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1.
Bioorg Med Chem Lett ; 29(4): 674-680, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30522953

RESUMO

The discovery of disease-modifying therapies for Parkinson's Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in LRRK2 are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. As such, research towards brain-permeable kinase inhibitors of LRRK2 has received much attention. In the course of a program to identify structurally diverse inhibitors of LRRK2 kinase activity, a 5-azaindazole series was optimized for potency, metabolic stability and brain penetration. A key design element involved the incorporation of an intramolecular hydrogen bond to increase permeability and potency against LRRK2. This communication will outline the structure-activity relationships of this matched pair series including the challenge of obtaining a desirable balance between metabolic stability and brain penetration.


Assuntos
Indazóis/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Descoberta de Drogas , Ligação de Hidrogênio
2.
J Med Chem ; 57(3): 921-36, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24354345

RESUMO

Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson's disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATP-binding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.


Assuntos
Encéfalo/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirimidinas/química , Animais , Linhagem Celular , Hepatócitos/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Macaca fascicularis , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade
3.
Bioorg Med Chem Lett ; 23(24): 6890-6, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24269482

RESUMO

This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.


Assuntos
Benzazepinas/química , Antagonistas dos Receptores Histamínicos H3/química , Receptores Histamínicos H3/química , Animais , Benzazepinas/farmacocinética , Cães , Meia-Vida , Haplorrinos , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/farmacocinética , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/farmacocinética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Histamínicos H3/metabolismo , Relação Estrutura-Atividade
4.
Bioorg Med Chem Lett ; 23(24): 6897-901, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24161834

RESUMO

This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.


Assuntos
Benzazepinas/química , Antagonistas dos Receptores Histamínicos H3/química , Receptores Histamínicos H3/química , Animais , Benzazepinas/síntese química , Benzazepinas/farmacocinética , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/farmacocinética , Humanos , Microssomos Hepáticos/metabolismo , Ligação Proteica , Ratos , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade
5.
ACS Med Chem Lett ; 4(1): 85-90, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24900567

RESUMO

The modulation of LRRK2 kinase activity by a selective small molecule inhibitor has been proposed as a potentially viable treatment for Parkinson's disease. By using aminopyrazoles as aniline bioisosteres, we discovered a novel series of LRRK2 inhibitors. Herein, we describe our optimization effort that resulted in the identification of a highly potent, brain-penetrant aminopyrazole LRRK2 inhibitor (18) that addressed the liabilities (e.g., poor solubility and metabolic soft spots) of our previously disclosed anilino-aminopyrimidine inhibitors. In in vivo rodent PKPD studies, 18 demonstrated good brain exposure and engendered significant reduction in brain pLRRK2 levels post-ip administration. The strategies of bioisosteric substitution of aminopyrazoles for anilines and attenuation of CYP1A2 inhibition described herein have potential applications to other drug discovery programs.

6.
J Med Chem ; 55(22): 9416-33, 2012 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-22985112

RESUMO

There is a high demand for potent, selective, and brain-penetrant small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether inhibition of LRRK2 kinase activity is a potentially viable treatment option for Parkinson's disease patients. Herein we disclose the use of property and structure-based drug design for the optimization of highly ligand efficient aminopyrimidine lead compounds. High throughput in vivo rodent cassette pharmacokinetic studies enabled rapid validation of in vitro-in vivo correlations. Guided by this data, optimal design parameters were established. Effective incorporation of these guidelines into our molecular design process resulted in the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity, metabolic stability, and brain penetration across multiple species. Advancement of GNE-7915 into rodent and higher species toxicity studies enabled risk assessment for early development.


Assuntos
Encéfalo/metabolismo , Morfolinas/farmacologia , Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Desenho de Fármacos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Morfolinas/síntese química , Morfolinas/farmacocinética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Bibliotecas de Moléculas Pequenas , Distribuição Tecidual
7.
J Neurol Sci ; 315(1-2): 110-4, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22129936

RESUMO

AIM: Histamine H(3) receptor antagonists have been proposed as a novel therapeutic approach for the symptomatic treatment of Alzheimer's disease (AD). However, it is unclear whether there is a neurochemical basis for extending their potential use in vascular and mixed dementias. In this study, we measured cortical H(3) receptors in patients with subcortical ischemic vascular dementia (SIVD) and mixed SIVD/AD (MIX). MATERIALS AND METHODS: Radioligand binding assays using [(3)H]GSK189254 were used to measure H(3) receptors in the postmortem frontal cortex, anterior cingulate gyrus and hippocampus of a cohort of longitudinally assessed SIVD, MIX and age-matched controls. RESULTS: H(3) receptor levels were unchanged in SIVD and MIX in all areas studied. Furthermore, frontal H(3) receptor densities negatively correlated with predeath assessment of cognition using Mini-Mental State Examination (MMSE) scores. CONCLUSION: Our data suggest that H(3) receptors are preserved in SIVD and MIX, thus supporting further assessments of H(3) antagonists as potential therapeutics in these dementias.


