Use Of Nitrogen-containing Bisphosphonates Research Articles

ATRAID regulates the action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

The Effects of Long-term Use of Nitrogen-containing Bisphosphonates on Fracture Healing.

Nitrogen-containing bisphosphonates (N-BPs) are pharmaceutical agents that have been used for many years to treat osteoporosis, multiple myeloma, Paget's disease, metastatic bone disease, and a variety of other diseases in which bone mineral density is reduced. Given that N-BPs inhibit bone resorption, an important stage in the fracture healing process, they have been extensively studied in preclinical models for their activity. In animal models, treatment with N-BPs is associated with a larger callus formation in fracture area and delay in remodeling from primary woven bone to lamellar bone, but there is no delay in formation of the fracture callus. In humans, all existing evidence suggest that initiating treatment with N-BPs, after upper and lower limb fractures, does not appear to have a significant effect on fracture healing. Rarely, patients with long-term use of N-BPs may develop “atypical fractures” and delay in their healing. Therefore, this clinical condition is not fully understood and many questions remain unanswered. Similarly, there are few studies about the benefits of stopping a long-term treatment with them, if a fracture occurs. Although most studies support that chronic N-BP therapy may lead to fracture healing delay, this is not fully documented. On the other hand, there are studies that are in complete disagreement with them. All of the above suggest that there is a need for more detailed future research into larger patient populations and different types of fractures, with sufficient data on the type, dosage, route and duration of administration of N-BPs, and the control methods of fracture healing, in order to have a safe final conclusion on the effect of their long-term administration in this highly complex process.

Nano-technology based carriers for nitrogen-containing bisphosphonates delivery as sensitisers of γδ T cells for anticancer immunotherapy.

Nitrogen containing bisphosphonates (N-BPs) including zoledronate (ZOL) and alendronate (ALD) inhibit farnesyl diphosphate synthase, and have been shown to have a cytotoxic affect against cancer cells as a monotherapy and to also sensitise tumour cells to destruction by γδ T cells. γδ T cells are a subset of human T lymphocytes and have a diverse range of roles in the immune system including the recognition and destruction of cancer cells. This property of γδ T cells can be harnessed for use in cancer immunotherapy through in vivo expansion or the adoptive transfer of ex vivo activated γδ T cells. The use of N-BPs with γδ T cells has been shown to have a synergistic effect in in vitro, animal and clinical studies. N-BPs have limited in vivo activity due to rapid clearance from the circulation. By encapsulating N-BPs in liposomes (L) it is possible to increase the levels of N-BPs at non-osseous tumour sites. L-ZOL and L-ALD have been shown to have different toxicological profiles than free ZOL or ALD. Both L-ALD and L-ZOL led to increased spleen weight, leucocytosis, neutrophilia and lymphocytopenia in mice after intravenous injection. L-ALD was shown to be better tolerated than L-ZOL in murine studies. Biodistribution studies have been performed in order to better understand the interaction of N-BPs and γδ T cells in vivo. Additionally, in vivo therapy studies have shown that mice treated with both L-ALD and γδ T cells had a significant reduction in tumour growth compared to mice treated with L-ALD or γδ T cells alone. The use of ligand-targeted liposomes may further increase the efficacy of this combinatory immunotherapy. Liposomes targeting the αvβ6 integrin receptor using the peptide A20FMDV2 had a greater ability than untargeted liposomes in sensitising cancer cells to destruction by γδ T cells in αvβ6 positive cancer cell lines.

Revival of nitrogen-containing bisphosphonate-induced inhibition of osteoclastogenesis and osteoclast function by water-soluble microfibrous borate glass

