RESUMEN
Accumulating evidence suggests that the non-receptor tyrosine kinase c-Abl plays an important role in the progression of Parkinson's disease (PD) and c-Abl inhibition could be neuroprotective in PD and related α-synucleinopathies. Nilotinib, a c-Abl inhibitor, has shown improved motor and cognitive symptoms in PD patients. However, issues concerning blood-brain barrier (BBB) penetration, lack of selectivity and safety still remain. Radotinib HCl is a selective Bcr-Abl kinase inhibitor that not only effectively access the brain, but also exhibits greater pharmacokinetic properties and safety profiles compared to Nilotinib and other c-Abl inhibitors. Here, we show the neuroprotective efficacy of Radotinib HCl, a brain penetrant c-Abl inhibitor, in a pre-clinical model of PD. Importantly, in vitro studies demonstrate that the treatment of Radotinib HCl protects the α-synuclein preformed fibrils (PFF)-induced neuronal toxicity, reduces the α-synuclein PFF-induced Lewy bodies (LB)/Lewy neurites (LN)-like pathology and inhibits the α-synuclein PFF-induced c-Abl activation in primary cortical neurons. Furthermore, administration of Radotinib HCl inhibits c-Abl activation and prevents dopaminergic neuron loss, neuroinflammation and behavioral deficits following α-synuclein PFF-induced toxicity in vivo. Taken together, our findings indicate that Radotinib HCl has beneficial neuroprotective effects in PD and provides an evidence that selective and brain permeable c-Abl inhibitors can be potential therapeutic agents for the treatment of PD and related α-synucleinopathies.
Asunto(s)
Encéfalo/efectos de los fármacos , Degeneración Nerviosa/tratamiento farmacológico , Enfermedad de Parkinson/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , alfa-Sinucleína/genética , Animales , Barrera Hematoencefálica , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Humanos , Cuerpos de Lewy/efectos de los fármacos , Ratones , Degeneración Nerviosa/genética , Fármacos Neuroprotectores/administración & dosificación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Proteínas Proto-Oncogénicas c-abl/genética , Pirimidinas/administración & dosificación , Sesquiterpenos/administración & dosificaciónRESUMEN
Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/administración & dosificación , Infecciones por Orthomyxoviridae/prevención & control , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Perros , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/farmacología , Células de Riñón Canino Madin Darby , Fusión de Membrana/efectos de los fármacos , Ratones , Mutación , Infecciones por Orthomyxoviridae/inmunología , Bibliotecas de Moléculas Pequeñas/farmacología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Tyrosine kinase (TK) plays a crucial role in the pathogenesis of idiopathic pulmonary fibrosis. Here, we aimed to investigate whether radotinib (Rb) could inhibit pulmonary fibrosis by inhibiting TK in vitro and in vivo. METHODS: The antifibrotic effects of Rb in transforming growth factor-ß (TGF-ß)1-stimulated A549 cells were determined using real-time polymerase chain reaction, western blotting, and immunocytochemistry assays. Rb inhibition of bleomycin-induced lung fibrosis in Sprague Dawley (SD) rats was determined by histopathological andâ immunohistochemical analyses. Rb-interfering metabolites were analyzed using LC-MS/MS. RESULTS: Rb concentrations of up to 1000 nM did not affect the viability of A549 cells, but Rb (30 nM) significantly reduced expression of TGF-ß1 (10 ng/mL)-induced ECM factors, such as Snail, Twist, and F-actin. Rb also regulated TGF-ß1-overexpressed signal cascades, such as fibronectin and α-smooth muscle actin. Furthermore, Rb attenuated the phosphorylation of Smad2 and phosphorylation of kinases, such as, extracellular signal-regulated kinase, and protein kinase B. In the inhibitory test against bleomycin (5 mg/kg)-induced lung fibrosis, the Rb (30 mg/kg/daily)-treated group showed a half-pulmonary fibrosis region compared to the positive control group. In addition, Rb significantly reduced collagen type I and fibronectin expression in the bleomycin-induced fibrotic region of SD rats. Further, the identified metabolite pantothenic acid was not altered by Rb. CONCLUSION: Taken together, these results indicate that Rb inhibits TGF-ß1-induced pulmonary fibrosis both in vitro and in vivo. These findings suggest that Rb may be an effective treatment for pulmonary fibrosis-related disorders and idiopathic pulmonary fibrosis.
Asunto(s)
Fibrosis Pulmonar Idiopática , Factor de Crecimiento Transformador beta , Ratas , Animales , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Fibronectinas , Reposicionamiento de Medicamentos , Cromatografía Liquida , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , BleomicinaRESUMEN
Aralia has been reported to exhibit various pharmacological properties, including anti-inflammatory, antidiabetic and antioxidant activities. We performed in vitro and in vivo analyses on the neuroprotective effects of an ethanolic extract of the aerial parts of Aralia cordata Thunb. (Araliaceae). In cultured cortical neurons from rats, A. cordata (5-20 microg/mL) inhibited 100 muM hydrogen peroxide (H(2)O(2))-induced apoptotic neuronal death, elevation of intracellular calcium concentration ([Ca(2+)](i)) and generation of reactive oxygen species (ROS). Since oleanolic acid isolated from A. cordata also inhibited H(2)O(2)-induced neuronal death, increase in [Ca(2+)](i) and ROS generation in cultured cortical neurons, some of the neuroprotective effects of A. cordata might be attributable to this compound. In rats, A. cordata prevented cerebral ischemic injury induced by 3 h of middle cerebral artery occlusion, followed by 24 h of reperfusion. Ischemic infarct and edema volumes were significantly reduced in rats that received A. cordata (50 mg/kg, orally). These animals exhibited a corresponding improvement in neurological function and a reduction of neuronal death, as determined histologically from the cortex and hippocampal regions. It is possible that the anti-oxidative properties of A. cordata may be responsible for its neuroprotective effects against focal cerebral ischemic injury. In future, A. cordata might play a therapeutic role in the prevention and treatment of neurodegeneration in stroke.
