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Background: Growth factors embedded in the extracellular matrix of the dentin play an important role in the migration, proliferation, and differentiation of dental pulp stem cells in regenerative endodontics. In regenerative endodontic treatments, the type of irrigation solution used is crucial for the release of growth factors (GFs) from the dentin matrix. This study evaluated the effectiveness of different irrigant activation techniques (IAT) using two different chelating agents, 17% ethylenediaminetetraacetic acid (EDTA) and 9% etidronic acid (HEDP), in terms of their GF release., Methods: Seventy-two mandibular premolar teeth were prepared to simulate an open apex. The root fragments were irrigated with 20 ml of 1.5% sodium hypochlorite and 20 ml of saline solution. Eight root fragments were randomly separated for the control group, and the remaining 64 fragments were randomly separated into eight groups based on two different chelating agents (17% EDTA and 9% HEDP) and four different IAT ((conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), sonic activation with EDDY, and XP-endo Finisher (XPF)). TGF-β1, VEGF-A, BMP-7 and IGF-1 release levels were determined using an ELISA, and statistical analysis was performed using the Kolmogorov-Smirnov test, ANOVA, and the Tukey test (p < .05)., Results: Compared to the control group, the experimental groups showed significantly higher GF release when using EDTA or HEDP. Among the activation groups, the EDDY group triggered the highest GF release, and the CNI group triggered the lowest., Conclusions: IAT with EDTA and HEDP can increase GF release, with EDDY being the most effective IAT method. Using chelating agents with IAT may be beneficial in regenerative endodontic treatments., (© 2024. The Author(s).)

Microbiologically influenced corrosion (MIC) caused by biofilm is a serious problem in many industries. D-amino acids could be a potential strategy to enhance traditional corrosion inhibitors due to their roles in biofilm reduction. However, the synergistic mechanism of D-amino acids and inhibitors remains unknown. In this study, D-Phenylalanine (D-Phe) and 1-hydroxyethane-1,1-diphosphonic acid (HEDP) were selected as the typical D-amino acid and corrosion inhibitor to evaluate their effect on the corrosion caused by Desulfovibrio vulgaris. The combination of HEDP and D-Phe obviously slowed down the corrosion process by 32.25%, decreased the corrosion pit depth and retarded cathodic reaction. SEM and CLSM analysis indicated that D-Phe reduced the content of extracellular protein and thus inhibited the biofilm formation. The molecular mechanism of D-Phe and HEDP on corrosion inhibition was further explored via transcriptome. The combination of HEDP and D-Phe down-regulated the gene expression of peptidoglycan, flagellum, electron transfer, ferredoxin and quorum sensing (QS) molecules, leading to less peptidoglycan synthesis, weaker electron transfer and stronger QS factor inhibition. This work provides a new strategy for improving traditional corrosion inhibitors, retarding MIC and mitigating subsequent water eutrophication., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: To investigate the effect of a rotary agitation method or ultrasonically activated irrigation on the antibiofilm effect of a mixture of sodium hypochlorite (NaOCl) and etidronate (1-hydroxyethylidene-1,1-bisphosphonate, HEBP) using a dual-species biofilm model in root canal system., Methods: Mature dual-species biofilms of Enterococcus faecalis and Streptococcus gordonii were formed in root canals of mandibular premolars. Teeth were randomly allotted (n = 12) to group 1, XP-endo Finisher (XPF); group 2, ultrasonically activated irrigation (UAI); group 3, syringe-and-needle irrigation (SNI). In all groups, canals were instrumented with a rotary instrument (XP-endo Shaper) prior to irrigant agitation/activation. A mixture containing 2.5% NaOCl and 9% HEBP was used throughout the experiment. Bacterial counts from the canal were determined using qPCR before preparation (S1), after preparation (S2), and after final irrigation agitation/activation (S3). Bacterial viability within the dentinal tubules in the coronal, middle and apical root-thirds was quantified using confocal microscopy after Live/Dead staining. The bacterial counts and viability were compared between groups using one-way ANOVA and post-hoc Tukey's tests. Paired t-test was used to compare the bacterial counts within groups., Results: Instrumentation alone could significantly reduce the microbial counts in all the groups (P < 0.0001). Subsequent agitation/activation resulted in significant microbial reduction only in XPF and UAI (P < 0.05), both of which reduced significantly more microbial counts than SNI (P < 0.05). Live/Dead staining revealed that XPF and UAI showed significantly greater percentage of dead bacteria within the dentinal tubules than SNI in the coronal third (P < 0.05); UAI resulted in the significantly highest percentage of dead bacteria in the middle third (P < 0.05); while there was no significant difference between the groups in the apical third (P > 0.05)., Conclusions: When using the sodium hypochlorite/etidronate mixture for irrigation, final irrigant agitation/activation with XP-endo Finisher or ultrasonic can improve disinfection of the main root canal space and the dentinal tubules in the coronal third, while ultrasonically activated irrigation appears to exhibit better disinfection within dentinal tubules in the middle third., (© 2022. The Author(s).)

Aim: To assess the effect of the bisphosphonate etidronate on choroidal neovascular (CNV) activity in patients with pseudoxanthoma elasticum (PXE)., Methods: This is an ancillary study in a single center, randomized, double-blind placebo-controlled trial (RCT) in which 74 patients with PXE were assigned to either one-year etidronate or placebo treatment. Spectral domain optical coherence tomography (SD-OCT) imaging and color fundus photography were performed every three months for one year and were systematically assessed on signs of CNV activity., Results: In the etidronate group, 11 (30%) of the patients had CNV activity at baseline, compared to 25 (67%) of the patients in the placebo group (P = 0.005). The proportion of eyes with CNV activity during the study ranged from 18-33% in the etidronate group and 42-56% in the placebo group and no significant difference in improvement or worsening of CNV activity was found (P = 0.168). Using a generalized mixed model for repeated measures, there was a protective effect of etidronate in crude analysis (RR 0.86, 95% CI 0.75-0.98) that disappeared when adjusting for baseline CNV activity (RR 0.97, 95% CI 0.84-1.13)., Conclusion: In this post-hoc RCT analysis we did not observe a protecting or deteriorating effect of etidronate on CNV activity in patients with PXE after adjustment for baseline CNV., Competing Interests: UNI-Pharma SA provided the etidronate and placebo capsules for free, but was not involved in the design, the execution, the analysis, or the reporting of the Treatment of Ectopic Mineralization in Pseudoxanthoma Elasticum trial. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Lessons Learned: Results are consistent with MBC-11 targeting and treating cancer-induced bone lesions by concentrating cytarabine and etidronate at the site of disease.MBC-11 was well tolerated, with an maximum tolerated dose of 5 mg/kg per day and myelosuppression as the principal toxicity.Treatment significantly reduced cancer cell activity in over half of bone lesions detected at baseline.MBC-11 pharmacokinetic and pharmacodynamic parameters are consistent with the novel drug design goals, and encouraging results warrant further clinical development., Background: MBC-11 is a first-in-class conjugate of the bone-targeting bisphosphonate etidronate covalently linked to the antimetabolite cytarabine (araC). This first-in-human phase I dose escalation study assessed safety, tolerability, maximum tolerated dose (MTD), plasma pharmacokinetics, bone turnover, tumor biomarkers, and bone lesion activity by fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG-PET/CT) imaging., Methods: Fifteen patients with advanced solid cancers and cancer-induced bone disease (CIBD) were treated with 0.5-10 mg/kg per day of MBC-11 administered daily for 5 days of every 4 weeks for up to four cycles., Results: Grade 1-2 myelosuppression, involving all lineages, was the principal toxicity. Two of three patients treated with 10 mg/kg experienced dose-limiting grade 4 neutropenia and thrombocytopenia (adverse event [AE] duration ≤5 days); the MTD was 5 mg/kg. Four of five patients with pretreatment elevations of the bone resorption marker TRAP5b (tartrate resistant acid phosphatase-5b) had persistent decrements. Six of 13 patients who reported baseline pain noted a reduction after MBC-11. 18 F-FDG-PET/CT imaging demonstrated partial metabolic responses in three patients and stable metabolic responses in three other patients. SUV max (standard unit of emission normalized to total uptake) was reduced by at least 25% in 110 (52%) of 211 bone lesions. Significant activity was noted across all doses, and myelosuppression increased with dose., Conclusion: At MBC-11 doses that were well tolerated, substantial reductions in metabolic activity of bone-associated cancer cells provide a foundation for further disease-directed efficacy studies., (© AlphaMed Press; the data published online to support this summary are the property of the authors.)

