compared X-ray radiography and peripheral quantitative computed tomography (pQCT) in evaluating the healing callus of standardized tibial fractures in 141 female rats after a 4- or 8-week follow-up The results were compared with the tensile (4-week) and compressive (8-week) failure load of the callus The projectional size of callus, as defined from lateral ex vivo radiographs, correlated significantly with the pQCT-defined cross-sectional area (CSA) of mid-callus This relationship was dependent on the pQCT attenuation threshold, being higher for the CSA of compact bone (r = 085, P < 00001) than for the total bone CSA (r = 068, P < 00001) Radiographically defined callus projectional area also correlated strongly with bone mineral content (BMC) (r = 084–086, P < 00001) The mean optical density of the callus analyzed from the radiographs had only a weak correlation with the pQCT-defined bone mineral density (BMD) of callus A weak negative relationship was found between CSA and BMD The optical density analyzed from lateral radiographs did not correlate with the tensile or compressive failure load of callus Callus size, BMC, and BMD were associated with the compressive failure load, whereas both radiographs and pQCT were poor in explaining the failure load in tension

Comparison of Radiographic and pQCT Analyses of Healing Rat Tibial Fractures

Daily oral clodronate treatment was evaluated in Sprague-Dawley rats for its ability to inhibit estrogen-deficiency-induced changes in femoral neck, femoral diaphysis, and lumbar vertebrae (L4-L5). Six-month-old ovariectomized (OVX) rats were administered by gavage a vehicle (Veh) or clodronate (100 or 500 mg/kg/day). Sham-operated (SHAM) control rats received the vehicle (n = 15/group). Treatment was started on the day of operation and continued for 3 months. Trabecular bone volume (BV/TV) and structural variables (trabecular number, Tb.N; thickness, Tb.Th; separation, Tb.Sp; and trabecular bone pattern factor, Tb.Pf) were assessed on secondary spongiosa of the right femoral neck Furthermore, cantilever bending test of the left femoral neck and compression test of L4, ash weight of L5, and morphometric studies of femoral diaphysis were carried out, and serum and urinary markers of bone turnover were determined. The OVX/Veh group had higher levels of serum osteocalcin and alkaline phosphatase and higher urinary excretion of deoxypyridinoline/creatinine than the SHAM/Veh group at 3 months postsurgery, and clodronate reduced these changes. BV/TV of femoral neck, bone mass of L5, and the maximum loads of the femoral neck and L4 were lower after OVX than SHAM operation. Although clodronate prevented trabecular bone loss in the femoral neck and preserved Tb.Pf at the SHAM control level, it failed to preserve the mechanical strength at the femoral neck However, in lumbar vertebrae, clodronate prevented the loss of bone mass and mechanical properties. Furthermore, there was a good positive correlation between maximum load of L4 and the ash weight of L5 (n = 58, r = 0.69, p < 0.001). In the femoral neck (n = 55), Tb.Pf correlated negatively with BV/TV and Tb.N (r = -0.59 and r = -0.55;p < 0.001, respectively) and positively with Tb.Sp (r = 0.61, p < 0.001). In femoral mid-diaphysis, there were no significant changes in cortical bone geometry in any of the groups. We conclude that orally administered clodronate suppresses the enhanced bone turnover in adult OVX rats and preserves trabecular bone volume and connectivity in the femoral neck In the axial skeleton, clodronate has a beneficial effect on lumbar vertebral bone mass and strength.

Ritva Hannuniemi

Papers

Characteristics of Clodronate-Induced Apoptosis in Osteoclasts and Macrophages

Calcitonin promotes osteoclast survival in vitro

Long-term administration of clodronate does not prevent fracture healing in rats.

Comparison of Radiographic and pQCT Analyses of Healing Rat Tibial Fractures

Clodronate Prevents Osteopenia and Loss of Trabecular Connectivity in Estrogen-Deficient Rats†