RESUMEN
PURPOSE: The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. MATERIALS AND METHODS: Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. RESULTS: Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. CONCLUSIONS: Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.
Asunto(s)
Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/metabolismo , Exosomas/metabolismo , Paclitaxel/farmacología , Animales , Animales Modificados Genéticamente , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Permeabilidad Capilar , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Modelos Animales de Enfermedad , Doxorrubicina/química , Doxorrubicina/metabolismo , Endocitosis , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Xenoinjertos , Humanos , Trasplante de Neoplasias , Paclitaxel/química , Paclitaxel/metabolismo , Tamaño de la Partícula , Tecnología Farmacéutica/métodos , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Pez CebraRESUMEN
One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7 ± 2.0 nm and zeta potentials of 4.03 ± 0.69 mV, which had no significant change when stored at 4 °C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent.
Asunto(s)
Silenciador del Gen , Proteínas de Neoplasias/antagonistas & inhibidores , ARN Interferente Pequeño/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular , Humanos , Liposomas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tamaño de la Partícula , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mineralizing osteoblasts are regularly used to study osteogenesis and model in vivo bone formation. Thus, it is important to verify that the mineral and matrix being formed in situ are comparable to those found in vivo. However, it has been shown that histochemical techniques alone are not sufficient for identifying calcium phosphate-containing mineral. The goal of the present study was to demonstrate the use of Fourier transform infrared imaging (FTIRI) as a tool for characterizing the spatial distribution and colocalization of the collagen matrix and the mineral phase during the mineralization process of osteoblasts in situ. MC3T3-E1 mouse osteoblasts were mineralized in culture for 28 days and FTIRI was used to evaluate the collagen content, collagen cross-linking, mineralization level and speciation, and mineral crystallinity in a spatially resolved fashion as a function of time. To test whether FTIRI could detect subtle changes in the mineralization process, cells were treated with risedronate (RIS). Results showed that collagen deposition and mineralization progressed over time and that the apatite mineral was associated with a collagenous matrix rather than ectopic mineral. The process was temporarily slowed by RIS, where the inhibition of osteoblast function caused slowed collagen production and cross-linking, leading to decreased mineralization. This study demonstrates that FTIRI is a complementary tool to histochemistry for spatially correlating the collagen matrix distribution and the nature of the resultant mineral during the process of osteoblast mineralization. It can further be used to detect small perturbations in the osteoid and mineral deposition process.
Asunto(s)
Osteoblastos/patología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Células 3T3 , Animales , Calcificación Fisiológica/fisiología , Fosfatos de Calcio/metabolismo , Condrocitos/citología , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Cristalización , Difosfonatos/farmacología , Ácido Etidrónico/análogos & derivados , Ácido Etidrónico/farmacología , Ratones , Microscopía Fluorescente/métodos , Odontoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Ácido Risedrónico , Factores de TiempoRESUMEN
Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl(2)). Three-month-old ovariectomized rats were treated for 90 days with doses of 25â mg kg(-1) d(-1) and 150â mg kg(-1) d(-1) of SrR or SrCl(2) at low (0.1% Ca) or normal (1.19% Ca) Ca diet. Vertebral bone tissue was analysed by confocal synchrotron-radiation-induced micro X-ray fluorescence and by backscattered electron imaging. Principal component analysis and k-means clustering of the acquired elemental maps of Ca and Sr revealed that the newly formed bone exhibited the highest Sr fractions and that low Ca diet increased the Sr uptake by a factor of three to four. Furthermore, Sr uptake in bone of the SrCl(2)-treated animals was generally lower compared with SrR. The study clearly shows that inadequate nutritional calcium intake significantly increases uptake of Sr in serum as well as in trabecular bone matrix. This indicates that nutritional calcium intake as well as serum Ca levels are important regulators of any Sr treatment.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Huesos/metabolismo , Calcio/deficiencia , Compuestos Organometálicos/uso terapéutico , Estroncio/metabolismo , Estroncio/uso terapéutico , Tiofenos/uso terapéutico , Animales , Calcio de la Dieta/administración & dosificación , Femenino , Análisis de Componente Principal , Ratas , Ratas Sprague-Dawley , Columna Vertebral/metabolismoRESUMEN
Young mice overexpressing Runx2 specifically in cells of the osteoblastic lineage failed to gain bone mass and exhibited a dramatic increase in bone resorption, leading to severe osteopenia and spontaneous vertebral fractures. The objective of the current study was to determine whether treatment with a bisphosphonate (risedronate, Ris), which reduces fractures in postmenopausal as well as in juvenile osteoporosis, was able to improve bone quality and reduce vertebral fractures in mice overexpressing Runx2. Four-week-old female Runx2 mice received Ris at 2 and 10 µg/kg subcutaneously twice a week for 12 weeks. Runx2 and wild-type mice received vehicle (Veh) as control. We measured the number of new fractures by X-ray and bone mineral density (BMD) by DEXA. We evaluated bone quality by histomorphometry, micro-CT, and Fourier transform infrared imaging (FTIRI). Ris at 20 µg/kg weekly significantly reduced the average number of new vertebral fractures compared to controls. This was accompanied by significantly increased BMD, increased trabecular bone volume, and reduced bone remodeling (seen in indices of bone resorption and formation) in the vertebrae and femoral metaphysis compared to Runx2 Veh. At the femur, Ris also increased cortical thickness. Changes in collagen cross-linking seen on FTIRI confirmed that Runx2 mice have accelerated bone turnover and showed that Ris affects the collagen cross-link ratio at both forming and resorbing sites. In conclusion, young mice overexpressing Runx2 have high bone turnover-induced osteopenia and spontaneous fractures. Ris at 20 µg/kg weekly induced an increase in bone mass, changes in bone microarchitecture, and decreased vertebral fractures.
Asunto(s)
Huesos/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Ácido Etidrónico/análogos & derivados , Fracturas Óseas/prevención & control , Animales , Conservadores de la Densidad Ósea/farmacología , Conservadores de la Densidad Ósea/uso terapéutico , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Huesos/fisiología , Modelos Animales de Enfermedad , Ácido Etidrónico/farmacología , Ácido Etidrónico/uso terapéutico , Femenino , Fracturas Óseas/epidemiología , Fracturas Óseas/etiología , Fracturas Óseas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoporosis/complicaciones , Osteoporosis/tratamiento farmacológico , Osteoporosis/genética , Osteoporosis/patología , Ácido Risedrónico , Regulación hacia Arriba/genéticaRESUMEN
MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR-10b was up-regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR-10b correlated with low neurofibromin expression was found in a neuroectodermal cell line: Ewing's sarcoma SK-ES-1 cells. Antisense inhibiting miR-10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR-10b. Overexpression of miR-10b in 293T cells suppressed neurofibromin expression and activated RAS signaling. Antisense inhibition of miR-10b restored neurofibromin expression in SK-ES-1 cells, and decreased RAS signaling independent of neurofibromin in NF1 MPNST cells. These results suggest that miR-10b may play an important role in NF1 tumorigenesis through targeting neurofibromin and RAS signaling.
Asunto(s)
MicroARNs/genética , Neurofibromatosis 1/genética , Transducción de Señal , Proteínas ras/genética , Regiones no Traducidas 3'/genética , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , ARN sin Sentido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Schwann/metabolismo , Células de Schwann/patología , Células Tumorales Cultivadas , Proteínas ras/metabolismoRESUMEN
One of the key parameters for a successful treatment with any drug is the use of an optimal dose regimen. Bisphosphonates (BPs) have been in clinical use for over five decades and during this period clinical pharmacokinetic (PK) and pharmacodynamic (PD) evaluations have been instrumental for the identification of optimal dose regimens in patients. Ideal clinical PK and PD studies help drug developers explain variability in responses and enable the identification of a dose regimen with an optimal effect. PK and PD studies of the unique and rather complex pharmacological properties of BPs also help determine to a significant extent ideal dosing for these drugs. Clinical PK and PD evaluations of BPs preferably use study designs and assays that enable the assessment of both short- (days) and long-term (years) presence and effect of these drugs in patients. BPs are mainly used for metabolic bone diseases because they inhibit osteoclast-mediated bone resorption and the best way to quantify their effects in humans is therefore by measuring biochemical markers of bone resorption in serum and urine. In these very same samples BP concentrations can also be measured. Short-term serum and urine data after both intravenous (IV) and oral administration enable the assessment of oral bioavailability as well as the amount of BP delivered to the skeleton. Longer-term data provide information on the anti-resorptive effect as well as the elimination of the BP from the skeleton. Using PK-PD models to mathematically link the anti-resorptive action of the BPs to the amount of BP at the skeleton provides a mechanism-based explanation of the pattern of bone resorption during treatment. These models have been used successfully during the clinical development of BPs. Newer versions of such models, which include systems pharmacology and disease progression models, are more comprehensive and include additional PD parameters such as BMD and fracture risk. Clinical PK and PD studies of BPs have been useful for the identification of optimal dose regimens for metabolic bone diseases. These analyses will also continue to be important for newer research directions, such as BP use in the delivery of other drugs to the bone to better treat bone metastases and bone infections, as well as the potential benefit of BPs at non-skeletal targets for the prevention and treatments of soft tissue cancers, various fibroses, and other cardiovascular and neurodegenerative diseases, and reduction in mortality and extension of lifespan.
Asunto(s)
Enfermedades Óseas Metabólicas , Neoplasias Óseas , Resorción Ósea , Resorción Ósea/tratamiento farmacológico , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Humanos , OsteoclastosRESUMEN
At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688-1623 cm(-1)), carbonate (v(2)CO(3)(2-): 905-825 cm(-1)), and phosphate (v(4)PO(4)(3-): 650-500 cm(-1)) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.
Asunto(s)
Remodelación Ósea/fisiología , Huesos/química , Calcificación Fisiológica , Osteón , Espectroscopía Infrarroja por Transformada de Fourier , Animales , Huesos/anatomía & histología , Huesos/metabolismo , Carbonatos/análisis , Femenino , Osteón/química , Osteón/metabolismo , Fosfatos/análisis , ConejosRESUMEN
We describe for the first time functional clusters of genes that are modulated during the differentiation of osteoclasts. Pathway analysis was applied to gene array data generated from affymetrix chips hybridized to RNA isolated from RAW264.7 cells exposed to RANK-ligand (RANK-L) for 5 days. This analysis revealed major functional gene clusters that were either up- or down-regulated during osteoclastogenesis. Some of the genes within the clusters have known functions, while others do not. We discuss herein the relevance of these functional gene clusters and their modulation to biological processes underlying the formation, function, and fate of osteoclasts.
Asunto(s)
Familia de Multigenes/fisiología , Osteoclastos/citología , Osteoclastos/metabolismo , Proteoma/metabolismo , Animales , Diferenciación Celular , Línea Celular , Ratones , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
PURPOSE: The purpose of this study was to determine whether bisphosphonate treatment can prevent or delay osteocyte apoptosis in a cyclic fatigue animal model and if there are differences between two different bisphosphonates in their effects on osteocyte apoptosis. INTRODUCTION: Fatigue loading induces microdamage in long bones in rats and causes osteocyte apoptosis. In vitro data suggest that the bisphosphonates can prevent osteocyte apoptosis. MATERIALS AND METHODS: Six month old female Sprague-Dawley rats (n=72) were given a daily subcutaneous (sc) injection of saline vehicle, risedronate (RIS: 0.05 mug/kg per day) or alendronate (ALN: 0.1 mug/kg per day). On the 8th day of drug treatment, an axial compressive load was applied to the right ulna using a load-controlled electromagnetic device (17N, 6000 cycles, 2 Hz, 10% loss of stiffness approximately 1 h). Three, seven or ten days after loading, the animals were sacrificed. Immunohistochemistry for caspase-3 was performed to assess the extent of osteocyte apoptosis in loaded and non-loaded ulnas. RESULTS: Microdamage (Mdx) created by cyclic loading of the ulna induced a significant increase (p=0.03) in the number of apoptotic osteocytes compared to non-damaged regions of the same ulna, and compared to the contralateral non-loaded ulna. Risedronate and alendronate had an early effect (3 days after loading) on reducing load-induced osteocyte apoptosis. Risedronate significantly reduced the density of apoptotic osteocytes compared to vehicle-treated controls by approximately 50% in the Mdx area, whereas alendronate reduced it by approximately 40%. There were no differences among groups by seven days following loading. CONCLUSIONS: (1) Low doses of risedronate or alendronate suppressed osteocyte apoptosis induced by fatigue loading of the ulna in rats. (2) There was no difference between the effects of risedronate or alendronate on osteocyte apoptosis at these doses.