Influence of the surface characteristics of three stainless steels (SS304, 316L and 317) and presence of scale inhibitors on adhesion kinetics of sulfate reducing bacteria (SRB) in circulating cooling water, were investigated by evaluating surface free energy, adhesion kinetic constants in a parallel plate flow chamber. Results show that the surface free energy values of SS317, SS316L and SS304 are -31.69, -24.18 and -13.92 mJ m -2 , respectively. SS317 surface had higher surface hydrophobicity than SS316L and SS304. In the process of bacteria cells adhesion onto SS surfaces, electrostatic interaction for SS is slightly more than hydrophobic interaction. The number of adhering bacteria and the adhesion kinetic constants are different on the three types of stainless steel. The adhesion kinetic constants for SS317 and 316L are greater than that for SS304, which are 0.0354, 0.0282 and 0.0190 min -1 , respectively. Scale inhibitors of hydrosy ethyl fork phosphonic acid (HEDP) and phosphono butane-1, 2, 4-tricarboxylic acid (PBTCA) have a certain influence on the initial adhesion of bacteria cell and adhesion kinetics constants are reduced in the presence of HEDP and PBTCA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The ability of (4,4)-armchair CNT and its three phosphate functionalized forms (CNT-nH 2 PO 4 , n=1-3) were evaluated as novel drug delivery systems (DDSs) for the two commercially well-known anti-osteoporosis drugs namely alendronate (AL) and etidronate (ET). For this purpose, the DFT calculations were accomplished at both B3LYP and B3PW91 levels using 6-31g(d) basis set. The binding energy was increased by increasing number of H 2 PO 4 moieties attached on the CNT with the most negative binding energy was measured for the carrier containing three phosphate groups. The dipole moments of all phosphate containing CNTs were much greater (∼2.2-4.4D) than that of pristine CNT (∼0D). The contour maps proved that when the CNT was functionalized by H 2 PO 4 groups, the symmetric distribution of electric charge was vanished with the charge distribution was the highest asymmetric for the CNT-2H 2 PO 4 while it was the lowest asymmetric for CNT-3H 2 PO 4 leading to the greatest dipole moment for the CNT-2H 2 PO 4 (4.177D) while the smallest dipole moment for the CNT-3H 2 PO 4 (1.614D). Among all compounds, those containing the CNT-3H 2 PO 4 exhibited the smallest band gap energy, chemical potential and hardness but the greatest electronegativity and electrophilicity index which were all suitable and effective for the attachment of drugs onto the bone surface (having partial positive charge due to the presence of Ca 2+ as CaCO 3 ) and therefore inhibiting the osteoporosis. Consequently, it was established that the drug-CNT-3H 2 PO 4 was the most appropriate drug-carrier compound for both of the AL and ET drugs and it could be used as the most effective drug vehicle. The attachment of AL, ET drugs as well as the AL-CNT-3H 2 PO 4 and ET-CNT-3H 2 PO 4 drug-carrier systems to the bone tissue was modelled by optimization of the structures of these compounds bonded to the hydroxyapatite (HA)-17water (w). It was found that among these four systems, the AL-CNT-3H 2 PO 4 could be suggested as the most suitable DDS for application in the treatment of osteoporosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Bisphosphonates (BPs) are typical anti-bone-resorptive drugs, with nitrogen-containing BPs (N-BPs) being stronger than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs have inflammatory/necrotic effects, while the non-N-BPs clodronate and etidronate lack such side effects. Pharmacological studies have suggested that clodronate and etidronate can (i) prevent the side effects of N-BPs in mice via inhibition of the phosphate transporter families SLC20 and/or SLC34, through which N-BPs enter soft-tissue cells, and (ii) also inhibit the phosphate transporter family SLC17. Vesicular transporters for the pain transmitters glutamate and ATP belong to the SLC17 family. Here, we examined the hypothesis that clodronate and etidronate may enter neurons through SLC20/34, then inhibit SLC17-mediated transport of glutamate and/or ATP, resulting in their decrease, and thereby produce analgesic effects. We analyzed in mice the effects of various agents [namely, intrathecally injected clodronate, etidronate, phosphonoformic acid (PFA; an inhibitor of SLC20/34), and agonists of glutamate and ATP receptors] on the nociceptive responses to intraplantar injection of capsaicin. Clodronate and etidronate produced analgesic effects, and these effects were abolished by PFA. The analgesic effects were reduced by N-methyl-D-aspartate (agonist of the NMDA receptor, a glutamate receptor) and α,β-methylene ATP (agonist of the P2X-receptor, an ATP receptor). SLC20A1, SLC20A2, and SLC34A1 were detected within the mouse lumbar spinal cord. Although we need direct evidence, these results support the above hypothesis. Clodronate and etidronate may be representatives of a new type of analgesic drug. Such drugs, with both anti-bone-resorptive and unique analgesic effects without the adverse effects associated with N-BPs, might be useful for osteoporosis.

Generalized arterial calcification of infancy is an intractable ectopic mineralization disorder caused by mutations in the ENPP1 gene, resulting in reduced plasma inorganic pyrophosphate (PPi) levels. We previously characterized the Enpp1(asj) mutant mouse as a model of generalized arterial calcification of infancy, and we have now explored the potential efficacy of bisphosphonates, nonhydrolyzable PPi analogs, in preventing ectopic mineralization in these mice. The mice were maintained on either basic diet (control) or diets containing etidronate or alendronate in three different concentrations (experimental). Considering low bioavailability of bisphosphonates when administered orally, subsequent studies tested the mice with subcutaneous injections of etidronate. The treatments were initiated at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks of age by quantitation of calcium deposits in the muzzle skin containing dermal sheath of vibrissae and in aorta. We found that bisphosphonate treatments significantly reduced mineralization in skin and aorta. These changes in treated mice were accompanied with restoration of their bone microarchitecture, determined by microcomputed tomography. The inhibitory capacity of bisphosphonates, with mechanistic implications, was confirmed in a cell-based mineralization assay in vitro. Collectively, these results suggest that bisphosphonate treatment may be beneficial by a dual effect for preventing ectopic soft tissue mineralization while correcting decreased bone mineralization in generalized arterial calcification of infancy caused by ENPP1 mutations., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurofibromatosis Type 1 (NF1) is a common hereditary disease characterized by disorders regarding the skin, neural, and skeletal systems. Osteoporosis is one of the skeletal manifestations of NF1, which is associated with increased fracture risk. The management of NF1-related low bone mass has been less studied in the literature. We present a 19-year-old patient with severe low bone mass complicating NF1. The patient received 1-year course of 35 mg risedronate sodium once per week along with a daily regimen of 1200 mg calcium and 800 IU vitamin D. Significant improvement with regard to the Z-scores and bone mineral density values was achieved. Besides, rapid favourable biochemical response was obtained. The patient experienced 24·4 and 15·0% improvements in bone mineral density at the lumbar site and hip, respectively, at the first year of therapy. No adverse effect was observed. Since increased bone turnover is the primary contributor of osteoporosis in NF1, antiresorptive agents such as bisphosphonates can be considered for treatment. Despite the lack of consensus on the treatment of osteoporosis in NF1, risedronate may hold a promise as a potential therapy for osteoporosis complicating NF1. This is the first report of risedronate therapy in a case with NF1-associated low bone mass in the literature.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by ABCC6 mutations by feeding Abcc6(-/-) mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the ABCC6 gene, when combined with careful monitoring of efficacy and potential side-effects.

Background: Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy., Method: MDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2., Results: Treatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1., Conclusions: In summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members as good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy.

Low to moderate ethanol consumption has been associated with protective effects in autoimmune diseases such as rheumatoid arthritis, RA. An expansion of γδ T cells induced by isopentenyl pyrophosphate, IPP, likewise seems to have a protective role in arthritis. The aim of this project was to test the hypothesis that low doses of ethanol can enhance IPP-induced expansion of synovial fluid γδ T cells from patients with arthritis and may thereby potentially account for the beneficial effects of ethanol on symptoms of the arthritic process. Thus, mononuclear cells from synovial fluid (SF) from 15 patients with arthritis and from peripheral blood (PB) from 15 healthy donors were stimulated with low concentrations of ethanol and IPP for 7 days in vitro. IPP in combination with ethanol 0.015%, 2.5 mM, equivalent to the decrease per hour in blood ethanol concentration due to metabolism, gave a significantly higher fractional expansion of SF γδ T cells compared with IPP alone after 7 days (ratio 10.1+/-4.0, p<0.0008, n = 12) in patients with arthritis. Similar results were obtained for PB γδ T cells from healthy controls (ratio 2.0+/-0.4, p<0.011, n = 15). The augmented expansion of γδ T cells in SF is explained by a higher proliferation (p = 0.0034, n = 11) and an increased survival (p<0.005, n = 11) in SF cultures stimulated with IPP plus ethanol compared to IPP alone. The synergistic effects of IPP and ethanol indicate a possible allosteric effect of ethanol. Similar effects could be seen when stimulating PB with ethanol in presence of risedronate, which has the ability to increase endogenous levels of IPP. We conclude that expansion of γδ T cells by combinatorial drug effects, possibly in fixed-dose combination, FDC, of ethanol in the presence of IPP might give a protective role in diseases such as arthritis.

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression., (© 2014. Published by The Company of Biologists Ltd.)

Bisphosphonates (BPs) are pyrophosphate analogs. They are widely used against enhanced bone-resorption in various diseases. Nitrogen-containing BPs (N-BPs) exhibit strong anti-bone-resorptive effects but have inflammatory and necrotic side effects. The non-nitrogen-containing BPs (non-N-BPs) etidronate and clodronate lack such side effects, but their anti-bone-resorptive effects are weak. In mice, etidronate and clodronate reduce the inflammatory/necrotic effects of N-BPs, even those of zoledronate, the N-BP with the strongest anti-bone-resorptive effect yet reported and the highest risk of inflammation/necrosis. Here, to explore the mechanisms underlying this protection, we used a mouse model in which a single reagent or a mixture of two reagents was injected subcutaneously into ear-pinnas. These reagents included zoledronate, four non-N-BPs, pyrophosphate, and inhibitors of various organic-anion-transporters. Pyrophosphate and two of the four non-N-BPs (not etidronate or clodronate) had inflammatory/necrotic effects. These effects were reduced by etidronate and clodronate, but not by phosphonoformate, an inhibitor of two of the three known phosphate-transporter families. Phosphonoformate reduced the inflammatory/necrotic effects of zoledronate, but not those of pyrophosphate or of non-N-BPs. Conversely, pyrophosphate, at non-inflammatory/necrotic concentrations, reduced the inflammatory/necrotic effects of non-N-BPs, but not those of zoledronate. The efficacies of the protective effects against the inflammatory/necrotic effects of zoledronate were clodronate > etidronate > phosphonoformate. These findings suggest that (i) the N-BP zoledronate may enter soft-tissue cells via phosphonoformate-inhibitable phosphate-transporters, (ii) other phosphate-transporters may carry pyrophosphate and inflammatory/necrotic non-N-BPs into such cells, and (iii) etidronate and clodronate inhibit all these transporters, and they ameliorate the side effects of zoledronate by inhibiting phosphonoformate-inhibitable phosphate-transporters.

Unlabelled: An increase in dentin roughness, associated with surface composition, contributes to bacterial adherence in recontaminations. Surface roughness is also important for micromechanical interlocking of dental materials to dentin, and understanding the characteristics of the surface is essential to obtain the adhesion of root canal sealers that have different physico-chemical characteristics., Objectives: To evaluate the effects of sodium hypochlorite (NaOCl), ethylenediaminetetraacetic (EDTA), etidronic (HEBP), and citric acid (CA) associated with different irrigation regimens on root dentin roughness., Material and Methods: Forty-five root halves of anterior teeth were used. The root parts were sectioned in thirds, embedded in acrylic resin and polished to a standard surface roughness. Initially, the samples of each third were randomly assigned into 3 groups and treated as follows: G1 - saline solution (control); G2 - 5% NaOCl+18% HEBP mixed in equal parts; and G3 - 2.5% NaOCl. After initial measuments, the G3 samples were distributed into subgroups G4, G5 and G6, which were subjected to 17% EDTA, 10% CA and 9% HEBP, respectively. Following the new measuments, these groups received a final flush with 2.5% NaOCl, producing G7, G8 and G9. The dentin surface roughness (Ra) was determined before and after treatments using a profilometer. The Wilcoxon test (α<0.05) was used to compare the values before and after treatments, and the Friedman test (α<0.05) to detect any differences among root thirds., Results: (i) NaOCl did not affect the surface roughness; (ii) there was a significant increase in roughness after the use of chelating agents (P<0.01); and (iii) only the G3 group showed a difference in surface roughness between apical third and other thirds of the teeth (P<0.0043)., Conclusion: Only the irrigation regimens that used chelating agents altered the roughness of root dentin.

Background: Statins are not effective in reducing atherosclerotic plaques of the abdominal aorta, and accumulating evidence suggests that bisphosphonates have the potential to induce the regression of atherosclerotic plaques of the abdominal aorta., Methods and Results: A prospective, randomized, open-label, blinded-end-point trial involving 108 participants with hypercholesterolemia was conducted. Participants received 20 mg atorvastatin daily, 400 mg etidronate daily, or both drugs daily. The primary end point was the percent change in maximal vessel wall thickness of atherosclerotic plaques in the thoracic and abdominal aortas as measured by magnetic resonance imaging after 12 months of treatment. In both the combination therapy and atorvastatin groups, maximal vessel wall thickness of the thoracic aorta was reduced by 13.8% (95% confidence interval, -16.4 to -11.3) and 12.3% (95% confidence interval, -14.9 to -9.7), respectively. These reduction rates were comparable between groups (P=0.61). Meanwhile, in the etidronate group, maximal vessel wall thickness of the thoracic aorta remained unchanged (2.2%; 95% confidence interval, -0.3 to 4.8). Conversely, maximal vessel wall thickness of the abdominal aorta was reduced more effectively in the combination therapy group (-11.4%) than in the atorvastatin group (-0.9%; P<0.001) and the etidronate group (5.5%; P=0.006)., Conclusions: Atorvastatin plus etidronate combination therapy for 12 months significantly reduced both thoracic and abdominal aortic plaques, whereas atorvastatin monotherapy reduced only thoracic aortic plaques and etidronate monotherapy reduced only abdominal aortic plaques. The effectiveness of combination therapy in reducing atherosclerotic plaques in the abdominal aorta was significantly greater than for both atorvastatin and etidronate monotherapy., Clinical Trial Registration: URL: http://www.umin.ac.jp/ctr/. Unique identifier: UMIN 000002635.

