Concentrations Of Zoledronic Acid Research Articles

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43 kJ.mol−1 when compared with the pure hydrogel formulation (PL), suggesting a water structurer role of ZA, which is withdrawn when glass particles are added to the system (PL-BG5Ho-ZA100).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.

Dose-dependent effects of zoledronic acid on the osteogenic differentiation of human bone marrow stem cells (hBMSCs)

Recent studies have shown that bisphosphonates can also impact osteoblasts besides osteoclasts. This study aimed to evaluate the effects of different concentrations of Zoledronic acid (ZA) during the osteogenic differentiation of human Bone Marrow Stem Cells (hBMSCs) in vitro. Thus, osteogenic differentiation of hBMSCs was conducted with different concentrations of Zoledronic Acid (ZA) (0, 0.1, 1.0, and 5.0 μM) for the first 3 days. Cell metabolism was quantified at 1-, 3-, 7-, and 14 days. At 7- and 14-days, the following analyses were performed: 1) mineralization nodule assay, 2) LIVE/DEAD™, 3) cell adhesion and spreading, 4) alkaline phosphatase (ALP) activity, and 5) qPCR analysis for RUNX-2), ALPL, and COL1 A1. Data were analyzed by ANOVA 2-way, followed by Tukey's post hoc test (p < 0.05). Cell metabolism (3-, 7-, and 14-days) (p < 0.001), mineralization (7-, 14-days) (p < 0.001), and ALP activity (14-days) (p < 0.001) were reduced in ZA 5.0 µM when compared to control (no ZA). Also, ZA 5.0 µM downregulated the expression of RUNX2 at 7- and 14-days (p < 0.001). It is possible to conclude that ZA (5.0 µM) can impair hBMSC differentiation into osteoblasts and interferes with its mineralization phase.

Zoledronic acid affects the process of Porphyromonas gingivalis infecting oral mucosal epithelial barrier: An in-vivo and in-vitro study.

Zoledronic acid (ZA), one of the commonly used bisphosphonates, is mainly used for bone-metabolic diseases. Studies proved that ZA has adverse effects on oral soft tissues. As the first line of innate immunity, the gingival epithelium could be infected by periodontal pathogens, which is a key process of the initiation of periodontal diseases. Yet, how ZA affects the periodontal pathogens infecting the epithelial barrier remains unclear. This study aimed to investigate the influences of ZA on the process of Porphyromonas gingivalis (P. gingivalis) infecting the gingival epithelial barrier via in-vitro and in-vivo experiments. In the in-vitro experiments, under the condition of different concentrations of ZA (0, 1, 10, and 100 μM), P. gingivalis was used to infect human gingival epithelial cells (HGECs). The infections were detected by transmission electron microscope and confocal laser scanning microscope. Besides, the internalization assay was applied to quantify the P. gingivalis, which infected the HGECs, in the different groups. To evaluate the expression levels of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and IL-8, by infected HGECs, real-time quantitative reverse transcription-polymerase chain reactions were applied. In the in-vivo experiments, rats were given ZA solution (ZA group) or saline (control group) by tail intravenous injection for 8 weeks. Subsequently, we put ligatures around the maxillary second molars of all the rats and inoculated P. gingivalis to the gingiva every other day from day 1 to day 13. The rats were sacrificed on days 3, 7, and 14 for micro-CT and histological analyses. The in-vitro results manifested that the quantity of P. gingivalis that had infected HGECs increased with the ZA concentrations. Pro-inflammatory cytokines expression by HGECs were significantly increased by 100 μM ZA. In the in-vivo study, compared to the control group, more P. gingivalis was detected in the superficial layer of gingival epithelium in the ZA group. Besides, ZA significantly increased the expression level of IL-1β on day 14 and IL-6 on days 7 and 14 in gingival tissues. These findings suggest that the oral epithelial tissues of patients who receive high-dose ZA treatment may be more susceptible to periodontal infections, resulting in severe inflammatory conditions.

