Nitrogen-containing Bisphosphonates Research Articles

Peptides as modulators of FPPS enzyme: A multifaceted evaluation from the design to the mechanism of action

Bone diseases are medical conditions caused by the loss of bone homeostasis consecutive to increased osteoclast activity and diminished osteoblast activity. The mevalonate pathway (MVA) is crucial for maintaining this balance since it drives the post-translational prenylation of small guanosine triphosphatases (GTPases) proteins. Farnesyl pyrophosphate synthase (FPPS) plays a crucial role in the MVA pathway. Consequently, in the treatment of bone-related diseases, FPPS is the target of FDA-approved nitrogen-containing bisphosphonates (N-BPs), which have tropism mainly for bone tissue due to their poor penetration in soft tissues. The development of inhibitors targeting the FPPS enzyme has garnered significant interest in recent decades due to FPPS's role in the biosynthesis of cholesterol and other isoprenoids, which are implicated in cancer, bone diseases, and other conditions. In this study, we describe a multidisciplinary approach to designing novel FPPS inhibitors, combining computational modeling, biochemical assays, and biophysical techniques. A series of peptides and phosphopeptides were designed, synthesized, and evaluated for their ability to inhibit FPPS activity. Molecular docking was employed to predict the binding modes of these compounds to FPPS, while Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) spectroscopy experiments – based on Saturation Transfer Difference (STD) and an enzymatic NMR assay – were used to measure their binding affinities and kinetics. The biological activity of the most promising compounds was further assessed in cellular assays using murine colorectal cancer (CRC) cells. Additionally, genomics and metabolomics profiling allowed to unravel the possible mechanisms underlying the activity of the peptides, confirming their involvement in the modulation of the MVA pathway. Our findings demonstrate that the designed peptides and phosphopeptides exhibit significant inhibitory activity against FPPS and possess antiproliferative effects on CRC cells, suggesting their potential as therapeutic agents for cancer.

Non-nitrogen-containing bisphosphonates and nitrogen-containing bisphosphonates for the treatment of atherosclerosis and vascular calcification: A meta-analysis.

The role of non-nitrogen-containing bisphosphonates (non-N-BPs) and nitrogen-containing bisphosphonates (N-BPs) in the treatment of atherosclerosis (AS) and vascular calcification (VC) is uncertain. This meta-analysis was conducted to evaluate the efficacy of non-N-BPs and N-BPs in the treatment of AS and VC. The PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases were searched from their inception to July 5th, 2023. Eligible studies comparing bisphosphonates (BPs) versus no BPs in the treatment of AS and VC were included. The data were analyzed using Review Manager Version 5.3. Seventeen studies were included in this meta-analysis. Twelve were randomized control trials (RCTs), and 5 were nonrandomized studies. Overall, 813 patients were included in the BPs group, and 821 patients were included in the no BPs group. Compared with no BP treatment, non-N-BP or N-BP treatment did not affect serum calcium (P > .05), phosphorus (P > .05) or parathyroid hormone (PTH) levels (P > .05). Regarding the effect on serum lipids, non-N-BPs decreased the serum total cholesterol (TC) level (P < .05) and increased the serum triglyceride (TG) level (P < .01) but did not affect the serum low-density lipoprotein cholesterol (LDL-C) level (P > .05). N-BPs did not affect serum TC (P > .05), TG (P > .05) or LDL-C levels (P > .05). Regarding the effect on AS, non-N-BPs did not have a beneficial effect (P > .05). N-BPs had a beneficial effect on AS, including reducing the intima-media thickness (IMT) (P < .05) and plaque area (P < .01). For the effect on VC, non-N-BPs had a beneficial effect (P < .01), but N-BPs did not have a beneficial effect (P > .05). Non-N-BPs and N-BPs did not affect serum calcium, phosphorus or PTH levels. Non-N-BPs decreased serum TC levels and increased serum TG levels. N-BPs did not affect serum lipid levels. Non-N-BPs had a beneficial effect on VC, and N-BPs had a beneficial effect on AS.

Alendronate modulates cytokine responses in healthy young individuals after BCG vaccination

Bacillus Calmette–Guérin (BCG) vaccination induces memory characteristics in innate immune cells and their progenitors, a process called trained immunity mediated by epigenetic and metabolic reprogramming. Cholesterol synthesis plays an amplifying role in trained immunity through mevalonate release. Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, can inhibit cholesterol synthesis. We explored their effects on trained immunity induced by BCG in a placebo-controlled clinical study (NL74082.091.20) in young, healthy individuals. Participants receiving single-dose oral alendronate on the day of BCG vaccination had more neutrophils and plasma cells one month after treatment. Alendronate led to reduced proinflammatory cytokine production by PBMCs stimulated with heterologous bacterial and viral stimuli one month later. Furthermore, the addition of alendronate transcriptionally suppressed multiple immune response pathways in PBMCs upon stimulation. Our findings indicate that N-BPs modulate the long-lasting effects of BCG vaccination on the cytokine production capacity of innate immune cells.

Bisphosphonate's effect on the tongue in adult male albino rats and the possible protective role of rutin: light and scanning electron microscopic study.

Alendronate sodium (ALS) is a nitrogen-containing bisphosphonate used for the treatment of different bone disorders. However, its adverse effect on oral soft tissue has been detected. Rutin (RUT) is natural flavonoid with antioxidant and anti-inflammatory properties. This work aimed to investigate the possible effect of ALS on the tongue of adult male albino rats and to evaluate the possible protective role of RUT. Forty adult male albino rats were equally divided into four groups: group I (control), group II (RUT): Received RUT 50 mg/kg, group III (ALS): Received ALS 1 mg/kg, group IV (ALS+RUT): Received ALS and RUT with the same doses as pervious groups. The drugs were given once daily for 5 weeks. Tongue specimens were taken and processed for light and scanning electron microscopic inspection. ALS treated group revealed structural changes in the tongue in the form of decrease in the height of the filiform papillae with blunt ends, marked atrophy in some papillae with areas of focal loss, loss of some epithelial cells, pyknotic nuclei and cytoplasmic vacuoles in some epithelial cells. The lamina propria showed inflammatory cellular infiltration with congested blood vessels. Statistically, there were highly significant decrease in the number of proliferating cell nuclear antigen immunopositive cells, area percentage of Bcl-2 immunoexpression and highly significant increase in the collagen content compared to control group. Administration of RUT with ALS minimizes these changes. RUT protected the rat tongue against the histological and immunohistochemical changes induced by ALS through its antioxidant and anti-inflammatory properties.

Development of Innate-Immune-Cell-Based Immunotherapy for Adult T-Cell Leukemia-Lymphoma.

γδ T cells and natural killer (NK) cells have attracted much attention as promising effector cell subsets for adoptive transfer for use in the treatment of malignant and infectious diseases, because they exhibit potent cytotoxic activity against a variety of malignant tumors, as well as virus-infected cells, in a major histocompatibility complex (MHC)-unrestricted manner. In addition, γδ T cells and NK cells express a high level of CD16, a receptor required for antibody-dependent cellular cytotoxicity. Adult T-cell leukemia-lymphoma (ATL) is caused by human T-lymphotropic virus type I (HTLV-1) and is characterized by the proliferation of malignant peripheral CD4+ T cells. Although several treatments, such as chemotherapy, monoclonal antibodies, and allogeneic hematopoietic stem cell transplantation, are currently available, their efficacy is limited. In order to develop alternative therapeutic modalities, we considered the possibility of infusion therapy harnessing γδ T cells and NK cells expanded using a novel nitrogen-containing bisphosphonate prodrug (PTA) and interleukin (IL)-2/IL-18, and we examined the efficacy of the cell-based therapy for ATL in vitro. Peripheral blood samples were collected from 55 patients with ATL and peripheral blood mononuclear cells (PBMCs) were stimulated with PTA and IL-2/IL-18 for 11 days to expand γδ T cells and NK cells. To expand NK cells alone, CD3+ T-cell-depleted PBMCs were cultured with IL-2/IL-18 for 10 days. Subsequently, the expanded cells were examined for cytotoxicity against ATL cell lines in vitro. The proportion of γδ T cells in PBMCs was markedly low in elderly ATL patients. The median expansion rate of the γδ T cells was 1998-fold, and it was 12-fold for the NK cells, indicating that γδ T cells derived from ATL patients were efficiently expanded ex vivo, irrespective of aging and HTLV-1 infection status. Anti-CCR4 antibodies enhanced the cytotoxic activity of the γδ T cells and NK cells against HTLV-1-infected CCR4-expressing CD4+ T cells in an antibody concentration-dependent manner. Taken together, the adoptive transfer of γδ T cells and NK cells expanded with PTA/IL-2/IL-18 is a promising alternative therapy for ATL.

