RESUMEN
Because treatment for postsurgical pain (PSP) remains a major unmet medical need, the emergence of safe and innovative nonopioid drugs has been strongly coveted. Tetrahydrobiopterin (BH4) is an interesting molecule for gaining a better understanding the pathological mechanism of neuropathic pain. However, whether BH4 and its pathway are involved in the pathogenesis of PSP remains unclear. In this study, we found that early in a rat paw incision model, the gene expression of GTP cyclohydrolase 1 (GTPCH) and sepiapterin reductase (SPR), BH4-producing enzymes in the de novo pathway, were significantly increased in incised compared with naive paw skin. Although a significant increase in GTPCH protein levels was observed in incised paw skin until only 1 day after incision, a significant increase in BH4 levels was observed until 7 days after incision. In vivo, Spr-knockout mice showed an antinociceptive phenotype in the hind paw incision compared with the wild-type and Spr heterozygote groups. Furthermore, QM385, the SPR inhibitor, showed a significant dose-dependent, antinociceptive effect, which was supported by a reduction in BH4 levels in incised skin tissues, with no apparent adverse effects. Immunohistochemical analysis demonstrated that macrophages expressing GTPCH protein were increased around the injury site in the rat paw incision model. These results indicate that BH4 is involved in the pathogenesis of PSP, and that inhibition of the BH4 pathway could provide a new strategy for the treatment of acute PSP.
Asunto(s)
Dolor Postoperatorio , Animales , Biopterinas/análogos & derivados , GTP Ciclohidrolasa/genética , Ratones , Dolor Postoperatorio/tratamiento farmacológico , Ratas , RoedoresRESUMEN
Oxaliplatin, a platinum-based chemotherapeutic agent, is widely used to treat colorectal cancer, but it induces peripheral neuropathy as a serious dose-limiting side effect. Recently, thrombomodulin alfa, a recombinant human soluble thrombomodulin, was reported to prevent oxaliplatin-induced peripheral neuropathy in a clinical phase 2 study. Here we conducted preclinical pharmacology studies. Rats were given oxaliplatin (6 mg/kg) intravenously to induce mechanical hyperalgesia associated with peripheral neuropathy. Single intravenous administration of thrombomodulin alfa (0.1, 0.3, 1 mg/kg) dose dependently prevented the development of oxaliplatin-induced mechanical hyperalgesia, with no sex difference in the efficacy. The preventative effect of thrombomodulin alfa on mechanical hyperalgesia was attenuated by antithrombin or carboxypeptidase inhibitor. In addition, carboxypeptidase B, a homolog of activated thrombin-activatable fibrinolysis inhibitor (TAFI) and human-derived activated protein C, prevented mechanical hyperalgesia, whereas antithrombin or other anti-coagulants did not. These results suggest that thrombomodulin alfa prevents sensory symptoms of oxaliplatin-induced peripheral neuropathy through the activation of TAFI and protein C by modulating thrombin activity, but the effects are independent of an anticoagulant effect. On the other hand, thrombomodulin alfa did not affect the anti-cancer activity of oxaliplatin on human colon cancer cell lines or mice transplanted with HCT116 cells. These results indicate that thrombomodulin alfa prevents sensory symptoms of oxaliplatin-induced peripheral neuropathy without affecting the anti-tumor activity of oxaliplatin. Therefore, thrombomodulin alfa is a promising drug to prevent the symptoms of oxaliplatin-induced peripheral neuropathy.