Zoledronic acid targets chemo-resistant polyploid giant cancer cells

Although polyploid giant cancer cells (PGCCs) are known as a key source of failure of current therapies, sufficient drugs to target these cells are not yet introduced. Considering the similarities of polyploid cells in regeneration and cancer, we hypothesized that zoledronic acid (ZA), an osteoclast-targeting agent, might be used to eliminate PGCCs. The 5637-bladder cancer cell line was treated with various doses of cisplatin to enrich polyploid cells and the efficacy of different concentrations of ZA in reducing this population was assessed. The metabolic profile of PGCCs was investigated with gas chromatography-mass spectrometry. Lipid profiles, mitochondrial density, and ROS content were also measured to assess the response of the cells to ZA. Cancer cells surviving after three days of exposure with 6 μM cisplatin were mainly polyploid. These cells demonstrated special morphological features such as fusion with diploid or other polyploid cells and originated in daughter cells through budding. ZA could substantially eradicate PGCCs with the maximal effect observed with 50 μM which resulted in the drop of PGCC fraction from 60 ± 7.5 to 19 ± 1.7%. Enriched PGCCs after cisplatin-treatment demonstrated a drastic metabolic shift compared to untreated cancer cells with an augmentation of lipids. Further assays confirmed the high content of lipid droplets and cholesterol in these cells which were reduced after ZA administration. Additionally, the mitochondrial density and ROS increased in PGCCs both of which declined in response to ZA. Taken together, we propose that ZA is a potent inhibitor of PGCCs which alters the metabolism of PGCCs. Although this drug has been successfully exploited as adjuvant therapy for some malignancies, the current evidence on its effects on PGCCs justifies further trials to assess its potency for improving the success of current therapies for tackling tumor resistance and relapse.

Quantification of Low Amounts of Zoledronic Acid by HPLC-ESI-MS Analysis: Method Development and Validation.

Zoledronic acid (ZA) is used in the treatment of various bone pathologies, but it forms complexes with calcium ions present in body fluids, decreasing ZA bioavailability. Thereby, the study first describes the identification of ZA-calcium complexes that form in calcium-rich environments, in order to establish the bioavailable ZA concentration. Then, a new method for quantification of low ZA amounts in milieus that mimics in vivo conditions by using simulated body fluid and calcium sulfate hemihydrate was described. Almost all analytical methods of ZA quantification described in the literature require compound derivatization. At very low concentrations, derivatization is prone to analyte loss, therefore compromising the analytical results. In our study, we avoided ZA derivatization by using a high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system, conducting the investigation based on the fragmentation mass extracted ion chromatograms specific to the ZA protonated form. The method was validated by selectivity, precision, accuracy, linearity, signal to noise ratio, and limit of detection and limit of quantification calculation. Experimentally, this method can detect ranges of 0.1–0.5 ng/mL and precisely quantify ZA concentrations as low as 0.1 ng/mL. This method could provide the basis for quantifying low amounts of ZA in the blood during long-term administration.

Influences of Compressive Force and Zoledronic Acid on Osteoblast Proliferation and Differentiation: An In Vitro Study

This study investigates the effects of zoledronic acid (ZA) and compressive force on osteoblast functions, to elucidate the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). MC3T3-E1 cells were exposed to ZA (1, 10 and 100 µM) to evaluate the effects of ZA on cell proliferation. Furthermore, to investigate the influence of ZA with or without compressive force on osteoblast differentiation, real-time polymerase chain reaction and Alizarin Red S staining were performed. ZA concentrations &gt; 10 μM were highly cytotoxic to MC3T3-E1 cells. Combining 1-μM ZA with compressive force influenced expression levels of osteoblast-related genes and matrix mineralization. The inhibitory effects of ZA on cell proliferation and the combination of ZA and compressive force on osteoblast differentiation may contribute to the pathogenesis of MRONJ.

The effect of high concentration of zoledronic acid on tooth induced movement and its repercussion on root, periodontal ligament and alveolar bone tissues in rats

Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.