Assuntos
Isquemia Encefálica/metabolismo , Córtex Cerebral/metabolismo , Demência/metabolismo , Receptores Histamínicos H3/metabolismo , Idoso , Idoso de 80 Anos ou mais , Isquemia Encefálica/patologia , Córtex Cerebral/patologia , Demência/patologia , Demência Vascular/metabolismo , Demência Vascular/patologia , Feminino , Seguimentos , Humanos , Masculino , Ligação Proteica/fisiologia
8.
Free Radic Biol Med ; 50(5): 633-40, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21185368

RESUMO

Neuroinflammation and the activation of inducible nitric oxide synthase (iNOS) have been proposed to play a role in the pathogenesis of Parkinson disease (PD). In this study we investigated the effects of the selective iNOS inhibitor GW274150 in the 6-OHDA model of PD. 6-OHDA administration was associated with increased numbers of cells expressing iNOS. Administration of the iNOS inhibitor twice daily for 7 days, beginning 2 days after the 6-OHDA lesioning, led to a significant neuroprotection as shown by assessment of the integrity of the nigrostriatal system by tyrosine hydroxylase immunocytochemistry and HPLC assessment of striatal dopamine content. However, GW274150 displayed a bell-shaped neuroprotective profile, being ineffective at high doses. 6-OHDA lesioning was associated with an increase in microglial activation as assessed by the MHC II antigen OX-6 and the number of matrix metalloproteinase 9 (MMP-9)-immunopositive cells. NO is a known modulator of MMP-9, and iNOS inhibition was associated with decreased numbers of MMP-9-immunopositive cells, culminating in a reduction in the numbers of reactive microglia. Withdrawal of GW274150 for a further 7 days negated any neuroprotective effects of iNOS inhibition, suggesting that the damaging effects of inflammation last beyond 7 days in this model and the continued administration of the drug may be required.


Assuntos
Citoproteção , Fármacos Neuroprotetores/administração & dosagem , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Doença de Parkinson/enzimologia , Doença de Parkinson/patologia , Sulfetos/administração & dosagem , Animais , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Metaloproteinase 9 da Matriz/análise , Microglia/efeitos dos fármacos , Microglia/enzimologia , Microglia/patologia , Oxidopamina/metabolismo , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/análise
9.
J Pharmacol Exp Ther ; 332(1): 164-72, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19815811

RESUMO

After oral administration, the nonimidazole histamine H(3) receptor antagonist, 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254), increased histamine release from the tuberomammillary nucleus, where all histaminergic somata are localized, and from where their axons project to the entire brain. To further understand functional histaminergic circuitry in the brain, dual-probe microdialysis was used to pharmacologically block H(3) receptors in the tuberomammillary nucleus, and monitor histamine release in projection areas. Perfusion of the tuberomammillary nucleus with GSK189254 increased histamine release from the tuberomammillary nucleus, nucleus basalis magnocellularis, and cortex, but not from the striatum or nucleus accumbens. Cortical acetylcholine (ACh) release was also increased, but striatal dopamine release was not affected. When administered locally, GSK189254 increased histamine release from the nucleus basalis magnocellularis, but not from the striatum. Thus, defined by their sensitivity to GSK189254, histaminergic neurons establish distinct pathways according to their terminal projections, and can differentially modulate neurotransmitter release in a brain region-specific manner. Consistent with its effects on cortical ACh release, systemic administration of GSK189254 antagonized the amnesic effects of scopolamine in the rat object recognition test, a cognition paradigm with important cortical components.


Assuntos
Benzazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H3/farmacologia , Liberação de Histamina/efeitos dos fármacos , Niacinamida/análogos & derivados , Receptores Histamínicos H3/metabolismo , Acetilcolina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Benzazepinas/química , Encéfalo/metabolismo , Cognição/efeitos dos fármacos , Dopamina/metabolismo , Antagonistas dos Receptores Histamínicos H3/química , Região Hipotalâmica Lateral/efeitos dos fármacos , Região Hipotalâmica Lateral/metabolismo , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Microdiálise , Atividade Motora , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Niacinamida/química , Niacinamida/farmacologia , Perfusão , Ratos , Ratos Sprague-Dawley
10.
J Nucl Med ; 50(12): 2064-72, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19910432

