The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins.Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs.In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.     

Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates

Bisphosphonates (BPS) inhibit bone resorption and are divided into two classes according to their chemical structure and mechanism of action: nonnitrogen containing BPS such as etidronate and clodronate that are of low potency and inhibit osteoclast function via metabolism into toxic ATP-metabolites and nitrogen-containing BPS (NBPS), such as alendronate and risedronate that inhibit the enzyme of the mevalonate biosynthetic pathway farnesyl pyrophosphate synthase (FPPS), resulting in inhibition of the prenylation of small GTP-binding proteins in osteoclasts and disruption of their cytoskeleton. Previously, studies in various cell types suggested, however, that pamidronate functions by mechanism(s) additional or independent of the mevalonate pathway. To examine if such mechanism(s) are also involved in the action of NBPS on osteoclastic bone resorption, we examined the action of alkyl and heterocyclic NBPS with close structural homology on FPPS/isopentenyl pyrophosphate isomerase (IPPI) activity, on osteoclastic resorption, and on reversibility of this effect with GGOH. As expected, both pamidronate and alendronate suppressed bone resorption and FPPS/IPPI activity, the latter with greater potency than the first. Surprisingly, however, unlike alendronate, the antiresorptive effect of pamidronate was only partially reversible with GGOH, indicating the involvement of mechanism(s) of action additional to that of suppression of FPPS. Comparable results were obtained with the heterocyclic NBP NE-21650, a structural analog of risedronate. Thus, despite an effect on FPPS, the actions on bone resorption of some NBPS may involve mechanisms additional to suppression of FPPS. These findings may lead to identification of additional pathways that are important for bone resorption and may help to differentiate among members of the NBP class which are currently distinguished only according to their potency to inhibit bone resorption.     

Low concentration amino-bisphosphonates stimulate human keratinocyte proliferation and in vitro wound healing

Amino-bisphosphonates (N-BPs) are widely used to treat a great variety of clinical conditions involving altered calcium metabolism, as well as to prevent bone metastases. The use of N-BPs, however, display well-known side effects, including cellular toxicity, mainly at soft tissue and mucosal level, that arise from N-BPs ability to induce cell apoptosis when administered at clinically relevant concentrations. The aim of this study was to evaluate, in an in vitro wound healing model, the effect of N-BPs low concentration (10 nM-10 μM) stimulation on keratinocyte cellular behaviour. Human keratinocytes were treated with neridronate and zoledronate, two N-BPs with different chemical structure and clinical potency, but sharing a common pharmacological target: farnesyl pyrophosphate (FPP) synthase. Surprisingly, at the tested concentrations, both drugs stimulated keratinocytes proliferation, upregulating cytokeratin 5 while downregulating filaggrin expression, and wound healing ability, without any significant effect on matrix metalloproteinase (MMP)-9 activity. The lack of N-BPs effect on MMP-9 activity indicates that wound closure, in our experimental model, is mainly due to an increase in cell proliferation rather than to an increase in cell migration. Therefore, it can be hypothesised that the observed wound healing results could be ascribed to an N-BPs mediated reduction of FPP endogenous levels, thus suggesting new possible clinical applications for these compounds.     

In Vitro and In Vivo Responses to High and Low Doses of Nitrogen-Containing Bisphosphonates Suggest Engagement of Different Mechanisms for Inhibition of Osteoclastic Bone Resorption

The effects of nitrogen-containing bisphosphonates (N-BPs) on osteoclasts (Ocs) may differ with dose and regimen. N-BPs reduce Oc bone resorption by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), an effect counteracted by geranylgeraniol (GGOH), which restores geranylgeranylation downstream of FPPS. We assessed GGOH effects on inhibition of bone resorption by the N-BPs alendronate (ALN), ibandronate (IBN), and zoledronate (ZOL) in an assay of rabbit Oc resorption of bovine cortical bone. GGOH blocked inhibition of resorption at low, but not high, N-BP concentrations, with a 14- to 20-fold increase in IC₅₀ values for each N-BP. In vivo, growing male rats were administered doses calculated to mimic bioavailable exposures in daily (ALN, IBN), weekly (ALN), monthly (IBN), and yearly (ZOL) clinical regimens. Tibiae were harvested at 48 h, and metaphyses were analyzed. With lower ALN and IBN doses, Oc numbers rose by 26–48 %, morphology was normal, and there was no increase in apoptotic Ocs. In contrast, with higher IBN and ZOL doses, bone-associated Ocs were generally rounded in appearance and numbers of nuclei/Oc versus vehicle increased 42 and 31 %, respectively (P < 0.05). With ZOL, there was no rise in Oc number, but there was a 6.5-fold increase in apoptotic Ocs versus vehicle and a ≥13.5-fold increase versus lower-dose ALN or IBN (P < 0.05). With higher-dose IBN there was no rise in Oc number but 7- and 14-fold increases in Oc apoptosis versus low-dose ALN and IBN (P < 0.02). These results suggest that different mechanisms may come into play across the dosing spectrum of N-BPs.     

Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy

Summary Bisphosphonates (BPs) are well established as the leading drugs for the treatment of osteoporosis. There is new knowledge about how they work. The differences that exist among individual BPs in terms of mineral binding and biochemical actions may explain differences in their clinical behavior and effectiveness. Introduction The classical pharmacological effects of bisphosphonates (BPs) appear to be the result of two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Discussion There is new information about both properties. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The antiresorptive effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) appear to result from their inhibition of the enzyme farnesyl pyrophosphate synthase (FPPS) in osteoclasts. FPPS is a key enzyme in the mevalonate pathway, which generates isoprenoid lipids utilized for the post-translational modification of small GTP-binding proteins that are essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. BPs share several common properties as a drug class. However, as with other families of drugs, there are obvious chemical, biochemical, and pharmacological differences among the individual BPs. Each BP has a unique profile that may help to explain potential clinical differences among them, in terms of their speed and duration of action, and effects on fracture reduction.     

The Pharmacological Profile of a Novel Highly Potent Bisphosphonate,OX14 (1‐Fluoro‐2‐(Imidazo‐[1,2‐α]Pyridin‐3‐yl)‐Ethyl‐Bisphosphonate)

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen‐containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1‐fluoro‐2‐(imidazo‐[1,2 alpha]pyridin‐3‐yl)‐ethyl‐bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short‐term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3‐NSG murine model of myeloma‐induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

New role for an established drug? Bisphosphonates as potential anticancer agents

As a result of their ability to effectively reduce the risk of skeletal-related events, bisphosphonates (BPs) were incorporated into clinical practice over a decade ago, leading to a new treatment paradigm for patients with skeletal involvement from advanced cancer. BPs are now a well-established treatment option in this setting. Our review of the literature found that in addition to maintaining bone health in patients with malignant bone lesions and patients at risk for cancer therapy-induced bone loss, emerging preclinical and clinical data suggest that BPs may also have anticancer activity. Later generation, nitrogen-containing BPs (N-BPs), such as zoledronic acid (ZOL), inhibit the mevalonate pathway, subsequently inhibiting a number of cellular functions in bone-resorbing osteoclasts. In addition, N-BPs inhibit cancer cell proliferation, viability, motility, invasion and angiogenesis; induce cancer cell apoptosis; and act in synergy with antineoplastic agents. N-BPs, especially ZOL, may be useful as anticancer agents. As evidence continues to emerge, another shift in cancer treatment paradigms, in which N-BPs are considered for their anticancer activity as well as palliative effects, may be approaching.     

Altered expression of farnesyl pyrophosphate synthase in prostate cancer: evidence for a role of the mevalonate pathway in disease progression?

Background

Preclinical studies demonstrated effects of drugs inhibiting the mevalonate pathway including nitrogen-containing bisphosphonates (N-BPs) and statins on tumor growth and progression. The exact role of this pathway in prostate cancer (PC) has not been identified yet. Herein, we evaluate the expression of farnesyl pyrophosphate synthase (FPPS), the key enzyme of the mevalonate pathway, in PC.