An imbalance of immunosuppressive cells and cytotoxic cells plays an important role in inhibiting the antitumor immune response of the tumor-bearing host. We previously reported the profiles of tumor infiltrating leukocytes in cutaneous angiosarcoma (AS) and suggested that a combination of docetaxel (DTX) with bisphosphonate risedronate sodium (RS) might be effective for MMP9-expressing AS by targeting immunosuppressive cells such as M2 macrophages. To further confirm the effect of this combination therapy, in this report we investigated the immunomodulatory effect of DTX and RS on CD163(+) arginase 1 (Arg1)(+) M2 macrophages in vitro. Interestingly, our present study demonstrated that DTX in combination with RS significantly upregulated the mRNA expression of CXCL10 on M2 macrophages and significantly decreased the mRNA expression of CCL17 and Arg1. Moreover, the production of CXCL10 and CXCL11 from M2 macrophages was significantly increased by DTX with RS though there was no effect of DTX with RS on the production of CCL5 and CCL17. Furthermore, DTX with RS significantly decreased the production of CCL18, which was previously reported to correlate with the severity and prognosis in cancer patients. Our present report suggests one of the possible mechanisms of DTX with RS in the supportive therapy for angiosarcoma.

Purpose: Nitrogen-containing bisphosphonates (N-BP) such as zoledronate and risedronate exhibit antitumor effects. They block the activity of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, leading to intracellular accumulation of mevalonate metabolites (IPP/ApppI), which are recognized as tumor phosphoantigens by Vγ9Vδ2 T cells. However, mechanisms responsible for Vγ9Vδ2 T-cell recognition of N-BP-treated tumors producing IPP/ApppI remain unclear., Experimental Design: The effects of N-BPs on Vγ9Vδ2 T-cell expansion and anticancer activity were evaluated in vitro and in animal models of human breast cancers. The modalities of recognition of breast tumors by Vγ9Vδ2 T cells in N-BP-treated animals were also examined., Results: We found a strong correlation between Vγ9Vδ2 T-cell anticancer activity and intracellular accumulation of IPP/ApppI in risedronate-treated breast cancer cells in vitro. In addition, following risedronate treatment of immunodeficient mice bearing human breast tumors, human Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels but not those expressing low IPP/ApppI levels. The combination of doxorubicin with a N-BP improved, however, Vγ9Vδ2 T-cell cytotoxicity against breast tumors expressing low IPP/ApppI levels. Moreover, Vγ9Vδ2 T-cell cytotoxicity in mice treated with risedronate or zoledronate did not only depend on IPP/ApppI accumulation in tumors but also on expression of tumor cell surface receptor intercellular adhesion molecule-1 (ICAM-1), which triggered the recognition of N-BP-treated breast cancer cells by Vγ9Vδ2 T cells in vivo., Conclusion: These findings suggest that N-BPs can have an adjuvant role in cancer therapy by activating Vγ9Vδ2 T-cell cytotoxicity in patients with breast cancer that produces high IPP/ApppI levels after N-BP treatment., (©2012 AACR.)

Bisphosphonates may prevent or treat the bone loss promoted by the immunosuppressive regimens used in renal transplantation. Risedronate is a commonly used third-generation amino-bisphosphonate, but little is known about its effects on the bone health of renal transplant recipients. We randomly assigned 42 new living-donor kidney recipients to either 35 mg of risedronate weekly or placebo for 12 months. We obtained bone biopsies at the time of renal transplant and after 12 months of protocol treatment. Treatment with risedronate did not affect bone mineral density (BMD) in the overall cohort. In subgroup analyses, it tended to preserve BMD in female participants but did not significantly affect the BMD of male participants. Risedronate did associate with increased osteoid volume and trabecular thickness in male participants, however. There was no evidence for the development of adynamic bone disease. In summary, further study is needed before the use of prophylactic bisphosphonates to attenuate bone loss can be recommended in renal transplant recipients.

We used Raman and Fourier transform infrared microspectroscopy (FTIRM) analysis to examine the intrinsic bone material properties at actively bone-forming trabecular surfaces in iliac crest biopsies from women with postmenopausal osteoporosis (PMO) who were treated with either alendronate (ALN) or risedronate (RIS). At eight study sites, women were identified who had postmenopausal osteoporosis (PMO), were at least 5 years postmenopause, and had been on long-term therapy (either 3-5 years or >5 years) with daily or weekly ALN or RIS. Following standard tetracycline labeling, biopsies were collected from 102 women (33 treated with ALN for 3-5 years [ALN-3], 35 with ALN for >5 years [ALN-5], 26 with RIS for 3-5 years [RIS-3], and 8 with RIS for >5 years [RIS-5]) and were analyzed at anatomical areas of similar tissue age in bone-forming areas (within the fluorescent double labels). The following outcomes were monitored and reported: mineral to matrix ratio (corresponding to ash weight), relative proteoglycan content (regulating mineralization commencement), mineral maturity (indicative of the mineral crystallite chemistry and stoichiometry, and having a direct bearing on crystallite shape and size), and the ratio of two of the major enzymatic collagen cross-links (pyridinoline/divalent). In RIS-5 there was a significant decrease in the relative proteoglycan content (-5.83% compared to ALN-5), while in both RIS-3 and RIS-5 there was significantly lower mineral maturity/crystallinity (-6.78% and -13.68% versus ALN-3 and ALN-5, respectively), and pyridinoline/divalent collagen cross-link ratio (-23.09% and -41.85% versus ALN-3 and ALN-5, respectively). The results of the present study indicate that ALN and RIS exert differential effects on the intrinsic bone material properties at actively bone-forming trabecular surfaces., (Copyright © 2012 American Society for Bone and Mineral Research.)

We report the results of an in vitro screening assay targeting the intraerythrocytic form of the malaria parasite Plasmodium falciparum using a library of 560 prenyl-synthase inhibitors. Based on "growth-rescue" and enzyme-inhibition experiments, geranylgeranyl diphosphate synthase (GGPPS) is shown to be a major target for the most potent leads, BPH-703 and BPH-811, lipophilic analogs of the bone-resorption drugs zoledronate and risedronate. We determined the crystal structures of these inhibitors bound to a Plasmodium GGPPS finding that their head groups bind to the [Mg(2+)](3) cluster in the active site in a similar manner to that found with their more hydrophilic parents, whereas their hydrophobic tails occupy a long-hydrophobic tunnel spanning both molecules in the dimer. The results of isothermal-titration-calorimetric experiments show that both lipophilic bisphosphonates bind to GGPPS with, on average, a ΔG of -9 kcal mol(-1), only 0.5 kcal mol(-1) worse than the parent bisphosphonates, consistent with the observation that conversion to the lipophilic species has only a minor effect on enzyme activity. However, only the lipophilic species are active in cells. We also tested both compounds in mice, finding major decreases in parasitemia and 100% survival. These results are of broad general interest because they indicate that it may be possible to overcome barriers to cell penetration of existing bisphosphonate drugs in this and other systems by simple covalent modification to form lipophilic analogs that retain their enzyme-inhibition activity and are also effective in vitro and in vivo.

Objective: To investigate the effects of low-dose risedronate sodium on the in vitro cellular profile of osteoblasts, adipocytes, osteocytes and osteoclasts in a rat model of abrupt oestrogen deficiency., Methods: Oestrogen deficiency was induced by ovariectomy in 24 female rats. The rats were treated with low-dose (0.24 μg/kg) or high-dose (2.4 μg/kg) risedronate sodium for 4 days presurgery, continuing every 3 days until 15 days postsurgery. Osteogenic and adipogenic differentiation were determined in cultured bone marrow cells by alkaline phosphatase and Oil Red O staining, respectively, and by osteogenic and adipogenic gene expression. Osteoclast formation was measured in bone marrow cells stimulated with macrophage colony-stimulating factor and receptor activator of nuclear factor κB ligand, and stained with tartrate-resistant acid phosphatase. Osteocyte apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assay and B-cell lymphoma-2 (Bcl-2) immunohistochemistry., Results: Low-dose risedronate sodium enhanced osteoblast differentiation, suppressed adipocyte differentiation and osteoclast formation, and reduced osteocyte apoptosis through regulation of Bcl-2 and Bcl-2-associated X protein., Conclusions: Low-dose risedronate sodium may have clinical benefit in protecting against bone loss after abrupt oestrogen deficiency.

