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.

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.

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.

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.

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.

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.

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.

Liposome-encapsulated zoledronate increases inflammatory macrophage population in TNBC tumours.

BACKGROUND: Tumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilised an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation. METHODS: Triple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumour resection) and intraperitoneally (without tumour resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR. RESULTS: Liposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL. CONCLUSION: Results show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumour effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.

Novel insights into the pharmacological modulation of human periodontal ligament stem cells by the amino-bisphosphonate Alendronate.

Alendronate (ALN) is a second-generation bisphosphonate widely used for osteoporosis and cancer-induced bone lesions. Many studies have confirmed a strong relationship between osteonecrosis of the jaws (ONJ) development and oral bisphosphonates, especially ALN, although the molecular mechanisms underlying this pathology have not yet been elucidated. The reduction in bone turnover and vascularization usually observed in ONJ are the result of ALN action on different cell types harboured in oral microenvironment, such as osteoclasts, endothelial cells, and periodontal ligament stem cells (PDLSCs). In this perspective, the present study aims to investigate the effects of different ALN concentrations (2 µM, 5 µM, 10 µM, 25 µM, 50 µM) on the phenotype and functional properties of human PDLSCs (hPDLSCs). hPDLSCs showed a decrease in cell viability (MTT assay) only when treated with ALN concentration of 10 µM or larger for 48 h and 72 h. Cell cycle analysis revealed a moderate increase in proportion of S-phase cells after exposure to low ALN concentration (2-5 µM), an effect that was reverted after exposure to 10-50 µM ALN. Conversely, cell death was evidenced via Annexin V/PI assay at very high concentration of ALN (50 µM) after 4 days of treatment. In addition, we explored whether the effects of ALN on hPDLSCs growth and survival can be mediated by its ability to modulate oxidative stress. To this, we quantified the intracellular ROS amount and lipid peroxidation by using DCF probe and Bodipy staining, respectively. Flow cytometry analysis showed that ALN induced a dose-dependent reduction of intracellular oxidative stress and lipid peroxidation upon treatment with low concentrations at both 48 h and 72 h. Increased levels of oxidative stress was reported at 50 µM ALN and was also confirmed via TEM analysis. Despite the stability of the cellular immunophenotype, hPDLSCs showed impaired mobility after ALN exposure. Chronic exposure (7-14 days) to ALN in the range of 2-10 µM significantly decreased the expression of the differentiation-related factors ALP, RUNX2, COLI, and OPN as well as the osteogenic ability of hPDLSCs compared with untreated cells. Conversely, higher doses were found to be neutral. Our findings indicated that the effects of ALN on hPDLSCs behavior are dose-dependent and suggest a role for oxidative stress in ALN-induced cell death that may lead to novel therapeutic approaches for ONJ.

High-Affinity Extended Bisphosphonate-Based Coordination Polymers as Promising Candidates for Bone-Targeted Drug Delivery.