RESUMO

UNLABELLED: The histamine H(3) receptor is a G-protein-coupled presynaptic auto- and heteroreceptor whose activation leads to a decrease in the release of several neurotransmitters including histamine, acetycholine, noradrenaline, and dopamine. H(3) receptor antagonists such as 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) can increase the release of these neurotransmitters and thus may offer potential therapeutic benefits in diseases characterized by disturbances of neurotransmission. The aim of this study was to synthesize and evaluate (11)C-labeled GSK189254 ((11)C-GSK189254) for imaging the histamine H(3) receptor in vivo by PET. METHODS: GSK189254 exhibits high affinity (0.26 nM) and selectivity for the human histamine H(3) receptor. Autoradiography experiments were performed using (3)H-GSK189254 to evaluate its in vitro binding in porcine brain tissues. GSK189254 was labeled by N-alkylation using (11)C-methyl iodide in good yields, radiochemical purity, and specific activity. A series of PET experiments was conducted to investigate (11)C-GSK189254 binding in the porcine brain. RESULTS: In vitro autoradiography demonstrated specific (3)H-GSK189254 binding in the porcine brain; therefore, (11)C-GSK189254 was evaluated in vivo in pigs and showed good brain penetration and high uptake in regions such as the striatum and cortices, known to contain high densities of the histamine H(3) receptors. The radioligand kinetics were reversible, and quantitative analysis was achieved with a 2-tissue-compartmental model yielding the distribution volume as the outcome measure of interest. The distribution volume was reduced to a homogeneous level in all regions after blocking by the coadministration of either unlabeled GSK189254 or ciproxifan, a structurally distinct histamine H(3) antagonist. Further coadministration studies allowed for the estimation of the radioligand affinity (0.1 nM) and the density of histamine H(3) receptor sites in the cerebellum (0.74 nM), cortex (2.05 nM), and striatum (2.65 nM). CONCLUSION: These findings suggest that (11)C-GSK189254 possesses appropriate characteristics for the in vivo imaging of the histamine H(3) receptor by PET.


Assuntos
Benzazepinas/metabolismo , Sistema Nervoso Central/diagnóstico por imagem , Sistema Nervoso Central/metabolismo , Imagem Molecular/métodos , Niacinamida/análogos & derivados , Receptores Histamínicos H3/metabolismo , Animais , Autorradiografia , Benzazepinas/administração & dosagem , Benzazepinas/química , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Cinética , Ligantes , Niacinamida/administração & dosagem , Niacinamida/química , Niacinamida/metabolismo , Tomografia por Emissão de Pósitrons , Radioquímica , Suínos , Trítio/metabolismo
11.
Neuropsychopharmacology ; 34(12): 2585-600, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19657331

RESUMO

To further understand the procognitive actions of GSK189254, a histamine H(3) receptor antagonist, we determined its influence on the modulation of hippocampal neural cell adhesion molecule (NCAM) polysialylation (PSA) state, a necessary neuroplastic mechanism for learning and memory consolidation. A 4-day treatment with GSK189254 significantly increased basal expression of dentate polysialylated cells in rats with the maximal effect being observed at 0.03-0.3 mg/kg. At the optimal dose (0.3 mg/kg), GSK189254 enhanced water maze learning and the associated transient increase in NCAM-polysialylated cells. The increase in dentate polysialylated cell frequency induced by GSK189254 was not attributable to enhanced neurogenesis, although it did induce a small, but significant, increase in the survival of these newborn cells. GSK189254 (0.3 mg/kg) was without effect on polysialylated cell frequency in the entorhinal and perirhinal cortex, but significantly increased the diffuse PSA staining observed in the anterior, ventromedial, and dorsomedial aspects of the hypothalamus. Consistent with its ability to enhance the learning-associated, post-training increases in NCAM PSA state, GSK189254 (0.3 mg/kg) reversed the amnesia induced by scopolamine given in the 6-h post-training period after training in an odor discrimination paradigm. Moreover, GSK189254 significantly improved the performance accuracy of a delayed match-to-position paradigm, a task dependent on the prefrontal cortex and degree of cortical arousal, the latter may be related to enhanced NCAM PSA-associated plasticity in the hypothalamus. The procognitive actions of H3 antagonism combined with increased NCAM PSA expression may exert a disease-modifying action in conditions harboring fundamental deficits in NCAM-mediated neuroplasticity, such as schizophrenia and Alzheimer's disease.


Assuntos
Benzazepinas/farmacologia , Aprendizagem por Discriminação/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H3/farmacologia , Memória/efeitos dos fármacos , Moléculas de Adesão de Célula Nervosa/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Niacinamida/análogos & derivados , Amnésia/induzido quimicamente , Amnésia/tratamento farmacológico , Amnésia/fisiopatologia , Animais , