RESUMEN
Hybrid compounds containing structural fragments of the Rho kinase inhibitor fasudil and the NRF2 inducers caffeic and ferulic acids were designed with the aid of docking and molecular mechanics studies. Following the synthesis of the compounds using a peptide-coupling methodology, they were characterized for their ROCK2 inhibition, radical scavenging, effects on cell viability (MTT assay), and NRF2 induction (luciferase assay). One of the compounds (1d) was selected in view of its good multitarget profile and good tolerability. It was able to induce the NRF2 signature, promoting the expression of the antioxidant response enzymes HO-1 and NQO1, via a KEAP1-dependent mechanism. Analysis of mRNA and protein levels of the NRF2 pathway showed that 1d induced the NRF2 signature in control and SOD1-ALS lymphoblasts but not in sALS, where it was already increased in the basal state. These results show the therapeutic potential of this compound, especially for ALS patients with a SOD1 mutation.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Ácidos Cumáricos/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/síntesis química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Anciano , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ácidos Cumáricos/síntesis química , Ácidos Cumáricos/toxicidad , Femenino , Depuradores de Radicales Libres/síntesis química , Depuradores de Radicales Libres/toxicidad , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/agonistas , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/toxicidad , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system characterized by oligodendrocyte loss and progressive neurodegeneration. The cuprizone (CPZ)-induced demyelination is widely used to investigate the demyelination/remyelination. Here, we explored the therapeutic effects of Hydroxyfasudil (HF), an active metabolite of Fasudil, in CPZ model. HF improved behavioral abnormality and reduced myelin damage in the corpus callosum. Splenic atrophy and myelin oligodendrocyte glycoprotein (MOG) antibody were observed in CPZ model, which were partially restored and obviously inhibited by HF, therefore reducing pathogenic binding of MOG antibody to oligodendrocytes. HF inhibited the percentages of CD4+IL-17+ T cells from splenocytes and infiltration of CD4+ T cells and CD68+ macrophages in the brain. HF also declined microglia-mediated neuroinflammation, and promoted the production of astrocyte-derived brain derived neurotrophic factor (BDNF) and regeneration of NG2+ oligodendrocyte precursor cells. These results provide potent evidence for the therapeutic effects of HF in CPZ-induced demyelination.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Enfermedades Desmielinizantes/tratamiento farmacológico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Linfocitos T CD4-Positivos/efectos de los fármacos , Cuprizona , Citocinas/inmunología , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/patologíaRESUMEN
This study investigates the respirability and efficacy of peptide-micelle hybrid nanoparticles as carriers for inhalational therapy of pulmonary arterial hypertension (PAH). CARSKNKDC (CAR), a cell-penetrating and lung-homing peptide, conjugated polyethylene glycol-distearoyl-phosphoethanolamine micelles containing fasudil, an investigational anti-PAH drug, were prepared by solvent evaporation method and characterized for various physicochemical properties. The pharmacokinetics and pharmacological efficacy of hybrid particles containing fasudil were evaluated in healthy rats and monocrotaline-induced PAH rats. CAR micelles containing fasudil had an entrapment efficiency of approximately 58%, showed controlled release of the drug, and were monodispersed with an average size of approximately 14 nm. Nuclear magnetic resonance scan confirmed the drug's presence in the core of peptide-micelle hybrid particles. Compared with plain micelles, CAR peptide increased the cellular uptake by approximately 1.7-fold and extended the drug half-life by approximately fivefold. The formulations were more prone to accumulate in the pulmonary vasculature than in the peripheral blood, which is evident from the ratio of the extent of reduction of pulmonary and systemic arterial pressures. On the whole, this study demonstrates that peptide-polymer hybrid micelles can serve as inhalational carriers for PAH therapy.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Antihipertensivos/administración & dosificación , Presión Arterial/efectos de los fármacos , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos , Etanolaminas/química , Hipertensión Pulmonar/tratamiento farmacológico , Oligopéptidos/metabolismo , Polietilenglicoles/química , Arteria Pulmonar/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Administración por Inhalación , Animales , Antihipertensivos/química , Antihipertensivos/farmacocinética , Antihipertensivos/toxicidad , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Arteriolas/fisiopatología , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/toxicidad , Química Farmacéutica , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Etanolaminas/toxicidad , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Inyecciones Intravenosas , Espectroscopía de Resonancia Magnética , Masculino , Micelas , Monocrotalina , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Oligopéptidos/toxicidad , Tamaño de la Partícula , Polietilenglicoles/toxicidad , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Ratas Sprague-Dawley , Solubilidad , Tecnología Farmacéutica/métodosRESUMEN