Extended bisphosphonate-based coordination polymers (BPCPs) were produced when 1,1'-biphenyl-4,4'-bisphosphonic acid (BPBPA), the analogue of 1,1'-biphenyl-4,4'-dicarboxylic acid (BPDC), reacted with bioactive metals (Ca2+, Zn2+, and Mg2+). BPBPA-Ca (11 Å × 12 Å), BPBPA-Zn (10 Å × 13 Å), and BPBPA-Mg (8 Å × 11 Å) possess channels that allow the encapsulation of letrozole (LET), an antineoplastic drug that combined with BPs treats breast-cancer-induced osteolytic metastases (OM). Dissolution curves obtained in phosphate-buffered saline (PBS) and fasted-state simulated gastric fluid (FaSSGF) demonstrate the pH-dependent degradation of BPCPs. Specifically, the results show that the structure of BPBPA-Ca is preserved in PBS (∼10% release of BPBPA) and collapses in FaSSGF. Moreover, the phase inversion temperature nanoemulsion method yielded nano-Ca@BPBPA (∼160 d. nm), a material with measurably higher (>1.5x) binding to hydroxyapatite than commercial BPs. Furthermore, it was found that the amounts of LET encapsulated and released (∼20 wt %) from BPBPA-Ca and nano-Ca@BPBPA are comparable to those of BPDC-based CPs [i.e., UiO-67-(NH2)2, BPDC-Zr, and bio-MOF-1], where other antineoplastic drugs have been loaded and released under similar conditions. Cell viability assays show that, at 12.5 µM, the drug-loaded nano-Ca@BPBPA exhibits higher cytotoxicity against breast cancer cells MCF-7 and MDA-MB-231 [relative cell viability (%RCV) = 20 ± 1 and 45 ± 4%] compared with LET (%RCV = 70 ± 1 and 99 ± 1%). At this concentration, no significant cytotoxicity was found for the hFOB 1.19 cells treated with drug-loaded nano-Ca@BPBPA and LET (%RCV = 100 ± 1%). Collectively, these results demonstrate the potential of nano-Ca@BPCPs as promising drug-delivery systems to treat OM or other bone-related diseases because these present measurably higher affinity, allowing bone-targeted drug delivery under acidic environments and effecting cytotoxicity on estrogen receptor-positive and triple-negative breast cancer cell lines known to induce bone metastases, without significantly affecting normal osteoblasts at the metastatic site.

Lesion Expansion in Gnathic Fibrous Dysplasia: Natural History, Indicators of Progression, and Response to Bisphosphonates.

Fibrous dysplasia (FD) is characterized by expansile fibro-osseous lesions that may occur in association with endocrinopathies as part of McCune-Albright syndrome (MAS). Craniofacial FD is a significant source of morbidity and most commonly involves the gnathic bones. There is a critical need to understand the natural history and risk factors for gnathic FD progression to develop preventative trials and identify candidates for intervention. The purpose of this study was to characterize gnathic FD lesion expansion and to identify risk factors associated with lesion growth. Patients with gnathic FD and serial CT imaging were evaluated. Volumetric analyses of CT scans were performed using MIM Encore software. Generalized mixed model analysis was used to account for intra-subject correlation, with FD lesion volume as the dependent variable. In addition to age, effects of MAS-associated endocrinopathies, sex, disease severity, and bisphosphonate treatment were evaluated. A total of 104 total lesions in 52 patients were characterized longitudinally. Median age at initial scan was 8.8 years (range 3.4-18.8), and median age at final scan was 16.8 years (range 6.9-33.4 years). The median number of scans per subject was 4 (range 2-14). FD lesion volume increased with age (2.50 cm3 /yr, 95% confidence interval [CI] 1.95-3.04, p < 0.001). However, lesion expansion rate decreased over time (-0.05 cm3 /yr, 95% CI -0.07 to 0.04, p < 0.001). Mandibular lesions tended to expand at a greater rate than maxillary lesions (p < 0.001). Growth hormone excess was associated with accelerated expansion rate (p = 0.002). Other MAS-associated endocrinopathies, pubertal status, sex, weight, lesion density, disease severity, and bisphosphonate treatment were not associated with lesion volume or expansion. Gnathic FD lesion expansion is most rapid in younger children and declines as patients approach adulthood. The availability of quantitative natural history data will guide clinicians in identifying patients who are candidates for medical and surgical interventions and clinical trials for preventative therapies. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Recent advance of small-molecule drugs for clinical treatment of osteoporosis: A review.

Osteoporosis is a metabolic bone disorder typified by a reduction in bone mass and structural degradation of bone tissue, leading to heightened fragility and vulnerability to fractures. The incidence of osteoporosis increases with age, making it a significant public health challenge. The pathogenesis of osteoporosis involves an imbalance between osteoblast-mediated bone formation and resorption. The current treatment options for osteoporosis include bisphosphonates, hormone replacement therapy (HRT), selective estrogen receptor modulators (SERMs), and denosumab. The recent advances in small-molecule drugs for the clinical treatment of osteoporosis offer promising options for improving bone health and reducing fracture risk. This review aims to provide an overview of the clinical applications and synthetic routes of representative small-molecule drugs for the treatment of osteoporosis. A comprehensive understanding of the synthetic methods of drug molecules for osteoporosis may inspire the development of new, more effective, and practical synthetic techniques for treating this condition.

