RESUMEN
Monocytes, macrophages, and inflammation play a key role in the process of neointimal proliferation and restenosis. The present study evaluated whether systemic and transient depletion of monocytes could be obtained by a single intravenous (IV) injection of simvastatin liposomes, for the inhibition of neointima formation. Balloon-injured carotid artery rats (n = 30) were randomly assigned to treatment groups of free simvastatin, simvastatin in liposomes (3 mg/kg), and saline (control). Stenosis and neointima to media ratio (N/M) were determined 14 days following single IV injection at the time of injury by morphometric analysis. Depletion of circulating monocytes was determined by flow cytometry analyzes of blood specimens. Inhibition of RAW264.7, J774, and THP-1 proliferation by simvastatin-loaded liposomes and free simvastatin was determined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide assay. Simvastatin liposomes were successfully formulated and were found to be 1.5-2 times more potent than the free drug in suppressing the proliferation of monocytes/macrophages in cell cultures of RAW 264.7, J774, and THP-1. IV injection of liposomal simvastatin to carotid-injured rats (3 mg/kg, n = 4) resulted in a transient depletion of circulating monocytes, significantly more prolonged than that observed following treatment with free simvastatin. Administration to balloon-injured rats suppressed neointimal growth. N/M at 14 days was 1.56 +/- 0.16 and 0.90 +/- 0.12, control and simvastatin liposomes, respectively. One single systemic administration of liposomal simvastatin at the time of injury significantly suppresses neointimal formation in the rat model of restenosis, mediated via a partial and transient depletion of circulating monocytes.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hiperplasia/tratamiento farmacológico , Simvastatina/administración & dosificación , Simvastatina/uso terapéutico , Animales , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Traumatismos de las Arterias Carótidas/patología , Cateterismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Química Farmacéutica , Sistemas de Liberación de Medicamentos , Oclusión de Injerto Vascular/tratamiento farmacológico , Oclusión de Injerto Vascular/prevención & control , Liposomas , Masculino , Monocitos/efectos de los fármacos , RatasRESUMEN
Liposomes containing bisphosphonates have been shown to deplete circulating monocytes and reduce experimental restenosis. However, acceptable shelf life was not achieved, and the disruption extent and rate of the vesicles in the circulation has not been examined. Designing an optimal liposomal formulation in general, and for an anti-inflammatory effect in particular, requires careful consideration of the factors that contribute to their in vitro stability and integrity in the blood after injection. An improved liposomal alendronate formulation was prepared by a modified thin lipid film hydration technique followed by extrusion, resulting in relatively smaller size vesicles, narrow size distribution, and low drug to lipid ratio in comparison to the reverse phase evaporation method. In order to rule out premature leakage of the drug, the integrity of the vesicles was examined by means of size-exclusion chromatography in vitro and in vivo, with subsequent analysis of size, drug (fractions of encapsulated and free) and lipid concentrations. Vesicles were found to be stable in serum, with 15 +/- 3% leakage of the drug after 10 min in rabbit's circulation, and intact liposomes were detected in the circulation 24 h following administration. It is concluded that the new formulation results in increased stability (2.5 years) as determined by the insignificant changes in vesicle size, drug leakage, lipid and drug stability, in vitro bioactivity (macrophages inhibition), as well as in vivo in depleting circulating monocytes and inhibition of restenosis in rabbits. Our in vitro stability results regarding dilution in serum paralleled in vivo data. Thus, in vitro assessment may provide a valuable tool in assessing in vivo integrity of liposomal formulations.
Asunto(s)
Alendronato/farmacología , Liposomas , Monocitos/fisiología , Alendronato/sangre , Animales , Antiinflamatorios/farmacología , Conservadores de la Densidad Ósea/farmacología , División Celular/efectos de los fármacos , Línea Celular , Estabilidad de Medicamentos , Liposomas/sangre , Liposomas/química , Macrófagos , Ratones , Monocitos/efectos de los fármacos , FosfatidilglicerolesRESUMEN
Although drug-eluting stents (DES) are successfully utilized for restenosis therapy, the development of local and systemic therapeutic means including nanoparticles (NP) continues. Lack of correlation between in vitro and in vivo studies is one of the major drawbacks in developing new drug delivery systems. The present study was designed to examine the applicability of the arterial explant outgrowth model, and of smooth muscle cells (SMC) cultures for prescreening of possible drugs. Elucidation of different species sensitivity (rat, rabbit, porcine and human) to diverse drugs (tyrphostins, heparin and bisphsophonates) and a delivery system (nanoparticles) could provide a valuable screening tool for further in vivo studies. The anticipated sensitivity ranking from the explant outgrowth model and SMC mitotic rates (porcine>rat>>rabbit>human) do not correlate with the observed relative sensitivity of those animals to antiproliferative therapy in restenosis models (rat>/=rabbit>porcine>human). Similarly, the inhibitory profile of the various antirestenotic drugs in SMC cultures (rabbit>porcine>rat>>human) do not correlate with animal studies, the rabbit- and porcine-derived SMC being highly sensitive. The validity of in vitro culture studies for the screening of controlled release delivery systems such as nanoparticles is limited. It is suggested that prescreening studies of possible drug candidates for restenosis therapy should include both SMC cell cultures of rat and human, appropriately designed with a suitable serum.
RESUMEN
Many drugs are not able to enter the brain due to the presence of the blood-brain barrier (BBB) and therefore cannot be used in the treatment of diseases of the brain. Since it is now known that the brain is under immunological surveillance, we hypothesized that phagocytic cells of the innate immune system, mainly neutrophils and monocytes, can be exploited as transporters of drugs to the brain. To target circulating mononuclear phagocytic cells, negatively-charged nano-sized liposomes were formulated encapsulating serotonin, a BBB impermeable neurological drug. Brain uptake, biodistribution, and the mechanism of brain transport were examined in vitro and in rats and rabbits by utilizing double-radiolabeled (3)H (in the membrane) and (14)C-serotonin (in the core), and liposomes with fluorescent markers (membrane and core). The brain uptake of liposomal serotonin was significantly higher (0.138%+/-0.034 and 0.097%+/-0.011, vs. 0.068%+/-0.02 and 0.057%+/-0.01, 4 h and 24 h after IV administration in rats, serotonin liposomes and in solution, respectively). The same brain uptake of both empty and serotonin liposomes, the co-localization in the brain of both markers, and the unchanged ratio of (3)H:(14)C suggest that intact liposomes entered the brain. Since treatment of animals by liposomal alendronate resulted with inhibition of monocytes but not of neutrophils, and with no brain delivery, it is suggested that monocytes are the main transporters of liposomes to the brain.
Asunto(s)
Encéfalo/metabolismo , Liposomas/farmacocinética , Monocitos/metabolismo , Fagocitosis/fisiología , Serotonina/administración & dosificación , Animales , Química Encefálica , Movimiento Celular/fisiología , Colesterol/química , Sistemas de Liberación de Medicamentos/métodos , Endocitosis/fisiología , Humanos , Inyecciones Intravenosas , Liposomas/química , Liposomas/metabolismo , Masculino , Monocitos/citología , Monocitos/fisiología , Neutrófilos/citología , Neutrófilos/metabolismo , Neutrófilos/fisiología , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Conejos , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , Serotoninérgicos/administración & dosificación , Serotoninérgicos/metabolismo , Distribución TisularRESUMEN
Monocytes/macrophages play a pivotal role in the formation of neointinal hyperplasia following vascular injury. Transient depletion of circulating monocytes by particulate delivery systems containing bisphosphonates, such as alendronate, results in restenosis inhibition. We hypothesized that a self-suspendable nanoparticulate dosage form, with a minimum amount of expients, could be formulated by complexing the negatively charged alendronate with gallium or gadolinium. We further hypothesized that a synergistic biological effect could be obtained by nanosuspensions of alendronate with these counter ions. Nanosuspensions (150-250 nm) of alendronate-gallium and alendronate-gadolinium were successfully formulated with no additives except for the active agents and HCl for pH adjustment. Both nanosuspensions exhibited macrophage cell line growth inhibition in a dose-response relationship in comparison to the various agents in solution and in liposomes. A synergistic effect of the nanosuspensions was observed in the inhibition of raw264 macrophages, and in reducing IL-1beta and TNF-alpha secretion in cell culture. Single IV administration at the time of injury, of alendronate-gallium or alendronate-gadolinium nanosuspensions resulted in inhibition of neointimal hyperplasia and stenosis in the rat model of vascular injury. The results correlated with the significant reduction of circulating monocytes. The nanosuspensions possess the advantages of no additives for minimal provocation of side effects, and the potential of immunomodulating inflammatory disorders.
