RESUMEN
Liposomal fasudil as a treatment for cerebral ischemia/reperfusion (I/R) injury has been demonstrated to be effective in animal models due to the high accumulation of liposomes in damaged brain tissue. However, it is still unclear what effect drug release rate has on the treatment of I/R injury, where pathology progresses dramatically in a short time. In the present study, we assessed four formulations of liposomal fasudil. The results of an in vitro drug release assay showed that the release properties of fasudil were changed by varying the lipid composition and internal phase of the liposomes. Based on these results, differences in the transition of fasudil plasma concentration were monitored after the administration of each type of liposomal fasudil in normal rats. A pharmacokinetic study showed that higher levels of drug retention in liposomal fasudil resulted in higher fasudil plasma concentration. Finally, treatment of I/R injury model rats with liposomal fasudil revealed that a mid-level release rate of fasudil from liposomes resulted in the greatest therapeutic effect among the formulations. In conclusion, these results demonstrate that an optimized drug release rate from liposomes enhances the therapeutic effect of fasudil for the treatment of cerebral I/R injury.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Liposomas/química , Daño por Reperfusión/tratamiento farmacológico , 1,2-Dipalmitoilfosfatidilcolina/química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Sulfato de Amonio/química , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Ácido Cítrico/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Composición de Medicamentos , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Liposomas/farmacocinética , Masculino , Fosfatidilcolinas/química , Compuestos de Amonio Cuaternario/química , Ratas Wistar , Daño por Reperfusión/patología , Resultado del TratamientoRESUMEN
Fasudil hydrochloride as an intracellular calcium ion antagonist that dilates blood vessels has exhibited a very potent pharmacological effect in the treatment of angina pectoris. The purpose of this study was to determine the absorption, distribution, and excretion profiles of fasudil in rats and beagle dogs, respectively, to clarify its pharmacokinetic pattern. A sensitive and reliable LC-MS/MS method has been developed and established and successfully applied to pharmacokinetic study, including absorption, tissue distribution, and excretion. The results revealed that in the range of 2-6 mg/kg, the pharmacokinetic behavior for instance, AUC and Cmax , in rats was observed in a dose dependent manner. However, the plasma concentrations were indicative of a significant gender difference in the pharmacokinetics of fasudil in rats, in terms of absolute bioavailability and excretion. Interestingly, the resulting data obtained from beagle dogs showed that there was no gender difference in the absolute bioavailability of fasudil hydrochloride after single or repeated administrations. In conclusion, this study characterized the pharmacokinetic pattern fasudil both in rats and beagle dogs through absorption, tissue distribution and excretion study. The findings may be valuable and provide a rationale for further study and its safe use in clinical practice.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Inhibidores de Proteínas Quinasas/farmacocinética , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/orina , Animales , Área Bajo la Curva , Bilis/química , Cromatografía Liquida , Perros , Heces/química , Femenino , Masculino , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/orina , Ratas Wistar , Espectrometría de Masas en Tándem , Distribución TisularRESUMEN
Dysfunction of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) is a primary characteristic of multiple sclerosis (MS). We evaluated the protective effects of fasudil, a selective ROCK inhibitor, in a model of experimental autoimmune encephalomyelitis (EAE) that was induced by guinea-pig spinal cord. In addition, we studied the effects of fasudil on BBB and BSCB permeability. We found that fasudil partly alleviated EAE-dependent damage by decreasing BBB and BSCB permeability. These results provide rationale for the development of selective inhibitors of Rho kinase as a novel therapy for MS.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Barrera Hematoencefálica/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/prevención & control , Encefalomielitis Autoinmune Experimental/fisiopatología , Médula Espinal/irrigación sanguínea , Médula Espinal/inmunología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/fisiopatología , Permeabilidad de la Membrana Celular/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/fisiopatología , Esclerosis Múltiple/prevención & control , Ocludina , Proyectos Piloto , Inhibidores de Proteínas Quinasas/farmacología , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Médula Espinal/enzimología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/biosíntesisRESUMEN
