RESUMO
A series of piperazine ureas was designed, synthesized, and evaluated for their potential as novel orally available fatty acid amide hydrolase (FAAH) inhibitors that are therapeutically effective against pain. We carried out an optimization study of the lead compound 3 to improve its DMPK profile as well as in vitro potency. We identified the thiazole compound 60j with potent inhibitory activity, high brain permeability, and good bioavailability. Compound 60j showed a potent and dose-dependent anti-nociceptive effect in the acetic acid-induced writhing test in mice.
Assuntos
Amidoidrolases/antagonistas & inibidores , Analgésicos/química , Analgésicos/uso terapêutico , Piperazinas/química , Piperazinas/uso terapêutico , Ureia/análogos & derivados , Ureia/uso terapêutico , Amidoidrolases/metabolismo , Analgésicos/farmacocinética , Animais , Humanos , Camundongos , Simulação de Acoplamento Molecular , Dor/tratamento farmacológico , Dor/enzimologia , Piperazina , Piperazinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacocinética , Tiazóis/uso terapêutico , Ureia/farmacocinéticaRESUMO
Hypothermia is the only neuroprotective therapy proven to be clinically effective. Identifying the molecules that play important roles in the efficacy of hypothermia, we developed a multi-channel computer-controlled system, in which the brain temperatures of freely moving rats were telemetrically monitored and maintained below 35 degrees C, and examined the time window necessary to exert its significant neuroprotective effects. Eight-week-old SD rats were subjected to a 2h middle cerebral artery occlusion (MCAO) with an intraluminal filament, and post-ischemic hypothermia was introduced at 0, 2, 4, or 6h after reperfusion until the rats were killed 2 days after MCAO. Since a significant protection was observed when hypothermia was started within 4h after reperfusion, it was concluded that the therapeutic time window of mild hypothermia lasts for 4h after reperfusion in our model. On the basis of the window, comprehensive gene expression analyses using oligonucleotide microarrays were conducted and identified potential genes related to the efficacy of hypothermia, which included inflammatory genes like osteopontin, early growth response-1, or macrophage inflammatory protein-3alpha. Therefore, the neuroprotective effects of post-ischemic mild hypothermia were strongly suggested to be mainly associated with the reduction of neuronal inflammation.
Assuntos
Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Infarto Cerebral/terapia , Citoproteção/genética , Encefalite/terapia , Expressão Gênica/fisiologia , Hipotermia Induzida/métodos , Animais , Temperatura Corporal/fisiologia , Isquemia Encefálica/metabolismo , Sobrevivência Celular/genética , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Infarto Cerebral/fisiopatologia , Infarto Cerebral/prevenção & controle , Quimiocina CCL20 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Encefalite/fisiopatologia , Encefalite/prevenção & controle , Hipotermia Induzida/normas , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Mediadores da Inflamação/metabolismo , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Masculino , Degeneração Neural/fisiopatologia , Degeneração Neural/prevenção & controle , Degeneração Neural/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Osteopontina/genética , Osteopontina/metabolismo , Ratos , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/terapia , Fatores de Tempo , Regulação para Cima/fisiologiaRESUMO
Although hypothermia is one of the most robust neuroprotectants clinically available, its underlying mechanisms remain unclear. Through microarray gene expression analysis, we previously identified several key molecules potentially involved in the efficacy of hypothermia in a 2h middle cerebral artery occlusion (MCAO) rat model, including cytokine and chemokine genes. The present study demonstrated that the expressions of 2 genes, macrophage inflammatory protein-3alpha (MIP-3alpha) and its receptor, CC-chemokine receptor 6 (CCR6), were upregulated in the model and were suppressed by hypothermia. To investigate the role of cerebral MIP-3alpha, it was administered into the rat striatum; dose- and time-dependent induction of CCR6 gene expression was observed. Interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha injection also induced sequential expressions of MIP-3alpha and CCR6. MIP-3alpha was found to be produced by proinflammatory cytokines in rat astrocytes, while it was suppressed by hypothermia. In turn, MIP-3alpha stimulated IL-1beta and inducible nitric oxide synthase expressions in rat microglia and rat brains. Furthermore, intracerebroventricular administration of an anti-rat MIP-3alpha-neutralizing antibody significantly reduced the infarct in MCAO rat brains. These findings suggest that MIP-3alpha plays a pivotal role in inflammatory cascades in ischemic brains, and may be a novel therapeutic target for cerebral ischemia.
Assuntos
Encéfalo/imunologia , Quimiocina CCL20/metabolismo , Infarto da Artéria Cerebral Média/imunologia , Receptores CCR6/metabolismo , Animais , Anticorpos , Astrócitos/imunologia , Encéfalo/patologia , Células Cultivadas , Citocinas/metabolismo , Hipotermia/imunologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Interleucina-1beta/metabolismo , Masculino , Microglia/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores CCR1/metabolismo , Receptores CCR2/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.