Design of Extended Bisphosphonate-Based Coordination Polymers as Bone-Targeted Drug Delivery Systems for Breast Cancer-Induced Osteolytic Metastasis and Other Bone Therapies.

The coordination between benzene 1,4-bis(bisphosphonic acid) (BBPA), the bisphosphonate (BP) analogue of benzene 1,4-dicarboxylic acid (BDC), and bioactive metals led to the formation of extended bisphosphonate-based coordination polymers (BPCPs). Four distinct crystalline phases were obtained, namely, BBPA-Ca forms I and II, BBPA-Zn, and BBPA-Mg. Among these, BBPA-Ca forms I (7 × 9 Å2) and II (8 × 12 Å2) possess channels large enough to encapsulate 5-fluorouracil (5-FU), a drug prescribed in combination with BPs to treat breast cancer-induced osteolytic metastases (OM). Dissolution curves show a 14% release of BBPA from BBPA-Ca form II in phosphate-buffered saline, while ∼90% was released in fasted-state simulated gastric fluid. These results suggest that this material is relatively stable in neutral environments yet collapses in acidic conditions. Moreover, the phase inversion temperature method decreased the particle size of BBPA-Ca form II, resulting in nano-Ca@BBPA (∼134 d.nm). Binding assays showed a higher affinity of nano-Ca@BBPA (∼97%) to hydroxyapatite than BBPA (∼70%) and significantly higher binding than commercial BPs, zolendronic (3.0×), and risedronic (2.4×) acids after 24 h. Furthermore, both BBPA-Ca form II and nano-Ca@BBPA presented comparable drug loading and release (∼30 wt % 5-FU) relative to BDC-based CCs (UiO-66, MIL-53, and BDC-Zr) where other pharmaceutical compounds (caffeine, ibuprofen, aspirin, and α-cyano-4-hydroxycinnamic acid) have been encapsulated. Cell viability assays established that drug-loaded nano-Ca@BBPA increases the cytotoxicity of a triple-negative human breast cancer cell line (MDA-MB-231) when compared to 5-FU (%RCV = 8 ± 5 vs 75 ± 1% at a 100 µM). At the same concentration, no significant decrease in cell viability was observed for normal human osteoblast-like hFOB 1.19 cells (%RCV = 85 ± 1%). Collectively, these results demonstrate the feasibility of nano-Ca@BBPA as a potential drug delivery system (DDS), with high affinity to bone tissue, to treat bone-related diseases such as OM.

Polymeric Nanoformulation of Zoledronic Acid Rescues Osteoblasts from the Harmful Effect of its Native Form: An In Vitro Investigation of Cytotoxic Potential on Osteoblasts and Osteosarcoma Cells.

Osteosarcoma (OS) is a malignant tumor, fatal for pediatric patients who do not respond to chemotherapy, alternative therapies and drugs can provide better outcomes. Zoledronic acid (Zol) belonging to the class of bisphosphonates (BPs) has a direct antitumor ability to prevent Ras GTPases modification and stimulate apoptosis. Despite advances in maintaining balance in skeletal events and direct anticancer properties, Zol causes cytotoxicity to normal healthy pre-osteoblast cells, hampering mineralization and differentiation. The study reports the preparation and evaluation of a nanoformulation that can diminish the existing drawbacks of native Zol. The cytotoxic effect is evaluated on bone cancer cells and healthy bone cells with three different cell lines namely, K7M2 (mouse OS cell line), SaOS2 (human OS cell line), and MC3T3E1 (healthy cell counterpart). It is observed that Zol nanoformulation is uptaken more (95%) in K7M2 whereas in MC3T3E1, the percent population internalizing nanoparticles (NPs) is 45%. Zol has a sustained release of 15% after 96 h from the NP which leads to a rescuing effect on the normal pre-osteoblast cells. In conclusion, it can be stated that Zol nanoformulation can be used as a good platform for a sustained release system with minimum side effects to normal bone cells.