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious skeletal complication associated with the long-term oral or intravenous use of nitrogen-containing bisphosphonates (N-BPs). Here, we investigated the effects of an ionic cocktail prepared from water-soluble microfibrous borate glass on neutralizing the inhibitory effects of two heterocyclic N-BPs, risedronate or zoledronic acid, on osteoclastogenesis, apoptosis of differentiated osteoclasts and osteoclast function. Cell growth and proliferation assays were first performed on RAW 264.7 cells to optimize the concentrations of the ionic cocktail and N-BPs to be used for static cell culture. The pre-osteoclasts were then stimulated with RANKL to differentiate into osteoclasts. The effects of the ionic cocktail and N-BPs on osteoclast differentiation, apoptosis and function were subsequently examined using 3 series of experiments conducted at the gene, protein, morphological and functional levels. After concentration optimization, the ionic cocktail was found to partially reverse N-BP-induced inhibition of osteoclastogenesis, stimulation of osteoclasts apoptosis and reduction of osteoclast resorptive activity. Ultrastructural examination of osteoclasts that had been exposed to either N-BP identified classical features of late apoptosis and secondary necrosis, while osteoclasts exposed simultaneously to the concentration-optimized ionic cocktail and N-BPs exhibited only signs of early apoptosis that were possibly reversible. Taken together, the results of the 4 series of experiments indicate that the ionic cocktail produced from dissolution of borate glass dressings has the potential to rescue the adverse effects of heterocyclic N-BPs on osteoclast differentiation and function. These results warrant further confirmation using dynamic cell culture and small animal BRONJ models. Statement of significanceLong-term oral and intravenous use of nitrogen-containing bisphosphonates (N-BPs) may result in bisphosphonate-related osteonecrosis of the jaw (BRONJ) due to the suppression of normal bone turnover. There is no effective treatment for such a complication to date. This work reported the use of an ionic cocktail derived from water-soluble microfibrous borate glass to revert heterocyclic N-BP-induced inhibition of osteoclastogenesis, stimulation of osteoclasts apoptosis and reduction of osteoclasts resorption in static cell culture condition. This ionic cocktail may have the potential to be further developed into a new adjunctive treatment for BRONJ.

A mechanistic study of the interaction of water-soluble borate glass with apatite-bound heterocyclic nitrogen-containing bisphosphonates

Long-term oral and intravenous use of nitrogen-containing bisphosphonates (N-BPs) is associated with osteonecrosis of the jaw. Although N-BPs bind strongly to bone surfaces via non-covalent bonds, it is possible for extrinsic ions to dissociate bound N-BPs from mineralized bone by competitive desorption. Here, we investigate the effects and mechanism of using an ionic cocktail derived from borate bioactive glass for sequestration of heterocyclic N-BPs bound to apatite. By employing solid-state and solution-state analytical techniques, we confirmed that sequestration of N-BPs from bisphosphonate-bound apatite occurs in the presence of the borate-containing ionic cocktail. Simulations by density functional theory computations indicate that magnesium cation and borate anion are well within the extent of the risedronate or zoledronate anion to form precipitate complexes. The sequestration mechanism is due to the borate anion competing with bisphosphonates for similar electron-deficient sites on the apatite surface for binding. Thus, application of the borate-containing ionic cocktail represents a new topical lavage approach for removing apatite-bound heterocyclic N-BPs from exposed necrotic bone in bisphosphonate-related osteonecrosis of the jaw. Statement of significanceLong-term oral consumption and injections of nitrogen-containing bisphosphonates (N-BPs) may result in death of the jaw bone when there is traumatic injury to the bone tissues. To date, there is no effective treatment for such a condition. This work reported the use of an ionic cocktail derived from water-soluble borate glass microfibers to displace the most potent type of N-BPs that are bound strongly to the mineral component on bone surfaces. The mechanism responsible for such an effect has been identified to be cation-mediated complexation of borate anions with negatively-charged N-BPs, allowing them to be released from the mineral surface. This borate-containing cocktail may be developed into a novel topical rinse for removing mineral-bound N-BPs from exposed dead bone.

Osteomalacia: The Missing Link in the Pathogenesis of Bisphosphonate-Related Osteonecrosis of the Jaws?

