BoneKEy-Osteovision | Meeting Reports
Meeting report from what is new in bisphosphonates: Sixth workshop on bisphosphonates
DOI:10.1138/2002041
Summary of the preclinical sessions Michael J. RogersThe sixth workshop on bisphosphonates attracted 600 participants from academia and the pharmaceutical industry. 35 abstracts covering preclinical studies on bisphosphonates were presented. The “new” issues arising from the 35 poster sessions and 20 oral presentations can be grouped into 5 main topics:
Internalization of Bisphosphonates by Osteoclasts and Other Cells Inhibition of Farnesyl Diphosphate Synthase and Anti-Resorptive Potency Effects on Protein Prenylation and Osteoclast Function Anti-Tumor and Anti-Angiogenic Effects of Bisphosphonates Long-term Effects on Bone and Resistance to Bisphosphonates
Internalization of bisphosphonates by osteoclasts and other cellsFor many years it has been assumed that bisphosphonates (BPs) specifically affect bone-resorbing osteoclasts in vivo owing to the ability of BPs to bind to bone mineral, whereupon they are released in the acidic environment of the resorption lacuna beneath resorbing osteoclasts. Uptake of the BPs most likely occurs by fluid-phase endocytosis, resulting in the internalization of drug within endocytic vacuoles in the cell. In order to enter the osteoclast cytoplasm (the site of action) BPs must cross the vacuolar membrane or the plasma membrane. Since all BPs are negatively charged other than at very low pH it is unlikely that entry into the cytosol is achieved by passive diffusion, suggesting the involvement of a membrane transport protein. Frith and colleagues (Univ of Aberdeen) () presented new data that strongly suggests the presence of such a transport mechanism, since clodronate and nitrogen-containing BPs (N-BPs, alendronate or ibandronate) appeared to compete for cellular uptake by rabbit osteoclasts and J774 macrophages cultured in vitro. In the presence of molar excess of clodronate the uptake of radiolabelled N-BP was reduced, the inhibitory effect of N-BPs on protein prenylation was prevented, and the induction of J774 apoptosis by N-BPs was attenuated. The exact transport mechanism involved in the uptake of BPs remains to be identified.
Inhibition of farnesyl diphosphate synthase and anti-resorptive potencyIt was at the 4th Bisphosphonate Workshop in Davos in 1998 that it was finally agreed that N-BPs inhibit bone resorption by disrupting the mevalonate pathway in osteoclasts. Since then it has become clear that the molecular target is farnesyl diphosphate (FPP) synthase, an enzyme in the mevalonate pathway that has two substrate binding sites for the isoprenoid lipids DMAPP, IPP and GPP. However, the exact mechanism by which N-BPs inhibit FPP synthase is not known. Computer modelling studies had previously been used to suggest that N-BPs may act as transition state analogues of GPP. However, Ebetino and colleagues from Procter & Gamble () presented new computer modelling data suggesting that N-BPs may bind to FPP synthase at the binding site for IPP, with the nitrogen of the BP side chain forming a salt bridge with the phosphate groups of GPP. Furthermore, changes in the chemical structure of the side chain that affected the potency for inhibiting FPP synthase also affected the orientation of the bisphosphonates in the IPP binding site. Hence, N-BPs may inhibit FPP synthase by preventing the binding of IPP. This conclusion was supported to some extent by enzyme kinetic analysis presented by Dunford and coworkers from the Univ of Aberdeen (). Using recombinant human FPP synthase, risedronate was shown to be a competitive inhibitor with respect to GPP, but an uncompetitive inhibitor with respect to IPP at low concentrations of risedronate and a non-competitive inhibitor at higher concentrations of risedronate. Clearly, the mode of inhibition of FPP synthase is very complex. While N-BPs probably act partly by competing with GPP for binding, they also influence the binding of IPP, and it may be the latter effect that has the major influence on inhibitory potency. Ultimately, the exact mode of inhibition of FPP synthase may not be clarified without X-ray crystallographic studies.
The differences in overall anti-resorptive potency of BPs observed in vivo is likely a reflection of subtle differences in pharmacokinetic profiles (e.g. affinity for bone mineral) as well as the ability to inhibit FPP synthase. Nancollas et al. (Procter & Gamble) showed that different BPs have different adsorption affinity constants for hydroxyapatite, in the rank order zoledronate>alendronate>ibandronate>risedronate >etidronate>clodronate (). The greater affinity of zoledronate for bone mineral (and hence possible readsorption of circulating BP released by osteoclasts during bone resorption) may help explain the basis for the prolonged effects of a single infusion of zoledronate on bone turnover and bone density in postmenopausal women, recently reported by Reid et al. () The latter effects were supported by data presented by Gasser and Green (Novartis), showing that a single IV injection of zoledronic acid was sufficient to prevent cancellous and cortical bone loss in ovariectomized rats for up to 32 weeks ().