Asunto(s)
Alendronato/farmacología , Inhibidores de la Angiogénesis , Gadolinio/farmacología , Galio/farmacología , Oclusión de Injerto Vascular/prevención & control , Hiperplasia/prevención & control , Neovascularización Patológica/prevención & control , Túnica Íntima/crecimiento & desarrollo , Alendronato/administración & dosificación , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Citocinas/metabolismo , Portadores de Fármacos , Gadolinio/administración & dosificación , Galio/administración & dosificación , Liposomas , Masculino , Ratones , Monocitos/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Ratas , Espectrofotometría Atómica , Suspensiones , Túnica Íntima/efectos de los fármacosRESUMEN
The present study explored a novel strategy for attenuation of restenosis after arterial injury by a bisphosphonate encapsulated in polymeric nanoparticles (NP) for transient selective depletion of macrophages. A bisphosphonate (BP), 2-(2-Aminopyrimidino) ethyldiene-1,1-bisphosphonic acid betaine (ISA), was successfully formulated in 400 nm sized polylactide/glycolide-based NP with high yield (69%) and entrapment efficiency (60% w/w). ISA NP, but not blank NP or free ISA, exhibited specific and significant cytotoxic effect on macrophages-like RAW 264 cells, in a dose-dependent manner, with no inhibitory effect on the growth of smooth muscle cells (SMCs). Fluorescent pyrene-labeled NP were shown to be taken up by RAW 264 cells, but not by SMCs. Intravenously (i.v.) administered ISA NP (15 mg/kg, single dose on day-1) resulted in a significant attenuation of neointima to media area ratio (N/M) by 40% and stenosis by 45% 14 days after rat carotid injury, in comparison to animals treated with free ISA, buffer or blank NP. However, the effect was not preserved 30 days post injury, and an insignificant reduction of neointimal formation was observed. Neointimal hyperplasia was also significantly suppressed after subcutaneous (SC) injection of ISA NP (15 mg/kg, single dose on day-1), reducing both N/M and stenosis. Intraperitoneal (i.p.) injection of silica, a known selective toxin for macrophages, (1000 mg/kg), also resulted in a significant inhibition of N/M and stenosis, which further reinforces the cause-effect relationship of macrophage-inactivation and the prevention of neointima formation. Biocompatible and biodegradable NP loaded with ISA characterized by high colloidal stability, reproducible activity, and high drug entrapment warrant further consideration for restenosis therapy, and may be useful in other disease processes involving monocytes/macrophages.
Asunto(s)
Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Enfermedades de las Arterias Carótidas/patología , Difosfonatos/administración & dosificación , Nanoestructuras/química , Animales , Difosfonatos/química , Portadores de Fármacos/química , Hiperplasia/tratamiento farmacológico , Masculino , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Ratas , Resultado del TratamientoRESUMEN
Systemic transient depletion of monocytes and macrophages by liposome-encapsulated bisphosphonates (BPs), reduces neointimal formation in experimental restenosis. The aim of this study was to examine the antirestenotic effect of a polymeric nanoparticulate formulation containing the BP alendronate (ALN). The BP was successfully formulated in polylactide-co-glycolide (PLGA) nanoparticles (NP). ALN NP with negative charge, size of 223+/-64 nm, and high entrapment efficiency (55.1%) have been formulated. ALN NP exhibited a significant cytotoxic effect, in a dose-response relationship, on macrophage-like RAW264 cells in cell culture. Subcutaneously (SC) administrated ALN NP (1.5 mg/kg on days -1 and +6) resulted in a significant attenuation of neointima to media ratio (N/M) by 52.7% and stenosis by 39.7% 28 days after balloon injury in the hypercholesterolemic rabbit model. Moreover, a good correlation was found between macrophage abundance in the injured arteries and the extent of stenosis. ALN NP treatment resulted in the reduction of both interleukin-1beta and matrix metalloproteinases (2 and 9). It is concluded that a particulated dosage form of polymeric NP loaded with ALN reduce neointimal formation in vivo by systemic transient depletion of monocytes.
Asunto(s)
Alendronato/toxicidad , Constricción Patológica/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Nanoestructuras/química , Alendronato/administración & dosificación , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/patología , Inyecciones Subcutáneas , Ácido Láctico/química , Macrófagos/patología , Ratones , Monocitos/patología , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Conejos , Factores de TiempoRESUMEN
The actual number of particles in formulations of nanoparticles (NP) is of importance for quality assurance, comprehensive physicochemical characterization, and pharmacodynamics. Some calculation methods that have been previously employed are limited because they rely on several assumptions and are not applicable for certain preparations. Currently there are no validated experimental methods for determining the particle number-concentration (Nc) of liposomal and polymeric nanoparticulate preparations (<500 nm). This study examines a new empirical method for counting the number of particles in nanoparticulate formulations including drug-containing liposomes and polymeric NP. In the new method, suspended NP are nebulized to form aerosol droplets which are dried and counted using a scanning mobility particle sizer (SMPS). Experiments were conducted with three different preparations, empty liposomes (200 and 400 nm), drug-loaded liposomes (200 nm), and polymeric NP (150 nm). It was verified that no detrimental morphological or structural changes of the formulations have been induced by the SMPS technique, and that the obtained Nc values represent the original particles. It is concluded that nano-formulations with concentrations of up to 10(7) particles per 1 cm3 air, corresponding to approximately 10(12) particles per 1 ml solution, can be directly counted within the size range of 30-900 nm. The measured values are compared to newly developed theoretical calculations to assess the viability of these calculations.
