Bisphosphonate type-dependent cell viability suppressive effects of carbon nanohorn-calcium phosphate-bisphosphonate nanocomposites.

In the process of bone metastasis, tumor cells spread to the bones to activate osteoclasts, which cause pathological bone resorption and destruction. Bisphosphonates (BPs) inhibit osteoclast activation to resorb bone, reducing bone pain and fracture. We previously developed a nanocomposite for potential localized treatment of bone metastasis by loading a BP compound, ibandronate, onto oxidized carbon nanohorns (OxCNHs), a next-generation drug carrier, using calcium phosphates (CaPs) as mediators to generate OxCNH-CaP-BP nanocomposites. The objective of the present study was to determine nanocomposite formation and biological properties of nanocomposites constructed from two BPs, zoledronate and pamidronate. In vitro tests using murine macrophages (RAW264.7 cells) and osteoclasts differentiated from RAW264.7 cells revealed that the resulting OxCNH-CaP-BP nanocomposites suppressed cell viability in a BP type-dependent manner and more effectively than OxCNHs or BPs alone. The mechanism for the potent and BP type-dependent suppression of cell viability by OxCNH-CaP-BP nanocomposites, based on their relative cellular uptake and reactive oxygen species generation, is also discussed. The present study supports the conclusions that BPs can be loaded onto OxCNHs using CaPs as mediators, and that OxCNH-CaP-BP nanocomposites are putative medicines for localized treatment of metastatic bone destruction.

The Effects of Polyphenol, Tannic Acid, or Tannic Acid in Combination with Pamidronate on Human Osteoblast Cell Line Metabolism.

BACKGROUND: This study investigates the effect of tannic acid (TA) combined with pamidronate (PAM) on a human osteoblast cell line. METHODS: EC50 for TA, PAM, and different combination ratios of TA and PAM (25:75, 50:50, 75:25) were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The combination index value was utilized to analyze the degree of drug interaction, while trypan blue assay was applied to analyze the cells proliferation effect. The mineralization and detection of bone BSP and Osx genes were determined via histochemical staining and PCR test, respectively. RESULTS: The EC50 of osteoblasts treated with TA and a 75:25 ratio of TA and PAM were more potent with lower EC50 at 0.56 µg/mL and 0.48 µg/mL, respectively. The combination of TA and PAM (75:25) was shown to have synergistic interaction. On Day 7, both TA and PAM groups showed significantly increased proliferation compared with control and combination groups. On Day 7, both the TA and combination-treated groups demonstrated a higher production of calcium deposits than the control and PAM-treated groups. Moreover, on Day 7, the combination-treated group showed a significantly higher expression of BSP and Osx genes than both the TA and PAM groups. CONCLUSION: Combination treatment of TA and PAM at 75:25 ameliorated the highest enhancement of osteoblast proliferation and mineralization as well as caused a high expression of BSP and Osx genes.

The effect of pamidronate delivery in bisphosphonate-naïve patients on neutrophil chemotaxis and oxidative burst.

The pathogenesis of medication-related osteonecrosis of the jaw (MRONJ), a morbid condition associated with bisphosphonate administration, has not been fully elucidated. Recent research utilizing a murine model has revealed that the neutrophil becomes dysfunctional following exposure to bisphosphonates. Accordingly, the impairment of neutrophil function could play an important role in the pathogenesis of MRONJ via an infectious mechanism mediated by the suppression of the innate immune system. Currently, the existing human data are insufficient to substantiate this theory. To investigate, we isolated neutrophils from blood and oral rinse samples from bisphosphonate-naïve patients who were recently diagnosed with multiple myeloma both prior to and one month following their initial infusion of pamidronate, an intravenous bisphosphonate agent. Stimulated blood and oral neutrophil superoxide production and chemotactic capabilities were found to be impaired relative to baseline values. These results suggest that impaired neutrophil function may partially contribute to the aetiology underlying the pathophysiological processes linked to the development of MRONJ. Further, as the functional status of circulating neutrophils was reflected in the oral cavity where sampling can be accomplished in a non-invasive fashion, it is conceivable that neutrophil function could serve as a potential biomarker for MRONJ prognostication.

Valproic acid enhances pamidronate-sensitized cytotoxicity of Vδ2+ T cells against EBV-related lymphoproliferative cells.

Therapeutic options for Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative diseases (PTLD) are currently limited, accompanying with some off-target toxicities. We previously demonstrated that early recovery of Vδ2+ T cells inversely correlated to EBV reactivation after allogeneic hematopoietic cell transplantation. Studies in vitro and in the mouse models showed the cytotoxic activity of Vδ2+ T cells on EBV-transformed lymphoproliferative cells, but the efficacy was moderate. Bisphosphonate, such as pamidronate (PAM), have been reported as a sensitizer to trigger tumor cells for Vδ2+ T cells recognition. Valproic acid (VPA) has attracted attentions due to its adjuvant anti-tumor effect with chemotherapy or immunotherapy. Whether PAM and VPA facilitate the immunogenicity of EBV-infected cells towards Vδ2+ T cells cytotoxicity remains unknown. Herein, we demonstrated that lower dosage of VPA and/or PAM did not induce apoptosis of EBV-transformed B lymphoblastoid cell lines (EBV-LCLs) or Vδ2+ T cells. Notably, pre-treatment with PAM significantly increased the cell death of EBV-LCLs after co-culture with Vδ2+ T cells at different ratios. Combining treatment with VPA reinforced the sensitizing effect of PAM. This efficacy was through inducing the accumulation of mevalonate pathway intermediates and dependent on the γδ T cell receptor of Vδ2+ T cells. Similar sensitizing effects of PAM and PAM plus VPA were also demonstrated on the primary PTLD cells. These results highlight the roles of PAM and VPA in the enhancement of immune surveillance and expand the fields of these two drugs in the treatment of different types of malignancies.

Effect of timing of bisphosphonate administration on canine osteosarcoma cells undergoing radiation therapy.

The effects of radiation therapy may be potentiated by combining radiation therapy with secondary therapies. Clinically, radiation therapy has been combined with bisphosphonates for treatment of canine appendicular osteosarcoma for years. The objective of this study was to determine if the timing of administration of bisphosphonates in relation to radiation therapy alters clonogenic survival or cell viability of canine osteosarcoma cells in vitro. Canine osteosarcoma cells were treated before administration of radiation, concurrent with radiation, or after radiation. Reduction in clonogenic survival was identified when bisphosphonates were administered post-radiation compared with pre-radiation. No significant differences were identified for cell viability at any time points. Further investigation of the cellular effects of bisphosphonates on canine osteosarcoma cells is warranted. Consideration may be given to administering bisphosphonates 24 h after radiation to reduce replication of canine osteosarcoma cells and possibly prolong the analgesic effects of both treatments.

Les effets de la radiothérapie peuvent être potentialisés en associant la radiothérapie à des thérapies secondaires. Cliniquement, la radiothérapie est associée aux bisphosphonates pour le traitement de l'ostéosarcome appendiculaire canin depuis des années. L'objectif de cette étude était de déterminer si le moment choisi pour l'administration de bisphosphonates en relation avec la radiothérapie altère la survie clonogénique ou la viabilité cellulaire de cellules d'ostéosarcome canin in vitro. Les cellules d'ostéosarcome canin ont été traitées avant l'administration d'un rayonnement, concomitant avec le rayonnement ou après l'administration d'un rayonnement. La réduction de la survie clonogénique a été identifiée lorsque les bisphosphonates étaient administrés post-irradiation par rapport à l'irradiation. Aucune différence significative n'a été identifiée pour la viabilité des cellules à aucun moment. Une étude plus approfondie des effets cellulaires des bisphosphonates sur les cellules d'ostéosarcome canin est justifiée. L'administration de bisphosphonates peut être envisagée 24 heures après l'administration de radiations afin de réduire la réplication des cellules d'ostéosarcome canin.(Traduit par les auteurs).

Two new, near-infrared, fluorescent probes as potential tools for imaging bone repair.

A precise imaging technique to evaluate osteogenesis, osteodifferentiation, and osseointegration following peri-implant surgery is in high clinical demand. Herein, we report the generation of two new, near-infrared (NIR) fluorescent probes for use in the molecular imaging of bone repair. The first probe aims to monitor the in vitro differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. A NIR fluorochrome was conjugated to a cyclic peptide that binds to integrin α5ß1, a factor that promotes osteogenesis in MSCs and therefore functioned as an osteoblast-specific marker. The second probe aims to monitor osteogenesis, and was generated by conjugating the drug pamidronate to a NIR fluorescent gold nanocluster. Pamidronate specifically binds to hydroxyapatite (HA), a mineral present in bone that is produced by osteoblasts, and therefore provides a functional marker for new bone formation. Our results show that both probes bind to their specific targets in vitro-differentiated osteoblasts, and not to undifferentiated MSCs, and emit NIR fluorescence for functional detection. This in vitro work demonstrates the ability of these probes to bind to active osteoblasts and their mineral deposits and highlight their potential utility as clinical tools for the imaging of the osseointegration process at the molecular level.

Prenylation-dependent Ras inhibition by pamidronate inhibits pediatric acute myeloid leukemia stem and differentiated cell growth and survival.

The clinical management of pediatric acute myeloid leukemia (AML) is still challenging and identification of drugs that can enhance the efficacy of standard of care is a potential therapeutic strategy. We show that pamidronate, a FDA-approved drug used for bone disorders, is an attractive candidate for AML treatment. Pamidronate inhibits proliferation and induces apoptosis of AML cells regardless of cellular and genetic heterogeneity. Pamidronate displays selective anti-AML activity by preferentially inhibiting survival and colony formation of AML CD34+ cells while normal bone marrow CD34+ cells are largely unaffected. Importantly, pamidronate remarkably enhances the inhibitory effects of all tested AML standard of care at subtoxic concentration. Mechanism studies show that pamidronate inhibits protein prenylation via dual action on geranylgeranylation and farnesylation, and subsequently decreases Ras activity. The rescue studies using overexpression of constitutively active Ras further confirm that pamidronate augments the efficacy of AML standard of care through inhibiting Ras. Since pamidronate is already used in clinic, our preclinical findings suggest that it may be an effective addition to treatment armamentarium for AML.

Evaluation of effects of radiation therapy combined with either pamidronate or zoledronate on canine osteosarcoma cells.

Canine osteosarcoma is a devastating disease with an overall poor prognosis. Radiation therapy and bisphosphonates are currently used in combination for palliative treatment, despite a paucity of literature that investigates their combined use. The objectives of this study were to assess the in vitro effects of radiation therapy and bisphosphonates on canine osteosarcoma cells when used in combination. Canine osteosarcoma cell lines D17 and Dharma were treated with radiation and pamidronate or zoledronate, both alone and in combination. The effects of these treatments were assessed using clonogenic survival and cell viability assays. Dose-dependent decreases in clonogenic survival and cell viability were observed for both radiation and bisphosphonate treatment. Combination index analysis revealed antagonistic interactions when radiation and bisphosphonates were used in combination at specific doses for both D17 and Dharma osteosarcoma cells. Further investigation of the combined effects of radiation and bisphosphonates for the palliative treatment of canine osteosarcoma is warranted.