Zoledronic Acid Is Not Equally Potent on Osteoclasts Generated From Different Individuals.

ABSTRACTZoledronic acid is a bisphosphonate commonly used to treat bone diseases such as osteoporosis and cancer‐induced bone disease. Patients exhibit a variable sensitivity to zoledronic acid; the underlying explanation for this remains unclear. The objective of this study was to obtain more knowledge in this regard. We hypothesized that osteoclasts generated from different individuals would show a variable sensitivity to zoledronic acid in vitro. Osteoclasts were generated using monocytes from 46 healthy female blood donors (40 to 66 years). Matured osteoclasts were reseeded onto bone slices precoated with different concentrations of zoledronic acid. IC50 values were determined based on total eroded bone surface after 3 days of resorption. The IC50 for inhibition of osteoclastic bone resorption varied from 0.06 to 12.57μM zoledronic acid; thus, a more than 200‐fold difference in sensitivity to zoledronic acid among osteoclasts from different individuals was observed. Multiple linear regression analyses showed that the determined IC50 correlated with smoking status, and the average number of nuclei per osteoclast in vitro. Further analyses showed that: (i) increasing protein levels of mature cathepsin K in osteoclast cultures rendered the osteoclasts less sensitive to zoledronic acid; (ii) surprisingly, neither the gene nor the protein expression of farnesyl diphosphate synthase was found to correlate with the IC50; and (iii) trench‐forming osteoclasts were found to be more sensitive to zoledronic acid than pit‐forming osteoclasts within the same cell culture. Thus, we conclude that there indeed is a high degree of variation in the potency of zoledronic acid on osteoclasts when generated from different individuals. We propose that our findings can explain some of the varying clinical efficacy of zoledronic acid therapy observed in patients, and may therefore be of clinical importance, which should be investigated in a clinical trial combining in vitro and in vivo investigations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Bifunctional effect of Zoledronic Acid (ZA) on human mesenchymal stem cells (hMSCs) based on the concentration level

BackgroundTreatment of massive bone defects is a great challenge. Mesenchymal stem cells (MSCs) enhance bone regeneration by differentiating into osteoblasts. Bisphosphonates (BPs) are antiresorptives reducing bone resorption. Despite Medication-related osteonecrosis of the jaw (MRONJ) is a known side effect of antiresorptives, evidences suggest that BPs have positive effect on bone formation. The aims of this study were to investigate the effect of zoledronic acid (ZA) and geranylgeraniol (GGOH) on human mesenchymal stem cells (hMSCs) being a part of the bone microenvironment and evaluate whether low dose of bisphosphonate has enhanced osteogenic differentiation of hMSCs. Materials and methodsThe effect of ZA and GGOH on MSCs was investigated in addition to the effect of low doses of ZA on osteogenic differentiation of MSCs and analysed by WST-1, Live/Dead staining and coefficient of drug index (CDI). The osteogenic differentiation of the cells was confirmed by ALP activity, xylenol orange and alizarin red staining, microarray and PCR with levels of statistical significance indicated at *P<0.05, **P<0.01 and ***P<0.0001. Main findingsAlthough, high concentration of ZA had significantly decreased the cell viability in MSCs, GGOH reversed the action of ZA on the cells while at very high concentration; it caused severe reduction in the cell viability. CDI showed antagonism or synergism depending on the concentrations of ZA and GGOH. ConclusionThe treatment of cells with ZA has increased the mineralization and osteogenic differentiation of MSCs. Our study supported the hypothesis that zoledronic acid plays a bifunctional role depending on the concentration.

Spectrophotometric and visual determination of zoledronic acid by using a bacterial cell-derived nanopaper doped with curcumin.