Patients and methods

Prostate cancer (PC) and benign prostate tissue of 114 men who underwent radical prostatectomy were constructed to a tissue microarray. Immunohistochemical staining of FPPS was quantified by the Remmele/Stegner immunoreactivity-score. Patients’ clinical follow-up was assessed. IRS was correlated to pathological and clinical data. The impact of FPPS expression on clinical course was assessed univariate and multivariate.

Results

Mean IRS in PC and benign tissue was 5.7 (95% CI 5.0–6.5) and 2.6 (2.1–3.0, p < 0.0001). Mean IRS in PC tissue of patients with organ-confined and locally advanced disease (pT ≥ 3) was 5.09 (4.22–5.96) and 6.87 (5.57–8.17, p = 0.035). IRS of PC tissue significantly correlated with Gleason score (p = 0.03). Patients with PC tissue IRS >3 showed shorter recurrence-free survival compared to the remaining (p = 0.01). Increased FPPS expression is an independent risk factor for early biochemical recurrence (p = 0.032).

Conclusions

This is the first study on FPPS in PC specimens. The association of FPPS with established histopathological risk parameters and biochemical recurrence implicates a contribution of the mevalonate pathway to PC progression. Further functional analysis is required to explore the role of this pathway in PC and to investigate whether FPPS expression affects the response of PC cells to N-BPs.     

The level of ATP analog and isopentenyl pyrophosphate correlates with zoledronic acid-induced apoptosis in cancer cells in vitro

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat excessive bone resorption associated, e.g., with bone metastases. They have also antitumor activity. However, it is unclear whether this reflects an indirect effect via inhibition of bone resorption or a direct antitumor effect.Nitrogen-containing bisphosphonates (N-BPs), including zoledronic acid (ZOL), act by inhibiting farnesyl pyrophosphate synthase (FPPS). The mevalonate pathway is blocked and the accumulation of isopentenyl pyrophosphate (IPP) consequently occurs. IPP is conjugated to AMP to form a novel ATP analog (ApppI). The present study was undertaken to clarify whether IPP and/or ApppI has a direct involvement in apoptosis caused by ZOL in different cancer cell lines.There are marked differences in ZOL-induced ApppI formation between different cancer cell lines. On this basis, we selected three cancer cell lines that differ significantly from each other in their ZOL-induced IPP and ApppI accumulation: human estrogen-dependent (MCF7) and estrogen-independent (MDA-MB 436) breast cancer cell lines and a human myeloma cell line (RPMI 8226).The amount of IPP/ApppI correlated with the capacity of cells to undergo apoptosis. Geranylgeraniol (GGOH), an intermediate of mevalonate metabolism, blocks both IPP and ApppI formation and to some degree ZOL-induced apoptosis in a cell line-dependent manner. In addition, lovastatin (LOV), an inhibitor of the enzyme HMGCoA reductase, completely blocks IPP/ApppI formation as determined by mass spectrometry analysis, but enhances apoptosis.In conclusion, the current data suggest that ZOL-induced IPP/ApppI formation can contribute to ZOL-induced apoptosis. This mechanism and the inhibition of protein prenylation, both outcomes of FPPS inhibition in mevalonate pathway, seem to act in concert in ZOL-induced apoptosis in cancer cells.     

Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy

Potent bisphosphonates (BPs) preferentially bind bone at sites of active osteoclastic bone resorption, where they are taken up by the osteoclast and inhibit resorption. We tested the hypothesis that BP affinity to human bone affects antiresorptive potency. [(1)(4)C]-Alendronate binding to human bone was saturable and reversible with an apparent Kd of 72 microM by Scatchard analysis. In competition binding assays, unlabeled alendronate (Ki: 61 microM) was slightly more potent than pyrophosphate (Ki = 156 microM) in blocking [(1)(4)C]-alendronate binding. Likewise, most tested BPs, including etidronate (Ki: 91 microM), ibandronate (116 microM), pamidronate (83 microM), risedronate (85 microM) and zoledronate (81 microM), showed comparable affinities. Interestingly, tiludronate (173 microM; P < 0.05 vs. all other BPs) and especially clodronate (806 microM; P > 0.0001 vs. all other BPs) displayed significantly weaker affinity for bone. The weak affinity of clodronate translated into a requirement for 10-fold higher dosing in in vitro bone resorption assays when bone was pretreated with BP and subsequently washed prior to adding osteoclasts. In stark contrast, neither alendronate nor risedronate lost any efficacy after washing the bone surface. These findings suggest that most clinically tested BPs may have similar affinities for human bone. For those with reduced affinity, this may translate into lower potency that necessitates higher dosing.     

Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway,impairing Ras membrane localization in prostate cancer cells

PURPOSE: Metastasis to bone is an important cause of morbidity in advanced prostate cancer. Despite the typically sclerotic nature of prostatic bone metastases osteolysis has a significant role in the pathogenesis of this disease. The nitrogen containing bisphosphonates (N-BPs), such as pamidronate and zoledronic acid, have greatly enhanced potency for inhibiting bone resorption and inducing apoptosis in osteoclasts. We investigated the effects of N-BPs on prostate cancer cells. MATERIALS AND METHODS: Cell viability was determined with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay. Cell cycle analysis, DNA fragmentation and caspase 3 activity were assessed using flow cytometry. Ras, Bcl-2 and Bax were quantified by Western blotting. RESULTS: Pamidronate and zoledronic acid decreased cell viability in the 3 human cell lines DU145, PC3 and LNCaP. These effects were associated with changes in cell cycle distribution, induction of DNA fragmentation and a decrease in the Bcl-2-to-Bax ratio, which are features of apoptotic cell death. Pre-incubation with caspase inhibitors attenuated the effects of zoledronic acid and caspase 3 activity was demonstrated in treated DU145 cells. Zoledronic acid induced loss of cell viability in DU145 cells was prevented by co-treatment with farnesol, suggesting that N-BPs cause inhibition of the mevalonate pathway and Ras prenylation. A decrease in active, membrane bound Ras in zoledronic acid treated DU145 cells was shown by Western blot analysis. CONCLUSIONS: N-BPs induce apoptosis in prostate cancer via a caspase dependent mechanism. They have effects on protein prenylation via inhibition of the mevalonate pathway and impair membrane localization of Ras in prostate cancer cells.     

Bisphosphonates: the first 40 years

The first full publications on the biological effects of the diphosphonates, later renamed bisphosphonates, appeared in 1969, so it is timely after 40years to review the history of their development and their impact on clinical medicine. This special issue of BONE contains a series of review articles covering the basic science and clinical aspects of these drugs, written by some of many scientists who have participated in the advances made in this field. The discovery and development of the bisphosphonates (BPs) as a major class of drugs for the treatment of bone diseases has been a fascinating story, and is a paradigm of a successful journey from 'bench to bedside'. Bisphosphonates are chemically stable analogues of inorganic pyrophosphate (PPi), and it was studies on the role of PPi as the body's natural 'water softener' in the control of soft tissue and skeletal mineralisation that led to the need to find inhibitors of calcification that would resist hydrolysis by alkaline phosphatase. The observation that PPi and BPs could not only retard the growth but also the dissolution of hydroxyapatite crystals prompted studies on their ability to inhibit bone resorption. Although PPi was unable to do this, BPs turned out to be remarkably effective inhibitors of bone resorption, both in vitro and in vivo experimental systems, and eventually in humans. As ever more potent BPs were synthesised and studied, it became apparent that physico-chemical effects were insufficient to explain their biological effects, and that cellular actions must be involved. Despite many attempts, it was not until the 1990s that their biochemical actions were elucidated. It is now clear that bisphosphonates inhibit bone resorption by being selectively taken up and adsorbed to mineral surfaces in bone, where they interfere with the action of the bone-resorbing osteoclasts. Bisphosphonates are internalised by osteoclasts and interfere with specific biochemical processes. Bisphosphonates can be classified into at least two groups with different molecular modes of action. The simpler non-nitrogen containing bisphosphonates (such as etidronate and clodronate) can be metabolically incorporated into non-hydrolysable analogues of ATP, which interfere with ATP-dependent intracellular pathways. The more potent, nitrogen-containing bisphosphonates (including pamidronate, alendronate, risedronate, ibandronate and zoledronate) are not metabolised in this way but inhibit key enzymes of the mevalonate/cholesterol biosynthetic pathway. The major enzyme target for bisphosphonates is farnesyl pyrophosphate synthase (FPPS), and the crystal structure elucidated for this enzyme reveals how BPs bind to and inhibit at the active site via their critical N atoms. Inhibition of FPPS prevents the biosynthesis of isoprenoid compounds (notably farnesol and geranylgeraniol) that are required for the post-translational prenylation of small GTP-binding proteins (which are also GTPases) such as rab, rho and rac, which are essential for intracellular signalling events within osteoclasts. The accumulation of the upstream metabolite, isopentenyl pyrophosphate (IPP), as a result of inhibition of FPPS may be responsible for immunomodulatory effects on gamma delta (γδ) T cells, and can also lead to production of another ATP metabolite called ApppI, which has intracellular actions. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of BPs have been made, and more than a dozen have been studied in man. As reviewed elsewhere in this issue, bisphosphonates are established as the treatments of choice for various diseases of excessive bone resorption, including Paget's disease of bone, the skeletal complications of malignancy, and osteoporosis. Several of the leading BPs have achieved 'block-buster' status with annual sales in excess of a billion dollars. As a class, BPs share properties in common. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various BPs. Each BP has a unique profile in terms of mineral binding and cellular effects that may help to explain potential clinical differences among the BPs. Even though many of the well-established BPs have come or are coming to the end of their patent life, their use as cheaper generic drugs is likely to continue for many years to come. Furthermore in many areas, e.g. in cancer therapy, the way they are used is not yet optimised. New 'designer' BPs continue to be made, and there are several interesting potential applications in other areas of medicine, with unmet medical needs still to be fulfilled. The adventure that began in Davos more than 40 years ago is not yet over.     

The relationship between the chemistry and biological activity of the bisphosphonates

The ability of bisphosphonates ((HO)(2)P(O)CR(1)R(2)P(O)(OH)(2)) to inhibit bone resorption has been known since the 1960s, but it is only recently that a detailed molecular understanding of the relationship between chemical structures and biological activity has begun to emerge. The early development of chemistry in this area was largely empirical and based on modifying R(2) groups in a variety of ways. Apart from the general ability of bisphosphonates to chelate Ca(2+) and thus target the calcium phosphate mineral component of bone, attempts to refine clear structure-activity relationships had led to ambiguous or seemingly contradictory results. However, there was increasing evidence for cellular effects, and eventually the earliest bisphosphonate drugs, such as clodronate (R(1)=R(2)=Cl) and etidronate (R(1)=OH, R(2)=CH(3)), were shown to exert intracellular actions via the formation in vivo of drug derivatives of ATP. The observation that pamidronate, a bisphosphonate with R(1)=OH and R(2)=CH(2)CH(2)NH(2), exhibited higher potency than previously known bisphosphonate drugs represented the first step towards the later recognition of the critical importance of having nitrogen in the R(2) side chain. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates took place particularly in the 1980s, but still with an incomplete understanding of their structure-activity relationships. A major advance was the discovery that the anti-resorptive effects of the nitrogen-containing bisphosphonates (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their potency as inhibitors of the enzyme farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids utilized in sterol synthesis and for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular targets, such as osteocytes, may also be important. Over the years many hundreds of bisphosphonates have been synthesized and studied. Interest in expanding the structural scope of the bisphosphonate class has also motivated new approaches to the chemical synthesis of these compounds. Recent chemical innovations include the synthesis of fluorescently labeled bisphosphonates, which has enabled studies of the biodistribution of these drugs. As a class, bisphosphonates share common properties. However, as with other classes of drugs, there are chemical, biochemical, and pharmacological differences among the individual compounds. Differences in mineral binding affinities among bisphosphonates influence their differential distribution within bone, their biological potency, and their duration of action. The overall pharmacological effects of bisphosphonates on bone, therefore, appear to depend upon these two key properties of affinity for bone mineral and inhibitory effects on osteoclasts. The relative contributions of these properties differ among individual bisphosphonates and help determine their clinical behavior and effectiveness.     

Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42, and Rho GTPases.

N-BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP-bound, unprenylated Rho family GTPases in macrophages and osteoclasts. In macrophages, this also leads to sustained, Rac-mediated activation of p38. The antiresorptive activity of N-BPs may therefore be caused at least in part, by the accumulation of unprenylated small GTPases, causing inappropriate activation of downstream signaling pathways. INTRODUCTION: Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of bone resorption that act by inhibiting farnesyl diphosphate synthase, thereby indirectly preventing the prenylation of Rho family GTPases that are required for the function and survival of bone-resorbing osteoclasts. However, the effect that these drugs have on the activity of Rho family GTPases has not been determined. MATERIALS AND METHODS: The effect of N-BPs on the activity of Rho family GTPases in J774 macrophages and osteoclasts was measured using a pull-down assay to isolate the GTP-bound forms. The effect of N-BPs, or decreasing Rac expression using siRNA, on downstream p38 activity was evaluated by Western blotting and apoptosis assessed by measurement of caspase 3/7 activity. RESULTS: Rather than inhibiting GTPase function, loss of prenylation after treatment with N-BPs caused an increase in the GTP-bound form of Rac, Cdc42, and Rho in J774 cells and osteoclast-like cells, which paralleled the rate of accumulation of unprenylated small GTPases. Activation of Rac also occurred with other inhibitors of prenylation of Rho-family proteins, such as mevastatin and the geranylgeranyl transferase I inhibitor GGTI-298. The Rac-GTP that increased after N-BP treatment was newly translated, cytoplasmic unprenylated protein, because it was not labeled with [(14)C] mevalonate, and the increase in Rac-GTP was prevented by cycloheximide. Furthermore, this unprenylated Rac-GTP retained at least part of its functional activity in J774 cells, because it mediated N-BP-induced activation of p38. Paradoxically, although risedronate induces apoptosis of J774 macrophages by inhibiting protein prenylation, the p38 inhibitor SB203580 enhanced N-BP-induced apoptosis, suggesting that Rac-induced p38 activation partially suppresses the pro-apoptotic effect of N-BPs in these cells. CONCLUSIONS: N-BP drugs may disrupt the function of osteoclasts in vivo and affect other cell types in vitro by inhibiting protein prenylation, thereby causing inappropriate and sustained activation, rather than inhibition, of some small GTPases and their downstream signaling pathways.     

Clinical efficacy of bisphosphonates and monoclonal antibodies on bone mineral density following skeletal fractures

BackgroundBisphosphonates and monoclonal antibodies are drugs primarily developed to inhibit osteoclast-mediated bone resorption and are used to treat an array of skeletal pathologies. Their use is aimed at increasing bone health and therefore reducing fracture risks. The aim of this study was to evaluate the effectiveness of bone protection therapy on improving bone mineral density (BMD) in patients following a fracture.MethodsInclusion criteria consisted of patients who sustained a skeletal fracture and were subsequently commenced on bone protection therapy. Dual-energy X-ray Absorptiometry (DEXA) scans were performed at baseline and following a consented period of drug therapy. Bone health data included T-Scores, Z-Scores, FRAX Major, FRAX Hip and BMD. The clinical effectiveness of four bisphosphonates (alendronate, risedronate, pamidronate and zoledronate) and one monoclonal antibody (denosumab) were evaluated.ResultsA total of 100 patients were included in the study. Overall, bone protection therapy significantly improved Z-score Hip, Z-score Spine, T-score Spine and BMD Spine (p < 0.05). There was a marked difference between drug therapies. Denosumab and zoledronate were associated with the greatest treatment effect size. Alendronate only improved Z-score Spine and Z-score Hip (p < 0.05). Pamidronate and risedronate did not demonstrate any statistically significant improvement across any DEXA parameter.ConclusionOverall, bisphosphonates/monoclonal antibodies confer beneficial effects on bone health as measured by DEXA scans in patients following skeletal fractures. However, the magnitude of improvement varies among the commonly used drugs. Alendronate, zoledronate and denosumab were associated with greatest therapeutic benefit. Bone protection therapy did not improve fracture risk of patients (FRAX scores).     

Protein geranylgeranylation is required for osteoclast formation, function, and survival: inhibition by bisphosphonates and GGTI-298.

Bisphosphonates are the important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Although their molecular mechanism of action has not been fully elucidated, recent studies have shown that the nitrogen-containing bisphosphonates can inhibit protein prenylation in macrophages in vitro. In this study, we show that the nitrogen-containing bisphosphonates risedronate, zoledronate, ibandronate, alendronate, and pamidronate (but not the non nitrogen-containing bisphosphonates clodronate, etidronate, and tiludronate) prevent the incorporation of [14C]mevalonate into prenylated (farnesylated and geranylgeranylated) proteins in purified rabbit osteoclasts. The inhibitory effect of nitrogen-containing bisphosphonates on bone resorption is likely to result largely from the loss of geranylgeranylated proteins rather than loss of farnesylated proteins in osteoclasts, because concentrations of GGTI-298 (a specific inhibitor of geranylgeranyl transferase I) that inhibited protein geranylgeranylation in purified rabbit osteoclasts prevented osteoclast formation in murine bone marrow cultures, disrupted the osteoclast cytoskeleton, inhibited bone resorption, and induced apoptosis in isolated chick and rabbit osteoclasts in vitro. By contrast, concentrations of FTI-277 (a specific inhibitor of farnesyl transferase) that prevented protein farnesylation in purified rabbit osteoclasts had little effect on osteoclast morphology or apoptosis and did not inhibit bone resorption. These results therefore show the molecular mechanism of action of nitrogen-containing bisphosphonate drugs in osteoclasts and highlight the fundamental importance of geranylgeranylated proteins in osteoclast formation and function.     

The Effects of Zoledronic Acid on Serum Lipids in Multiple Myeloma Patients

Nitrogen-containing bisphosphonates (N-BPs) inhibit osteoclast-mediated bone resorption and are widely used for tumor-associated osteolysis. The mechanism of action of these drugs has not been completely clarified, but it has been observed that N-BPs may inhibit squalene synthase or farnesyl pyrophosphate synthase. Zoledronic acid (ZA) represents a novel N-BP which also has antitumor activity. To explore the effects of ZA on serum lipids, we studied 26 patients with smoldering myeloma at diagnosis. Sixteen patients were treated with ZA (4 mg) at baseline and at months 1, 2, 4, and 6. The remaining 10 served as controls. In all subjects, total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and C-terminal telopeptide of type I collagen (CTX) were measured at baseline and after 1, 3, and 6 months. In treated patients, we observed a progressive and significant reduction of TC, with a maximum decrease of 13% at 6 months. Moreover LDL-C decreased by 21% at 6 months, while no significant difference was appreciated in HDL-C and TGs. Also, the indexes of cardiovascular risk improved after ZA administration: TC/HDL-C ratio progressively decreased by 17% and HDL-C/LDL-C ratio increased by 36%, showing an effect that appears to be cumulative. In conclusion, ZA given intravenously at high doses in patients with smoldering myeloma seems to be able to modify the lipid profile with an improvement of atherosclerotic risk index.     

Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite

Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis. Although bisphosphonates act directly on osteoclasts, and interfere with specific biochemical processes such as protein prenylation, their ability to adsorb to bone mineral also contributes to their potency and duration of action. The aim of the present study was to compare the binding affinities for hydroxyapatite (HAP) of 6 bisphosphonates currently used clinically and to determine the effects of these bisphosphonates on other mineral surface properties including zeta potential and interfacial tension. Affinity constants (K(L)) for the adsorption of bisphosphonates were calculated from kinetic studies on HAP crystal growth using a constant composition method at 37 degrees C and at physiological ionic strength (0.15 M). Under conditions likely to simulate bisphosphonate binding onto bone, there were significant differences in K(L) among the bisphosphonates for HAP growth (pH 7.4) with a rank order of zoledronate > alendronate > ibandronate > risedronate > etidronate > clodronate. The measurements of zeta potential show that the crystal surface is modified by the adsorption of bisphosphonates in a manner best explained by molecular charges related to the protonation of their side-chain moieties, with risedronate showing substantial differences from alendronate, ibandronate, and zoledronate. The studies of the solid/liquid interfacial properties show additional differences among the bisphosphonates that may influence their mechanisms for binding and inhibiting crystal growth and dissolution. The observed differences in kinetic binding affinities, HAP zeta potentials, and interfacial tension are likely to contribute to the biological properties of the various bisphosphonates. In particular, these binding properties may contribute to differences in uptake and persistence in bone and the reversibility of effects. These properties, therefore, have potential clinical implications that may be important in understanding differences among potent bisphosphonates, such as the apparently more prolonged duration of action of alendronate and zoledronate compared with the more readily reversible effects of etidronate and risedronate.     

Zoledronate reduces unwanted bone resorption in intercalary bone allografts

Bone allografts are often hampered by graft incorporation and poor host bone formation. Bisphosphonates, synthetic pyrophosphate analogs, have shown promise in inhibiting bone resorption in human and animal trials. Some in vitro studies have suggested that high dose bisphosphonate may also inhibit bone formation, leading to our hypothesis that an ideal dose of bisphosphonate in allografts could protect allografts from resorption. We transplanted intercalary allografts in to the segmental defect of the rat femurs after soaking each allograft in zoledronate solution (30 μM) and then analysed bone density of the allografts six to 12 weeks after transplantation. At six and 12 weeks, the bone mineral density was higher in the experimental group compared with the control group. Qualitative radiographic and histological analysis also revealed more allograft resorption in the control group than in the zoledronate-treated group. Our data indicate that pharmacological modification of intercalary allografts with zoledronate solution can decrease osteoclast-mediated allograft resorption.     

Treatment with pyrophosphate inhibits uremic vascular calcification

Pyrophosphate, which may be deficient in advanced renal failure, is a potent inhibitor of vascular calcification. To explore its use as a potential therapeutic, we injected exogenous pyrophosphate subcutaneously or intraperitoneally in normal rats and found that their plasma pyrophosphate concentrations peaked within 15 min. There was a single exponential decay with a half-life of 33 min. The kinetics were indistinguishable between the two routes of administration or in anephric rats. The effect of daily intraperitoneal pyrophosphate injections on uremic vascular calcification was then tested in rats fed a high-phosphate diet containing adenine for 28 days to induce uremia. Although the incidence of aortic calcification varied and was not altered by pyrophosphate, the calcium content of calcified aortas was significantly reduced by 70%. Studies were repeated in uremic rats given calcitriol to produce more consistent aortic calcification and treated with sodium pyrophosphate delivered intraperitoneally in a larger volume of glucose-containing solution to prolong plasma pyrophosphate levels. This maneuver significantly reduced both the incidence and amount of calcification. Quantitative histomorphometry of bone samples after double-labeling with calcein indicated that there was no effect of pyrophosphate on the rates of bone formation or mineralization. Thus, exogenous pyrophosphate can inhibit uremic vascular calcification without producing adverse effects on bone.