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a well-documented adverse event from treatment with nitrogen-containing bisphosphonates (NBPs). During a preliminary histomorphometric study aimed at assessing the rate of bone remodeling in the jaws of patients with surgically resected BRONJ, we found a defect of bone mineralization (unpublished data). We hypothesized that osteomalacia could be a risk factor for BRONJ in patients taking NBPs. Therefore, we looked for static and dynamic histomorphometric evidence of osteomalacia in biopsies from subjects with and without BRONJ. This case-control study used histomorphometric analysis of bone specimens of patients using NBPs (22 patients with BRONJ and 21 patients without BRONJ) who required oral surgical interventions for the treatment/prevention of osteonecrosis. Patients were given tetracycline hydrochloride according to a standardized protocol before taking bone biopsies from their jaws. Biopsies with evidence of osteomyelitis or necrosis at histology were excluded from the study. Osteomalacia was defined as a mineralization lag time >100 days, a corrected mean osteoid thickness >12.5 mm, and an osteoid volume >10%. In all, 77% of patients with BRONJ were osteomalacic compared with 5% of patients without BRONJ, according to histomorphometry. Because osteomalacia was found almost exclusively in NBP users with BRONJ, this is likely to be a generalized process in which the use of NBPs further deteriorates mechanisms of bone repair. Osteomalacia represents a new and previously unreported risk factor for disease development. This finding may contribute to a better understanding of the pathogenesis of this disease and help with the development of strategies to increase the safety of NBP administration.

Oncologic doses of zoledronic acid induce osteonecrosis of the jaw-like lesions in rice rats (Oryzomys palustris) with periodontitis

Though osteonecrosis of the jaw (ONJ) is temporally-associated with the use of nitrogen-containing bisphosphonates (N-BPs), a cause-and-effect relationship has not yet been established. We hypothesize that ONJ is a two-stage process in which: (1) risk factors initiate pathologic processes in the oral cavity that lead to a supranormal rate of hard tissue necrosis; and (2) powerful antiresorptives reduce the rate of removal of necrotic bone sufficiently to allow its net accumulation in the jaw. To test this hypothesis, we used the rice rat model of periodontitis. At age 28 days, rats (n = 15/group) were placed on a high-sucrose and casein diet to exacerbate the development of periodontitis. Animals were injected subcutaneously (SC) biweekly with vehicle or alendronate (ALN, 15 µg/kg), or IV once monthly with vehicle, a low dose (LD) of zoledronic acid (ZOL), or a high dose (HD) of ZOL and sacrificed after 6, 12, 18, and 24 weeks. Mandibles and maxillae were analyzed to determine the effects on the: (1) progression of periodontitis; (2) integrity of alveolar bone; (3) status of bone resorption and formation; (4) vascularity; and (5) osteocyte viability. We found that only HD-ZOL induced ONJ-like lesions in mandibles of rice rats after 18 and 24 weeks of treatment. These lesions were characterized by areas of exposed necrotic alveolar bone, osteolysis, a honeycomb-like appearance of the alveolar bone, presence of bacterial colonies, and periodontal tissue destruction. In addition, inhibition of bone formation, a paradoxical abolition of the antiresorptive effect of only HD-ZOL, increased osteocyte necrosis/apoptosis, and decreased blood vessel number were found after 18 and/or 24 weeks. Our study suggests that only HD-ZOL exacerbates the inflammatory response and periodontal tissue damage in rice rats, inducing bone lesions that resemble ONJ.

Matrix Metalloproteinase-9 Expression in Alveolar Extraction Sockets of Zoledronic Acid–Treated Rats

The use of nitrogen-containing bisphosphonates (n-bis) is associated with necrosis of the jaws, also known as bisphosphonate-related osteonecrosis of the jaws (BRONJ); however, the pathophysiology is unknown. Matrix metalloproteinase-9 (MMP-9) expression is essential for normal bone healing and is also required for angiogenesis. N-bis alters MMP-9 expression in vitro and in vivo; therefore, we hypothesized that n-bis alters MMP-9 expression during oral wound healing after tooth extraction. A total accumulated dose of 2.25 mg/kg (n = 20) of Zoledronic acid (ZA) Zometa or saline (control, n = 20) was administered to Sprague-Dawley male rats. Next, both groups had maxillary molar teeth extracted. Rats were sacrificed at postoperative day 1, 3, 7, or 21. Western blotting or multiplex ELISA was used to evaluate proteins of interest. Real-time polymerase chain reaction was used to assess the relative quantities of target gene mRNA. MMP-9 enzymatic activity was assessed by zymography. The ZA group showed a statistically significant reduction in bone mineralization rate 21 days after tooth extraction compared with the control group (Student t test, P = .005). Moreover, ZA-treated animals showed a statistically significant increase in MMP-9-specific mRNA at postoperative days 3 (P = .003), 7 (P < .0001), and 21 (P < .0001) and protein on postoperative days 3 (P = .005) and 7 (P < .0001). MMP-9 enzymatic activity was also increased in ZA-treated rats compared with control animals (Student t test, P = .014). We also evaluated the extraction sockets for the presence of tissue inhibitor of MMP-1 (TIMP1), which is an inhibitor of MMP-9 enzymatic activity. TIMP1-specific mRNA and protein were not significantly altered by ZA treatment at the times tested (P > .05). Receptor of NF-κB ligand (RANKL) is known to regulate the expression of MMP-9; we therefore assessed the RANKL expression in our experimental oral wound-healing model. The ZA-treated animals had significantly increased RANKL mRNA at postoperative days 3 (P = .02) and 21 (P = .004), while the protein expression was significantly increased at postoperative days 1 (P < .0001), 7 (P = .02), and 21 (P = .03) compared with the control group. ZA reduced bone mineralization within tooth extraction sockets, suggesting aberrant bone healing. ZA increases the amount and enzymatic activity of MMP-9, while apparently not altering the amount of TIMP1 within extraction sockets. RANKL is increased in ZA-treated rats, which suggests that increased MMP-9 expression is due, in part, to augmented RANKL expression.