Asunto(s)
Aralia/química , Isquemia Encefálica/prevención & control , Fármacos Neuroprotectores/farmacología , Ácido Oleanólico/farmacología , Extractos Vegetales/farmacología , Animales , Calcio/metabolismo , Técnicas de Cultivo de Célula , Muerte Celular/efectos de los fármacos , Peróxido de Hidrógeno , Infarto de la Arteria Cerebral Media , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Ácido Oleanólico/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Previous work has shown that the Smilacis chinae rhizome (SCR) markedly inhibits amyloid beta protein (25-35)-induced neuronal cell damage in cultured rat cortical neurons. The present study was conducted to further verify the neuroprotective effect of SCR on excitotoxic and cerebral ischemic injury using both in vitro and in vivo studies. Exposure of cultured cortical neurons to 1 mM N-methyl-D-aspartate (NMDA) for 12 h induced neuronal cell death. SCR (10 and 50 microg/ml) inhibited NMDA-induced neuronal death, elevation of intracellular calcium ([Ca(2+)](i)), and generation of reactive oxygen species (ROS) in primary cultures of rat cortical neurons. In vivo, SCR prevented cerebral ischemic injury induced by 3-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct was significantly reduced in rats that received SCR (30 and 50 mg/kg, orally), with a corresponding improvement in neurological function. Moreover, SCR treatment significantly decreased the histological changes observed following ischemia. Oxyresveratrol and resveratrol isolated from SCR also inhibited NMDA-induced neuronal death, increase in [Ca(2+)](i), and ROS generation in cultured cortical neurons, suggesting that the neuroprotective effect of SCR may be attributable to these compounds. Taken together, these results suggest that the neuroprotective effect of SCR against focal cerebral ischemic injury is due to its anti-excitotoxic effects and that SCR may have a therapeutic role in neurodegenerative diseases such as stroke.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/toxicidad , Infarto de la Arteria Cerebral Media/terapia , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Daño por Reperfusión/prevención & control , Smilax , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Masculino , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/uso terapéutico , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Preparaciones de Plantas/química , Preparaciones de Plantas/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Resveratrol , Rizoma , Smilax/química , Estilbenos/aislamiento & purificación , Estilbenos/farmacología , Factores de TiempoRESUMEN
Our previous studies reported that methanol extract of Sanguisorbae radix from Sanguisorba officinalis L. (Rosaceae) prevented neuronal cell damage induced by Abeta (25-35) in vitro. The present study was carried out to investigate the effect of gallic acid isolated from Sanguisorbae radix on Abeta (25-35)-induced neurotoxicity using cultured rat cortical neurons. Gallic acid (0.1, 1 microM) showed a concentration-dependent inhibition on Abeta (25-35) (10 microM)-induced apoptotic neuronal death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33,342 staining. Pretreatment of gallic acid inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) and generation of reactive oxygen species (ROS), which were measured by fluorescent dyes. Gallic acid also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC. These results suggest that gallic acid prevents Abeta (25-35)-induced apoptotic neuronal death by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release and generation of ROS, and that these effects of gallic acid may be partly associated with the neuroprotective effect of Sanguisorbae radix.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Corteza Cerebral/efectos de los fármacos , Ácido Gálico/farmacología , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Sanguisorba/química , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Femenino , Ácido Glutámico/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Sanguisorbae radix (SR), the root of Sanguisorba officinalis L. (Rosaceae), has been traditionally used for its anti-inflammatory, anti-infectious and analgesic activities in Korea. Previous work has shown that SR prevents neuronal cell damage induced by Abeta (25--35) in cultured rat cortical neurons. The present study was carried out to further investigate the neuroprotective effect of SR on oxidative stress-induced toxicity in primary culture of rat cortical neurons, and on ischemia-induced brain damage in rats. SR, over a concentration range of 10--50 microg/ml, inhibited H2O2 (100 microM)-induced neuronal death, which was significantly inhibited by MK-801 (5 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, and verapamil (20 microM), an L-type Ca2+ channel blocker. Pretreatment of SR (10-50 microg/ml), MK-801 (5 microM), and verapamil (20 microM) inhibited H2O2-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) measured by a fluorescent dye, Fluo-4 AM. SR (10-50 microg/ml) inhibited H2O2-induced glutamate release into medium measured by HPLC, and generation of reactive oxygen species (ROS) measured by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In vivo, SR prevented cerebral ischemic injury induced by 2-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct and edema were significantly reduced in rats that received SR (10, 30 mg/kg, orally), with a corresponding improvement in neurological function. Catechin isolated from SR inhibited H2O2-induced neuronal death in cultures. Taken together, these results suggest that SR inhibits H2O2-induced neuronal death by interfering with the increase of [Ca2+]i, and inhibiting glutamate release and generation of ROS, and that the neuroprotective effect of SR against focal cerebral ischemic injury is due to its anti-oxidative effects. Thus SR might have therapeutic roles in neurodegenerative diseases such as stroke.