L'ostéosarcome canin est une maladie dévastatrice, avec un mauvais pronostic global. La radiothérapie et les bisphosphonates sont actuellement utilisés en combinaison pour le traitement palliatif, malgré une littérature limitée qui étudie leur utilisation combinée. Les objectifs de cette étude étaient d'évaluer les effets in vitro de la radiothérapie et des bisphosphonates sur des cellules d'ostéosarcome canin en combinaison. Les lignées cellulaires d'ostéosarcome canin D17 et Dharma ont été traitées par irradiation, le pamidronate et le zolédronate seuls et en association. Les effets de ces traitements ont été évalués en utilisant des tests de survie clonogénique et de viabilité cellulaire. Des diminutions dépendantes de la dose de la survie clonogénique et de la viabilité cellulaire ont été observées pour le traitement par rayonnement et par bisphosphonate. L'analyse de l'indice de combinaison a révélé des interactions antagonistes lorsque les radiations et les bisphosphonates étaient utilisés en association à des doses spécifiques, pour les cellules de D17 et d'ostéosarcome de Dharma. Une étude plus approfondie des effets combinés des rayonnements et des bisphosphonates pour le traitement palliatif de l'ostéosarcome canin est justifiée.(Traduit par les auteurs).

Pamidronate decreases bilirubin-impaired cell death and improves dentinogenic dysfunction of stem cells from human deciduous teeth.

BACKGROUND: Hyperbilirubinemia that occurs in pediatric liver diseases such as biliary atresia can result in the development of not only jaundice in the brain, eyes, and skin, but also tooth abnormalities including green pigmentation and dentin hypoplasia in the developing teeth. However, hyperbilirubinemia-induced tooth impairments remain after liver transplantation. No effective dental management to prevent hyperbilirubinemia-induced tooth impairments has been established. METHODS: In this study, we focused on pamidronate, which is used to treat pediatric osteopenia, and investigated its effects on hyperbilirubinemia-induced tooth impairments. We cultured stem cells from human exfoliated deciduous teeth (SHED) under high and low concentrations of unconjugated bilirubin in the presence or absence of pamidronate. We then analyzed the effects of pamidronate on the cell death, associated signal pathways, and dentinogenic function in SHED. RESULTS: We demonstrated that a high concentration of unconjugated bilirubin induced cell death in SHED via the mitochondrial pathway, and this was associated with the suppression of AKT and extracellular signal-related kinase 1 and 2 (ERK1/2) signal pathways and activation of the nuclear factor kappa B (NF-κB) signal pathway. The high concentration of unconjugated bilirubin impaired the in vitro and in vivo dentinogenic capacity of SHED, but not the low concentration. We then demonstrated that pamidronate decreased the bilirubin-induced cell death in SHED via the altered AKT, ERK1/2, and NF-κB signal pathways and recovered the bilirubin-impaired dentinogenic function of SHED. CONCLUSIONS: Our findings suggest that pamidronate may prevent tooth abnormalities in pediatric patients with hyperbilirubinemia.

In Vitro Investigation of the Antimicrobial Effect of Three Bisphosphonates Against Different Bacterial Strains.

PURPOSE: Since the first descriptions of medication-related osteonecrosis of the jaw (MRONJ) in 2003, the pathogenesis has remained unanswered. Recent histomorphometric studies have found several microorganisms, including Actinomyces, Bacillus, Fusobacterium, Staphylococcus, Streptococcus, Selenomonas, Treponema, and Candida albicans in necrotic bone. Polymerase chain reaction studies have recently confirmed the occurrence of 48 genera. Only a few studies have examined the antimicrobial effect of bisphosphonates (BPs). The influence of bacterial growth on the etiology remains unclear. The aim of the present study was the in vitro investigation of the antimicrobial effect of 3 BPs against different bacterial strains. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 48 strains from 40 species were determined in microdilution assays against pamidronic, ibandronic, and zoledronic acid. RESULTS: Growth of gram-positive oral microbiota, which account for most microorganisms in MRONJ, was present for 2 of 22 species; 6 of 26 gram-negative species and 9 of 13 anaerobes were inhibited. The MIC values were compared with the BP bone concentrations from previous reports. Of the 48 strains, 9 had an MIC or MBC less than the bone concentrations. CONCLUSIONS: The results of the present study have demonstrated that BPs have an inhibitory effect on selected bacterial species and might inhibit the growth of some relevant pathogens in osteonecrosis. However, most of the species tested were unaffected at the concentration levels assumed present in the human jawbone. The clinical relevance of these in vitro data will better be clarified with reliable data on the BP concentrations in the human jawbone. The present study has provided a first approach toward the assessment of the interaction of oral bacteria and BPs.