Nitrogen-containing bisphosphonate was associated with a lower risk of dementia in patients with fragility fractures: A population-based cohort study in Hong Kong

Nitrogen-containing bisphosphonate was associated with a lower risk of dementia in patients with fragility fractures: A population-based cohort study in Hong Kong

THU442 A Case Of Early Medication Related Osteonecrosis Of The Jaw In Patient Receiving Intravenous Zoledronate For Treatment Of Osteoporosis

Abstract Disclosure: V. Ekpe: None. X. Au: None. M. Golson: None. L.E. McGuire: None. L.E. Aguirre: None. Introduction: Medication related Osteonecrosis of the jaw (MRONJ) is a rare side effect in patients receiving nitrogen-containing bisphosphonates intravenously and other anti-bone resorptive agents such as denosumab . The incidence is 0.1% in patients with metabolic bone disease1. Annual infusions of zoledronic acid have been recommended in order to improve patient compliance, optimize therapeutic effects and to minimize side effects in selected patients. Risk factors include history of malignancy, smoking, poor dental health, prior oral surgery, high dose exposure to zoledronic acid and longer duration of treatment with anti-resorptive therapy. Increasing usage of bisphosphonates and other antiresorptive drugs, have led to an increased incidence of MRONJ. Clinical Case: We report the case of a 63-year-old Caucasian female suffering from osteoporosis who developed early stage MRONJ. She has a history of several years of oral bisphosphonate treatment and three annual administrations of zoledronic acid. Patient presented with a new complaint of gum pain and a sore above her tooth. Prior routine dental exams have all been unremarkable. She was promptly referred and evaluated by a dental provider. Dental photographs showed a resorptive defect at the gumline. Patient was diagnosed with medication related osteonecrosis of the jaw (MRONJ) in the gum area above tooth number 9. Our patient was treated with oral amoxicillin 500mg twice daily and chlorhexidine gluconate mouth rinse twice daily. The intravenous zoledronic acid was discontinued. Her gum infection resolved. Discussion: Early detection of MRONJ is possible in patients potentially at risk for this rare complication. Providers should be aware of MRONJ and refer those patients on antiresorptive drugs to the dentist for frequent evaluation and assessment for any drug-related jaw changes.

The bisphosphonates alendronate and zoledronate induce adaptations of aerobic metabolism in permanent human endothelial cells

Nitrogen-containing bisphosphonates (NBPs), compounds that are widely used in the treatment of bone disorders, may cause side effects related to endothelial dysfunction. The aim of our study was to investigate the effects of chronic 6-day exposure to two common bone-preserving drugs, alendronate and zoledronate, on endothelial function and oxidative metabolism of cultured human endothelial cells (EA.hy926). NBPs reduced cell viability, induced oxidative stress and a pro-inflammatory state and downregulated the prenylation-dependent ERK1/2 signaling pathway in endothelial cells. In addition, NBPs induced increased anaerobic respiration and slightly increased oxidative mitochondrial capacity, affecting mitochondrial turnover through reduced mitochondrial fission. Moreover, by blocking the mevalonate pathway, NBPs caused a significant decrease in the level of coenzyme Q10, thereby depriving endothelial cells of an important antioxidant and mitochondrial electron carrier. This resulted in increased formation of reactive oxygen species (ROS), upregulation of antioxidant enzymes, and impairment of mitochondrial respiratory function. A general decrease in mitochondrial respiration occurred with stronger reducing fuels (pyruvate and glutamate) in NBP-treated intact endothelial cells, and significantly reduced phosphorylating respiration was observed during the oxidation of succinate and especially malate in NBP-treated permeabilized endothelial cells. The observed changes in oxidative metabolism caused a decrease in ATP levels and an increase in oxygen levels in NBP-treated cells. Thus, NBPs modulate the energy metabolism of endothelial cells, leading to alterations in the cellular energy state, coenzyme Q10 redox balance, mitochondrial respiratory function, and mitochondrial turnover.