In Vitro Cytotoxicity of Antiresorptive and Antiangiogenic Compounds on Oral Tissues Contributing to MRONJ: Systematic Review.

BACKGROUND: Invasive dental treatment in patients exposed to antiresorptive and antiangiogenic drugs can cause medication-related osteonecrosis of the jaw (MRONJ). Currently, the exact pathogenesis of this disease is unclear. METHODS: In March 2022, Medline (Ovid), Embase (Ovid), Scopus, and Web of Science were screened to identify eligible in vitro studies investigating the effects of antiresorptive and antiangiogenic compounds on orally derived cells. RESULTS: Fifty-nine articles met the inclusion criteria. Bisphosphonates were used in 57 studies, denosumab in two, and sunitinib and bevacizumab in one. Zoledronate was the most commonly used nitrogen-containing bisphosphonate. The only non-nitrogen-containing bisphosphonate studied was clodronate. The most frequently tested tissues were gingival fibroblasts, oral keratinocytes, and alveolar osteoblasts. These drugs caused a decrease in cell proliferation, viability, and migration. CONCLUSIONS: Antiresorptive and antiangiogenic drugs displayed cytotoxic effects in a dose and time-dependent manner. Additional research is required to further elucidate the pathways of MRONJ.

Denosumab use in bone fibrous dysplasia refractory to bisphosphonate: A retrospective multicentric study.

INTRODUCTION: Increased RANKL expression is observed in the bone tissue of fibrous dysplasia of bone/McCune-Albright syndrome (FD/MAS). In one animal model of FD/MAS, the inhibition of RANKL reduced tumor volume. A beneficial effect of denosumab on pain in patients refractory to bisphosphonates has been reported, but without systematic quantification of pain improvement. This work describes the clinical experience of our group on the efficacy on pain of denosumab treatment, along with safety, in FD/MAS patients refractory to bisphosphonates. MATERIALS AND METHODS: We have conducted a retrospective multicenter study in 6 academic rheumatology centers in France. We have collected patients and FD/MAS characteristics, duration of prior exposure to bisphosphonates, denosumab treatment modalities (dosage - administration regimen - number of courses); evolution of pain evaluated by Visual Analogic Scale (VAS). RESULTS: 13 patients were included (10 women and 3 men) 45 years on average, 5 MAS, 4 monostotic and 4 polyostotic forms. The average duration post-diagnosis of FD/MAS was 25 years and the mean duration of prior exposure to bisphosphonates was 4.7 years. Pain could be analyzed in 7 patients, showing a significant improvement from a mean VAS of 7.8 to 2.9 (-4.9 points, p = 0.003). In one patient with fronto-orbital FD/MAS, a 30 % decrease in lesional volume, assessed by MRI, was observed within 6 months of treatment, that was sustained over the following 12 months. Treatment regimens were heterogeneous. No hypercalcemia was observed after treatment cessation and the clinical tolerance was good. DISCUSSION: This study suggests that denosumab reduces pain in patients with DF/MAS refractory to bisphosphonates, and quantifies this improvement for the first time in a multicenter study. In our cohort, no patients who discontinued denosumab developed hypercalcemia and clinical tolerance was overall good. This study also provides encouraging data regarding lesion volume control. Further controlled studies are required to determine the place and modalities of the denosumab treatment of FD/MAS. CONCLUSION: Denosumab significantly decreased pain in FD/MAS refractory to bisphosphonate. This study paves the way for a randomized clinical trial to validate and standardize the prescription of denosumab in FD/MAS.