Background: The aim of this study was to assess the efficacy, tolerability and safety of risedronate in adults with CF., Methods: Patients with a lumbar spine (LS), total hip (TH) or femoral neck (FN) bone mineral density (BMD) Z-score of -1 or less were randomised to receive risedronate 35 mg weekly or placebo, and calcium (1g)+vitamin D(3) (800IU)., Results: At baseline, BMD Z-scores in the risedronate (n=17) and placebo (n=19) groups were similar. By 24 months, 7/17 risedronate patients vs 0/19 placebo patients stopped the study medication due to bone pain. After 24 months treatment, the mean difference (95% CI) in change in LS, TH and FN BMD between the risedronate vs placebo groups was 4.3% (0.4, 8.2) p=0.03; 4.0% (-0.5, 8.6) p=0.08; and 2.4% (-3.5, 8.2) p=0.41., Conclusions: After two years treatment there was a significant increase in LS BMD with weekly risedronate compared to placebo., (Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Objective: Nitrogen-containing bisphosphonates are one of the most successful therapeutics for osteoporosis. The aim of this study was to elucidate the functional mechanism of one of the typical nitrogen-containing bisphosphonates, risedronate., Methods: Osteoclasts generated from murine bone marrow macrophages were treated with risedronate in vitro, and its effects on apoptosis and bone-resorbing activity were examined. The mechanism of action of risedronate was examined by gene induction of constitutively active Akt-1 and constitutively active MEK-1, and by gene deletion of Bim. Bim(-/-) mice, in which osteoclasts were resistant to apoptosis, were treated with risedronate and analyzed radiographically, biochemically, and histologically., Results: Risedronate induced osteoclast apoptosis through the mitochondria-dependent pathway with an increased expression of Bim, and the proapoptotic effect of risedronate was suppressed by Bim deletion and constitutively active MEK-1 introduction. In contrast, the risedronate-induced suppression of bone resorption was completely reversed by inducing constitutively active Akt-1, but not by Bim deletion or constitutively active MEK-1 introduction. These results suggested that apoptosis and bone-resorbing activity of osteoclasts were regulated through the ERK/Bim axis and the Akt pathway, respectively, both of which were suppressed by risedronate. Although osteoclast apoptosis in response to risedronate administration was suppressed in the Bim(-/-) mice, risedronate treatment increased bone mineral density in Bim(-/-) mice at a level equivalent to that in wild-type mice., Conclusion: Our findings indicate that the antiresorptive effect of risedronate in vivo is mainly mediated by the suppression of the bone-resorbing activity of osteoclasts and not by the induction of osteoclast apoptosis., (Copyright © 2011 by the American College of Rheumatology.)

Androgen deprivation therapy (ADT) for prostate cancer (PCa) causes bone loss. Although we reported previously that risedronate significantly recovers bone mineral density (BMD) for up to 12 months, there have been no reports with longer follow-up periods to date. This study extended our earlier series extending the follow-up period to 24 months. Eligible patients had histologically confirmed PCa without lumbar spine metastasis and underwent ADT. Lumbar spine BMD, urinary deoxypyridinoline (uDPD) and serum bone alkaline phosphatase were measured at 6, 12 and 24 months. Among the total of 96 patients, we analyzed 26 and 18 patients in risedronate administration and control groups, respectively. BMD relative to the young adult mean ratio, uDPD and serum bone alkaline phosphatase of the risedronate administration group recovered significantly after 24 months compared with the control group (P<0.0001, P=0.0001, and P<0.0001, respectively). Transient blurred vision, malaise and vertigo were observed in 1 patient each among the 46 patients treated with risedronate within 28 days after first administration. Oral administration of risedronate is safe and effective for the recovery of ADT-induced bone loss in PCa patients even at 24 months after commencement of treatment.

Vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis and end-stage renal disease. The in vitro calcification of primary mouse, human and bovine vascular smooth muscle cells (VSMCs) is commonly employed to examine the mechanisms of vascular calcification. However, to date, no published studies have utilised a murine cell line to investigate this process. In the present study, we aimed to determine whether the mouse VSMC line MOVAS-1 can calcify in vitro. We established that the calcification of MOVAS-1 cells can be induced in the presence of calcifying medium (containing β-glycerophosphate and ascorbic acid), as detected by Alizarin Red and von Kossa staining, and quantification of calcium deposition and alkaline phosphatase activity. We also showed that the time course of MOVAS-1 calcification is comparable to that of the primary murine aortic VSMCs, establishing the MOVAS-1 cells as a feasible and relevant model. Significant increases in the mRNA expression profile of key genes associated with vascular calcification (Ocn, Akp2 and PiT-1) were observed in MOVAS-1 cells cultured under calcifying conditions, with similar changes in expression in murine aortic VSMCs. Furthermore, a significant reduction in calcification was observed in MOVAS-1 cells following treatment with levamisole and etidronate, known inhibitors of calcification. In conclusion, we demonstrated that the MOVAS-1 line is a reliable, convenient and economical system in which to investigate vascular calcification in vitro, and will make a useful contribution to increasing our understanding of this pathological process.

Objective: To determine whether treatment with an antiresorptive drug in combination with an antiinflammatory drug reduces periarticular bone and soft tissue adaptations associated with the progression of posttraumatic secondary osteoarthritis (OA)., Methods: We used in vivo microfocal computed tomography (micro-CT) to map bony adaptations and in vivo micro-magnetic resonance imaging (micro-MRI) to examine joint inflammation in a rat model of surgically induced OA secondary to knee triad injury. We examined the arthroprotective effects of the bisphosphonates alendronate and risedronate and the nonsteroidal antiinflammatory drug (NSAID) meloxicam., Results: Micro-CT revealed reduced levels of periarticular trabecular bone loss in animals with knee triad injury treated with the bisphosphonate drugs alendronate or risedronate, or the NSAID meloxicam, compared with untreated animals. Alendronate treatment reduced bony osteophyte development. While risedronate as a monotherapy did not positively impact osteophytogenesis, combination therapy with risedronate and meloxicam reduced osteophyte severity somewhat. Micro-MRI revealed an increased, diffuse water signal in the epiphyses of untreated rats with knee triad injury 8 weeks after surgery, suggestive of a bone marrow lesion-like stimulus. In contrast, meloxicam-treated rats showed a significant reduction in fluid signal compared with both bisphosphonate-treated groups 8 weeks after surgery. Histologic analysis qualitatively confirmed the chondroprotective effect of both bisphosphonate treatments, showing fewer degradative changes compared with untreated rats with knee triad injury., Conclusion: Our findings indicate that select combinations of bisphosphonate and NSAID drug therapy in the early stages of secondary OA preserve trabecular bone mass and reduce the impact of osteophytic bony adaptations and bone marrow lesion-like stimulus. Bisphosphonate and NSAID therapy may be an effective disease-modifying drug regimen if administered early after the initial injury.

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and have demonstrated clinical utility in the treatment of patients with osteolytic bone metastases. They also exhibit direct antitumor activity in vitro and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo. However, whether such effects are caused by a direct action of bisphosphonates on tumor cells or indirectly through inhibition of bone resorption remains unclear. To address this question, we used here a structural analog of the bisphosphonate risedronate, NE-58051, which has a bone mineral affinity similar to that of risedronate, but a 3000-fold lower bone antiresorptive activity. In vitro, risedronate and NE-58051 inhibited proliferation of breast cancer and melanoma cell lines. In vivo, risedronate and NE-58051 did not inhibit the growth of subcutaneous B02 breast tumor xenografts or the formation of B16F10 melanoma lung metastasis. In contrast to NE-58051, risedronate did inhibit B02 breast cancer bone metastasis formation by reducing both bone destruction and skeletal tumor burden, indicating that the antitumor effect of bisphosphonates is achieved mainly through inhibition of osteoclast-mediated bone resorption.

