Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes.

Synthesis and preliminary anticancer evaluation of photo-responsive prodrugs of hydroxymethylene bisphosphonate alendronate.

The antitumoral activity of hydroxymethylene bisphosphonates (HMBP) such as alendronate or zoledronate is hampered by their exceptional bone-binding properties and their short plasmatic half-life which preclude their accumulation in non-skeletal tumors. In this context, the use of lipophilic prodrugs represents a simple and straightforward strategy to enhance the biodistribution of bisphosphonates in these tissues. We describe in this article the synthesis of light-responsive prodrugs of HMBP alendronate. These prodrugs include lipophilic photo-removable nitroveratryl groups which partially mask the highly polar alendronate HMBP scaffold. Photo-responsive prodrugs of alendronate are stable in physiological conditions and display reduced toxicity compared to alendronate against MDA-MB-231 cancer cells. However, the antiproliferative effect of these prodrugs is efficiently restored after cleavage of their nitroveratryl groups upon exposure to UV light. In addition, substitution of alendronate with such photo-responsive substituents drastically reduces its bone-binding properties, thereby potentially improving its biodistribution in soft tissues after i.v. administration. The development of such lipophilic photo-responsive prodrugs is a promising approach to fully exploit the anticancer effect of HMBPs on non-skeletal tumors.

Heterogeneous osteoimmune profiles via single-cell transcriptomics in osteoporotic patients who fail bisphosphonate treatment.

Postmenopausal osteoporosis arises from imbalanced osteoclast and osteoblast activity, and mounting evidence suggests a role for the osteoimmune system in bone homeostasis. Bisphosphonate (BP) is an antiresorptive agent, but its treatment failure rate can be as high as 40%. Here, we performed single-cell RNA sequencing on peripheral immune cells from carefully selected postmenopausal women: non-osteoporotic, osteoporosis improved after BP treatment, and BP-failed cases. We found an increase in myeloid cells in patients with osteoporosis (specifically, T cell receptor+ macrophages). Furthermore, lymphoid lineage cells varied significantly, notably elevated natural killer cells (NKs) in the BP-failed group. Moreover, we provide fruitful lists of biomarkers within the immune cells that exhibit condition-dependent differences. The existence of osteoporotic- and BP-failure-specific cellular information flows was revealed by cell-cell interaction analysis. These findings deepen our insight of the osteoporosis pathology enhancing comprehension of the role of immune heterogeneity in postmenopausal osteoporosis and BP treatment failure.

Inhibition of cysteine protease disturbs the topological relationship between bone resorption and formation in vitro.

INTRODUCTION: Osteoporosis is a global health issue. Bisphosphonates that are commonly used to treat osteoporosis suppress both bone resorption and subsequent bone formation. Inhibition of cathepsin K, a cysteine proteinase secreted by osteoclasts, was reported to suppress bone resorption while preserving or increasing bone formation. Analyses of the different effects of antiresorptive reagents such as bisphosphonates and cysteine proteinase inhibitors will contribute to the understanding of the mechanisms underlying bone remodeling. MATERIALS AND METHODS: Our team has developed an in vitro system in which bone remodeling can be temporally observed at the cellular level by 2-photon microscopy. We used this system in the present study to examine the effects of the cysteine proteinase inhibitor E-64 and those of zoledronic acid on bone remodeling. RESULTS: In the control group, the amount of the reduction and the increase in the matrix were correlated in each region of interest, indicating the topological and quantitative coordination of bone resorption and formation. Parameters for osteoblasts, osteoclasts, and matrix resorption/formation were also correlated. E-64 disrupted the correlation between resorption and formation by potentially inhibiting the emergence of spherical osteoblasts, which are speculated to be reversal cells in the resorption sites. CONCLUSION: These new findings help clarify coupling mechanisms and will contribute to the development of new drugs for osteoporosis.

α-Amino bisphosphonate triazoles serve as GGDPS inhibitors.

