RESUMEN
Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Descubrimiento de Drogas , Macaca mulatta , Ratones , Ratones Transgénicos , Ratas , Ratas Sprague-DawleyRESUMEN
Alzheimer's disease is a major unmet medical need with pathology characterized by extracellular proteinaceous plaques comprised primarily of ß-amyloid. γ-Secretase is a critical enzyme in the cellular pathway responsible for the formation of a range of ß-amyloid peptides; one of which, Aß42, is believed to be responsible for the neuropathological features of the disease. Herein, we report 4,4 disubstituted piperidine γ-secretase inhibitors that were optimized for in vitro cellular potency and pharmacokinetic properties in vivo. Key agents were further characterized for their ability to lower cerebral Aß42 production in an APP-YAC mouse model. This structural series generally suffered from sub-optimal pharmacokinetics but hypothesis driven lead optimization enabled the discovery of γ-secretase inhibitors capable of lowering cerebral Aß42 production in mice.
Asunto(s)
Amidas/síntesis química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Piperidinas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Amidas/farmacología , Péptidos beta-Amiloides/biosíntesis , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Ratones , Fragmentos de Péptidos/biosíntesisRESUMEN
The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Fragmentos de Péptidos/antagonistas & inhibidores , Purinas/química , Purinas/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Animales , Humanos , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/metabolismo , Purinas/farmacología , Receptores Notch/metabolismo , Relación Estructura-ActividadRESUMEN
We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.
Asunto(s)
Acetatos/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Flúor/química , Piperidinas/química , Acetatos/síntesis química , Acetatos/farmacocinética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Compuestos de Diazonio/química , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Piperidinas/síntesis química , Piperidinas/farmacocinética , Ratas , Receptores Notch/metabolismoRESUMEN
BACKGROUND: Neuropathic pain and radicular low back pain both have a major impact on human health worldwide. Microarray gene analysis on central nervous system tissues holds great promise for discovering novel targets for persistent pain modulation. METHODS: Rat models of lumbar radiculopathy (L5 nerve root ligation) and neuropathy (L5 spinal nerve transection) were used for these studies. The authors measured mechanical allodynia followed by analysis of global gene expression in the lumbar spinal cord at two time points (7 days and 14 days) after surgery using the Affymetrix RAE230A GeneChip(R) (Santa Clara, CA). The expression patterns of several genes of interest were subsequently confirmed using real-time reverse transcriptase polymerase chain reaction. RESULTS: The authors observed similarly robust mechanical allodynia in both models. Second, they observed significant differences in lumbar spinal cord gene expression across chronic pain models. There was little overlap between genes altered in each injury model, suggesting that the site and type of injury produce distinct spinal mechanisms mediating the observed mechanical allodynia. The authors further confirmed a subset of the genes using reverse transcriptase polymerase chain reaction and identified several genes as either neuropathy-associated genes or radiculopathy-associated genes. CONCLUSIONS: These two models of persistent pain produce similar allodynic outcomes but produce differential gene expression. These results suggest that diverging mechanisms lead to a common behavioral outcome in these pain models. Furthermore, these distinct pathophysiologic mechanisms in neuropathic versus radicular pain may implicate unique drug therapies for these types of chronic pain syndromes.
Asunto(s)
Perfilación de la Expresión Génica , Dolor de la Región Lumbar/metabolismo , Neuralgia/metabolismo , Radiculopatía/metabolismo , Médula Espinal/metabolismo , Animales , Antígenos de Histocompatibilidad Clase II/análisis , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Neuropathic pain remains a prevalent and persistent clinical problem because of our incomplete understanding of its pathogenesis. This study demonstrates for the first time, to our knowledge, a critical role for CNS innate immunity by means of microglial Toll-like receptor 4 (TLR4) in the induction phase of behavioral hypersensitivity in a mouse and rat model of neuropathy. We hypothesized that after L5 nerve transection, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4. To test this hypothesis, experiments were undertaken to assess tactile and thermal hypersensitivity in genetically altered (i.e., TLR4 knockout and point-mutant) mice after L5 nerve transection. In a complementary study, TLR4 antisense oligodeoxynucleotide (ODN) was administered intrathecally to L5 spinal nerve injured rats to reduce the expression of spinal TLR4. Both the genetically altered mice and the rats treated with TLR4 antisense ODN displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines as compared with their respective control groups. This finding shows that TLR4 contributes to the initiation of CNS neuroimmune activation after L5 nerve transection. Further understanding of this early, specific, innate CNS/microglial response and how it leads to sustained glial/neuronal hypersensitivity may point to new therapies for the prevention and treatment of neuropathic pain syndromes.