The purpose of the present study was to discuss the effects of risedronate on osteoarthritis (OA) of the knee by reviewing the existing literature. The literature was searched with PubMed, with respect to prospective, double-blind, randomized placebo-controlled trials (RCTs), using the following search terms: risedronate, knee, and osteoarthritis. Two RCTs met the criteria. A RCT (n = 231) showed that risedronate treatment (15 mg/day) for 1 year improved symptoms. A larger RCT (n = 1,896) showed that risedronate treatment (5 mg/day, 15 mg/day, 35 mg/week, and 50 mg/week) for 2 years did not improve signs or symptoms, nor did it alter radiological progression. However, a subanalysis study (n = 477) revealed that patients with marked cartilage loss preserved the structural integrity of subchondral bone by risedronate treatment (15 mg/day and 50 mg/week). Another subanalysis study (n = 1,885) revealed that C-terminal crosslinking telopeptide of type II collagen (CTX-II) decreased with risedronate treatment in a dose-dependent manner, and levels reached after 6 months were associated with radiological progression at 2 years. The results of these RCTs show that risedronate reduces the marker of cartilage degradation (CTX-II), which could contribute to attenuation of radiological progression of OA by preserving the structural integrity of subchondral bone. The review of the literature suggests that higher doses of risedronate (15 mg/day) strongly reduces the marker of cartilage degradation (CTX-II), which could contribute to attenuation of radiological progression of OA by preserving the structural integrity of subchondral bone.

Nonvertebral fractures account for 80% of all fractures and their accompanying morbidity and mortality. Despite this, the effect of drug therapy on cortical morphology has received limited attention, partly because cortical bone is believed to remodel less and decrease less with age than trabecular bone. However, the haversian canals traversing the cortex provide a surface for remodeling that produces bone loss, porosity, and cortical fragility. We developed a new method of 3D micro-computed tomography (microCT) to quantify intracortical porosity and the effects of treatment. Women with osteoporosis randomized to risedronate (5 mg/day, n = 28) or placebo (n = 21) had paired transiliac biopsies at baseline and 5 years imaged using 3D microCT. Pores determined from 8 to 12 slices were stratified by their minor axis length into those 25 to 100 microm (closing cone of haversian canals), 100 to 300 microm (cutting cone of haversian canals), and >300 microm (coalescent cavities). Porosity was analyzed as pore area (percent bone area) and pore density (pore number/mm(2)). Medians are reported. Risedronate reduced pore area in the 25 to 100, 100 to 300, and 300 to 500 microm ranges over 5 years (p = .0008, .04, NS, respectively) corresponding to an 18% to 25% reduction. In the placebo group, pore area was unchanged. At 5 years, pore area and pore number/mm(2) in the 25 to 100 microm range were each 17% lower in the risedronate group than in the placebo group (p = .02 and .04, respectively). Risedronate is likely to maintain bone strength and reduce nonvertebral fracture risk in part by reducing remodeling and therefore the number and size of intracortical cavities., (2010 American Society for Bone and Mineral Research)

Introduction: The purpose of this study was to evaluate the effects of risedronate (Ris) in the modulation of bone formation in rats with glucocorticoid (GC)-induced osteoporosis by histomorphometric, immunohistochemical and gene expression analyses., Methods: We analyzed structure, turnover and microarchitecture, cyclooxygenase 2 (COX-2) levels and osteocyte apoptosis in 40 female rats divided as follows: 1) vehicle of methylprednisolone (vGC) + vehicle of risedronate (vRis); 2) Ris 5 μg/Kg + vGC; 3) methylprednisolone (GC) 7 mg/Kg + vRis; 4) GC 7 mg/Kg +Ris 5 μg/Kg. In addition, we evaluated cell proliferation and expression of COX-2 and bone alkaline phosphatase (b-ALP) genes in bone marrow cells and MLO-y4 osteocytes treated with Ris alone or in co-treatment with the selective COX-2 inhibitor NS-398 or with dexametasone., Results: Ris reduced apoptosis induced by GC of osteocytes (41% vs 86%, P < 0.0001) and increased COX-2 expression with respect to controls (Immuno-Hystochemical Score (IHS): 8.75 vs 1.00, P < 0.0001). These positive effects of Ris in bone formation were confirmed by in vitro data as the viability and expression of b-ALP gene in bone marrow cells resulted increased in a dose dependent manner., Conclusions: These findings suggest a positive effect of Ris in bone formation and support the hypothesis that the up-regulation of COX-2 could be an additional mechanism of anabolic effect of Ris.

Objective: The aim of this study was to investigate the responses of serum osteocalcin (OC), undercarboxylated osteocalcin (ucOC) and N-terminal telopeptide of type I collagen (NTx) to corticosteroids, and to examine the effects of risedronate therapy with or without vitamin K(2) supplementation on bone metabolic markers in corticosteroid-treated patients., Methods: Sixteen patients on corticosteroid therapy for neuromuscular disorders were assigned randomly to 2 groups (A: risedronate monotherapy, n=8; B: combined risedronate and vitamin K(2) therapy, n=8) and treated for 1 year. Another 6 patients who received intravenous steroid pulse therapy were assigned to group C for investigation of the effects of corticosteroids on OC and ucOC 1 month after pulse therapy., Results: Serial measurements revealed that significant decreases of OC, ucOC and NTx persisted with a similar time course profile during 1 year of treatment in groups A and B, and between-group analysis failed to demonstrate any additional effects of vitamin K(2) on risedronate therapy. Intravenous steroid pulse therapy induced a transient depression of OC and ucOC within 1 week in group C., Conclusion: These results indicate that serum concentrations of OC and ucOC become consistently low during corticosteroid administration despite risedronate therapy with or without vitamin K(2) supplementation, and the serum ucOC level may not be a reliable indicator of vitamin K status under corticosteroid administration.

The effect of risedronate (2.5 mg once daily) on femoral strength was evaluated using Advanced Hip Assessment (AHA) for the first time in Japan. In total, 104 patients with primary osteoporosis and available data on bone mineral density (BMD; lumbar spine/proximal femur), urinary NTx (cross-linked N-telopeptides of type I collagen) and AHA-based parameters collected before and after 4 months of risedronate therapy were included in the analyses. Change and percentage change from baseline in these parameters were determined. Percentage change in femur strength index was 7.9 +/- 21.1% and 5.5 +/- 18.0% for the right and left femurs, respectively; both increases were statistically significant. Cross-sectional moment of inertia, cross-sectional area and mean neck width in the femoral neck region of interest also increased significantly in both femurs. Percentage change in lumbar spine BMD (L2 - L4) was 3.0 +/- 3.7%, and proximal femoral BMD was 1.1 +/- 3.1% and 0.7 +/- 3.2% in the right and left femurs, respectively, all showing a significant increase from baseline. Percentage change in urinary NTx was -41.5 +/- 30.5%, which was a significant decrease. Using AHA, this study showed that, in patients with primary osteoporosis, risedronate improved BMD and bone quality, thereby enhancing femoral strength as early as 4 months after treatment initiation.