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Analgésicos/uso terapéutico , Antineoplásicos/efectos adversos , Neoplasias del Colon/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Oxaliplatino/efectos adversos , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Trombomodulina/uso terapéutico , Analgésicos/farmacología , Animales , Antineoplásicos/uso terapéutico , Carboxipeptidasa B2/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/patología , Femenino , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Oxaliplatino/uso terapéutico , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Proteína C/metabolismo , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Trombomodulina/genética , TactoRESUMEN
Clinical studies have reported that teriparatide (TPTD), a human parathyroid hormone analog, reduces back pain in osteoporotic patients. However, the mechanistic insights of this pharmacological action remain elusive. This study investigated the antinociceptive effect of TPTD mainly on primary sensory neurons in ovariectomized (OVX) rats. The plantar test showed thermal hyperalgesia in the OVX rats, which was significantly, but not fully, recovered immediately after the initial TPTD administration. The von Frey test also demonstrated reduced withdrawal threshold in the OVX rats. This was partially recovered by TPTD. Consistently, the number and size of spinal microglial cells were significantly increased in the OVX rats, while TPTD treatment significantly reduced the number but not size of these cells. RNA sequencing-based bioinformatics of the dorsal root ganglia (DRG) demonstrated that changes in neuro-protective and inflammatory genes were involved in the pharmacological effect of TPTD. Most neurons in the DRG expressed substantial levels of parathyroid hormone 1 receptor. TPTD treatment of the cultured DRG-derived neuronal cells reduced the cAMP level and augmented the intracellular calcium level as the concentration increased. These findings suggest that TPTD targets neuronal cells as well as bone cells to exert its pharmacological action.
Asunto(s)
Analgésicos/farmacología , Hiperalgesia/tratamiento farmacológico , Ovariectomía/efectos adversos , Teriparatido/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Conservadores de la Densidad Ósea/farmacología , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/etiología , Microglía/efectos de los fármacos , Dolor/tratamiento farmacológico , Hormona Paratiroidea/metabolismo , Ratas Sprague-Dawley , Receptor de Hormona Paratiroídea Tipo 1/genética , Médula Espinal/citologíaRESUMEN
INTRODUCTION: Disseminated intravascular coagulation (DIC), a deadly complication characterized by uncontrolled hypercoagulation, causes a decrease in the platelet count and impairs platelet aggregation. Thrombomodulin (TM) alfa, a recombinant human soluble TM, reduces hypercoagulation in DIC patients. However, the effects of TM alfa on impaired platelet aggregation remain to be determined. In this study, we aim to investigate the effects of TM alfa on platelet aggregation using lipopolysaccharide (LPS)-induced and tissue factor (TF)-induced DIC rat models. MATERIALS AND METHODS: Sprague-Dawley rats were administered TF or LPS intravenously, with or without TM alfa before the injection. Six hours after LPS injection or 1â¯h after TF infusion, blood samples were obtained, and platelet-rich plasma was prepared. Collagen or adenosine diphosphate-induced platelet aggregation was measured using an aggregometer. In the other experiments, platelets were transfused 1â¯h after the TF infusion. Five minutes after transfusion, collagen-induced platelet aggregation was also measured. RESULTS: The amplitude of platelet aggregation in platelet-rich plasma was decreased in LPS- and TF-treated rats. TM alfa inhibited the decrease in platelet aggregation in a dose-dependent manner. The washed platelet aggregation amplitude was not decreased in TF-treated rats. Suspension of normal platelets in plasma obtained from TF-treated rats reduced platelet aggregation. Platelet transfusion for TF-treated rats increased the platelet count but was unable to improve platelet aggregation. CONCLUSIONS: TM alfa attenuated impairment of platelet aggregation in LPS- and TF-induced DIC rat models. The changes in plasma composition played a role in the decrease of platelet aggregation in TF-treated rats.
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Coagulación Intravascular Diseminada/tratamiento farmacológico , Agregación Plaquetaria/efectos de los fármacos , Trombomodulina/uso terapéutico , Animales , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/patología , Humanos , Ratas , Ratas Sprague-DawleyRESUMEN
INTRODUCTION: Extracellular histones are reported to increase thrombin generation in the plasma and induce endothelial cell death in vitro. These effects of histones were suggested to involve histone-induced inhibition of TM-dependent activated protein C (APC) generation. Therefore, we hypothesized that TM alfa, a recombinant human soluble TM, attenuates these effects of histones by promoting the generation of APC. In the present study, we investigated the effects of TM alfa on the histone-induced decrease in APC generation, an increase in thrombin generation, and endothelial cell death in vitro. METHODS: APC generation was investigated using a chromogenic substrate based assay. Thrombin generation in plasma was studied by using a calibrated automated thrombogram method. Histone cleavage was detected by western blot analysis. Histone-induced endothelial cell death was evaluated by the trypan blue exclusion test. RESULTS: Histones decreased APC generation and increased thrombin generation in the presence of endothelial cells. TM alfa increased APC generation and decreased thrombin generation in the presence of histones and endothelial cells. TM alfa with thrombin and protein C cleaved histone H3, and attenuated histone-induced endothelial cell death. Antithrombin, an endogenous thrombin inhibitor, and gabexate mesilate, a synthetic protease inhibitor, inhibited thrombin generation, decreased APC generation, and did not have any effect on histone H3 cleavage or histone-induced endothelial cell death. CONCLUSIONS: TM alfa attenuated the histone-induced increase in thrombin generation and endothelial cell death by promoting APC generation in vitro.
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Anticoagulantes/uso terapéutico , Histonas/metabolismo , Proteína C/metabolismo , Trombomodulina/metabolismo , Anticoagulantes/farmacología , HumanosRESUMEN
We investigated the anxiolytic effects and mechanism of action of a new anxiolytic drug, (R)-piperonyl-1,2,3,4-tetrahydro[1]benzothieno[2,3-c]pyridine-3- carboxamide hydrochloride (AP521). AP521 showed equal or more potent anxiolytic-like effects compared with diazepam, a benzodiazepine receptor agonist, or tandospirone, a partial 5-hydroxytryptamine (5-HT)1A receptor agonist, in three rat anxiety models; the Vogel-type conflict test, elevated plus maze test, and conditioned fear stress test. Although AP521 did not bind to the benzodiazepine receptor, it did bind to 5-HT1A, 5-HT1B, 5-HT1D, 5-HT5A and 5-HT7 receptors, and showed agonist activity for the human 5-HT1A receptor expressed in HEK293 cells. Tandospirone, which can stimulate the presynaptic 5-HT1A receptors in the raphe, tended to decrease extracellular 5-HT concentration in the medial prefrontal cortex (mPFC) in rats. In contrast, AP521 increased extracellular 5-HT concentration. In addition, AP521 enhanced the anti-freezing effect of citalopram, a selective serotonin reuptake inhibitor, in the fear conditioning model in rats and enhanced the citalopram-caused increase of the extracellular 5-HT concentration in the mPFC. These results suggest that AP521 exhibits potent anxiolytic effects by acting as a postsynaptic 5-HT1A receptor agonist and by enhancing serotonergic neural transmission in the mPFC by a novel mechanism of action.
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Ansiolíticos/farmacología , Piridinas/farmacología , Serotonina/metabolismo , Transmisión Sináptica/efectos de los fármacos , Tiofenos/farmacología , Animales , Ansiolíticos/uso terapéutico , Ansiedad/tratamiento farmacológico , Células Cultivadas , Citalopram/farmacología , Diazepam/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Humanos , Isoindoles/farmacología , Masculino , Piperazinas/farmacología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Piridinas/uso terapéutico , Pirimidinas/farmacología , Ratas , Receptores de Serotonina/metabolismo , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Tiofenos/uso terapéuticoRESUMEN
There is growing evidence that Rho-kinase contributes to cardiovascular disease, which has made Rho-kinase a target for the treatment of human diseases. To date, the only Rho-kinase inhibitor employed clinically in humans is fasudil, which has been used for the prevention of cerebral vasospasm and subsequent ischemic injury after surgery for subarachnoid hemorrhage (SAH). A number of pathological processes, in particular hemodynamic dysfunctions and inflammatory reactions, are thought to be related in the pathogenesis of delayed cerebral vasospasm and subsequent ischemic injury after SAH. This review focuses on fasudil's pleiotropic therapeutic effects: amelioration of hemodynamic dysfunction and inflammation, and discusses in detail the clinical studies on fasudil administered after the occurrence of SAH.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Inhibidores de Proteínas Quinasas/uso terapéutico , Hemorragia Subaracnoidea/tratamiento farmacológico , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/enzimología , Ensayos Clínicos como Asunto/métodos , Evaluación Preclínica de Medicamentos/métodos , Pleiotropía Genética/efectos de los fármacos , Pleiotropía Genética/fisiología , Humanos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Hemorragia Subaracnoidea/enzimología , Resultado del Tratamiento , Quinasas Asociadas a rho/metabolismoRESUMEN
Using a cellular approach, the present study examined whether fasudil and active metabolite hydroxyfasudil, Rho-kinase inhibitors, exert a direct protective effect on endothelin-induced cardiac myocyte hypertrophy in vitro. Treatment with endothelin (10nM) caused significant hypertrophy of cultured neonatal rat cardiomyocytes by a 21.2% increase in cell surface area. Fasudil (1-10 µM) and hydroxyfasudil (0.3-10 µM) significantly prevented endothelin-induced cardiomyocyte hypertrophy. The present results suggest that inhibition of cardiac hypertrophy by fasudil is, at least in part, due to direct protection of cardiomyocytes from hypertrophy.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Cardiomegalia/prevención & control , Citoprotección , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/enzimología , Células Cultivadas , Endotelinas/farmacología , Miocitos Cardíacos/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the anti-vasospastic potential of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg-1·30 min-1) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and ß at a concentration of 10 µM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 µM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.
RESUMEN
We investigated the anti-vasospastic potential of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg(-1)·30 min(-1)) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and ß at a concentration of 10 µM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 µM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Isquemia Encefálica/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Hemorragia Subaracnoidea/tratamiento farmacológico , Vasoespasmo Intracraneal/tratamiento farmacológico , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Viscosidad Sanguínea/efectos de los fármacos , Isquemia Encefálica/enzimología , Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Perros , Femenino , Hematócrito , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Hemorragia Subaracnoidea/fisiopatología , Vasoespasmo Intracraneal/fisiopatologíaRESUMEN
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.
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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
The aim of this study was to investigate the possible effects of the Rho-kinase inhibitor, fasudil, on the lysophosphatidic acid (LPA)-induced neurite retraction in N1E-115 cells. In cultured N1E-115 cells, LPA produced a marked increase in the population of rounded cells. Fasudil or hydroxyfasudil, an active metabolite of fasudil, blocked cell rounding in a concentration-dependent manner at levels between 1 and 10 µM, with IC50 values of 1.7 or 1.6 µM, respectively. Fasudil or hydroxyfasudil concentration-dependently inhibited phosphorylation of the myosin binding subunit of myosin phosphatase in N1E-115 cells. These results indicate that Rho-kinase is essential for LPA-induced neurite retraction in N1E-115 cells and that inactivation of Rho-kinase by a Rho-kinase inhibitor, such as fasudil, eliminates cell rounding and promotes neurite outgrowth, thus improving neurological function in the brain damage.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Línea Celular/efectos de los fármacos , Lisofosfolípidos/farmacología , Neuritas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Humanos , Ratones , Neuritas/ultraestructura , Neuronas/citología , Quinasas Asociadas a rho/metabolismoRESUMEN
We investigated the neuroprotective effects of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, in a rat stroke model in which endothelial damage and subsequent thrombotic occlusion were selectively induced in perforating arteries. By examining the effects on the endothelial damage/dysfunction, we thought to explore the mechanism of Rho-kinase inhibitors. Hydroxyfasudil (10mg/kg, i.p., once daily for 3 days) significantly improved neurological functions and reduced the size of the infarct area produced by internal carotid artery injection of sodium laurate in a rat cerebral microthrombosis model. Treatment with fasudil or hydroxyfasudil concentration-dependently inhibited tumor necrosis factor alpha-induced tissue factor expression on the surface of cultured human umbilical vein endothelial cells. They also inhibited thrombin-induced endothelial hyperpermeability. The present findings suggest that hydroxyfasudil is efficacious in preventing brain damage associated with cerebral ischemia, and is partially responsible for fasudil's cytoprotective potential. The results also suggest that the therapeutic benefits against ischemic injury of Rho-kinase inhibitors are attributed, at least in part, to activity upon endothelial damage/dysfunction.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Isquemia Encefálica/tratamiento farmacológico , Endotelio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fármacos Neuroprotectores/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/patología , Isquemia Encefálica/patología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio/metabolismo , Endotelio/patología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Humanos , Técnicas In Vitro , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacocinética , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Tromboplastina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.