Depletion of cellular levels of geranylgeranyl diphosphate by inhibition of the enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential strategy for disruption of protein transport by limiting the geranylgeranylation of the Rab proteins that regulate intracellular trafficking. As such, there is interest in the development of GGDPS inhibitors for the treatment of malignancies characterized by abnormal protein production, including multiple myeloma. Our previous work has explored the structure-function relationship of a series of isoprenoid triazole bisphosphonate-based GGDPS inhibitors, with modifications having impact on enzymatic, cellular and in vivo activities. We have synthesized a new series of α-amino bisphosphonates to understand the impact of modifying the alpha position with a moiety that is potentially linkable to other agents. Bioassays evaluating the enzymatic and cellular activities of these compounds demonstrate that incorporation of the α-amino group affords compounds with GGDPS inhibitory activity which is modulated by isoprenoid tail chain length and olefin stereochemistry. These studies provide further insight into the complexity of the structure-function relationship and will enable future efforts focused on tumor-specific drug delivery.

Zoledronate reduces loading-induced microdamage in cortical ulna of ovariectomized rats.

As a daily physiological mechanism in bone, microdamage accumulation dissipates energy and helps to prevent fractures. However, excessive damage accumulation might bring adverse effects to bone mechanical properties, which is especially problematic among the osteoporotic and osteopenic patients treated by bisphosphonates. Some pre-clinical studies in the literature applied forelimb loading models to produce well-controlled microdamage in cortical bone. Ovariectomized animals were also extensively studied to assimilate human conditions of estrogen-related bone loss. In the present study, we combined both experimental models to investigate microdamage accumulation in the context of osteopenia and zoledronate treatment. Three-month-old normal and ovariectomized rats treated by saline or zoledronate underwent controlled compressive loading on their right forelimb to create in vivo microdamage, which was then quantified by barium sulfate contrast-enhanced micro-CT imaging. Weekly in vivo micro-CT scans were taken to evaluate bone (re)modeling and to capture microstructural changes over time. After sacrifice, three-point-bending tests were performed to assess bone mechanical properties. Results show that the zoledronate treatment can reduce cortical microdamage accumulation in ovariectomized rats, which might be explained by the enhancement of several bone structural properties such as ultimate force, yield force, cortical bone area and volume. The rats showed increased bone formation volume and surface after the generation of microdamage, especially for the normal and the ovariectomized groups. Woven bone formation was also observed in loaded ulnae, which was most significant in ovariectomized rats. Although all the rats showed strong correlations between periosteal bone formation and microdamage accumulation, the correlation levels were lower for the zoledronate-treated groups, potentially because of their lower levels of microdamage. The present study provides insights to further investigations of pharmaceutical treatments for osteoporosis and osteopenia. The same experimental concept can be applied in future studies on microdamage and drug testing.

Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Assessing bone formation on hydrophilic and hydrophobic implant surfaces in a murine model treated with bisphosphonates.

OBJECTIVE: To evaluate the osseointegration of implants with hydrophobic (HFB) and hydrophilic (HFL) surfaces in a murine model of high-dose bisphosphonates (BPs). MATERIALS AND METHODS: Sixty-four rats were randomly allocated into four groups: control group with HFB implants (CG-HFB), control group with HFL implants (CG-HFL), BP group with HFB implants (BP-HFB), and BP group with HFL implants (BP-HFL). Animals were euthanized after 15 and 45 days (n=8). The dependent variables assessed were the removal torque (biomechanical analysis), the bone volume around the implants (%BV/TV) (microtomographic analysis), the bone-implant contact (%BIC), the bone between the threads (%BBT) (histomorphometric analysis), and the expression of bone metabolism markers (immunohistochemistry analysis). RESULTS: The CG-HFL and BP-HFL groups presented higher removal torque than the CG-HFB and BP-HFB implants. The %BIC of the CG-HFL surfaces was slightly higher than that of the CG-HFB implants. The BP-HFB and BP-HFL groups presented a higher %BIC than that of the CG-HFB and CG-HFL groups (p<0.001). BP therapy also increased the %BBT at both implant surfaces. Higher levels of ALP were observed in the matrix region of bone tissue on the HFL surfaces than on the HFB surfaces. CONCLUSION: Both surfaces enable osseointegration in rats under BP therapy. CLINICAL RELEVANCE: The study demonstrates that hydrophobic (HFB) and hydrophilic (HFL) implant surfaces can promote osseointegration in rats undergoing bisphosphonate therapy. The HFL surfaces exhibited improved biomechanical performance, higher bone-implant contact, and increased bone volume, suggesting their potential clinical relevance for implant success in individuals on bisphosphonate treatment.

The role of neridronate in the management of osteoporosis: A meta-analysis.

BACKGROUND: It is estimated that 1 in 3 women and 1 in 5 men over the age of 50 worldwide will experience an osteoporosis fracture during their lives. Neridronate is a third-generation bisphosphonate with established efficacy in metabolic bone disease. It can be used in the treatment of osteoporosis. OBJECTIVES: We aimed to conduct a meta-analysis of the effect of neridronate on the treatment of osteoporosis. MATERIAL AND METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations were used to guide the present study. We searched PubMed and the Cochrane Central Register of Controlled Trials (CENTRAL) for reports published until August 31, 2021, related to neridronate and osteoporosis. The modification of the bone mineral density (BMD, g/cm2) of the patient is the core indicator for neridronate treatment. RESULTS: Significant increases in the BMD of the lumbar spine (mean difference (MD) = 5.99, 95% confidence interval (95% CI): 3.96-8.02), femoral neck (MD = 4.51, 95% CI: 2.01-7.01) and total hip (MD = 2.55, 95% CI: 2.10-3.00) were found. Greater improvement in the BMD of the lumbar spine and femoral neck could also be detected in patients with postmenopausal osteoporosis than with other causes of osteoporosis. Moreover, significant decreases in serum C-telopeptide of collagen type I (sCTX, standardized mean difference (SMD) = -0.84, 95% CI: -1.32--0.37) and bone alkaline phosphatase (ALP, MD = -5.29, 95% CI: -7.31--3.26) levels were observed. CONCLUSION: The pool analysis of the selected clinical trials indicates the great benefit of neridronate in improving the condition of patients with osteoporosis of all causes, particularly patients with postmenopausal osteoporosis, which causes an increase in BMD as well as in sCTX and bone ALP levels.

Ozone Improved Bone Dynamic of Female Rats Using Zoledronate.

The aim of this study was to analyze the effect of ozone (OZN) therapy on the dynamics of bone tissue in ovariectomized rats treated with zoledronic acid (ZOL). Female Wistar rats aged 6 months (n = 110) were subjected to bilateral ovariectomy (OVX). At month 3 post-OVX, 10 animals were euthanized to characterize the bone tissue architecture using microtomography (micro-CT). The remaining animals were divided into two groups: ZOL group, administered with ZOL (100 µg/kg body weight); saline (SAL) group (0.45 mL of SAL solution), both for 28 days. At month 3 post-treatment, 10 animals from each group were euthanized to characterize the bone architecture using micro-CT. The remaining animals were divided into the following groups: ZOL (n = 20), ZOL + OZN (n = 20); SAL (n = 20), and SAL + OZN (n = 20). The animals in ZOL + OZN and SAL + OZN groups were intraperitoneally administered with OZN (0.7 mg/kg body weight) once every 2 days. On days 30 and 60, six animals from each group were euthanized for analysis and structural characterization of bones in the femoral head and spine. Some samples of the femoral neck were subjected to biomechanical tests, while some samples were analyzed under a laser confocal microscope. The other samples collected from the femoral neck and spine were analyzed for area of neoformed bone and used for performing inflammatory cell and osteocyte counts. Data were submitted to statistical analysis considering a significance level of p < 0.05. Bone volume percentage and osteocyte and inflammatory cell counts were upregulated in the femoral head region of the ZOL + OZN group. Biomechanical analysis of the femoral neck revealed that the modulus of elasticity was similar between the ZOL and ZOL + OZN groups but differed significantly between the SAL and SAL + OZN groups. The positive areas for calcein and alizarin in the ZOL and ZOL + OZN groups were higher than those in the SAL and SAL + OZN groups. This suggested a positive synergistic effect of OZN and ZOL on the maintenance of bone mass and restoration of bone tissue vitality in ovariectomized rats.