Potential pathophysiological mechanisms in osteonecrosis of the jaw

Bisphosphonates are used in the treatment of hypercalcemia of malignancy, skeletal complications associated with metastastic bone disease, Paget's disease, and osteoporosis. Osteonecrosis of the jaw (ONJ) is a recently described clinical condition that has been associated with the use of nitrogen-containing bisphosphonates. Reports describing this entity first appeared in the literature in 2003. While there have been significant numbers of case reports and a limited number of retrospective and prospective studies examining risk factors associated with ONJ, the pathophysiology of this condition remains elusive. In this review, we explore proposed mechanisms underlying ONJ development and identify potential areas for future investigation.

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

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. 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. 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. These results suggest that alendronate-mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF-κB and Smad3 activity.

Outcome of Patients With Early Breast Cancer Receiving Nitrogen-Containing Bisphosphonates: A Comparative Analysis From the Metropolitan Detroit Cancer Surveillance System

Background Preclinical data suggest that bisphosphonates exhibit antitumor activity. However, clinical studies indicated conflicting results. In this study, we compared the overall survival (OS) of postmenopausal patients with nonmetastatic invasive breast cancer who received any of the second-generation nitrogen-containing bisphosphonates for osteopenia or osteoporosis, with the survival of those who did not. Patients and Methods We conducted a retrospective study at the Wayne State University/Karmanos Cancer institute (KCI) in Detroit, Michigan and extracted data from the Metropolitan Detroit Cancer Surveillance System (MDCSS), a Surveillance, Epidemiology and End Results (SEER) registry. Patients > 50 years of age with stage I, II, or III invasive breast cancer between the years 2000 through 2003 were included. Information regarding medications was extracted from the patients' medical records. Results A total of 696 women with stage I-III breast cancer were included. Ninety-seven women (14%) used nitrogen-containing bisphosphonates. The difference in OS between bisphosphonate users and nonusers was not statistically significant ( P = .32) at 3 years. After adjusting for differences between the groups in age, stage of disease, hormone receptor status, endocrine therapy, vitamin D, and calcium use, there was a marginally significant ( P = .07) difference in survival; bisphosphonate users had poorer survival than nonusers. Conclusion Our results indicate that the use of nitrogen-containing bisphosphonates is not associated with improved OS in patients with nonmetastatic breast cancer, even after adjusting for known prognostic factors, but with a marginally worse OS. Further research is awaited to clarify the role of bisphosphonates in the adjuvant setting.

Zoledronic Acid Induces MMP9 Overexpression Within Tooth Extraction Sockets in Rats

Statement of the Problem: The use of nitrogen-containing bisphosphonates (BIS) is associated with necrosis of the jaws a.k.a. bisphosphonate-related osteonecrosis of the jaws (BRONJ). The incidence of BRONJ increases with the number of BIS infusions and duration of BIS treatment. BRONJ is associated with invasive dental procedures, such as dental extractions(1); however, the pathophysiology that may explain the pathogenesis of jaw necrosis is unknown. In other experimental systems BIS inhibits bone resorption and angiogenesis, which are required for normal healing, such as after tooth extraction.