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Antiinflamatorios/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fenilacetatos/química , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/química , Pirimidinas/química , Tiofenos/química , Animales , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Área Bajo la Curva , Descubrimiento de Drogas , Humanos , Concentración 50 Inhibidora , Ratones , Fenilacetatos/farmacocinética , Fenilacetatos/farmacología , Inhibidores de Fosfodiesterasa/farmacocinética , Inhibidores de Fosfodiesterasa/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-Actividad , Tiofenos/farmacocinética , Tiofenos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Vómitos/inducido químicamenteRESUMEN
BACKGROUND: Rho-kinase (ROCK) is a downstream effector of Rho GTPase that is known to regulate various pathological processes important to the development of ischemic stroke, such as thrombus formation, inflammation, and vasospasm. Inhibition of ROCK leads to decreased infarct size in animal models of ischemic stroke. This study tests the hypothesis that ROCK activity increases during the acute phase of ischemic stroke. METHODS: Serial blood samples were drawn from 10 patients with acute ischemic stroke presenting within 24 h of symptom onset and with NIHSS scores >or=4. Samples were taken at 24, 48, and 72 h. Leukocyte ROCK activity was determined by immunoblotting leukocyte lysates with antibodies to the phosphorylated form of myosin-binding subunit (P-MBS) of myosin light chain phosphatase (MLCP). MBS and P-MBS contents were normalized to alpha-tubulin, and ROCK activity was expressed as the ratio of P-MBS to MBS. ROCK activities in these 10 patients were compared to baseline ROCK activities in 10 control subjects without acute illness and matched for sex, age, and number of vascular risk factors using a two-tailed Student's t-test. RESULTS: The mean NIHSS score in patients with stroke was 15.4. ROCK activity was significantly increased at 24 and 48 h in patients after acute ischemic stroke when compared to control values, with peak elevations at 48 h after stroke onset. There was no apparent correlation between ROCK activity and stroke severity based on NIHSS. CONCLUSIONS: Leukocyte ROCK activity is increased in patients after acute ischemic stroke with maximal activity occurring about 48 h after stroke onset. These findings suggest that activation of ROCK may play a role in the pathogenesis of ischemic stroke in humans.
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Isquemia Encefálica/enzimología , Leucocitos/enzimología , Accidente Cerebrovascular/enzimología , Quinasas Asociadas a rho/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Anciano , Western Blotting , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Whether Rho-kinase activity is really associated with the pathogenesis of cerebral infarction remains unclear. To consider this question, we investigated correspondences between severity of neurological deficit, infarct size, amount of various marker proteins, and Rho-kinase activity in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere in rats. We prepared rats with various severities of neurological deficit (mild to severe) 3 days after injection of laurate, then measured infarct size and amount of various marker proteins, phosphorylation of substrates of Rho-kinase, myosin-binding subunit (MBS), myosin light chain (MLC), ezrin/radixin/moesin (ERM), and adducin using Western blot methods. First, infarct size increased corresponding to the severity of neurological deficit. Second, amounts of activating transcription factor 3, nestin, CD68, proliferating cell nuclear antigen, and heat shock protein 70 were increased, whereas neurofilament and myelin-associated glycoprotein were decreased corresponding to the severity of neurological deficit and infarct size. Finally, Rho-kinase activity (phospho-MBS/MBS, phospho-MLC/MLC, phospho-ERM/ERM, and phospho-adducin/adducin) was increased corresponding to the severity of neurological deficit and infarct size. Rho-kinase thus appears to play a crucial role in the pathogenesis of cerebral infarction.
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Infarto Cerebral/patología , Trombosis Intracraneal/patología , Quinasas Asociadas a rho/metabolismo , Animales , Biomarcadores/metabolismo , Infarto Cerebral/metabolismo , Infarto Cerebral/fisiopatología , Trombosis Intracraneal/metabolismo , Trombosis Intracraneal/fisiopatología , Masculino , Modelos Animales , Ratas , Ratas Sprague-DawleyRESUMEN
Evidence that Rho-kinase is involved in cerebral infarction has accumulated. However, it is uncertain whether Rho-kinase is activated in the brain parenchyma in cerebral infarction. To answer this question, we measured Rho-kinase activity in the brain in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere. At 6 h after injection, increase of activating transcription factor 3 (ATF3) and c-Fos was found in the ipsilateral hemisphere, suggesting that neuronal damage occurs. At 0.5, 3, and 6 h after injection of laurate, Rho-kinase activity in extracts of the cerebral hemispheres was measured by an ELISA method. Rho-kinase activity in extracts of the ipsilateral hemisphere was significantly increased compared with that in extracts of the contralateral hemisphere at 3 and 6 h but not 0.5 h after injection of laurate. Next, localization of Rho-kinase activity was evaluated by immunohistochemical analysis in sections of cortex and hippocampus including infarct area 6 h after injection of laurate. Staining for phosphorylation of myosin-binding subunit (phospho-MBS) and myosin light chain (phospho-MLC), substrates of Rho-kinase, was elevated in neuron and blood vessel, respectively, in ipsilateral cerebral sections, compared with those in contralateral cerebral sections. These findings indicate that Rho-kinase is activated in neuronal and vascular cells in a rat cerebral infarction model, and suggest that Rho-kinase could be an important target in the treatment of cerebral infarction.
Asunto(s)
Encéfalo/enzimología , Infarto Cerebral/enzimología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Factor de Transcripción Activador 3/metabolismo , Amidas/farmacología , Animales , Western Blotting , Encéfalo/patología , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Infarto Cerebral/patología , Inhibidores Enzimáticos/farmacología , Hipocampo/enzimología , Hipocampo/patología , Inmunohistoquímica , Masculino , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Extractos de Tejidos/química , Extractos de Tejidos/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.
Asunto(s)
Endotelio/citología , Neovascularización Patológica , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Capilares/citología , Adhesión Celular , División Celular , Movimiento Celular , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Técnicas de Transferencia de Gen , Humanos , Isquemia , Flujometría por Láser-Doppler , Óxido Nítrico Sintasa/metabolismo , Compuestos Nitrosos/farmacología , Oxadiazoles/farmacología , Fosforilación , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-akt , Quinoxalinas/farmacología , Ratas , Proteínas Recombinantes/metabolismo , S-Nitrosoglutatión/farmacología , Factores de TiempoRESUMEN
The effects of the repeated administration of milnacipran, a serotonin (5-HT)-noradrenaline reuptake inhibitor (SNRI), on the functional status of somatodendritic 5-HT1A receptors, and postsynaptic 5-HT1A receptors were explored using electrophysiological approaches in rats. In-vitro electrophysiological recordings in the dorsal raphe nucleus showed that 5-HT inhibited the firing of serotonergic neurones, and the selective 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), reversed the inhibitory effect of 5-HT. The potency of 5-HT to inhibit the firing of serotonergic neurones was slightly attenuated after 3 days of treatment with milnacipran (30 mg/kg, p.o., twice daily), and significantly attenuated after 7 or 14 days treatment at the same dose. The tricyclic antidepressant, imipramine, did not significantly modify the inhibitory effect of 5-HT. After 7 days treatment at 30 mg/kg, p.o., once daily, milnacipran reduced the potency of 5-HT to inhibit the firing of serotonergic neurones, whereas the selective serotonin reuptake inhibitors, fluvoxamine and fluoxetine (60 and 30 mg/kg, p.o., once daily, respectively), did not modify it under these conditions. Treatment with milnacipran (30 mg/kg, p.o., twice daily) for 14 days did not change the inhibition of the CA1 field potential in rat hippocampal slices by 5-HT. These data suggest that somatodendritic 5-HT1A receptors, but not postsynaptic 5-HT1A receptors, rapidly desensitize in response to the repeated administration of milnacipran.