Association of prior treatment with nitrogen-containing bisphosphonates on outcomes of COVID-19 positive patients.

Widely used in osteoporosis treatment, nitrogen-containing bisphosphonates (N-BP) have been associated with reduced mortality and morbidity among older adults. Based on prior studies, we hypothesized that prior treatment with N-BP might reduce intensive care unit (ICU) admission, ventilator use, and death among older adults diagnosed with COVID-19. This retrospective analysis of the PCORnet Common Data Model across 4 academic medical centers through 1 September 2021 identified individuals age >50 years with a diagnosis of COVID-19. The composite outcome included ICU admission, ventilator use, or death within 15, 30, and 180 days of COVID-19 diagnosis. Use of N-BP was defined as a prescription within 3 years prior. ICU admission and ventilator use were determined using administrative codes. Death included both in-hospital and out-of-hospital events. Patients treated with N-BP were matched 1:1 by propensity score to patients without prior N-BP use. Secondary analysis compared outcomes among those prescribed zoledronic acid (ZOL) to those prescribed oral N-BPs. Of 76,223 COVID-19 patients identified, 1,853 were previously prescribed N-BP, among whom 559 were prescribed ZOL. After propensity score matching, there were no significant differences in the composite outcome at 15 days (HR 1.22, 95% CI: 0.89-1.67), 30 days (HR 1.24, 95% CI: 0.93-1.66), or 180 days (HR 1.17, 95% CI: 0.93-1.48), comparing those prescribed and not prescribed N-BP. Compared to those prescribed oral N-BP, there were no significant differences in outcomes among those prescribed ZOL. Among older COVID-19 patients, prior exposure to N-BP including ZOL was not associated with a reduction in ICU admission, ventilator use, or death.

Alendronate induces skeletal alterations in the chicken embryonic development model

Alendronate, a nitrogen-containing bisphosphonate, has reported long-term clinical success in the management of distinct bone-related conditions, particularly in the modulation of post-menopausal osteoporosis. Nonetheless, whether the inhibitory activity over osteoclastic cells' functionality is widely acknowledged, contradictory evidence arises from the assessment of alendronate activity over osteoblastic populations. This may be of particular relevance in situations in which bone formation exceeds bone resorption, with further emphasis on embryonic development, since alendronate can cross the placental barrier and alendronate-based therapies are being extended into women of reproductive age. Accordingly, the present study aims to assess the effects of alendronate, at distinct concentrations (1.5E−10M to 1.5E−7M) on bone tissue development, within a translational animal model – the embryonic chicken development model. Embryos, at the beginning of osteogenesis (day 7) were exposed to different alendronate concentrations for 4 days. Embryos were following characterized for skeletal development by histomorphometric analysis upon histochemical staining, microtomographic analysis, and gene expression assessment of genes related to osteoclastogenic/osteoclastic and osteoblastogenic/osteogenic differentiation, as well as to the immuno-inflammatory activation. The findings revealed that exposure to alendronate had a dose-dependent impact on skeletal growth and mineralization. This effect was evidenced by diminished bone volume and reduced bone surface parameters, with the 1.5E−7M concentration leading to a remarkable reduction of over 50%. Additionally, a decreased osteoclastogenic/osteoclastic gene expression was verified, associated with a diminished osteoblastogenic/osteogenic program – within the 30–50% range for 1.5E-7 M, supporting the diminished bone formation process. An increased inflammatory activation may contribute, at least in part, to the attained outcomes. Overall present findings suggest a negative influence of alendronate on the embryonic bone development process in a dose-dependent manner, highlighting the potential risk of alendronate use during embryonic development.

CD73 regulates zoledronate-induced lymphocyte infiltration in triple-negative breast cancer tumors and lung metastases.

Bisphosphonates (BPs) are bone-protecting osteoclast inhibitors, typically used in the treatment of osteoporosis and skeletal complications of malignancies. When given in the adjuvant setting, these drugs may also prevent relapses and prolong overall survival in early breast cancer (EBC), specifically among postmenopausal patients. Because of these findings, adjuvant nitrogen-containing BPs (N-BPs), such as zoledronate (ZOL), are now the standard of care for high-risk EBC patients, but there are no benefit-associated biomarkers, and the efficacy remains low. BPs have been demonstrated to possess anti-tumor activities, but the mechanisms by which they provide the beneficial effects in EBC are not known. We used stably transfected 4T1 breast cancer cells together with suppression of CD73 (sh-CD73) or control cells (sh-NT). We compared ZOL effects on tumor growth and infiltrating lymphocytes (TILs) into tumors and lung metastases using two mouse models. B cell depletion was performed using anti-CD20 antibody. Sh-CD73 4T1 cells were significantly more sensitive to the growth inhibitory effects of n-BPs in vitro. However, while ZOL-induced growth inhibition was similar between the tumor groups in vivo, ZOL enhanced B and T lymphocyte infiltration into the orthotopic tumors with down-regulated CD73. A similar trend was detected in lung metastases. ZOL-induced tumor growth inhibition was found to be augmented with B cell depletion in sh-NT tumors, but not in sh-CD73 tumors. As an internal control, ZOL effects on bone were similar in mice bearing both tumor groups. Taken together, these results indicate that ZOL modifies TILs in breast cancer, both in primary tumors and metastases. Our results further demonstrate that B cells may counteract the growth inhibitory effects of ZOL. However, all ZOL-induced TIL effects may be influenced by immunomodulatory characteristics of the tumor.

WHAT ARE THE MAIN CLINICAL FEATURES OF ONCOLOGICAL PATIENTS WITH MRONJ? A SYSTEMATIC REVIEW AND META-ANALYSIS

Objective To integrate the data published on patients with cancer who underwent antiresorptive drug (ARD) therapy and developed MRONJ into a comprehensive analysis of clinical features, and to evaluate if some clinical factors could influence MRONJ staging. Study Design An electronic search was performed, followed by descriptive analyses of the clinical pathological data. Poisson regression was used to evaluate the predictors of the MRONJ stage. Results In total, 1,598 cases were included. Most cases were reported in women, and the mean age was 66.45±10.5 years. Integumentary system cancer was the main reason for using ARDs, and metastasis occurred in 65.5% (n=436) patients. The most common etiology of MRONJ was traumatic events. MRONJ stage 2 was the most prevalent. Bisphosphonate monotherapy or combination therapy was used in 77.3% (n: 419) of the cases, while denosumab was used in 14.4% cases (n=77). Poisson regression analysis demonstrated that the MRONJ stage was not affected by comorbidities, metastasis, duration of ARD use, or the primary neoplasm site. Conclusion Nitrogen-containing bisphosphonates (used alone or in combination) are the most common ARDs associated with MRONJ in patients with cancer. Moreover, the primary neoplasm site, as well as metastasis, do not interfere in MRONJ staging. To integrate the data published on patients with cancer who underwent antiresorptive drug (ARD) therapy and developed MRONJ into a comprehensive analysis of clinical features, and to evaluate if some clinical factors could influence MRONJ staging. An electronic search was performed, followed by descriptive analyses of the clinical pathological data. Poisson regression was used to evaluate the predictors of the MRONJ stage. In total, 1,598 cases were included. Most cases were reported in women, and the mean age was 66.45±10.5 years. Integumentary system cancer was the main reason for using ARDs, and metastasis occurred in 65.5% (n=436) patients. The most common etiology of MRONJ was traumatic events. MRONJ stage 2 was the most prevalent. Bisphosphonate monotherapy or combination therapy was used in 77.3% (n: 419) of the cases, while denosumab was used in 14.4% cases (n=77). Poisson regression analysis demonstrated that the MRONJ stage was not affected by comorbidities, metastasis, duration of ARD use, or the primary neoplasm site. Nitrogen-containing bisphosphonates (used alone or in combination) are the most common ARDs associated with MRONJ in patients with cancer. Moreover, the primary neoplasm site, as well as metastasis, do not interfere in MRONJ staging.