Effects on protein prenylation and osteoclast functionInhibition of FPP synthase in osteoclasts prevents the synthesis of isoprenoid lipids required for post-translational prenylation of small GTPases such as Rho, Rac and Rabs. However, it is not yet known how the loss of prenylated GTPases leads to disruption of osteoclast function and osteoclast apoptosis. Coxon et al. (Univ of Aberdeen) showed that inhibition of prenylation of Rab GTPases by risedronate (or a specific Rab GGTase inhibitor) causes changes in the subcellular distribution of Rab6 in rabbit osteoclasts, and accumulation of Rab6 and Rab27 in the cytosolic fraction rather than the membrane fraction of macrophages in vitro (). Hence, the anti-resorptive effects of N-BPs are likely to be due in part to the disruption of vesicular trafficking owing to loss of Rab function. Reszka and colleagues (Merck) also presented evidence that p70S6 kinase signalling is an important survival pathway in osteoclasts (). The anti-apoptotic cytokines M-CSF, sRANKL and TNF caused activation of p70S6K in murine osteoclasts, an effect that was reduced following inhibition of protein geranylgeranylation by GGTI. Furthermore, rapamycin, an inhibitor of p70S6 activation, caused osteoclast apoptosis. Hence, N-BPs may cause osteoclast apoptosis by inhibiting protein prenylation and thereby suppressing the activation of the pro-survival p70S6 pathway.
Anti-tumor and anti-angiogenic effects of bisphosphonatesSeveral abstracts were presented confirming recent reports that BPs can affect breast, prostate and myeloma tumor cells in vitro, by decreasing cell invasion, migration and proliferation or by causing apoptosis (,,,). In particular, Croucher and coworkers (Univ of Oxford) showed that treatment with zoledronic acid (previously called zoledronate) prevented osteolytic disease and bone loss in the 5T2MM model of multiple myeloma (). Zoledronic acid also caused a decrease in paraprotein levels, tumor burden and angiogenesis, and importantly also increased the survival of tumor-bearing animals. Fournier et al. (INSERM) also showed that zoledronic acid treatment suppressed osteolytic disease and decreased tumor burden in bone in nude mice inoculated with GFP-expressing MDA-MB-231 breast cancer cells (). In addition to indirect or direct anti-tumor effects, several recent studies have indicated that zoledronic acid may have anti-angiogenic effects. The effects of zoledronic acid on an in vivo model of angiogenesis were described by Boissier and colleagues (INSERM), who suggested that zoledronic acid may affect angiogenesis at extra-skeletal sites (). Zoledronic acid treatment significantly inhibited vascular regrowth in the ventral prostate of testosterone-treated castrated rats, and caused apoptosis of endothelial cell in vitro. The molecular mechanisms underlying these effects on endothelial cells remain to be determined.
Long-term effects on bone and resistance to bisphosphonatesConcerns have been raised regarding the long-term effects of BPs on bone remodelling and hence bone quality. Addressing this question in an rat model in vivo, Seedor and Rodan (Merck) showed that the formation of cancellous bone that occurs following marrow ablation was not affected by up to 28-fold the human therapeutic dose of alendronate (). The equivalent therapeutic dose in human also did not affect subsequent resorption of the newly-formed bone, and 25-fold higher doses were required to slow the rate of resorption by 50%. Similarly, Seeherman et al. (Genetics Institute) () showed that systemic treatment with ibandronate did not affect bone formation following implantation of BMP2/absorbable collagen sponge into femoral core defects in experimental animals, but did inhibit the initial increase in osteoclastic bone resorption that occurred following implantation.
Resistance to the anti-resorptive effects of BPs, for example in patients with Paget's disease and hypercalcemia, also remains a poorly-understood phenomenon. Thompson et al. (Univ of Aberdeen) described the isolation of a BP-resistant strain of J774 macrophages (). The resistant cells appeared to internalise N-BPs to the same extent as the parental cells, but were markedly resistant to the cytotoxic effects of N-BPs and protein prenylation was less affected than in parental cells. The exact basis for the resistance of these cells to BPs, and the possible relevance to mechanisms of resistance in vivo, remain to be determined.
Summary of the clinical sessions R. Graham Russell and Nelson B. WattsThe plenary presentations on Thursday morning March 21 began with Phillippe Clezardin (Lyon) who reviewed the evidence that bisphosphonates might have direct anti-tumor effects that contribute to their clinical value in treating bone metastases. Reported effects include induction of apoptosis, as well as interference with tumor cell adhesion and migration, proliferation, and production of metalloproteinases (MMPs 2, 9 and 12). Several of these effects are mediated by inhibition of the mevalonate pathway, and occur at low concentrations, suggesting that they may supplement the better known effects of BPs on osteoclast-mediated bone resorption.