The cytokine erythropoietin (Epo) initiates adaptive cellular responses to both moderate environmental challenges and tissue damaging insults in various non-hematopoietic mammalian tissues including the nervous system. Neuroprotective and neuroregenerative functions of Epo in mammals are mediated through receptor-associated Janus kinase 2 and intracellular signaling cascades that modify the transcription of Epo-regulated genes. Signal transducers and activators of transcription (STAT) and phosphoinositol-3-kinase (PI3K) represent key components of two important Epo-induced transduction pathways. Our previous study on insects revealed neuroprotective and regenerative functions of recombinant human Epo (rhEpo) similar to those in mammalian nervous tissues. Here we demonstrate that rhEpo effectively rescues primary cultured locust brain neurons from apoptotic cell death induced by hypoxia or the chemical compound H-7. The Janus kinase inhibitor AG-490 and the STAT inhibitor sc-355797 abolished protective effects of rhEpo on locust brain neurons. In contrast, inhibition of PI3K with LY294002 had no effect on rhEpo-mediated neuroprotection. The results indicate that rhEpo mediates the protection of locust brain neurons through interference with apoptotic pathways by the activation of a Janus kinase-associated receptor and STAT transcription factor(s). The involvement of similar transduction pathways in mammals and insects for the mediation of neuroprotection and support of neural regeneration by Epo indicates that an Epo/Epo receptor-like signaling system with high structural and functional similarity exists in both groups of animals. Epo-like signaling involved in tissue protection appears to be an ancient beneficial function shared by vertebrates and invertebrates.
Asunto(s)
Apoptosis/efectos de los fármacos , Eritropoyetina/farmacología , Saltamontes/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Animales , Apoptosis/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Saltamontes/fisiología , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia/fisiopatología , Proteínas de Insectos/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Morfolinas/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Tirfostinos/farmacologíaRESUMEN
Numerous data indicate that Rho kinase inhibitors, such as Fasudil, may constitute a novel therapy for cardiovascular and metabolic diseases. We evaluated long-term effects of exposure to Fasudil during late gestation (10 mg/day) in male rat offspring from birth until 9 months. We also analyzed its effects in offspring from hypertensive mothers treated with a nitric oxide synthesis inhibitor (L-NAME; 50 mg/day). Prenatal exposure to Fasudil did not affect birth weight, but increased body weight from postnatal day 7 (P7) to 9 months. In intrauterine growth-restricted (IUGR) fetuses exposed to L-NAME, maternal Fasudil treatment increased birth weight. At P42 and P180, rats exposed to Fasudil and L-NAME showed alterations of their food intake as well as an increased basal glycemia associated with mild glucose intolerance at 6 months which was also observed in Fasudil-exposed rats. In 9 month-old rats, exposure to Fasudil increased the daily food intake as well as hypothalamic mRNA level of the orexigenic NPY peptide without modulation of the anorexigenic POMC gene expression. Altogether, our data suggest that prenatal Fasudil exposure alleviates fetal growth in IUGR rats, but programs long-term metabolic disturbances including transient perturbations of glucose metabolism, a persistent increase of body weight gain, hyperphagia and an augmented expression of hypothalamic NPY orexigenic gene. We postulate that Fasudil treatment during perinatal periods may predispose individuals to the development of metabolic disorders.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Desarrollo Fetal/efectos de los fármacos , Hiperfagia/inducido químicamente , Sobrepeso/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Factores de Edad , Animales , Femenino , Desarrollo Fetal/fisiología , Retardo del Crecimiento Fetal/metabolismo , Retardo del Crecimiento Fetal/patología , Retardo del Crecimiento Fetal/prevención & control , Hiperfagia/metabolismo , Hiperfagia/fisiopatología , Masculino , Sobrepeso/metabolismo , Sobrepeso/fisiopatología , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Distribución Aleatoria , Ratas , Ratas Wistar , Factores SexualesRESUMEN