Comparative Evaluation of the Effect of Different Chelating Agents on Mineral Content and Erosion of Radicular Dentine: A FESEM-EDS Analysis.

OBJECTIVE: Irrigating solutions play an important role in the debridement and disinfection of the root canal space, and thus, it is crucial to comprehend their effects on the composition and surface structure of radicular dentine. This study evaluated and compared the effects of 17% ethylenediaminetetraacetic acid (EDTA), 9% 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and 0.2% chitosan on the mineral content and erosion of radicular dentine when used as a final rinse. METHODS: Sixty extracted human mandibular premolar teeth were decoronated and instrumented to ProTaper size F2. After final instrumentation, the samples were randomly divided into 4 groups (n=15) according to the type of final irrigant used: Normal saline (control), 17% EDTA, 9% HEBP and 0.2% chitosan. Field emission scanning electron microscopy was used to assess the erosion of radicular dentine, and energy dispersive X- ray spectroscopy was used to quantify the radicular dentine mineral composition at the coronal, middle and apical levels of all the prepared samples after final irrigation. The one-way analysis of variance was used for intra-group and inter-group comparisons of means, the Kruskal Wallis test for intra-group and inter-group comparisons of medians and Tukey's post hoc test for pairwise comparisons. RESULTS: There was no significant difference in the levels of calcium (Ca), phosphorus (P) and Ca/P ratio after final rinse with 17% EDTA, 9% HEBP and 0.2% chitosan at all three root levels (p>0.05); except at the coronal level, where 0.2% chitosan caused significantly less alteration in Ca levels and Ca/P ratio than 17% EDTA and 9% HEBP respectively (p<0.05). 17% EDTA, 9% HEBP and 0.2% chitosan caused no erosion at the middle and apical levels. Meanwhile, 17% EDTA and 9% HEBP caused moderate erosion at the coronal level. CONCLUSION: Alternatives to 17% EDTA during final irrigation can be 9% HEBP and 0.2% chitosan. (EEJ-2023-06-078).

Effect of bisphosphonates and statins on the in vitro radiosensitivity of breast cancer cell lines.

BACKGROUND: Early-stage breast cancer is usually treated with breast-conserving surgery followed by adjuvant radiation therapy. Acute skin toxicity is a common radiation-induced side effect experienced by many patients. Recently, a combination of bisphosphonates (zoledronic acid) and statins (pravastatin), or ZOPRA, was shown to radio-protect normal tissues by enhancing DNA double-strand breaks (DSB) repair mechanism. However, there are no studies assessing the effect of ZOPRA on cancerous cells. The purpose of this study is to characterize the in vitro effect of the zoledronic acid (ZO), pravastatin (PRA), and ZOPRA treatment on the molecular and cellular radiosensitivity of breast cancer cell lines. MATERIALS: Two breast cancer cell lines, MDA MB 231 and MCF-7, were tested. Cells were treated with different concentrations of pravastatin (PRA), zoledronate (ZO), as well as their ZOPRA combination, before irradiation. Anti-γH2AX and anti-pATM immunofluorescence were performed to study DNA DSB repair kinetics. MTT assay was performed to assess cell proliferation and viability, and flow cytometry was performed to analyze the effect of the drugs on the cell cycle distribution. The clonogenic assay was used to assess cell survival. RESULTS: ZO, PRA, and ZOPRA treatments were shown to increase the residual number of γH2AX foci for both cell lines. ZOPRA treatment was also shown to reduce the activity of the ATM kinase in MCF-7. ZOPRA induced a significant decrease in cell survival for both cell lines. CONCLUSIONS: Our findings show that pretreatment with ZOPRA can decrease the radioresistance of breast cancer cells at the molecular and cellular levels. The fact that ZOPRA was previously shown to radioprotect normal tissues, makes it a good candidate to become a therapeutic window-widening drug.

Magnesium-Organic Framework-Loaded Bisphosphonate-Functionalized Gel Scaffolds for Enhanced Bone Regeneration.