Rho kinase (ROCK), one of the serine/threonine kinases, is involved in pathologic conditions, and its activation causes neuronal cell death. Fasudil, a selective ROCK inhibitor, has been reported to cause increased cerebral blood flow (CBF) in the ischemic brain and protect against neuronal cell death by inhibiting ROCK. Ozagrel, a thromboxane A(2) synthase inhibitor, inhibits platelet aggregation and causes vasodilatation, thereby increasing CBF in cerebral thrombosis. The present study evaluates the combination therapy of fasudil and ozagrel on focal brain ischemia induced by middle cerebral artery occlusion (MCAO) in mice. Each monotherapy of fasudil at 10 mg/kg i.p. and ozagrel at 30 mg/kg i.p. significantly reduced cerebral infarction. The combination therapy of fasudil (3 mg/kg i.p.) and ozagrel (10 mg/kg i.p.), which are noneffective doses, resulted in reduction of cerebral infarction, and the protective effect was observed up to 5 min, but not 3 h, after reperfusion. Regional CBF after MCAO and phosphorylation of endothelial nitric-oxide synthase (NOS) significantly increased in response to the combination therapy, whereas these effects were not observed with monotherapy of either drug. The protective effect of combination treatment was antagonized by the treatment of a NOS inhibitor, nitro-l-arginine methyl ester hydrochloride. These findings indicate that the combination treatment of fasudil and ozagrel exhibits additive effects for neuroprotection after MCAO. These findings indicate that the combination treatment of fasudil and ozagrel may be useful as a potential therapeutic strategy for the treatment of stroke.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Infarto Cerebral/prevención & control , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Metacrilatos/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , Animales , Células Cultivadas , Infarto Cerebral/sangre , Infarto Cerebral/etiología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Metacrilatos/metabolismo , Ratones , Distribución AleatoriaRESUMEN
A fast and sensitive method to quantify fasudil hydrochloride (FH) and its active metabolite hydroxyfasudil (M3) in human plasma using HPLC-MS/MS has been developed and validated in present study. The method involved simple sample preparation with methanol as protein precipitation (3:1, v/v) and ranitidine as an internal standard (IS). The analytes and IS were separated using a gradient elution procedure on the analytical column ZORBAX StableBond-C18 (5 microm, 150 mm x 4.6mm). Detection was performed by an AB 3200 QTRAP tandem mass spectrometer equipped with a Turbo IonSpray ionization source set in positive ion mode. Multiple reaction monitoring (MRM) using the precursor to product ion was m/z 292.2/99.2 for fasudil, m/z 308.2/99.2 for M3 and m/z for 315.3/176.2 for IS. The linear range of the method was from 0.4 to 250 ng/mL for both fasudil and M3. The lower limit of quantification was 0.4 ng/mL for both fasudil and M3. The intra- and inter-day relative standard deviation over the entire concentration range was less than 7.11% for fasudil and 10.6% for M3, respectively. The validated method was successfully applied for the evaluation of pharmacokinetic of fasudil hydrochloride after administration of 30 mg fasudil hydrochloride by continuous intravenous infusion over 30 min in 12 healthy Chinese volunteers.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Cromatografía Liquida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , Femenino , Humanos , Masculino , Estructura Molecular , Control de Calidad , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/métodos , Factores de TiempoRESUMEN
The neuroprotective potential and therapeutic time window for fasudil, a Rho-kinase inhibitor (RKI), were evaluated for delayed neuronal death in gerbils. A preliminary screening was done on fasudil, ozagrel, and edaravone using a single administration in a delayed neuronal death study. Intraperitoneal (i.p.) administration of edaravone, a free radical scavenger (3, 10 mg/kg) immediately after re-circulation did not reduce neuronal degeneration. We previously reported that ozagrel, a thromboxane A(2) synthetase inhibitor (30 mg/kg) also did not reduce neuronal degeneration, while fasudil (3, 30 mg/kg) significantly protected against the ischemia-induced neuronal loss. To clarify the therapeutic time window of fasudil, which showed a positive effect in a preliminary screening, animals received their first i.p. administration of fasudil (10 mg/kg) 24 or 48 h after ischemia. Administration of fasudil twice daily was continued until day 6. Fasudil significantly protected against the ischemia-induced delayed neuronal death when the treatment was started 24 h after ischemia. In gerbils, hydroxyfasudil, an active metabolite of fasudil, was found following an i.p. administration of fasudil (10 mg/kg), and the value of the area under the plasma level curve of hydroxyfasudil was 7 times higher than that of fasudil. Hydroxyfasudil may contribute to the potency of fasudil. The present findings indicate that the RKI fasudil reduces ischemic neuronal damage with a wide therapeutic time window in gerbil, and may be useful in the treatment of acute ischemic stroke in humans.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Isquemia , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Antipirina/análogos & derivados , Antipirina/uso terapéutico , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Edaravona , Depuradores de Radicales Libres/uso terapéutico , Gerbillinae , Isquemia/sangre , Isquemia/complicaciones , Isquemia/tratamiento farmacológico , Isquemia/patología , Masculino , Neuronas/patología , Fármacos Neuroprotectores/sangre , Fármacos Neuroprotectores/farmacocinética , Factores de TiempoRESUMEN
BACKGROUND: Rho-kinase has been implicated as an important regulator of inflammatory responses mediated by cytokines and chemokines. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined whether long-term blockade of Rho-kinase suppresses LV remodeling in a mouse model of MI in vivo. METHODS AND RESULTS: Mice underwent ligation of the left coronary artery and were treated with a Rho-kinase inhibitor, fasudil (100 mg x kg(-1) x d(-1) in tap water), for 4 weeks, starting 1 day after the surgery. At 4 weeks, LV infarct size was histologically comparable between the 2 groups. LV cavity dilatation and dysfunction evaluated by echocardiography were significantly suppressed in the fasudil group (P<0.05, n=15 to 28). The beneficial effects of fasudil were accompanied by suppression of cardiomyocyte hypertrophy and interstitial fibrosis (both P<0.01, n=6). The expression of inflammatory cytokines, including transforming growth factor (TGF)-beta2, TGF-beta3, and macrophage migration inhibitory factor, was upregulated in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (both P<0.05, n=10 to 11). Rho-kinase activity as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, was significantly increased in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (P<0.05, n=5). CONCLUSIONS: These results indicate that Rho-kinase is substantially involved in the pathogenesis of LV remodeling after MI associated with upregulation of proinflammatory cytokines, suggesting a therapeutic importance of the molecule for the prevention of post-MI heart failure.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Hipertrofia Ventricular Izquierda/prevención & control , Infarto del Miocardio/tratamiento farmacológico , Disfunción Ventricular Izquierda/prevención & control , Remodelación Ventricular/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Proteínas de Unión al ADN/metabolismo , Esquema de Medicación , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Oxidorreductasas Intramoleculares , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Factores Inhibidores de la Migración de Macrófagos/genética , Masculino , Ratones , Infarto del Miocardio/complicaciones , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Tamaño de los Órganos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta2 , Factor de Crecimiento Transformador beta3 , UltrasonografíaRESUMEN
Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-kinase with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Inhibidores Enzimáticos/farmacología , Hipertensión Pulmonar/prevención & control , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/uso terapéutico , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/mortalidad , Hipertrofia Ventricular Derecha/fisiopatología , Hipertrofia Ventricular Derecha/prevención & control , Péptidos y Proteínas de Señalización Intracelular , Masculino , Monocrotalina , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Tasa de Supervivencia , Factores de Tiempo , Quinasas Asociadas a rhoRESUMEN
To date, the pharmacological approach to cerebral vasospasm following subarachnoid hemorrhage has been hampered in part by an inability to attain sufficiently high concentrations of vasodilator drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of protein kinase inhibitor Fasudil. Cerebral vasospasm in rats was induced by double-injection method. Treated rats received 0.417 mg liposome-entrapped Fasudil via the cisterna magna and control rats received drug-free liposomes in the same manner. The diameter of the basilar artery was assessed at 7 days after the initial blood injection. Vasoconstriction of the rat basilar artery was significantly reduced in group treated with liposomal Fasudil compared to the control group (treated group: 87.7 +/- 6.18%, n= 10; control group: 66.3 +/- 9.82%, n = 10; ***P< 0.001). This new approach for cerebral vasospasm may have significant potential for use in the clinical setting.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , Hemorragia Subaracnoidea/complicaciones , Vasodilatadores/farmacocinética , Vasoespasmo Intracraneal/tratamiento farmacológico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/líquido cefalorraquídeo , Animales , Arteria Basilar/efectos de los fármacos , Arteria Basilar/fisiología , Sistemas de Liberación de Medicamentos/métodos , Inyecciones Espinales , Liposomas/farmacocinética , Masculino , Ratas , Ratas Sprague-Dawley , Vasoconstricción/efectos de los fármacos , Vasodilatadores/sangre , Vasodilatadores/líquido cefalorraquídeo , Vasoespasmo Intracraneal/etiología , Vasoespasmo Intracraneal/prevención & controlRESUMEN
Long-term inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces coronary vascular remodeling in rats. To determine the pathogenic mechanism involved in vascular remodeling, we examined the effects of fasudil, a Rho-kinase inhibitor, on vascular lesion formation. In rats treated with L-NAME at 10 mg/kg/day, vascular remodeling was evident in both large and small coronary arteries at the fourth week. Fasudil (3 mg/kg, p.o., twice daily) markedly prevented the development of vascular remodeling in small coronary arteries. Coronary flow was measured in Langendorff perfused isolated heart preparations. Long-term treatment with L-NAME caused a significant decrease in coronary flow, which was significantly inhibited by fasudil. Fasudil suppressed the structural and functional changes in coronary arteries by chronic blockade of NO synthesis. Thus, the Rho-kinase pathway may be substantially involved in the pathogenesis of vascular remodeling in this rat model.