A nanopaper-based analytical device (NAD) is described for a colorimetric metal-complexing indicator-displacement assay (M-IDA) for zoledronic acid (ZA). Bacterial cellulose nanopaper was doped with curcumin to obtain a chemosensor on which hydrophilic test zones were patterned via laser printing of hydrophobic walls. The color intensity of the test zones decreases in the presence of Fe(III) due to the formation of Fe(III)-curcumin complex. However, upon addition of ZA, Fe(III) ions preferably binds ZA. Subsequently, the color of the zone changes from light yellow to dark yellow. The changes in the absorption (measured at 427nm) and of the color of the test stripe can be monitored visually, by using a digital camera, or by a spectrophotometer. Under optimal conditions, the analytical signals increase linearly in the 0.01-100μM ZA concentration range, and the detection limits are 8.8 and 8.0nM for smartphone and spectrophotometer-based methods, respectively. The method was employed to the determination of ZA in (spiked) urine, serum, saliva, and in pharmaceutical samples. Graphical abstract Schematic representation of a nanopaper-based analytical device based on curcumin-doped BC nanopaper (CDBC) integrated with smartphone for metal-complexing indicator-displacement assay of zoledronic acid (ZA). High affinity of ZA to Fe(III) on the NAD/CDBC leads to color change.

Radiation Therapy and Bone-Modifying Agents for Bone Metastases in Patients with Breast Cancer: Clinical and in Vitro Study

Bone-modifying agents (BMAs) are reportedly associated with a decrease in skeletal-related events (SREs), which include pathologic fractures, spinal cord compression, bone surgery, radiation therapy (RT) for bones, and hypercalcemia. Therefore, BMAs are frequently used for treating patients with bone metastases. Although RT is often used to prevent SREs, RT is itself a type of SRE. Here we evaluated the usefulness of RT combined with BMA compared with BMA alone for bone metastasis treatment. In this clinical study, osteolytic bone metastases of the spine or pelvic bone in patients with breast cancer were evaluated. Tomographic imaging was used to evaluate 28 lesions before and after RT + BMA and 15 lesions before and after BMAs alone. The median age of the participants was 62 (range, 33-82) years. None of the lesions were treated with chemotherapy or molecular targeted drugs during the follow-up period for evaluating local responses. For calculating the overall survival (OS) rates, the periods in which chemotherapy or molecular targeted drugs were used after RT and/or BMAs were included. The median follow-up period was 18 (range, 2-90) months. Response to therapy was evaluated based on the revised Response Evaluation Criteria in Solid Tumors guidelines version 1.1. Patients with complete or partial response were considered as responders, whereas those with stable or progressive disease were considered as non-responders. For the in vitro study, a breast cancer cell-line, MCF-7, was analyzed using a colony formation assay. Cells were incubated with various concentrations of zoledronic acid (ZA; 0-100 μM), followed by irradiation with 0 (control), 2, 4, and 8 Gy. After 14 days, the colonies were stained with crystal violet and counted. Most osteolytic lesions treated with RT + BMA showed bone reformation (75.0%). Decreased fluorodeoxyglucose uptake and improved signal abnormalities were also confirmed using magnetic resonance imaging. Although some osteolytic lesions improved after using BMAs alone (33.3%), most remained unchanged or gradually worsened. The rate of improvement with RT + BMA was significantly higher than that with BMAs alone (p = 0.001). The cumulative incidence rate of response at 6 months was 54.4% in the RT + BMA group and 27.5% in the BMAs alone group. The OS rate in the responder group (83.1% at 1 year) was significantly higher than that in the non-responder group (37.5% at 1 year; p = 0.029). Colony formation decreased in a ZA concentration- and dose-dependent manner.Abstract 3340; Table 1TreatmentCR or PRSD or PDBMA alone510RT + BMA217 Open table in a new tab RT + BMA is more useful than BMAs alone in both clinical and in vitro situations.

Evaluation of a New Hydroxyapatite Nanoparticle as a Drug Delivery System to Oral Squamous Cell Carcinoma Cells.