Background: Osteosarcoma is a highly malignant bone tumor and is the most commonly encountered malignant bone tumor in children and adolescents. Furthermore, significant numbers of patients eventually develop pulmonary metastases and succumb to the disease even after conventional multi-agent chemotherapy and surgical excision. Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs), and recently clinical trials have been initiated on MMP-inhibitors. On the other hand, bisphosphonates (BPs), which have a profound effect on bone resorption, are widely used to treat osteoclast-mediated bone diseases. BPs are also known to inhibit tumor growths and metastases in some tumors such as breast cancer, renal cell carcinoma, and prostate cancer., Methods: Two osteosarcoma cell lines (SaOS-2 and U2OS) were treated with risedronate (0, 0.1, 1, 10 microM) for 48 hours. Cell viabilities were determined using MTT assay, the mRNA levels of MMP-2 and MMP-9 were analyzed by reverse-transcription polymerase chain reaction, the amount of MMP-2 and MMP-9 protein were analyzed by Westernblot, the activities of MMP-2 and MMP-9 were observed by Gelatin zymography, and Matrigel invasion assays were used to investigate the invasive potential of osteosarcoma cell lines before and after risedronate treatment., Results: The invasiveness of osteosarcoma cell lines (SaOS-2, U2OS) were reduced in a dose dependent manner follow 48 hour treatment of up to 10 microM of the risedronate at which concentration no cytotoxicity occurred. Furthermore, the gelatinolytic activities and protein and mRNA levels of MMP-2 and MMP-9 were also suppressed by increasing risedronate concentrations., Conclusion: Given that MMP-2 and MMP-9 are instrumental in tumor cell invasion, our results suggest the risedronate could reduce osteosarcoma cell invasion.

Risedronate, a nitrogen-containing bisphosphonate, is widely used in the clinical field for the treatment of osteoporosis. Risedronate is known to exert its effects through binding to hydroxyapatite in bone tissue, inhibiting osteoclastic activity, and inducing apoptosis of osteoclasts. The purpose of this study was to determine the effects of risedronate on osteoclast differentiation in vitro and on an inflammatory bone loss model in vivo. Risedronate inhibited osteoclast differentiation in co-culture of bone marrow cells (BMCs) and osteoblasts, and suppressed receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from bone marrow-derived macrophages (BMMs) in a dose-dependent manner without toxicity. Risedronate significantly inhibited expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 induced by RANKL. To examine the effect of risedronate on bone loss in vivo, we used a mouse model of lipopolysaccharide (LPS)-mediated bone loss. Micro-CT analysis of the femurs showed that LPS treatment caused bone loss. However, bone loss was significantly attenuated in mice administered with risedronate. Taken together, we conclude that risedronate exerts beneficial effects on osteoporosis by inhibiting osteoclast differentiation both directly and indirectly. In infectious conditions, the inhibitory effect of risedronate on bone erosion was excellent. Thus risedronate could be a treatment option for osteoporosis caused by inflammatory and infectious conditions.

Background: We conducted the present study to investigate the therapeutic effects of a combination treatment of alfacalcidol (ALF) and risedronate (RIS) on the bone mechanical properties of bone and calcium (Ca) metabolism using an ovariectomized (OVX) rat model of osteoporosis., Methods: Female Wistar rats were OVX- or sham-operated at 40 weeks of age. Twelve weeks post-surgery, rats were randomized into seven groups: 1) sham + vehicle, 2) OVX + vehicle, 3) OVX + ALF 0.025 microg/kg/day, 4) OVX + ALF 0.05 microg, 5) OVX + RIS 0.3 mg, 6) OVX + RIS 3.0 mg, 7) OVX + ALF 0.025 microg + RIS 0.3 mg. Each drug was administered orally five times a week for 12 weeks. After treatment, we evaluated the mechanical properties of the lumbar vertebra and femoral midshaft. In the lumbar vertebra, structural and material analyses were performed using micro-computed tomography (micro-CT) and microbeam X-ray diffraction (micro-XRD), respectively. Biochemical markers in serum and urine were also determined., Results: (1) With respect to improvement in the mechanical strength of the lumbar spine and the femoral midshaft, the combination treatment of ALF and RIS at their sub-therapeutic doses was more effective than each administered as a monotherapy; (2) In the suppression of bone resorption and the amelioration of microstructural parameters, the effects of ALF and RIS were considered to be independent and additive; (3) The improvement of material properties, such as microstructural parameters and the biological apatite (Bap) c-axis orientation, contributed to the reinforcement of spinal strength; and (4) The combination treatment of ALF and RIS normalized urinary Ca excretion, suggesting that this treatment ameliorated the changes in Ca metabolism., Conclusion: These results demonstrate that the combination treatment of ALF and RIS at their sub-therapeutic doses can improve the mechanical properties of the spine as well as the femur and ameliorate changes in Ca metabolism in an animal model of osteoporosis, suggesting that the combination treatment of ALF and RIS has a therapeutic advantage over each monotherapy for the treatment of osteoporosis.

Purpose: To compare the effect of vitamin K(2) and risedronate on trabecular bone in glucocorticoid (GC)-treated rats., Materials and Methods: Forty-eight Sprague-Dawley female rats, 3 months of age, were randomized by the stratified weight method into 5 groups according to the following treatment schedule: age-matched control, GC administration, and GC administration with concomitant administration of vitamin K(2), risedronate, or vitamin K(2) + risedronate. GC (methylprednisolone sodium succinate, 5.0 mg/kg) and risedronate (10 microg/kg) were administered subcutaneously three and five times a week, respectively. Vitamin K(2) (menatetrenone, 30 mg/kg) was administered orally three times a week. At the end of the 8-week experiment, bone histomorphometric analysis was performed on trabecular bone of the tibial proximal metaphysis., Results: GC administration decreased trabecular bone mass compared with age-matched controls because of decreased bone formation (mineralizing surface, mineral apposition rate, and bone formation rate) and increased bone erosion. Vitamin K(2) attenuated GC-induced trabecular bone loss by preventing GC-induced decrease in bone formation (mineralizing surface) and subsequently reducing GC-induced increase in bone erosion. Risedronate prevented GC-induced trabecular bone loss by preventing GC-induced increase in bone erosion although it also suppressed bone formation (mineralizing surface, mineral apposition rate, and bone formation rate). Vitamin K(2) mildly attenuated suppression of bone formation (mineralizing surface) and bone erosion caused by risedronate without affecting trabecular bone mass when administered in combination., Conclusion: The present study showed differential effect of vitamin K(2) and risedronate on trabecular bone in GC-treated rats.

Male osteoporosis is increasingly recognized as a major public health issue. This multinational, 2-yr, randomized, double-blind, placebo-controlled study was conducted to determine the efficacy and safety of 35 mg once-a-week risedronate in men with osteoporosis. Patients had to be men >or=30 yr old, with lumbar spine T-score

Three auditory ossicles including the malleus, incus, and stapes conduct sound in the middle ear from the tympanic membrane to the inner ear. Auditory ossicles are massively resorbed by osteoclasts in Opg(-/-) mice, which lack osteoprotegerin (OPG), a soluble decoy receptor for the osteoclastogenic cytokine RANKL. Opg(-/-) mice exhibit progressive hearing loss and are a model for juvenile Paget's disease. However, effects of antiresorptive treatment on auditory ossicles and on hearing function in Opg(-/-) mice are unknown. We intraperitoneally injected Opg(-/-) mice with bisphosphonate risedronate 5 d/wk for 9 wk. Morphology of auditory ossicles was examined microscopically, radiographically, and histologically. Hearing function was monitored by measuring the auditory brain stem response (ABR). Control Opg(-/-) mice exhibited thinning of all three ossicles and tibia. In contrast, risedronate treatment significantly inhibited bone loss in auditory ossicles as well as in long bones of Opg(-/-) mice. Bony fusion of the junction between the stapes and the otic capsule was reduced after treatment. Moreover, ABR measurement showed that hearing in Opg(-/-) mice was significantly improved by risedronate treatment. These data suggest that hearing loss in pathologies characterized by excessive resorption of the auditory ossicles may be prevented by bisphosphonates.

Background: Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time., Results: With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle., Conclusions: The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.

Fragility fractures that occur as a result of osteoporosis are frequently associated with chronic pain and decreased quality of life as well as significant morbidity and mortality. Fracture reduction, however, is often less than optimal due to poor compliance with medications. Studies have demonstrated that risedronate, a heterocyclic nitrogen containing bisphosphonate can reduce vertebral, nonvertebral, and hip fracture incidence in postmenopausal women, in men, and in subsets of older patients at great risk of falls and fragility. The mechanism, efficacy, dosing options, and tolerability of risedronate are reviewed.

The heterogeneous distribution of mineral content in trabecular bone reflects the continuous renewal of bone material in bone remodeling and the subsequent increase in mineral content in the newly formed bone packets. The bone mineralization density distribution (BMDD) is typically used to describe this nonuniform mineral content of the bone matrix. Our mathematical model describes changes of the BMDD of trabecular bone as a function of bone resorption and deposition rates and the mineralization kinetics in a newly formed bone packet. Input parameters used in the simulations were taken from experimental studies. The simulations of the time evolution of the BMDD after increase in bone turnover (perimenopausal period) resulted in a shift of the BMDD toward lower values of the mineral content. Transiently, there was a broadening of the BMDD configuration partly showing two peaks, which points to a strongly heterogeneous distribution of the mineral. Conversely, when the remodeling rate was reduced (antiresorptive therapy), the BMDD shifted toward higher values of the mineral content. There was a transient narrowing of the distribution before broadening again to reach the new steady state. Results from this latter simulation are in good agreement with measurements of the BMDD of patients after 3 and 5 yr of treatment with risedronate. Based on available experimental data on bone remodeling, this model gives reliable predictions of changes in BMDD, an important factor of bone material quality. With the availability of medications with a known effect on bone turnover, this knowledge opens the possibility for therapeutic manipulation of the BMDD.

Objective: Glucocorticoid excess decreases bone mineralization and microarchitecture and leads to reduced bone strength. Both anabolic (parathyroid hormone [PTH]) and antiresorptive agents are used to prevent and treat glucocorticoid-induced bone loss, yet these bone-active agents alter bone turnover by very different mechanisms. This study was undertaken to determine how PTH and risedronate alter bone quality following glucocorticoid excess., Methods: Five-month-old male Swiss-Webster mice were treated with the glucocorticoid prednisolone (5 mg/kg in a 60-day slow-release pellet) or placebo. From day 28 to day 56, 2 groups of glucocorticoid-treated animals received either PTH (5 microg/kg) or risedronate (5 microg/kg) 5 times per week. Bone quality and quantity were measured using x-ray tomography for the degree of bone mineralization, microfocal computed tomography for bone microarchitecture, compression testing for trabecular bone strength, and biochemistry and histomorphometry for bone turnover. In addition, real-time polymerase chain reaction (PCR) and immunohistochemistry were performed to monitor the expression of several key genes regulating Wnt signaling (bone formation) and mineralization., Results: Compared with placebo, glucocorticoid treatment decreased trabecular bone volume (bone volume/total volume [BV/TV]) and serum osteocalcin, but increased serum CTX and osteoclast surface, with a peak at day 28. Glucocorticoids plus PTH increased BV/TV, and glucocorticoids plus risedronate restored BV/TV to placebo levels after 28 days. The average degree of bone mineralization was decreased after glucocorticoid treatment (-27%), but was restored to placebo levels after treatment with glucocorticoids plus risedronate or glucocorticoids plus PTH. On day 56, RT-PCR revealed that expression of genes that inhibit bone mineralization (Dmp1 and Phex) was increased by continuous exposure to glucocorticoids and glucocorticoids plus PTH and decreased by glucocorticoids plus risedronate, compared with placebo. Wnt signaling antagonists Dkk-1, Sost, and Wif1 were up-regulated by glucocorticoid treatment but down-regulated after glucocorticoid plus PTH treatment. Immunohistochemistry of bone sections showed that glucocorticoids increased N-terminal Dmp-1 staining while PTH treatment increased both N- and C-terminal Dmp-1 staining around osteocytes., Conclusion: Our findings indicate that both PTH and risedronate improve bone mass, degree of bone mineralization, and bone strength in glucocorticoid-treated mice, and that PTH increases bone formation while risedronate reverses the deterioration of bone mineralization.

Bisphosphonates bind avidly to bone mineral and are potent inhibitors of osteoclast-mediated bone destruction. They also exhibit antitumor activity in vitro. Here, we used a mouse model of human breast cancer bone metastasis to examine the effects of risedronate and NE-10790, a phosphonocarboxylate analogue of the bisphosphonate risedronate, on osteolysis and tumor growth. Osteolysis was measured by radiography and histomorphometry. Tumor burden was measured by fluorescence imaging and histomorphometry. NE-10790 had a 70-fold lower bone mineral affinity compared with risedronate. It was 7-fold and 8,800-fold less potent than risedronate at reducing, respectively, breast cancer cell viability in vitro and bone loss in ovariectomized animals. We next showed that risedronate given at a low dosage in animals bearing human B02-GFP breast tumors reduced osteolysis by inhibiting bone resorption, whereas therapy with higher doses also inhibited skeletal tumor burden. Conversely, therapy with NE-10790 substantially reduced skeletal tumor growth at a dosage that did not inhibit osteolysis, a higher dosage being able to also reduce bone destruction. The in vivo antitumor activity of NE-10790 was restricted to bone because it did not inhibit the growth of subcutaneous B02-GFP tumor xenografts nor the formation of B16-F10 melanoma lung metastases. Moreover, NE-10790, in combination with risedronate, reduced both osteolysis and skeletal tumor burden, whereas NE-10790 or risedronate alone only decreased either tumor burden or osteolysis, respectively. In conclusion, our study shows that decreasing the bone mineral affinity of bisphosphonates is an effective therapeutic strategy to inhibit skeletal tumor growth in vivo.

We evaluated the effect of the bisphosphonate, risedronate, on pain and cartilage metabolism in 33 patients with osteoarthritis of the knee, randomized into two groups. Group RC was treated with risedronate (2.5 mg/day) and calcium (900 mg/day); group C received calcium (900 mg/day) alone. Pain on exercise was estimated using a subjective visual rating scale (VRS) and an electroalgometric method of measuring decrease in skin impedance, previously shown to be indicative of pain. We measured urinary excretion of cartilage-specific collagen type II fragments as a marker of cartilage degradation. Multiple regression analysis revealed that pain alleviation as measured by skin impedance, but not VRS, was associated with a decrease in collagen fragment excretion. This suggests that, for pain evaluation, reduction in skin impedance may have a greater physiological basis compared with VRS-based evaluation. We consider that the chondroprotective and analgesic effects of risedronate may be related.

Tissue-nonspecific alkaline phosphatase (TNSALP) and Ca-ATPase are known to play roles in bone mineralization, but how these enzymes contribute to appositional mineralization has been illusive. Here we examined the active sites of these enzymes in appositional mineralization using the bones of young rats being administered with 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 5 days. The doses of HEBP totally abolished mineralization of newly formed bone matrix except in matrix vesicles (MVs), and hence allowed precise localization of MVs and phosphatase reactions within non-mineralized extracellular matrix. Intense TNSALP and ATPase reactions were confirmed along the limited portions of osteoblast membranes where intimate cell-cell contacts were maintained. Diffuse reactions of these enzymes were throughout the osteoid implicating efflux of TNSALP and ATPase molecules into extracellular matrix from the osteoblast membranes. Phosphatase reactions associated with MVs varied both in intensity and location among the individual vesicles; newly formed MVs were almost free of reactions but appeared to gain those activities later in the osteoid. These data suggest that TNSALP and ATPase are released from the osteoblast membrane and later integrated into MVs within the osteoid. The osteoblasts may thus regulate appositional mineralization of bone from a distance at least in part by providing phosphatases via MVs.