Asunto(s)
Alendronato/farmacología , Apoptosis/efectos de los fármacos , Conservadores de la Densidad Ósea/farmacología , Ácido Etidrónico/análogos & derivados , Osteocitos/citología , Osteocitos/efectos de los fármacos , Animales , Fenómenos Biomecánicos , Ácido Etidrónico/farmacología , Femenino , Osteocitos/fisiología , Ratas , Ratas Sprague-Dawley , Ácido Risedrónico , Estrés Mecánico , Cúbito/citología , Cúbito/efectos de los fármacos , Cúbito/fisiologíaRESUMEN
The study was designed to investigate if pre-treating rats with a therapeutic equivalent dose of risedronate blunted the anabolic effects of PTH, and whether a withdrawal period prior to PTH treatment would alter any effect of risedronate on PTH treatment. Skeletally mature rats were treated for 18 weeks with vehicle, risedronate, or risedronate for 8 weeks followed by vehicle for 10 weeks (withdrawal period). At the end of this period, animals were treated for a further 12 weeks with PTH or PTH vehicle. Trabecular and cortical bone mass were monitored by serial pQCT, or by DXA and microCT. Bone histomorphometry was performed on the proximal tibiae and tibial shafts for bone turnover parameters at week 40. Risedronate alone moderately increased while PTH alone markedly increased trabecular bone mass at the proximal tibial (35% and 200%, respectively) and lumbar vertebral body (14% and 36%, respectively). The maximum bone gains were similar with and without pretreatment with risedronate as compared to the PTH alone. Continuous administration of risedronate for 18 weeks prior to PTH treatment had lower percentage increases in proximal tibial BMD during the first 8 weeks of PTH treatments, and had lower active bone forming surface and bone formation rates after being treated with PTH 12 weeks as compared to the PTH alone group. However, with the 10-week withdrawal period, risedronate did not blunt the stimulatory effect of PTH on osteoblast activity as shown by similar bone formation rates as with PTH alone. Our findings suggest that while risedronate pretreatment may slow the bone anabolic response to PTH, a withdrawal period prior to PTH treatment allows osteoblastic activity to respond normally to PTH stimulation.
Asunto(s)
Factores de Edad , Anabolizantes/farmacología , Ácido Etidrónico/análogos & derivados , Hormona Paratiroidea/farmacología , Absorciometría de Fotón , Animales , Ácido Etidrónico/farmacología , Femenino , Hormona Paratiroidea/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Ácido Risedrónico , Tomografía Computarizada por Rayos XRESUMEN
The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.
Asunto(s)
Difosfonatos/química , Difosfonatos/farmacología , Osteoclastos/metabolismo , Animales , Neoplasias Óseas/secundario , Resorción Ósea , Huesos/metabolismo , Difosfonatos/uso terapéutico , Guanosina Trifosfato/química , Humanos , Modelos Biológicos , Modelos Químicos , Mieloma Múltiple/metabolismo , Metástasis de la Neoplasia , Nitrógeno/química , Osteocitos/metabolismo , Osteoporosis/terapia , Procesamiento Proteico-Postraduccional , Linfocitos T/metabolismo , Resultado del TratamientoRESUMEN
UNLABELLED: N-BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP-bound, unprenylated Rho family GTPases in macrophages and osteoclasts. In macrophages, this also leads to sustained, Rac-mediated activation of p38. The antiresorptive activity of N-BPs may therefore be caused at least in part, by the accumulation of unprenylated small GTPases, causing inappropriate activation of downstream signaling pathways. INTRODUCTION: Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of bone resorption that act by inhibiting farnesyl diphosphate synthase, thereby indirectly preventing the prenylation of Rho family GTPases that are required for the function and survival of bone-resorbing osteoclasts. However, the effect that these drugs have on the activity of Rho family GTPases has not been determined. MATERIALS AND METHODS: The effect of N-BPs on the activity of Rho family GTPases in J774 macrophages and osteoclasts was measured using a pull-down assay to isolate the GTP-bound forms. The effect of N-BPs, or decreasing Rac expression using siRNA, on downstream p38 activity was evaluated by Western blotting and apoptosis assessed by measurement of caspase 3/7 activity. RESULTS: Rather than inhibiting GTPase function, loss of prenylation after treatment with N-BPs caused an increase in the GTP-bound form of Rac, Cdc42, and Rho in J774 cells and osteoclast-like cells, which paralleled the rate of accumulation of unprenylated small GTPases. Activation of Rac also occurred with other inhibitors of prenylation of Rho-family proteins, such as mevastatin and the geranylgeranyl transferase I inhibitor GGTI-298. The Rac-GTP that increased after N-BP treatment was newly translated, cytoplasmic unprenylated protein, because it was not labeled with [(14)C] mevalonate, and the increase in Rac-GTP was prevented by cycloheximide. Furthermore, this unprenylated Rac-GTP retained at least part of its functional activity in J774 cells, because it mediated N-BP-induced activation of p38. Paradoxically, although risedronate induces apoptosis of J774 macrophages by inhibiting protein prenylation, the p38 inhibitor SB203580 enhanced N-BP-induced apoptosis, suggesting that Rac-induced p38 activation partially suppresses the pro-apoptotic effect of N-BPs in these cells. CONCLUSIONS: N-BP drugs may disrupt the function of osteoclasts in vivo and affect other cell types in vitro by inhibiting protein prenylation, thereby causing inappropriate and sustained activation, rather than inhibition, of some small GTPases and their downstream signaling pathways.
Asunto(s)
Difosfonatos/farmacología , Prenilación de Proteína/efectos de los fármacos , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Western Blotting , Línea Celular , Activación Enzimática , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
UNLABELLED: Long-term effects of risedronate on bone mineralization density distribution in triple transiliac crest biopsies of osteoporotic women were evaluated. In this double-blinded study, 3- and 5-year treatment with risedronate increased the degree and homogeneity of mineralization without producing hypermineralization. These changes at the material level of bone could contribute to risedronate's antifracture efficacy. INTRODUCTION: Risedronate, a nitrogen-containing bisphosphonate, is widely used in the treatment of osteoporosis. It reduces bone turnover, increases BMD, and decreases fracture risk. To date, there are no data available on the long-term effects of risedronate on bone mineralization density distribution (BMDD) in humans. MATERIALS AND METHODS: Osteoporotic women enrolled in the VERT-NA trial received either risedronate (5 mg/day, orally) or placebo for up to 5 years. All subjects received calcium and vitamin D supplementation if deficient at baseline. Triple iliac crest biopsies were collected from a subset of these subjects at baseline and 3 and 5 years. BMDD was measured in these biopsies using quantitative backscattered electron imaging, and the data were also compared with a normal reference group. RESULTS: At baseline, both risedronate and placebo groups had a lower degree and a greater heterogeneity of mineralization as well as an increase in low mineralized bone compared with the normal reference group. The degree of mineralization increased significantly in the risedronate as well as in the placebo group after 3- and 5-year treatment compared with baseline. However, the degree of mineralization did not exceed that of normal. Three-year treatment with risedronate significantly increased the homogeneity of mineralization and slightly decreased low mineralized bone compared with placebo. Surprisingly with 5-year risedronate treatment, heterogeneity of mineralization increased compared with 3-year treatment, which might indicate an increase in newly formed bone. CONCLUSIONS: Long-term treatment with risedronate affects the homogeneity and degree of mineralization without inducing hypermineralization of the bone matrix. These changes at the material level of the bone matrix may contribute to risedronate's antifracture efficacy in osteoporotic patients.
Asunto(s)
Conservadores de la Densidad Ósea/administración & dosificación , Densidad Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Ácido Etidrónico/análogos & derivados , Fracturas Óseas/prevención & control , Osteoporosis Posmenopáusica/tratamiento farmacológico , Biopsia , Ácido Etidrónico/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Osteoporosis Posmenopáusica/patología , Ácido Risedrónico , Factores de Riesgo , Factores de TiempoRESUMEN
UNLABELLED: Long-term effects of risedronate on bone mineral maturity/crystallinity and collagen cross-link ratio in triple iliac crest biopsies of osteoporotic women were evaluated. In this double-blinded study, 3- and 5-year treatment with risedronate arrested the tissue aging encountered in untreated osteoporosis and in osteoporosis treated with other antiresorptives. This effect may be contributing to risedronate's antifracture efficacy. INTRODUCTION: Risedronate is widely used in the treatment of osteoporosis. It reduces bone turnover, increases BMD, and decreases fracture risk. To date, there are no data available on the long-term effects of risedronate on bone material properties in humans. MATERIALS AND METHODS: Osteoporotic women enrolled in the VERT-NA trial received either risedronate (5 mg/day, orally) or placebo for up to 5 years. All subjects received calcium. They also received vitamin D supplementation if deficient at baseline. Triple iliac crest biopsies were collected from a subset of these subjects at baseline, 3 years, and 5 years. Mineral maturity/crystallinity and collagen cross-link ratio was measured in these biopsies using Fourier transform infrared imaging. RESULTS: Patients that received placebo exhibited increased mineral maturity/crystallinity and collagen cross-link ratio after 3 and 5 years compared with baseline values. On the contrary, patients that received risedronate retained baseline values in both bone material indices throughout. A more spatially detailed analysis revealed that this was achieved mainly through beneficial effects on active bone-forming areas. Surprisingly, patients that received risedronate achieved premenopausal values at bone-forming areas in both indices after 5 years of treatment. CONCLUSION: Long-term treatment with risedronate affects bone material properties (mineral maturity/crystallinity and collagen cross-link ratio) and arrests the tissue aging apparent in untreated osteoporosis. These changes at the material level of the bone matrix may contribute to risedronate's rapid and sustained antifracture efficacy in osteoporotic patients.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Densidad Ósea/efectos de los fármacos , Ácido Etidrónico/análogos & derivados , Ilion/patología , Biopsia , Conservadores de la Densidad Ósea/administración & dosificación , Calcificación Fisiológica/efectos de los fármacos , Calcio/uso terapéutico , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Ácido Etidrónico/administración & dosificación , Ácido Etidrónico/uso terapéutico , Femenino , Humanos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Ácido Risedrónico , Espectroscopía Infrarroja por Transformada de Fourier , Vitamina D/uso terapéuticoRESUMEN
One year of treatment with bisphosphonates at 5x the dose used for post-menopausal osteoporosis significantly increases failure load and microdamage, and decreases toughness at multiple skeletal sites in intact female beagles. The goal of this study was to determine if similar changes occur with doses equivalent to those used for post-menopausal osteoporosis treatment. Skeletally-mature female beagles were treated daily for 1 year with vehicle (VEH) or one of three doses of risedronate (RIS; 0.05, 0.10, 0.50 mg/kg/day) or alendronate (ALN; 0.10, 0.20, 1.00 mg/kg/day). Doses of ALN corresponded to treatment dose for PMO, 1/2 that dose, and 5x that dose on a mg/kg basis; RIS was given at a dose-equivalent to ALN. Vertebral density, geometry, percent ash, static/dynamic histology, microdamage, and biomechanical parameters were quantified. Trabecular bone activation frequency (Ac.f) was dose-dependently lower in RIS-treated groups (-40%, -66%, -84%, P < 0.05 vs. VEH) while the three ALN groups were all similarly lower compared to VEH (-65%, -71%, -76%; P <0.05). Crack surface density (Cr.S.Dn) was significantly higher than VEH for all doses of RIS and ALN (+2.9 to 5.4-fold vs. VEH). Stiffness was significantly increased with both agents while there were no significant changes in any other structural or estimated material properties. Cr.S.Dn and Ac.f exhibited a significant non-linear correlation (r(2) = 0.21; P < 0.001) while there was no relationship between Cr.S.Dn and any mechanical properties. These results document that 1 year of bisphosphonate treatment at clinical doses allows significant accumulation of microdamage in the vertebra but this is offset by increases in bone volume and mineralization such that there is no significant impairment of mechanical properties.
Asunto(s)
Alendronato/farmacología , Huesos/efectos de los fármacos , Ácido Etidrónico/análogos & derivados , Alendronato/administración & dosificación , Animales , Fenómenos Biomecánicos , Densidad Ósea/efectos de los fármacos , Huesos/patología , Huesos/fisiopatología , Calcificación Fisiológica/efectos de los fármacos , Difosfonatos/administración & dosificación , Difosfonatos/farmacología , Perros , Relación Dosis-Respuesta a Droga , Ácido Etidrónico/administración & dosificación , Ácido Etidrónico/farmacología , Femenino , Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/patología , Vértebras Lumbares/fisiopatología , Ácido Risedrónico , Factores de TiempoRESUMEN
Recent studies demonstrate that bisphosphonates suppress bone resorption by leading to apoptosis of the osteoclast and inhibiting the differentiation to mature osteoclasts. The influence of bisphosphonates on bone formation is unknown, although it has been hypothesized that bisphosphonates inhibit osteoblast apoptosis and stimulate osteoblast proliferation and differentiation in vitro, leading to increased bone formation. The purpose of this study was to investigate the effect of bisphosphonates on bone formation. We administered risedronate at 0.05, 0.5 or 5.0 microg/kg/day or alendronate at 0.1, 1.0 or 10 microg/kg/day subcutaneously for 17 days to 6-month-old female Sprague-Dawley rats. Control rats were given a daily subcutaneous injection of saline. Following sacrifice, the femoral and tibial mid-diaphyses were harvested and mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/BS) were measured on periosteal and endocortical surfaces. In the femur, periosteal MAR was significantly lower in all treatment groups (22-29% for risedronate, 26-36% for alendronate) than in control. In the tibia, periosteal MAR and BFR of all treatment groups were significantly lower (41-50% for risedronate, 43-52% for alendronate) than in the control group. Because the periosteal surfaces of these bones are only undergoing bone formation in modeling mode, our results show that bisphosphonates suppress bone formation independently of bone resorption. Because this effect is seen on periosteal MAR rather than on periosteal MS/BS, we hypothesize that bisphosphonates affect the activity of individual osteoblasts at the cell level. This may help to explain the reason that the anabolic effects of teriparatide are blunted when administered concurrently with or following a course of bisphosphonates in humans.
Asunto(s)
Difosfonatos/farmacología , Fémur/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Tibia/efectos de los fármacos , Alendronato/administración & dosificación , Alendronato/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Conservadores de la Densidad Ósea/administración & dosificación , Conservadores de la Densidad Ósea/farmacología , Remodelación Ósea/fisiología , Resorción Ósea/fisiopatología , Difosfonatos/administración & dosificación , Ácido Etidrónico/análogos & derivados , Ácido Etidrónico/farmacología , Femenino , Fémur/citología , Fémur/metabolismo , Inyecciones Subcutáneas , Osteoblastos/citología , Osteoblastos/metabolismo , Ratas , Ratas Sprague-Dawley , Ácido Risedrónico , Estrés Mecánico , Tibia/citología , Tibia/metabolismoRESUMEN
The primary goal of our study was to determine changes in bone mineralization in postmenopausal osteoporotic women treated for 3 years with risedronate or placebo. A secondary goal was to determine the relationship between mineralization and indices of bone turnover measured on the same biopsies. The degree of mineralization was measured by micro-computed tomography using Synchrotron radiation (Synchrotron microCT) in the trabecular bone of paired transiliac biopsies taken at baseline and after 3 years of treatment from patients receiving risedronate 5 mg daily (n=11) or placebo (n=8). In the risedronate-treated patients, the average mineralization (Avg-MIN) and peak mineralization (Peak-MIN) at 3 years were significantly increased from baseline by 4.7% (P<0.0001) and 5.4% (P=0.0003), respectively and showed significant negative correlation to turnover indices. In the placebo-treated patients, the increases in Avg-MIN (2.0%) and Peak-MIN (1.6%) were not significantly different from baseline and correlation to turnover indices was weaker. Risedronate significantly reduced the ratio of low- to high-mineralized bone fractions estimated by volume (BMR-V) and surface area (BMR-S) by 70.1% and 54.1%, respectively from baseline. These changes were consistent with the significant reduction of turnover from baseline assessed by reductions in mineralizing surface, MS/BS (-72.8%); activation frequency, Ac.F (-60.4%); and bone formation rate, BFR-BV (-63.6%) in the same biopsies in the risedronate-treated patients. Comparing the pair-wise changes from baseline, risedronate significantly reduced the low-mineralized bone fraction in comparison to placebo, as indicated by a larger reduction of BMR-V (P=0.015) and BMR-S (P=0.035). In the risedronate group, BMR-V and BMR-S showed significant positive correlation to MS/BS (R2: 0.83 and 0.92, respectively). The correlations to Ac.F and BFR-BV were also significant, with BMR-S showing a strong relation (R2: 0.77 and 0.79, respectively). The data suggest that BMR-V and BMR-S are markers of turnover of trabecular bone and may be used to assess treatment effect on turnover in bone biopsies. The results demonstrate that the reduction of turnover by risedronate increased the degree of mineralization and reduced the ratio of low- to high-mineralized bone fractions which may increase bone's resistance to fracture.