Geranylgeraniol Application in Human Osteoblasts and Osteoclasts for Reversal of the Effect of Bisphosphonates.

Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on human osteoblast and osteoclast activities suppressed by zoledronate was investigated in this study. The effect of GGOH on human osteoblasts and osteoclasts subjected to treatment with zoledronate was analyzed by assessing cell viability, osteoclast differentiation, resorption ability, gene expression, and protein synthesis. Cell viability suppressed by bisphosphonates in osteoblasts and osteoprogenitor cells was restored with GGOH. Osteoclast differentiation was analyzed by vitronectin receptor immunofluorescence staining, and the addition of GGOH to zoledronate significantly increased osteoclast differentiation compared with zoledronate alone. A trend of reversal of osteoclast resorption by GGOH was observed; however, it was not significant in all groups. The expression of ALP, type 1 collagen, and RUNX2 in osteoblasts was recovered by the addition of GGOH. Only CALCR expression in osteoclasts was significantly recovered by GGOH addition in the zoledronate group. Although the activities of osteoblasts and osteoclasts were not entirely restored, the possibility that the topical application of GGOH in MRONJ patients or patients with dental problems and bisphosphonates might lessen the risk of development and recurrence of MRONJ is shown.

Prevention and treatment of osteonecrosis of the jaws using Pentoxifylline and tocopherol: A case series of patients under antiresorptive or antiangiogenic agents inducing osteonecrosis of the jaws

Introduction: Medication-related osteonecrosis of a jaw can occur in patient taking antiresorptive medication mainly as Nitrogen-containing Bisphosphonates, longer and intravenous treatment by bisphosphonates and patient who received tyrosine kinase inhibitor. We report an evaluation of the association of Pentoxifylline-Tocopherol to reduce or to treat the side effects on jawbone of the antiresorptive medications. Patients and methods: Six patients were selected two of them with intractable cases of MRONJ due to antiresorptive medication (Nitrogen-containing bisphosphonates and tyrosine kinase inhibitor) and four of them regarded as risky patients because taking Nitrogen-containing Bisphosphonates for at least two years and having to undergo jawbone surgery. Results: following the prescription of pentoxifylline -tocopherol, to prevent the occurrence of an MRONJ or to protect the surgical procedure for surgical removal of necrotic exposed bone, at the last follow up no adverse effects were identified and clinical appreciation was confirmed by radiological examinations. Conclusion: the association Pentoxifylline -Tocopherol has proven its efficacy in preventing or treating MRONJ bu. It was also critical to highlight the MRONJ risk in patients under monoclonal antibodies at the time of these molecules are rising, and to emphasize on the action of pentoxifylline-tocopherol in the management of the side effects of various monoclonal antibodies on the jaw bone

Towards Controlling the Local Bone Tissue Remodeling-Multifunctional Injectable Composites for Osteoporosis Treatment.