This was followed by Pierre Meumier (Lyon), who addressed the importance of mineralization of bone. Newly formed bone undergoes primary mineralization (~100 days) but requires secondary mineralization (which requires months or years) to reach its full potential mineral content. Secondary mineralization is less in states of high turnover (such as osteoporosis) and improved under treatment with bisphosphonates. As measured by microradiography, secondary mineralization under the influence of alendronate accounted for a 7% to 10% increase in density, consistent with changes in BMD as measured by DXA ().
Pierre Delmas (Lyon) then reviewed studies that showed that increases in BMD explained only part of the anti-fracture effects of bisphosphonates (and other antiresorptive agents). Evidence for this includes similar reductions in vertebral fracture risk for agents that produce different changes in BMD (), similar reductions in vertebral fracture risk for patients who have smaller or greater gains in BMD on bisphosphonate treatment (), and different fracture rates for placebo and raloxifene-treated patients who have similar changes in BMD (). Changes in bone turnover (which reach maximum much earlier than changes in BMD) could account for some of the anti-fracture effect of bisphosphonates that is not explained by BMD ().
Two abstracts looked at the effect of intravenous ibandronate on BMD and bone turnover markers. Brenner and colleagues () treated 20 women who had primary osteoporosis with 2 mg ibandronate infusions every third month plus daily calcium and vitamin D. After 2 years, BMD increased significantly by 3.5% to 5.5% in the hip and spine and serum levels of C-telopeptide and P1NP decreased significantly. On the other hand, Ishida et al.() gave the same ibandronate treatment (but without calcium and vitamin D) to 10 Japanese women who had borderline low bone density. They found no increase in BMD after 6 months. Bone turnover was significantly reduced within 8 days of dosing but returned to baseline after 2 months (one month before the next infusion was due). It is likely that sustained suppression of bone turnover is necessary for optimal anti-fracture effect. Failure to achieve sustained suppression may account for the lack of antifracture effect in a large clinical trial of ibandronate () and could be due to either too low a dose of the drug, dosing less often than necessary, or lack of adequate calcium and vitamin D.
Francis Glorieux reviewed his experience using pamidronate to treat patients with osteogenesis imperfecta (). He and his colleagues have observed reductions in fractures, lessening of bone pain, and improvement of growth in children () and infants () with severe osteogenesis imperfecta.
Silvano Adami reviewed conditions where bisphosphonates have been used for conditions other than the main indications. He showed encouraging data using bisphosphonates for treatment of primary hyperparathyroidism (), fibrous dysplasia (), and reflex sympathetic dystrophy ().
Socrates Papapoulous discussed dosing of bisphosphonates. The daily dosing with several bisphosphonates has been shown to reduce the risk of fractures (). Intermittent dosing has not (Papapoulous considers once weekly dosing to be equivalent to daily dosing) (). Intravenous dosing of a potent bisphosphonate with high affinity for bone has recently shown promise ().
The meeting concluded with presentations of selected abstracts. Delmas and colleagues () randomized 139 postmenopausal women to take alendronate 10 mg daily 30 min before breakfast or 1 hr before lunch, or before dinner (with at least 4-h fast before the dose). Biochemical markers of bone turnover were reduced to a similar degree with all three regimens. On behalf of absent authors, Belch presented a study showing 60% to 70% suppression of bone turnover markers following monthly treatment with 4 mg zoledronic acid intravenously in men with prostate cancer metastatic to bone () and a second study from the same group, clinically-relevant benefits of zoledronic acid treatment were shown in men with both blastic skeletal metastases from prostate cancer (reduced proportion of patients with skeletal related events, reduced number with pathologic fractures, reduced mean skeletal morbidity rate) (). Brown et al. reported the potential value of biochemical markers in identifying and monitoring cancer patients during treatment with BPs ().
Neridronate has recently been approved for registration in Italy for the treatment of osteogenesis imperfecta, and the clinical effects of iv administration at 2mg/kg in 81 patients were reviewed by Adami et al. (). BMD gains of up to 50% were observed in younger patients, accompanied by a reduction in fracture rate.
The effect of BPs on the loosening and migration of joint protheses is attracting attention, and Aspenburg described a double-blind study showing the efficacy of clodronate in reducing the migration of knee protheses (). Finally, Rodan (on behalf of absent authors) presented data on fracture rates and height loss through 7 years from the Phase III study with alendronate in women with postmenopausal osteoporosis. Clinical vertebral fractures (reported as adverse events) and height loss were similar during the early (years 0-3) and late (years 6-7) phases of the trial ().
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