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Ácido Etidrónico/análogos & derivados , Osteoporosis Posmenopáusica/tratamiento farmacológico , Tomografía Computarizada por Rayos X/métodos , Anciano , Biopsia , Densidad Ósea , Conservadores de la Densidad Ósea/farmacología , Conservadores de la Densidad Ósea/uso terapéutico , Interpretación Estadística de Datos , Ácido Etidrónico/farmacología , Ácido Etidrónico/uso terapéutico , Femenino , Humanos , Ilion/efectos de los fármacos , Ilion/patología , Vértebras Lumbares/química , Persona de Mediana Edad , Osteoporosis Posmenopáusica/patología , Ácido Risedrónico , Sincrotrones , Factores de Tiempo , Tomografía Computarizada por Rayos X/instrumentación , Resultado del TratamientoRESUMEN
Bisphosphonates inhibit bone loss through inhibition of osteoclast-mediated bone resorption. At low doses, vitamin D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphosphonates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol), alone and in combination, for the prevention of ovariectomy-induced bone loss in rats. One hundred ten female 4-month-old Sprague-Dawley rats were used for this experiment. Ninety rats were bilaterally ovariectomized (OVX), 10 rats were sham-operated (SHAM), and 10 rats were killed at the time of surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of risedronate or 0.05 microg or 0.1 microg of calcitriol/kg body weight, alone and in combination. Both compounds were administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency-induced bone loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedronate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in calcitriol-treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphosphonates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs alone.
Asunto(s)
Calcitriol/farmacología , Ácido Etidrónico/análogos & derivados , Ácido Etidrónico/farmacología , Osteoporosis/prevención & control , Animales , Fenómenos Biomecánicos , Peso Corporal , Densidad Ósea , Calcitriol/administración & dosificación , Ácido Etidrónico/administración & dosificación , Femenino , Ovariectomía , Hormona Paratiroidea/sangre , Ratas , Ratas Sprague-Dawley , Ácido RisedrónicoRESUMEN
Risedronate reduces the risk of new vertebral fractures up to 70% within 1 year of treatment in patients with osteoporosis. Both increases in bone mass and preservation of bone architecture are thought to contribute to antifracture effects. Our objectives were to determine the effects of risedronate on trabecular bone mass and architecture and to determine the relative contributions of mass and architecture to strength in the vertebra of ovariectomized (OVX) minipigs. The minipigs were OVX at 18 months of age and were treated daily for 18 months with either vehicle or risedronate at doses of 0.5 mg/kg per day or 2.5 mg/kg per day. The three-dimensional (3D) bone architecture of the L4 vertebral cores of Sinclair S1 minipigs was evaluated by 3D microcomputed tomography (muCT). Compared with the OVX control, the vertebral bone volume (bone volume/tissue volume [BV/TV]) was higher in both treated groups (p < 0.05). The architectural changes were more significant at the 2.5-mg/kg dose and were more prevalent at the cranial-caudal ends compared with the midsection. At the higher dose, the trabecular thickness (Tb.Th), trabecular number (Tb.N), and connectivity were higher, and marrow star volume (Ma.St.V) and trabecular separation (Tb.Sp) were lower (p < 0.05). The trabecular separation variation index (TSVI), a new measure to approximate structural variations, was smaller in the 2.5-mg/kg-treated group (p < 0.05). In this group, a significant preservation of trabeculae orthogonal to the cranial-caudal axis was confirmed by a decrease in the degree of anisotropy (DA) and an increase in the percent Cross-strut (% Cross-strut; p < 0.05). Both normalized maximum load (strength) and normalized stiffness of the same vertebral cores were higher in the 2.5-mg/kg risedronate group compared with the OVX group (p < 0.05). BV/TV alone could explain 76% of the variability of the bone strength. The combination of bone volume and architectural variables explained >90% of the strength. The study showed that risedronate preserved trabecular architecture in the vertebra of OVX minipigs, and that bone strength is tightly coupled to bone mass and architecture.