Pharmacological treatment for cerebral ischemia cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. The objective of this study is the development of a liposomal drug delivery system that maintains effective concentrations of protein kinase inhibitors fasudil in the CSF, resulting in neuroprotection against cerebral ischemia. Focal cerebral ischemia in rats was induced by middle cerebral artery occlusion using an intraluminal suture technique. Treated rats received 0.25 mg liposome-entrapped fasudil via the cisterna magna 2 hours after ischemic insult. Control rats received drug-free liposomes. Neurological condition and the infarct size were assessed at 24 and 72 hours after ischemia. The concentration of liposome-entrapped fasudil in the CSF was measured before sacrifice. Treated animals showed significantly improved neurological outcomes after the 24-hour observation period compared to the control group (p < 0.001). Treatment with 0.25 mg liposomal fasudil resulted in a reduction in the infarct area (24 hours: 29.0 +/- 4.4%, 72 hours: 28.1 +/- 3.9% of total brain slices) compared to controls (49.6 +/- 4.6%, p < 0.001), but there was no statistical difference between 24 and 72 hours. At 24 hours post-administration, CSF concentrations of liposome-entrapped fasudil were 45.4 +/- 31.5 micrograms/ml (20% of the injected dose). A single intrathecal injection of liposomal fasudil can maintain a therapeutic drug concentration in the CSF over a period of time, significantly decreasing infarct size in a rat model of acute ischemia.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Animales , Daño Encefálico Crónico/etiología , Daño Encefálico Crónico/prevención & control , Isquemia Encefálica/etiología , Infarto Cerebral/etiología , Infarto Cerebral/patología , Infarto Cerebral/prevención & control , Cisterna Magna , Portadores de Fármacos , Evaluación Preclínica de Medicamentos , Infarto de la Arteria Cerebral Media/complicaciones , Inyecciones Espinales , Liposomas , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/toxicidad , Ratas , Ratas Sprague-DawleyRESUMEN
Spontaneous germ cell death during spermatogenesis is an important event, and the usefulness of the seminiferous epithelium as an in vivo model to study apoptosis has been evidenced. Nevertheless, the response of the testis to apoptogenic agents has not been analyzed. This study was designed to determine germ cell sensitivity to induction of apoptosis and to provide baseline data on the testis response to several apoptogenic agents. Induced apoptosis was assessed by in situ DNA 3'-end labeling and quantified at every stage of the spermatogenic cycle. The shortest response time for every agent was established based on morphological and quantitative criteria. Our results show significantly increased incidence of germ cell deaths after all treatments, mainly at stages I, XII, and XIV. These specific stages coincide with those at which the greatest numbers of spontaneous germ cell deaths occur in control animals. Moreover, the rapid and highly specific response of germ cells to all the apoptogenic agents used in the present study indicate that apoptosis must be tightly regulated at these stages of the seminiferous epithelium. As a consequence, we propose that the disruption of apoptosis control might be an important determinant for idiopathic male infertility.
Asunto(s)
Apoptosis , Células Germinativas/efectos de los fármacos , Túbulos Seminíferos/efectos de los fármacos , Testículo/citología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/toxicidad , Animales , Colchicina/toxicidad , Cicloheximida/toxicidad , Citocalasinas/toxicidad , Inhibidores Enzimáticos/toxicidad , Supresores de la Gota/toxicidad , Etiquetado Corte-Fin in Situ , Masculino , Microscopía Electrónica , Ésteres del Forbol/toxicidad , Inhibidores de la Síntesis de la Proteína/toxicidad , Ratas , Ratas Wistar , Túbulos Seminíferos/ultraestructura , Testículo/efectos de los fármacosRESUMEN
In membrane preparation of chick cerebral cortex, HA (histamine) did not affect both basal and forskolin-stimulated adenylyl cyclase activity. However, in slices of chick cerebral cortex prelabeled with [3H]adenine, HA, as well as 2-methylHA, 4-methylHA, and N alpha-methylHA, potently increased [3H]cyclic AMP accumulation in a concentration-dependent manner. The stimulatory effect of the HA-ergic compounds on cyclic AMP formation was antagonized by HA H2-receptor blockers (aminopotentidine, ranitidine), and by chelerythrine (50 microM), a potent and selective inhibitor of PKC (protein kinase C). Of the two other tested PKC inhibitors H-7 (100 microM) significantly reduced the HA action, while the effect of staurosporine (1 microM) did not reach the level of statistical significance. Preincubation of chick cerebral cortical slices with a PKC activator, PDB (4 beta-phorbol 12,13-dibutyrate, 1 microM), markedly enhanced the accumulation of cyclic AMP evoked by HA, 2-methylHA, 4-methylHA and N alpha-methylHA. 4 beta-Phorbol, inactive on PKC, was ineffective. A possible role of PKC in the regulation of HA-induced cyclic AMP synthesis in chick cerebrum has been discussed.