The development of magnesium-derived biomaterials is one of the most promising research in bone tissue engineering, and related strategies have been extensively used for tendon, skull, cartilage, and bone regeneration. Also, alendronate, a well-recognized drug for osteoporosis treatment, has recently attracted a great deal of attention for bone repair. However, rapid corrosion in vivo of Mg2+ and low systemic bioavailability of alendronate are the main limitations hampering their full exploitation. In this work, by means of physical and chemical cross-linking conjugating magnesium-metal-organic frameworks (Mg-MOFs) and bone-targeting alendronate to biocompatible gelatin scaffolds, a facile method is developed for the preparation of organic/inorganic nanocomposite gel scaffolds. The results affirmed that the nanocomposite gel scaffolds possessed excellent biocompatibility, continuous slow release of Mg2+ and alendronate, strong bone affinity, and bone regeneration. It is noteworthy that the continuous slow release of Mg2+ and alendronate could induce the macrophage switch to the M2 phenotype and promote osteogenic differentiation in the early stage, resulting in improved bone regeneration during implanting the scaffolds into the distal femoral. In summary, Mg-MOFs-loaded alendronate-modified gelatin gel scaffolds have been developed, exhibiting great potential for bone regenerative.

Discovery and Evaluation of C6-Substituted Pyrazolopyrimidine-Based Bisphosphonate Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase and Evaluation of Their Antitumor Efficacy in Multiple Myeloma, Pancreatic Ductal Adenocarcinoma, and Colorectal Cancer.

Novel C6-substituted pyrazolo[3,4-d]pyrimidine- and C2-substituted purine-based bisphosphonate (C6-PyraP-BP and C2-Pur-BP, respectively) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) were designed and evaluated for their ability to block the proliferation of multiple myeloma (MM), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) cells. Pyrazolo[3,4-d]pyrimidine analogs were identified that induce selective intracellular target engagement leading to apoptosis and downregulate the prenylation of Rap-1A in MM, PDAC, and CRC cells. The C6-PyraP-BP inhibitor RB-07-16 was found to exhibit antitumor efficacy in xenograft mouse models of MM and PDAC, significantly reducing tumor growth without substantially increasing liver enzymes or causing significant histopathologic damage, usually associated with hepatotoxicity. RB-07-16 is a metabolically stable compound in cross-species liver microsomes, does not inhibit key CYP 450 enzymes, and exhibits good systemic circulation in rat. Collectively, the current studies provide encouraging support for further optimization of the pyrazolo[3,4-d]pyrimidine-based GGPPS inhibitors as potential human therapeutics for various cancers.

Structural Diversity in Antiosteolytic Bisphosphonates: Deciphering Structure-Activity Trends in Ultra Long Controlled Release Phenomena.

Bisphosphonate (BP)-based treatments have been extensively prescribed for bone-related conditions, particularly for osteoporosis. Their low bioavailability creates the need for prescribed dosage increase to reach therapeutic levels but generates a plethora of undesirable side effects. A viable approach to alleviating these issues is to design and exploit controlled release strategies. Herein, the controlled release profiles of 15 structurally characterized BPs (actual drugs and structural analogs) were thoroughly studied from tablets containing three (cellulose, lactose, and silica) or two (cellulose, and silica) excipients in human stomach-simulated pH conditions. The BPs were of two types, alkyl-BPs and amino-BPs. Alkyl-BPs included four derivatives of etidronate (acid, disodium, tetra-sodium, and monopotassium forms), medronic acid, and three analogs of etidronate, in which the -CH3 group was replaced by the moieties -H, -CH2CH2CH3, and -CH2CH2CH2CH2CH3. Amino-BPs included the commercial drugs pamidronate, alendronate, neridronate, and ibandronate, as well as three analog compounds. Release curves were constructed based on data taken from 1H NMR peak integration and were expressed as "% BP release" vs time. The controlled release profiles (initial release rate, plateau value, etc.) were correlated with certain structural features (number of hydrogen and metal-oxygen bonds), showing that the molecular and crystal lattice features of each BP profoundly influence its release characteristics. It was concluded that for all BPs, in general, the initial rate became lower as the total number of lattice interactions increased. For the alkyl-BPs elongation of the alkyl side chain seems to decelerate the release. Amino-BPs, in general, show slower release than the alkyl-BPs. No adverse effects of alkyl- and amino-BP drugs on NIH3T3 cell viability were noted.