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Sistema Nervioso Central , Inmunidad Innata/fisiología , Neuroinmunomodulación/fisiología , Dolor/fisiopatología , Receptores Inmunológicos/metabolismo , Animales , Conducta Animal/fisiología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/fisiopatología , Citocinas/inmunología , Relación Dosis-Respuesta a Droga , Inyecciones Espinales , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microglía/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores Inmunológicos/genética , Nervios Espinales/patología , Receptor Toll-Like 4RESUMEN
Peripheral inflammation induces central sensitization characterized by the development of allodynia and hyperalgesia to mechanical and thermal stimuli. Recent evidence suggests that activation of glial cells and a subsequent increase in proinflammatory cytokines contribute to the development of behavioral hypersensitivity after nerve injury or peripheral inflammation. In the present study, we examined mRNA and protein expression of glial markers and proinflammatory cytokines at the lumbar spinal cord, brainstem and forebrain following intraplantar administration of complete Freunds adjuvant (CFA) in rats. Gene expression studied by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for microglial markers (Mac-1, TLR4 and CD14) showed a significant increase in their expression during all phases (acute, subacute and chronic) of inflammation. Conversely, up-regulation of astroglial markers [glial fibrillary acidic protein (GFAP) and S100B] was observed only at the subacute and chronic phases of inflammation. Increased immunoreactivity for OX-42 (CR3/CD11b) and GFAP at various brain regions was also observed after the acute and subacute phases of the inflammation, respectively. Quantification of proinflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) at the mRNA (by real-time RT-PCR) and protein level (by ELISA) revealed enhanced expression during the acute, subacute and chronic phases of CFA-induced peripheral inflammation. This study demonstrates that CFA-induced peripheral inflammation induces robust glial activation and proinflammatory cytokines both spinally and supraspinally. In addition, similar to nerve injury-induced behavioral hypersensitivity microglial activation preceded astrocytic activation following CFA-induced peripheral inflammation, supporting a role of microglia in the initiation phase and astrocytes in maintaining hypersensitivity. These findings further support a unifying theory that glial activation and enhanced cytokine expression at the CNS have a role in eliciting behavioral hypersensitivity.
Asunto(s)
Sistema Nervioso Central/patología , Citocinas/metabolismo , Adyuvante de Freund/farmacología , Expresión Génica/efectos de los fármacos , Neuroglía/efectos de los fármacos , Análisis de Varianza , Animales , Conducta Animal , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Adyuvante de Freund/efectos adversos , Lateralidad Funcional , Inflamación/inducido químicamente , Masculino , Neuroglía/metabolismo , Dimensión del Dolor/métodos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
One area that has emerged as a promising therapeutic target for the treatment and prevention of chronic pain and opioid tolerance/hyperalgesia is the modulation of the central nervous system (CNS) immunological response that ensues following injury or opioid administration. Broadly defined, central neuroimmune activation involves the activation of cells that interface with the peripheral nervous system and blood. Activation of these cells, as well as parenchymal microglia and astrocytes by injury, opioids, and other stressors, leads to subsequent production of cytokines, cellular adhesion molecules, chemokines, and the expression of surface antigens that enhance a CNS immune cascade. This response can lead to the production of numerous pain mediators that can sensitize and lower the threshold of neuronal firing: the pathologic correlate to central sensitization and chronic pain states. CNS innate immunity and Toll-like receptors, in particular, may be vital players in this orchestrated immune response and may hold the answers to what initiates this complex cascade. The challenge remains in the careful perturbation of injury/opioid-induced neuroimmune activation to down-regulate this process without inhibiting beneficial CNS autoimmunity that subserves neuronal protection following injury.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Neuroinmunomodulación/fisiología , Dolor/tratamiento farmacológico , Analgésicos Opioides/farmacología , Animales , Enfermedad Crónica , Citocinas/metabolismo , Tolerancia a Medicamentos/fisiología , Humanos , Hiperalgesia/inmunología , Hiperalgesia/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Neuroinmunomodulación/efectos de los fármacos , Dolor/inmunología , Dolor/metabolismoRESUMEN
Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/microbiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Staphylococcus aureus/fisiología , Animales , Femenino , Lipopolisacáridos/farmacología , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peptidoglicano/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptor Toll-Like 2 , Receptores Toll-LikeRESUMEN
The activation of glial cells and enhanced proinflammatory cytokine expression at the spinal cord has been implicated in the development of morphine tolerance, and morphine withdrawal-induced hyperalgesia. The present study investigated the effect of propentofylline, a glial modulator, on the expression of analgesic tolerance and withdrawal-induced hyperalgesia in chronic morphine-treated rats. Chronic morphine administration through repeated subcutaneous injection induced glial activation and enhanced proinflammatory cytokine levels at the lumbar spinal cord. Moreover, glial activation and enhanced proinflammatory cytokine levels exhibited a temporal correlation with the expression of morphine tolerance and hyperalgesia. Consistently, propentofylline attenuated the development of hyperalgesia and the expression of spinal analgesic tolerance to morphine. The administration of propentofylline during the induction of morphine tolerance also attenuated glial activation and proinflammatory cytokines at the L5 lumbar spinal cord. These results further support the hypothesis that spinal glia and proinflammatory cytokines contribute to the mechanisms of morphine tolerance and associated abnormal pain sensitivity.
Asunto(s)
Hiperalgesia/tratamiento farmacológico , Dependencia de Morfina/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Xantinas/uso terapéutico , Análisis de Varianza , Animales , Conducta Animal , Citocinas/genética , Interacciones Farmacológicas , Tolerancia a Medicamentos/genética , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hiperalgesia/etiología , Hiperalgesia/genética , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/genética , Inflamación/inmunología , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/metabolismo , Masculino , Morfina/farmacología , Dependencia de Morfina/genética , Dimensión del Dolor , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Síndrome de Abstinencia a Sustancias/complicaciones , Síndrome de Abstinencia a Sustancias/genéticaRESUMEN
Injury to peripheral nerves often produces non-physiological, long-lasting spontaneous pain, hyperalgesia and allodynia that are refractory to standard treatment and often insensitive to opioids, such as morphine. Recent studies demonstrate spinal glial activation and increased proinflammatory cytokines in animal models of neuropathic pain. When these data are considered together, a unifying hypothesis emerges which implicates a role of central neuroimmune processes in the etiology of neuronal and behavioral hypersensitivity. The present investigation assessed the influence of propentofylline, a glial modulating and anti-inflammatory agent, on the development of L5 spinal nerve transection-induced hyperalgesia and associated enhancement of spinal neuroimmune responses using real-time reverse transcription-polymerase chain reaction, RNase protection assay, enzyme-linked immunosorbent assay, and immunocytochemistry in rats. The results show that chronic propentofylline treatment attenuated the development of hyperalgesia and restored the analgesic activity of acute morphine in neuropathic rats. These findings directly correlated with the ability of propentofylline to inhibit glial activation and enhanced spinal proinflammatory cytokines following peripheral nerve injury. These findings along with our earlier observations of an anti-allodynic activity of propentofylline using the identical animal model of mononeuropathy supports the concept that modulation of glial and neuroimmune activation may be potential therapeutic targets to treat or prevent neuropathic pain. Further, restoration of the analgesic activity of morphine by propentofylline treatment suggests that increased glial activity and proinflammatory cytokine responses may account for the decreased analgesic efficacy of morphine observed in the treatment of neuropathic pain.
Asunto(s)
Citocinas/biosíntesis , Morfina/farmacología , Umbral del Dolor/efectos de los fármacos , Nervios Espinales/efectos de los fármacos , Xantinas/farmacología , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Citocinas/genética , Relación Dosis-Respuesta a Droga , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/genética , Hiperalgesia/metabolismo , Masculino , Morfina/uso terapéutico , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Umbral del Dolor/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Nervios Espinales/lesiones , Nervios Espinales/metabolismo , Xantinas/uso terapéuticoRESUMEN
Microglia, the intrinsic macrophages of the central nervous system, have previously been shown to be activated in the spinal cord in several rat mononeuropathy models. Activation of microglia and subsequent release of proinflammatory cytokines are known to play a role in inducing a behavioral hypersensitive state (hyperalgesia and allodynia) in these animals. The present study was undertaken to determine whether minocycline, an inhibitor of microglial activation, could attenuate both the development and existing mechanical allodynia and hyperalgesia in an L5 spinal nerve transection model of neuropathic pain. In a preventive paradigm (to study the effect on the development of hypersensitive behaviors), minocycline (10, 20, or 40 mg/kg intraperitoneally) was administered daily, beginning 1 h before nerve transection. This regimen produced a decrease in mechanical hyperalgesia and allodynia, with a maximum inhibitory effect observed at the dose of 20 and 40 mg/kg. The attenuation of the development of hyperalgesia and allodynia by minocycline was associated with an inhibitory action on microglial activation and suppression of proinflammatory cytokines at the L5 lumbar spinal cord of the nerveinjured animals. The effect of minocycline on existing allodynia was examined after its intraperitoneal administration initiated on day 5 post-L5 nerve transection. Although the postinjury administration of minocycline significantly inhibited microglial activation in neuropathic rats, it failed to attenuate existing hyperalgesia and allodynia. These data demonstrate that inhibition of microglial activation attenuated the development of behavioral hypersensitivity in a rat model of neuropathic pain but had no effect on the treatment of existing mechanical allodynia and hyperalgesia.