Zoledronic Acid Disrupts VEGFR2 Intracellular Trafficking in Endothelial Cells

Statement of the Problem: Long-term use of nitrogen-containing bisphosphonates (n-BISs) are associated with jaw necrosis. Tooth extraction is the most frequent precipitating factor. Proper healing after tooth extraction requires angiogenesis, which is inhibited by n-BIS in vitro and in vivo. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal for endothelial cells. N-BIS inhibit VEGF responses in endothelial cells (2). The molecular mechanism(s) that account for these attenuated responses to VEGF are not known.

Transient Disturbance in Physeal Morphology Is Associated With Long-Term Effects of Nitrogen-Containing Bisphosphonates in Growing Rabbits

Bisphosphonates have clinical benefit in children with severe osteogenesis imperfecta or osteoporosis and potential benefit in children with Perthes disease or undergoing distraction osteogenesis. However, there is concern about the effects of bisphosphonates on the physis and bone length. In 44 growing rabbits, zoledronic acid caused a transient disruption of physeal morphology, retention of cartilaginous matrix in trabeculae and cortical bone of the metaphysis, and a minor decrement in tibial bone length at maturity. Data from growing animal models suggest that bisphosphonates cause retention of longitudinal cartilaginous septa at the chondro-osseous junction, extension of trabeculae to the metaphyseal-diaphyseal junction, and varying dose-dependent effects on longitudinal growth. However, there is a lack of data regarding effects of intermittent use of nitrogen-containing bisphosphonates on the physis and on tibial length in models reaching maturity. Contralateral tibias of juvenile rabbits were examined after right tibial distraction osteogenesis from two previous studies. Animals were randomized to receive 0.1 mg/kg zoledronic acid (ZA) IV at 8 weeks of age (ZA*1) or 8 and 10 weeks of age (ZA*2) or saline. Body mass was analyzed from 5 to 44 weeks of age; tibial length and proximal physeal-metaphyseal histology and histomorphometry were analyzed at 8-52 weeks of age. Tibial length was 3% less at 14 weeks of age in the ZA*2-treated versus saline group (p<0.05) in both studies, and this difference persisted at maturity in the long-term study group (26 weeks of age, p<0.05). Total body mass gain from 5 to 26 weeks of age was 14% less in ZA*2-treated than saline animals (p<0.05). Rate of weight gain from 8 to 10 weeks of age was 76% less in ZA*2 compared with saline animals (p<0.05). Radiographs showed radiodense lines in the metaphyses of ZA-treated bones, corresponding to the number of doses. Histologically, lines resulting from the first dose of ZA contained longitudinal cartilaginous matrix cores surrounded by bone, whereas those from the second dose contained spherical cores of matrix caused by transient disruption of physeal morphology after the first dose of ZA. Resorption of these lines at later times was radiographically and histologically evident, but remnants of cartilaginous matrix remained in the cortical bone of ZA-treated animals. ZA treatment within the final 13.5% of the rabbit tibial growth period caused a transient disruption in physeal morphology and resorption associated with retention of cartilaginous matrix and coinciding with a persistent 3% decrement in tibial length. Disruption of physeal morphology and potential loss of bone length should be considered when administering nitrogen-containing bisphosphonates to children before closure of the major physes.

Gastric damage in the rat with nitrogen-containing bisphosphonates depends on pH.

The use of nitrogen-containing bisphosphonates (N-BPs) has been reported to be associated with gastrointestinal intolerance. The fasted, indomethacin-treated rat provides a model for assessing the gastrointestinal effects of these compounds. The aims of this study were to elucidate the effect of pH on N-BP-induced gastric damage, and to evaluate the structure-activity relationship between N-BP anti-resorptive and gastric effects. Fasted rats were dosed concomitantly with indomethacin (40 mg/kg, subcutaneously) and an N-BP (pamidronate, alendronate, or risedronate at 150 or 300 mg/kg, orally), with the N-BP dosing solutions adjusted to pH 2, 4 or 7. The aminopentane and aminohexane N-BPs (150, 225 or 300 mg/kg, orally) were only tested at pH 4 only. Nitrogen-containing bisphosphonate-induced gastric damage was pH-dependent, with increased damage at increasing pH. Gastric damage potential did not correlate with bone anti-resorptive effects, and the more potent anti-resorptive N-BPs were not necessarily more damaging to the stomach.