Effects of bisphosphonates and treadmill exercise on bone and kidney in adenine-induced chronic kidney disease rats.

INTRODUCTION: The increasing prevalence of osteoporosis and chronic kidney disease (CKD) due to the aging of society has highlighted the need for development of effective treatments for elderly patients. This study examined whether the combination of treadmill exercise therapy and alendronate (ALN) can improve bone mineral density (BMD) and bone strength without worsening renal function in adenine-induced CKD model rats. MATERIALS AND METHODS: 8-week-old male Wistar rats (n = 70) were divided into experimental groups based on the treatment protocol, i.e., non-CKD (control), vehicle only (CKD), ALN only, exercise only, and combined ALN plus exercise. A 0.75% adenine diet was used to induce CKD. Groups were killed at either 20 or 30 weeks of age. Comprehensive assessments included serum and urine biochemistry tests, renal histology, bone histomorphometry, BMD measurement, micro-computed tomography examinations, and biomechanical testing. RESULTS: Blood biochemistry tests, urine analyses and histological evaluations of the kidney demonstrated that ALN treatment did not worsen renal function or kidney fibrosis in moderate-stage CKD model rats. Both ALN and treadmill exercise significantly suppressed bone resorption (p < 0.05-p < 0.01). Moreover, ALN monotherapy and combined ALN and treadmill exercise significantly improved BMD of the lumbar spine and femur, bone microstructure, and trabecular bone strength (p < 0.05-p < 0.01). Treadmill exercise was also shown to decrease cortical porosity at the mid-diaphysis of the femur and improve kidney fibrosis. CONCLUSION: The combination of ALN and treadmill exercise is effective in improving BMD, the microstructure of trabecular and cortical bone, and bone strength, without compromising renal function in adenine-induced CKD model rats.

Bisphosphonates attenuate age-related muscle decline in Caenorhabditis elegans.

BACKGROUND: Age-related muscle decline (sarcopenia) associates with numerous health risk factors and poor quality of life. Drugs that counter sarcopenia without harmful side effects are lacking, and repurposing existing pharmaceuticals could expedite realistic clinical options. Recent studies suggest bisphosphonates promote muscle health; however, the efficacy of bisphosphonates as an anti-sarcopenic therapy is currently unclear. METHODS: Using Caenorhabditis elegans as a sarcopenia model, we treated animals with 100 nM, 1, 10, 100 and 500 µM zoledronic acid (ZA) and assessed lifespan and healthspan (movement rates) using a microfluidic chip device. The effects of ZA on sarcopenia were examined using GFP-tagged myofibres or mitochondria at days 0, 4 and 6 post-adulthood. Mechanisms of ZA-mediated healthspan extension were determined using combined ZA and targeted RNAi gene knockdown across the life-course. RESULTS: We found 100 nM and 1 µM ZA increased lifespan (P < 0.001) and healthspan [954 ± 53 (100 nM) and 963 ± 48 (1 µM) vs. 834 ± 59% (untreated) population activity AUC, P < 0.05]. 10 µM ZA shortened lifespan (P < 0.0001) but not healthspan (758.9 ± 37 vs. 834 ± 59, P > 0.05), whereas 100 and 500 µM ZA were larval lethal. ZA (1 µM) significantly improved myofibrillar structure on days 4 and 6 post-adulthood (83 and 71% well-organized myofibres, respectively, vs. 56 and 34% controls, P < 0.0001) and increased well-networked mitochondria at day 6 (47 vs. 16% in controls, P < 0.01). Genes required for ZA-mediated healthspan extension included fdps-1/FDPS-1 (278 ± 9 vs. 894 ± 17% population activity AUC in knockdown + 1 µM ZA vs. untreated controls, respectively, P < 0.0001), daf-16/FOXO (680 ± 16 vs. 894 ± 17%, P < 0.01) and agxt-2/BAIBA (531 ± 23 vs. 552 ± 8%, P > 0.05). Life/healthspan was extended through knockdown of igdb-1/FNDC5 (635 ± 10 vs. 523 ± 10% population activity AUC in gene knockdown vs. untreated controls, P < 0.01) and sir-2.3/SIRT-4 (586 ± 10 vs. 523 ± 10%, P < 0.05), with no synergistic improvements in ZA co-treatment vs. knockdown alone [651 ± 12 vs. 635 ± 10% (igdb-1/FNDC5) and 583 ± 9 vs. 586 ± 10% (sir-2.3/SIRT-4), both P > 0.05]. Conversely, let-756/FGF21 and sir-2.2/SIRT-4 were dispensable for ZA-induced healthspan [630 ± 6 vs. 523 ± 10% population activity AUC in knockdown + 1 µM ZA vs. untreated controls, P < 0.01 (let-756/FGF21) and 568 ± 9 vs. 523 ± 10%, P < 0.05 (sir-2.2/SIRT-4)]. CONCLUSIONS: Despite lacking an endoskeleton, ZA delays Caenorhabditis elegans sarcopenia, which translates to improved neuromuscular function across the life course. Bisphosphonates might, therefore, be an immediately exploitable anti-sarcopenia therapy.