Alendronate (ALN) is the most commonly prescribed oral nitrogen-containing bisphosphonate for osteoporosis therapy. However, its administration is associated with serious side effects. Therefore, the drug delivery systems (DDS) enabling local administration and localized action of that drug are still of great importance. Herein, a novel multifunctional DDS system based on the hydroxyapatite-decorated mesoporous silica particles (MSP-NH2-HAp-ALN) embedded into collagen/chitosan/chondroitin sulfate hydrogel for simultaneous osteoporosis treatment and bone regeneration is proposed. In such a system, the hydrogel serves as a carrier for the controlled delivery of ALN at the site of implantation, thus limiting potential adverse effects. The involvement of MSP-NH2-HAp-ALN in the crosslinking process was established, as well as the ability of hybrids to be used as injectable systems. We have shown that the attachment of MSP-NH2-HAp-ALN to the polymeric matrix provides a prolonged ALN release (up to 20 days) and minimizes the initial burst effect. It was revealed that obtained composites are effective osteoconductive materials capable of supporting the osteoblast-like cell (MG-63) functions and inhibiting osteoclast-like cell (J7741.A) proliferation in vitro. The purposely selected biomimetic composition of these materials (biopolymer hydrogel enriched with the mineral phase) allows their biointegration (in vitro study in the simulated body fluid) and delivers the desired physicochemical features (mechanical, wettability, swellability). Furthermore, the antibacterial activity of the composites in in vitro experiments was also demonstrated.

Nitrogen-Containing Bisphosphonates Downregulate Cathepsin K and Upregulate Annexin V in Osteoclasts Cultured In Vitro.

Bisphosphonates are widely used in the treatment of osteoporosis; however, they are associated with the serious adverse event of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The aim of this study is to assess the effects of nitrogen-containing bisphosphonates (N-PHs) on the synthesis of IL-1β, TNF-α, sRANKL, cathepsin K, and annexin V in bone cells cultured in vitro. Osteoblasts and bone marrow-derived osteoclasts were cultured in vitro, subjected to treatment with alendronate, risedronate, or ibandronate at a concentration of 10-5 M for 0 to 96 h and then assayed for IL-1β, sRANKL, and TNF-α production by ELISA. Cathepsin K and Annexin V-FITC staining in osteoclasts were assessed by flow cytometry. There was significant downregulation of IL-1β, sRANKL, and TNF-α in experimental osteoblasts compared to control cells, and there was upregulation of IL-1β and downregulation of RANKL and TNF-α in experimental osteoclasts. Furthermore, in osteoclasts, cathepsin K expression was downregulated at 48-72 h with alendronate treatment, while risedronate treatment resulted in upregulated annexin V expression at 48 h compared to the control treatment. Bisphosphonates added to bone cells inhibited osteoclastogenesis, which led to the downregulation of cathepsin K and induction of apoptosis in osteoclasts; these changes limited the capacity of bone remodelling and healing that may contribute to BRONJ induced by surgical dental procedures.

Effect of Bone Resorption Inhibitors on Serum Cholesterol Level and Fracture Risk in Osteoporosis: Randomized Comparative Study Between Minodronic Acid and Raloxifene

The positive link between osteoporosis and hypercholesterolemia has been documented, and bone resorption inhibitors, such as nitrogen-containing bisphosphonates (N-BP) and selective estrogen receptor modulators (SERMs), are known to reduce serum cholesterol levels. However, the relationship between the baseline cholesterol level and incident fracture rate under the treatment using the bone resorption inhibitors has not been documented. We investigated the relation between vertebral fracture incident and the baseline cholesterol levels and cholesterol-lowering effect of N-BP and SERM in osteoporosis through a prospective randomized open-label study design. Patients with osteoporosis (n = 3986) were allocated into two groups based on the drug used for treatment: minodronic acid (MIN) (n = 1624) as an N-BP and raloxifene (RLX) as an SERM (n = 1623). Serum levels of cholesterol and incidence of vertebral fracture were monitored for 2 years. The vertebral fracture rates between the two groups were compared using the pre-specified stratification factors. The patients receiving MIN with baseline low-density lipoprotein (LDL)-cholesterol level of ≥ 140 mg/dL, high-density lipoprotein cholesterol level < 40 mg/dL, age group of ≥ 75 years, and T score of BMD ≥ -3 SD had significantly lower vertebral fracture rates than those receiving RLX (incidence rate ratios (IRR) 0.45 [95% confidence interval (CI) 0.30 0.75, p = 0.001], 0.25 [95% CI 0.09 0.65, p = 0.005], 0.71 [95% CI 0.56 0.91, p = 0.006], 0.47 [95% CI 0.30 0.75, p = 0.0012], respectively). The cholesterol-lowering effect was stronger in the RLX group than in the MIN group, regardless of prior statin use. These results indicated that MIN treatment was more effective in reducing fracture risk in patients with higher LDL cholesterol levels, although its cholesterol-lowering ability was lesser than the RLX treatment.Trial registration University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR), No. UMIN000005433; date: April 13, 2011.

Rational design for MgO-modified porous carbon towards enhancing zoledronic acid adsorption

Nitrogen-containing bisphosphonates (N-BPs), namely zoledronic acid (ZA), are typically used as antiresorptive drugs by inhibiting osteoclast formation and functions. However, the long-term use of ZA may increase the risk of osteonecrosis of the jaw (ONJ) after tooth extraction. Hence, this study aims to develop a new sustainable adsorption material for capturing the exposed ZA. The porous activated carbon (AC) incorporated with MgO at various ratios was successfully developed and extensively characterized by several techniques. The adsorption behaviors correlated to their physicochemical properties for ZA removal were also carried out. Furthermore, density functional theory (DFT) was applied to investigate the adsorption mechanism of ZA adsorbed on a carbon surface functionalized with crystalline MgO. The results found that the well-dispersed MgO/AC provided a good adsorption capacity for ZA. The MgO with 15 % wt loading shows the highest performance (73 mg/g) on adsorbing ZA under a neutral condition compared to a bare AC (14 mg/g). The MgO/AC adsorbents were cytocompatible with human monocytic cells in vitro. The DFT result demonstrated that the MgO cluster is an active site for ZA adsorption. This evidence could imply that MgO/AC is a potential adsorbent for ZA removal in preventing ZA-related bone complications.

Nebulization of risedronate alleviates airway obstruction and inflammation of chronic obstructive pulmonary diseases via suppressing prenylation-dependent RAS/ERK/NF-κB and RhoA/ROCK1/MLCP signaling

BackgroundChronic obstructive pulmonary disease (COPD) is a progressive disorder that causes airway obstruction and lung inflammation. The first-line treatment of COPD is the bronchodilators of β2-agonists and antimuscarinic drugs, which can help control the airway obstruction, but the long-term use might render the drug tolerance. Bisphosphonates are widely used in osteoclast-mediated bone diseases treatment for decades. For drug repurposing, can delivery of a third generation of nitrogen-containing bisphosphonate, risedronate (RIS) ameliorate the progression of COPD?MethodsCOPD rats or mice models have been established through cigarette-smoking and elastase injection, and then the animals are received RIS treatment via nebulization. Lung deposition of RIS was primarily assessed by high-performance liquid chromatography (HPLC). The respiratory parameters of airway obstruction in COPD rats and mice were documented using plethysmography method and resistance-compliance system.ResultsHigh lung deposition and bioavailability of RIS was monitored with 88.8% of RIS input dose. We found that RIS could rescue the lung function decline of airspace enlargement and mean linear intercept in the COPD lung. RIS could curb the airway obstruction by suppressing 60% of the respiratory resistance and elevating the airway’s dynamic compliance, tidal volume and mid-expiratory flow. As an inhibitor of farnesyl diphosphate synthase (FDPS), RIS suppresses FDPS-mediated RAS and RhoA prenylation to obstruct its membrane localization in airway smooth muscle cells (ASMCs), leading to the inhibition of downstream ERK-MLCK and ROCK1-MLCP pathway to cause ASMCs relaxation. Additionally, RIS nebulization impeded pro-inflammatory cell accumulation, particularly macrophages infiltration in alveolar parenchyma. The NF-κB, tumor necrosis factor-alpha, IL-1β, IL-8, and IL-6 declined in microphages following RIS nebulization. Surprisingly, nebulization of RIS could overcome the tolerance of β2-agonists in COPD-rats by increasing the expression of β2 receptors.ConclusionsNebulization of RIS could alleviate airway obstruction and lung inflammation in COPD, providing a novel strategy for treating COPD patients, even those with β2-agonists tolerance.

Fine-tuning of nitrogen-containing bisphosphonate esters that potently induce degradation of HMG-CoA reductase

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.