Asunto(s)
Coloides/análisis , Liposomas/análisis , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Nanotubos/análisis , Polímeros/análisis , Ultrafiltración/métodos , Algoritmos , Coloides/química , Liposomas/química , Peso Molecular , Nanotecnología/métodos , Nanotubos/química , Tamaño de la Partícula , Polímeros/químicaRESUMEN
BACKGROUND: Innate immunity is of major importance in vascular repair. The present study evaluated whether systemic and transient depletion of monocytes and macrophages with liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation. METHODS AND RESULTS: Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment. Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting. Monocyte counts were reduced by >90% 24 to 48 hours after a single injection of liposomal alendronate, returning to basal levels at 6 days. This treatment significantly reduced intimal area at 28 days, from 3.88+/-0.93 to 2.08+/-0.58 and 2.16+/-0.62 mm2. Lumen area was increased from 2.87+/-0.44 to 3.57+/-0.65 and 3.45+/-0.58 mm2, and arterial stenosis was reduced from 58+/-11% to 37+/-8% and 38+/-7% in controls, rabbits treated with 3 mg/kg, and rabbits treated with 6 mg/kg, respectively (mean+/-SD, n=8 rabbits/group, P<0.01 for all 3 parameters). No drug-related adverse effects were observed. Reduction in neointimal formation was associated with reduced arterial macrophage infiltration and proliferation at 6 days and with an equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury. Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation. CONCLUSIONS: Systemic transient depletion of monocytes and macrophages, by a single liposomal bisphosphonates injection concurrent with injury, reduces in-stent neointimal formation and arterial stenosis in hypercholesterolemic rabbits.
Asunto(s)
Alendronato/farmacología , Oclusión de Injerto Vascular/prevención & control , Hiperplasia/prevención & control , Inmunidad Innata/efectos de los fármacos , Túnica Íntima/efectos de los fármacos , Alendronato/administración & dosificación , Animales , Recuento de Células , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Dieta Aterogénica , Modelos Animales de Enfermedad , Oclusión de Injerto Vascular/inmunología , Oclusión de Injerto Vascular/patología , Hiperplasia/inmunología , Hiperplasia/patología , Arteria Ilíaca/efectos de los fármacos , Arteria Ilíaca/lesiones , Arteria Ilíaca/patología , Recuento de Leucocitos , Liposomas , Macrófagos/efectos de los fármacos , Macrófagos/patología , Monocitos/efectos de los fármacos , Monocitos/patología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Conejos , Stents/efectos adversos , Túnica Íntima/inmunología , Túnica Íntima/patologíaRESUMEN
OBJECTIVES: Macrophage depletion by liposomal clodronate inhibits neointimal formation after balloon-injury. The present study examined bisphosphonates (BPs) potency-effect relationship and the role of systemic versus local monocytes in vascular repair. METHODS AND RESULTS: Liposomal preparations of clodronate, pamidronate, alendronate, and ISA-13-1 inhibited RAW-264 macrophages growth in a dose-response manner. Administration to balloon-injured rats suppressed neointimal growth. Neointima to media ratio (N/M) at 14 days was reduced from 1.35 +/- 0.22 (control) to 0.4 +/- 0.1 and 0.9 +/- 0.17 by liposomal alendronate (1.5 mg/kg, i.v.) and liposomal ISA-13-1 (15 mg/kg), respectively (n = 8-10, P < 0.05). Suppression of neointimal formation was preserved at 30 days. Subcutaneous administration of liposomal BP (LBP) was also effective in suppressing neointimal formation, while short local intraluminal application had no effect. Immunostaining for ED-1 and ED-2 revealed no resident macrophages in the arterial wall, and reduced macrophage infiltration in LBP-treated animals. Arterial PDGF-B chain and PDGF-beta receptor activation were reduced in LBP-treated animals and up-regulation of the PDGF receptor was noted. CONCLUSIONS: Systemic transient inactivation of monocytes and macrophages by LBPs reduced macrophage infiltration and neointimal formation in the rat carotid injury model. The findings demonstrate a BP potency-effect relationship, and highlight the role of circulating monocytes in vascular injury and repair.