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.

Pharmacological agents for bone fracture healing: talking points from recent clinical trials.

INTRODUCTION: Pharmacological strategies might influence bone healing in terms of time to union or quality of mature bone. This expert opinion discussed the current level I evidence on the experimental pharmacological agents used to favor bone fracture healing. AREAS COVERED: This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the 2020 PRISMA statement. In April 2023, the following databases were accessed: PubMed, Web of Science, Google Scholar, Embase. All the randomized clinical trials investigating pharmacological agents for bone fracture healing were accessed. No time constraint was set for the search. The search was restricted to RCTs. No additional filters were used in the database search. Data from 19 RCTs (4067 patients) were collected. 78% (3160 of 4067) were women. The mean length of the follow-up was 9.3 months (range, 1-26 months). The mean age of the patients was 64.4 years (range, 8-84 years). EXPERT OPINION: Calcitonin could favor bone fracture healing. Bisphosphonates (alendronate, zoledronate, clodronate), monoclonal antibodies (denosumab, romosozumab), statins, vitamin D and calcium supplementation, strontium ranelate, and ibuprofen did not influence bony healing. Concerning the effect of parathormone, current level I evidence is controversial, and additional studies are required. LEVEL OF EVIDENCE: Level I, systematic review of RCTs.

An optimized cultivation method for future in vivo application of γδ T cells.

γδ T cells, with their properties of both the innate and acquired immune systems, are suitable candidates for cellular immunotherapy in cancer. Because of their non-major histocompatibility complex (MHC) binding T cell receptor, allogenic transfer is feasible without relevant graft versus host reactions. In recent years, much experience has been gained with ex vivo expansion and stimulation of γδ T cells using bisphosphonates and Interleukin 2. Unfortunately, many current stimulation protocols are based on the use of xenogenic materials and other potentially hazardous supplements, which conflicts with basic principles of Good Manufacturing Practice (GMP). Adherence to the concept and current guidelines of GMP is state of the art for production of Advanced Therapy Medicinal Products (ATMP) like cell therapeutics and a necessity for clinical use under a regulatory perspective. In this study, we developed a new stimulation protocol that induces a marked increase of γδ T cell counts and allows for an easier transition from research to clinical applications with minimized regulatory workload. It reliably leads to a cell product with a purity of more than 90% γδ T cells and improved in vitro anti-tumor activity compared to our previous standard procedure. Furthermore, by investigating correlations between properties of unstimulated γδ T cells and proliferation rate as well as degranulation ability of stimulated γδ T cells, we can draw conclusions about suitable donors. Finally, we examined if expansion can be improved by pulsing zoledronate and/or using Interleukin 15 with or without Interleukin 2. Significant improvements can be achieved with respect to intrinsic and antibody-dependent cell-mediated cytotoxicity. Our results demonstrate that the stimulation protocol presented here leads to an improved γδ T cell product for future clinical applications.