Metastasis prevention with bone-targeted agents: a complex interaction between the microenvironment and tumour biology.

INTRODUCTION: The use of bone-targeted treatments has transformed the clinical care of many patients with metastatic breast cancer. In addition, due to the profound effects of bisphosphonates and denosumab on bone physiology and the bone microenvironment, the potential of bone-targeted agents to modify the process of metastasis has been studied extensively. FINDINGS: Many adjuvant trials with bisphosphonates in early breast cancer have been performed. Variable outcomes in terms of disease recurrence have been reported, with any treatment benefits apparently influenced by the age and menopausal status of the patients. Results show that in breast cancer the use of adjuvant bisphosphonates reduce bone metastases and breast-cancer deaths in postmenopausal women. These effects are in addition to the benefits associated with the use of standard adjuvant endocrine, cytotoxic and targeted treatments with prevention of one in six breast-cancer deaths at 10 years. Biomarkers that can predict patient benefit from the use of bone-targeted treatments in the adjuvant setting are being evaluated. Currently, tumour expression of the transcription factor, MAF, seems to be the most promising biomarker; benefits from adjuvant bisphosphonates are seen in the 80% of patients with normal levels of expression irrespective of menopausal status and age, while over expression is associated with a poor prognosis and a higher rate of visceral metastases. CONCLUSIONS: Adjuvant bisphosphonates are now part of standard clinical guidelines for postmenopausal women with early breast cancer at intermediate to high risk of recurrence. MAF testing of primary tumours may improve patient selection for treatment.

A colloidal hydrogel-based drug delivery system overcomes the limitation of combining bisphosphonates with bioactive glasses: in vitro evidence of a potential selective bone cancer treatment allied with bone regeneration.

Bisphosphonates are a class of drugs that induce bone cancer cell death and favor bone regeneration, making them suitable for bone cancer treatment. However, when combined with bioactive glasses to enhance bone regeneration, a chemical bond between biphosphonates and the glass surface inactivates their mechanism of action. A new colloidal hydrogel-based drug delivery system could overcome that limitation once bisphosphonates, such as zoledronic acid (ZA), are incorporated into hydrogel micelles, avoiding their interaction with the glass surface. In this work, we proposed formulations based on a poloxamer 407 thermo-responsive hydrogel matrix containing holmium-doped bioactive glass nanoparticles and different concentrations (0.05 and 5 mg/mL) of ZA. We characterized the influence of the glass and the ZA on the hydrogel properties. In addition, a drug concentration screening was performed, and biological characterizations evaluated the best result. The biological characterization consisted of evaluating cytotoxicity and in vitro bone regeneration ability through cell migration and quantification of genes related to osteogeneses through RT-PCR. The results suggest that the addition of glasses and ZA to the poloxamer did not significantly influence the sol-gel transition of the hydrogels (around 13 °C) regardless of the ZA content. However, the ZA at high concentration (PL-ZA100) decreased the enthalpy of gel formation from 68 to 43 kJ.mol-1 when compared with the pure hydrogel formulation (PL), suggesting a water structurer role of ZA, which is withdrawn when glass particles are added to the system (PL-BG5Ho-ZA100). Solid-state 31P nuclear resonance spectroscopy results showed that part of the ZA is chemically bonded to the glass surface, which explains the withdrawal in the water structurer role of ZA when the glasses were incorporated into the hydrogel. Besides, based on the drug release results, we proposed a model where part of the ZA is "free," encapsulated in the hydrogel matrix, while another part of the ZA is bonded to the glass surface. Finally, considering the in vitro results and our proposed model, the ratio between "free" and "bonded" ZA in our drug delivery systems showed in vitro evidence of a cancer treatment that selectively kills osteosarcoma cells while still favoring an osteogenic microenvironment. By overcoming the limitation of combining bisphosphonates with bioactive glasses, hydrogel-based drug delivery systems can be a solution for the development of new formulations proposed for bone cancer treatment in conjunction with bone regeneration.