Efficacy,effectiveness and side effects of medications used to prevent fractures

There is an increasing number of effective therapies for fracture prevention in adults at risk of osteoporosis. However, shortcomings in the evidence underpinning our management of osteoporosis still exist. Evidence of antifracture efficacy in the groups of patients who most commonly use calcium and vitamin D supplements is lacking, the safety of calcium supplements is in doubt, and the safety and efficacy of high doses of vitamin D give cause for concern. Alendronate, risedronate, zoledronate and denosumab have been shown to prevent spine, nonspine and hip fractures; in addition, teriparatide and strontium ranelate prevent both spine and nonspine fractures, and raloxifene and ibandronate prevent spine fractures. However, most trials provide little information regarding long‐term efficacy or safety. A particular concern at present is the possibility that oral bisphosphonates might cause atypical femoral fractures. Observational data suggest that the incidence of this type of fracture increases steeply with duration of bisphosphonate use, resulting in concern that the benefit–risk balance may become negative in the long term, particularly in patients in whom the osteoporotic fracture risk is not high. Therefore, reappraisal of ongoing use of bisphosphonates after about 5 years is endorsed by expert consensus, and ‘drug holidays’ should be considered at this time. Further studies are needed to guide clinical practice in this area.     

The increase in bone mineral density by bisphosphonate with active vitamin D analog is associated with the serum calcium level within the reference interval in postmenopausal osteoporosis

Objectives: To examine the factors associated with increase in lumbar spine bone mineral density (LS-BMD) by bisphosphonates (BPs) with active vitamin D analog (aVD).

Methods: Two independent postmenopausal osteoporotic patients treated by BPs with aVD for 24 months (Study 1: n?=?93, Study 2: n?=?99) were retrospectively analyzed.

Results: In Study 1, LS-BMD of the patients significantly increased for 24 m (5.4%, p?r²: 0.088, p?=?.02). While average sCa of the patients was 9.2?mg/dL before treatment, it increased time-dependently to 9.6?mg/dL for 24 m by treatment. As each patient had their LS-BMD five times during the study, there were four instances of %LS-BMD in each patient, resulting in 372 instances of %LS-BMD in Study 1. The smallest Akaike’s information criterion value for the most appropriate cut-off levels of sCa for %LS-BMD by treatment every 6 m was 9.3?mg/dL. The %LS-BMD by treatment for 6 m during 24 m period in patients with sCa ≥9.3?mg/dL (1.5%) was significantly higher than that in patients with sCa <9.3?mg/dL (0.8%, p?=?.038). The results of Study 2 were similar to those of Study 1, confirming the phenomena observed.

Conclusion: sCa was associated with an increased LS-BMD by BPs with aVD.     

Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts

Tamai R, Kiyoura Y, Sugiyama A. Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts. J Periodont Res 2011; 46: 13–20. © 2010 John Wiley & Sons A/S Background and Objective: Nitrogen‐containing bisphosphonates (NBPs) are widely used as anti‐bone‐resorptive drugs. However, use of NBPs results in inflammatory side‐effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. Methods: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)‐1β, IL‐6, IL‐8 and monocyte chemoattractant protein‐1 (MCP‐1) in culture supernatants were measured by ELISA. We also examined nuclear factor‐κB (NF‐κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. Results: Pretreatment of PBMCs with alendronate promoted lipid A‐induced production of IL‐1β and IL‐6, but decreased lipid A‐induced IL‐8 and MCP‐1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A‐induced production of IL‐6 and IL‐8, and increased NF‐κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate‐augmented IL‐6 and IL‐8 production by human gingival fibroblasts but up‐regulated alendronate‐decreased IL‐8 production by PBMCs. Conclusion: These results suggest that alendronate‐mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF‐κB and Smad3 activity.     

Zoledronic acid impairs re-epithelialization through down-regulation of integrin αvβ6 and transforming growth factor beta signalling in a three-dimensional in vitro wound healing model

This study examined the negative effects of zoledronic acid on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model. A living oral mucosa equivalent was constructed by seeding a mixture of primary human oral keratinocytes and fibroblasts, at a cell density of 1.5 × 10⁵ cm² each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2 mM Ca²⁺ EpiLife for 5 days, and then in an air–liquid interface for 14 days. The equivalent was wounded by excising a linear 2-mm-wide epithelial layer on day 8 and subsequently incubated with 10 μM zoledronic acid for an additional 11 days. Histological and immunohistochemical observations revealed zoledronic acid to significantly suppress the epithelial thickness and Ki-67-labelling index. Zoledronic acid also abolished integrin αvβ6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-β1) production into the supernatant, but down-regulated TGF-β receptor types I and II expression and Smad3 phosphorylation, as was also confirmed by immunofluorescence microscopy. This study therefore showed zoledronic acid to abrogate integrin αvβ6 expression, cause the down-regulation of TGF-β/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.     

Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE^−/− mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Atherosclerotic plaque rupture is the leading cause of myocardial infarction and stroke (1, 2). Studies assessing the correlation between calcium scores and cardiovascular events have demonstrated a predictive power that is superior to and independent from that of lipid scores (3, 4). Additionally, clinical imaging studies have revealed that the risk of plaque rupture is further heightened by the presence of small, “spotty” calcifications, or microcalcifications (5, 6), and cardiovascular risk is inversely correlated with the size of calcific deposits, quantified as a calcium density score (7). Indeed, computational modeling has demonstrated that, while large calcifications can reinforce the fibrous cap (8), microcalcifications (typically 5 to 15 μm in diameter) uniquely mediate an increase in mechanical stress of the relatively soft, collagen-rich fibrous cap (9–12).Histologic studies have revealed the presence of cell-derived vesicles within calcifying atherosclerotic lesions (13–16). The inflammatory environment of the atherosclerotic lesion can induce vascular smooth muscle cells (vSMCs) to take on an osteochondrogenic phenotype and release calcifying extracellular vesicles (EVs) (17–19). Macrophages have also been shown to release procalcifying vesicles (20, 21). Thus, just as bone formation is hypothesized to be an active, cell-driven process (22, 23), mediated by calcifying matrix vesicles, atheroma-associated calcification may similarly be initiated by the production and aggregation of calcifying EVs (11, 20, 24–28).One proposed strategy for halting pathologic calcification has been the use of bisphosphonates (BiPs). BiPs are analogs of pyrophosphate (29), a naturally occurring compound derived in vivo from adenosine triphosphate (ATP) (30). Pyrophosphate binds to calcium phosphate and inhibits calcification via physicochemical mechanisms, namely, by blocking calcium and phosphate ions from forming crystals, preventing crystal aggregation, and preventing mineral transformation from amorphous calcium phosphate to hydroxyapatite (29). BiPs were identified as pyrophosphate analogs that, unlike pyrophosphate itself, resist enzymatic hydrolysis. A second, distinct property of BiPs is the ability to inhibit bone resorption via biological activity directed against osteoclasts following osteoclast endocytosis of the BiP molecule adsorbed to the surface of bone (29, 31). First-generation, or nonnitrogen-containing BiPs, are incorporated into nonhydrolyzable ATP analogs, and induce osteoclast apoptosis by limiting ATP-dependent enzymes. In contrast, nitrogen-containing BiPs inhibit farnesyl pyrophosphate synthetase and thereby induce osteoclast apoptosis (31).In vivo animal investigations have been performed to explore the potential for BiPs to inhibit cardiovascular calcification. Studies of first-generation BiPs revealed that the doses required to inhibit cardiovascular calcification also critically compromised normal bone mineralization (29, 32). However, newer, nitrogen-containing BiPs effectively arrested cardiovascular calcification in animal models at doses that did not compromise bone formation (32). Further, while it has been proposed that BiP treatment modifies cardiovascular calcification via its impact on bone-regulated circulating calcium and phosphate levels, a study in uremic rats demonstrated that BiP treatment inhibited medial aortic calcification with no significant change in plasma calcium and phosphate levels (33). The same study demonstrated that BiP treatment inhibited calcification of explanted rat aortas, indicating that BiPs can act directly on vascular tissue, independent of bone metabolism (33).Retrospective clinical data examining the effect of BiP therapy on cardiovascular calcification has demonstrated conflicting findings and intriguing paradoxes. In women with chronic kidney disease, BiP therapy decreased the mortality rate for patients without a prior history of cardiovascular disease (34), but for those patients with a history of prior cardiovascular events, BiP therapy was associated with an increased mortality rate (35). In another study, BiP therapy correlated with a lower rate of cardiovascular calcification in older patients (>65 y), but a greater rate in younger patients (<65 y) (36). These clinical findings motivated our study, in which we sought to further understand how BiP therapy impacts cardiovascular outcomes. Given that cardiovascular calcification, and especially the presence of microcalcification, is a strong and independent risk factor for adverse cardiac events, and BiPs are prescribed to modulate pathologies of mineralization, we hypothesize that BiPs modulate cardiovascular outcomes by altering the dynamics of cardiovascular calcification.EVs are smaller than the resolution limits of traditional microscopy techniques, hindering studies into the mechanisms of calcification nucleation and growth. We previously developed an in vitro collagen hydrogel platform that allowed the visualization of calcific mineral development mediated by EVs isolated from vSMCs (24). Using superresolution microscopy, confocal, and electron microscopy techniques, we showed that calcification requires the accumulation of EVs that aggregate and merge to build mineral. Collagen serves as a scaffold that promotes associations between EVs that spread into interfibrillar spaces. The resultant mineral that forms within the collagen hydrogel appears spectroscopically similar to microcalcifications in human tissues and allows the study of these structures on the time scale of 1 wk. In this study, we utilized this three-dimensional (3D) acellular platform to examine the direct effect of ibandronate, a nitrogen-containing BiP, on the EV-directed nucleation and growth of microcalcifications, a process that cannot be isolated from cellular and tissue-level mechanisms in a more complex, in vivo system. In parallel, we utilized a mouse model of atherosclerosis to assess the effect of ibandronate therapy on plaque-associated calcification, comparing mineral morphologies between the in vitro and in vivo samples. We hypothesize that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Understanding the EV-specific action of BiPs is imperative both to develop anticalcific therapeutics targeting EV mineralization and to understand one potential mechanism driving the cardiovascular impact of BiPs used in clinical settings.