Due to its abilities of substance adsorption and intracellular transportation, hydroxyapatite is a potential carrier in drug delivery systems (DDS). This in vitro study investigated whether newly-developed, highly-dispersive calcined hydroxyapatite nanoparticles with an average grain diameter of 20 nm (nano-SHAP) were suitable as a DDS for the drugs zoledronic acid (ZA), cisplatin, and carboplatin. The effects of drug-bearing nano-SHAP on cell proliferation were assessed using three human oral squamous cell carcinoma cell lines (HSC-4, KOSC, and SAS) and one human breast cancer cell line (MCF-7). Nano-SHAP alone did not affect proliferation of any cell line until a concentration of 1 μg/ml was reached. Although the effective concentration of ZA in ZA-bearing nano-SHAP differed, it inhibited cell proliferation better than ZA alone. Cisplatin and carboplatin-bearing nano-SHAP had the same effect as these drugs alone. The nano-SHAP system is of potential use as a drug delivery system.

Development and validation of a method using ultra performance liquid chromatography coupled to tandem mass spectrometry for determination of zoledronic acid concentration in human bone

A method for the extraction and quantification of zoledronic acid (ZA) from human bone was set up and validated. This method allowed the quantification of ZA from jawbone sequestrations of patients affected by bisphosphonate-related osteonecrosis of the jaw (BRONJ) associated with ZA treatment. The analyte was extracted from the bone tissues with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetramethyl phosphonate was then quantified by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), showing high accuracy, repeatability and selectivity. Lower limits of quantification and detection (LLOQ and LLQD) were 3.4 ng/mL and 1 ng/mg, respectively. This study fully described the analytical process for the determination of ZA in human bone sequestrations, representing a pivotal step for further biomedical research on ZA and BRONJ.

Minimally effective concentration of zoledronic acid to suppress osteoclasts in vitro.

Zoledronic acid is regarded as the most potent bisphosphonate and is widely used in patients with osteoporosis; however, its side effects, including acute-phase reactions, gastrointestinal complaints, renal dysfunction and bisphosphonate-associated osteonecrosis impair the safety and quality of life of patients. The present study was designed to determine the minimal effective concentration of zoledronic acid through testing the dose-dependent effects of zoledronic acid on osteoclast suppression. A primary culture of bone marrow mononuclear cells obtained from C57 mice (age, 6 weeks) was established and induced to form osteoclasts. The number of multinuclear cells was determined by tartrate-resistant acid phosphatase staining and compared among cultured marrow cells treated with different concentrations of zoledronic acid. Furthermore, the cellular properties, including adhesion, migration and bone resorption, were compared at the minimal effective concentration. At a concentration of 1×10−6 mol/l, zoledronic acid significantly inhibited the formation of osteoclasts. This inhibitory effect was further enhanced at the concentration of 1×10−5 mol/l. However, the inhibitory effect of zoledronic acid tapered at the concentration of 1×10−4 mol/l and there was no further dose-dependent increase. In addition, the concentration of 1×10−6 mol/l was sufficient to alter cellular functions, including cell adhesion, migration and bone resorption. In conclusion, zoledronic acid was effective in reducing osteoclast formation and suppressing cellular functions. The minimal effective concentration of zoledronic acid in vitro was 1 µmol/l. Based on these results, a comparable dosage should be explored in clinical applications.

Zoledronic acid overcomes chemoresistance by sensitizing cancer stem cells to apoptosis

Unlike low tumorigenic bulk tumor cells (non-CSCs), cancer stem cells (CSCs) are a subset of tumor cells that can self-renew and differentiate into different cancer subtypes. CSCs are considered responsible for tumor recurrence, distant metastasis, angiogenesis, and drug or radiation resistance. CSCs also are resistant to apoptosis. Zoledronic acid (ZA) is a third generation bisphosphonate that reduces cell proliferation and exhibits anti-tumor effects by inducing cell death in some malignancies; however, the effects of ZA on CSCs are unclear. We investigated the anti-cancer effects of ZA on two epithelial cancer cell lines, prostate DU-145 and breast MCF7, focusing primarily on induction and activation of apoptosis. Cluster of differentiation (CD) 133+/CD44+ prostate CSCs and CD 44+/CD24 breast CSCs were isolated from the DU-145 human prostate cancer and MCF-7 human breast cancer cell lines, respectively, using FACSAria flow cytometry cell sorting. CSCs and non-CSCs were exposed to increasing concentrations of ZA for 24, 48 and 72 h to determine the IC50 dose. Annexin-V assay for detecting cell death and cell cycle was performed using the Muse™ Cell Analyzer. Prostate CSCs and non-CSCs were assayed by quantitative reverse transcription PCR (qRT-PCR) array for detecting 84 key apoptosis related genes. Gene regulation at the protein level was investigated by immunofluorescence. ZA caused a dose- and time-dependent decrease in cell viability. Treatment with ZA resulted in a concomitant increase in apoptosis and cell cycle arrest at S-phase in CSCs. Significant over/under-expressions were detected in seven of the genes of ZA-treated DU-145 CSCs cells. Expressions of CASP9, CASP4, BAX and BAD genes increased, while the expressions of BIRC3, BIRC2 and BCL2 genes decreased. In the DU-145 non-CSCs, five genes exhibited changes in gene expression after ZA treatment, two exhibited increased expression (CASP7 and BAD) and three exhibited decreased expression (BIRC3, BIRC2 and BCL2). ZA caused cell death of drug resistant breast MCF-7 and prostate DU-145 cancer stem cells by activating apoptosis. ZA can facilitate the intrinsic pathway of apoptosis in human prostate CSCs by down-regulating anti-apoptotic genes and up-regulating pro-apoptotic genes. ZA may be an effective therapeutic agent for targeting chemoresistance in CSCs.

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro.

BackgroundZoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro.Materials and methodsUsing assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol.ResultsIn the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively).ConclusionWe have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status.

Cytoprotective effects of melatonin on zoledronic acid-treated human osteoblasts

ObjectiveTo evaluate the cytoprotective effects of melatonin (MLT) on zoledronic acid (ZA)-treated human osteoblasts. MethodsHuman osteoblasts were exposed to ZA (1, 5, 10, 50, 100 and 300 μM) and MLT (1, 10, 50, 100 y 200 μM) for 24, 48 and 72 h of incubation, to evaluate their effects on cell viability. ResultsAs ZA concentration increased, greater reductions in cell viability of human osteoblasts were induced whether at 24, 48 or 72 h incubation. At 24 h incubation with MLT, greatest cell viability was obtained when low dose of MLT was applied (without significant differences); 48 and 72 h incubation presented the greatest cell viability with the highest MLT concentrations (100 and 200 μM). MLT at concentrations of 100 and 200 μM would appear to have a certain cytoprotective effect on ZA-treated human osteoblasts with low concentrations of ZA (1 y 5 μM), whether at 24, 48 or 72 h; however, at ZA concentrations ≥10 μM the possible cytoprotective effects of MLT were low at 24 h incubation. ConclusionsMLT has a cytoprotective effect on ZA-treated human osteoblasts and could represent a promising preventative alternative for patients at risk of bisphosphonate-related osteonecrosis of the jaw.

Safety, pharmacokinetics, and changes in bone metabolism associated with zoledronic acid treatment in Japanese patients with primary osteoporosis.

Although once-yearly intravenous administration of zoledronic acid has been reported to inhibit bone resorption and increase bone mineral density, no studies have evaluated its effectiveness in treating osteoporosis in Japanese patients. Therefore, the purpose of this study was to investigate the pharmacokinetics and assess the safety of and changes in bone metabolism associated with zoledronic acid treatment in Japanese patients with primary osteoporosis. This was a single-administration study with a single-blind parallel-group design. The study participants were 24 Japanese patients with primary osteoporosis. The patients were divided into two groups, with each group receiving a single injection of zoledronic acid (4 or 5mg). Pharmacokinetics and urinary excretion were then compared, and drug-related adverse events and changes in the levels of bone turnover markers were assessed at 12months. Mean plasma concentrations of zoledronic acid peaked in both groups immediately after administration, and decreased to 1% or less of peak levels after 24h. Noncompartmental analysis revealed that C max and the area under the curve from time zero to infinity increased in proportion to the dose. The levels of bone resorption and formation markers decreased from day 15 and from 3months after administration respectively, and suppression of these markers remained constant for the entire study period. No serious adverse events were reported. There was no large difference between the 4- and 5-mg groups in terms of pharmacokinetics, changes in the levels of bone turnover markers, and safety profiles. This study demonstrated acceptable pharmacokinetics and changes in bone metabolism associated with zoledronic acid treatment in female Japanese osteoporosis patients. Both the 4-mg dose and the 5-mg dose demonstrated acceptable safety and sustained antiresorptive effects for the duration of the study.

Osteoblast-like cells with different embryologic origins behave differently in increasing zoledronic acid concentrations: a pilot study in pigs

We compared the intraindividual effects of increasing zoledronic acid (ZA) concentrations on osteoblast-like cells with different embryologic origins. Cultured osteoblast-like cells from mandible and iliac crest bone samples of domestic pigs were exposed to increasing concentrations of ZA (0, 10-8, 10-6, and 10-4M). Proliferation was assessed by cell counting. Receptor activator of nuclear factor kB ligand and osteoprotegerin (OPG) messenger RNA expression were assessed at 0, 1, 4, 7, and 10days. The OPG expression level was higher in the iliac crest than in the mandible. Neither ZA concentration nor the cells' origin affected the expression of receptor activator of nuclear factor kB ligand. At 10-6M, OPG expression from both locations reached the same level after 7days of cultivation, as OPG expression increased to a greater extent in the mandible in comparison to the iliac crest. Cultured mandibular osteoblast-like cells reacted more sensitively to high ZA concentrations than did osteoblast-like cells from the iliac crest.

An intrapleural administration of zoledronic acid for inoperable malignant mesothelioma patients: a phase I clinical study protocol.

BackgroundThe third generation of bisphosphonates is clinically in use for patients of osteoporosis or malignancy-linked hypercalcemia. The agents can also produce anti-tumor effects on bone metastasis of several types of tumors. We recently found that one of the agents achieved cytotoxicity to mesothelioma in vitro and in an orthotopic animal model. Mesothelioma is resistant to a number of chemotherapeutic agents, and suppression of local tumor growth is beneficial to the patients since metastasis to extra-thoracic organs is relatively infrequent until a late stage.Methods/designWe demonstrated in an orthotopic mouse model that an intrapleural but not intravenous injection of zoledronic acid, one of the third generation bisphosphonates, at a clinically equivalent dose suppressed the tumor growth. Nevertheless, a high concentration of zoledronic acid administrated in the pleural cavity produced pleural adhesion. We also showed that zoledronic acid produced synergistic cytotoxic effects with cisplatin, the first-line chemotherapeutic agent for mesothelioma. We then planned to conduct a phase I clinical study to investigate any adverse effects and a possible clinical benefits produced by an intrapleural administration of zoledronic acid to mesothelioma patients who became resistant to the first-line chemotherapeutic agents. The clinical trial is a dose escalation study starting with 0.4, 1, 4, 8 and 16 mg per person since safety of administration of zoledronic acid into the pleural cavity remains unknown. Each dose group consists of three persons and the protocol allows to repeat administration of the same dose into the pleural cavity at a 4-weeks interval.DiscussionWe will conduct a possible combinatory study of intrapleural administration of zoledronic acid and systemic administration of the first-line agent to a chemotherapy-naïve patient based on the maximum tolerance dose of zoledronic acid determined by the present clinical trial. We propose that administration of bisphosphonates in a closed cavity is a treatment strategy for tumors developed in the cavity probably through the direct cytotoxic activity.Trial registration: UMIN clinical trials registry, Japan. Register ID: UMIN8093.