Use of pamidronate for osteoporosis treatment in public health care in Brazil / Uso de pamidronato para o tratamento da osteoporose no sistema público de saúde no Brasil

Abstract Purpose: The use of bisphosphonates for osteoporosis is effective in reducing the risk of fractures. However, oral formulations are sometimes not well tolerated or are contraindicated. Due to its availability in Brazilian public health system, pamidronate is frequently prescribed for osteoporosis, despite the lack of studies demonstrating its anti-fracture efficacy and the absence of FDA or EMEA approval for this purpose. The aim of this study was to evaluate the bone mineral density (BMD) response to pamidronate in a group of women with osteoporosis in a tertiary care hospital. Patients and methods: The medical records of women with osteoporosis who received pamidronate for up to two years of treatment were reviewed. Patients were stratified at high or intermediate risk of fracture. Results: A total of 70 women were in treatment with pamidronate. Among them, 74% were at high risk of fracture. A significant gain in spine BMD after 24 months of treatment was observed (p = 0.012). There was no difference between the groups of high and not high risk of fracture. At the femur, no significant increase in BMD was present, though, a strong negative correlation with high PTH levels (r = −0.61; p = 0.003) was seen. In the multivariate analysis BMI at 12 months had impact in the response to the treatment. Conclusion The intravenous pamidronate in a group of postmenopausal women with predominant high risk of fracture promoted an isolated gain in the spine BMD, even though, clinical randomized trials are needed to confirm its anti-fracture efficacy.

Resumo Justificativa: O uso de bisfosfonatos para a osteoporose é eficaz na redução do risco de fraturas. No entanto, as formulações orais às vezes não são bem toleradas ou são contraindicadas. Em razão da sua disponibilidade no sistema público de saúde brasileiro, o pamidronato é frequentemente prescrito para a osteoporose, apesar da falta de estudos que demonstrem a sua eficácia antifratura e da ausência de aprovação da Food and Drug Administration (FDA) ou da European Medicine Agency (Emea) para essa finalidade. O objetivo deste estudo foi avaliar a resposta da densidade mineral óssea (DMO) ao pamidronato em um grupo de mulheres com osteoporose em um hospital terciário. Pacientes e métodos: Revisaram-se os prontuários médicos de mulheres com osteoporose que receberam pamidronato por até dois anos de tratamento. As pacientes foram estratificadas em risco alto ou intermediário de fratura. Resultados: Estavam em tratamento com pamidronato 70 mulheres. Entre elas, 74% tinham alto risco de fratura. Observou-se um ganho significativo na DMO da coluna vertebral após 24 meses de tratamento (p = 0,012). Não houve diferença entre os grupos de risco de fratura alto e não alto. No fêmur, não foi encontrado aumento significativo na massa óssea; contudo, observou-se uma forte correlação negativa com altos níveis de PTH (r = −0,61; p = 0,003). Na análise multivariada, o IMC aos 12 meses tinha impacto na resposta ao tratamento. Conclusão O pamidronato intravenoso em um grupo de mulheres na pós-menopausa predominantemente com alto risco de fratura promoveu um ganho isolado na DMO da coluna vertebral, embora sejam necessários ensaios clínicos randomizados para confirmar sua eficácia antifratura.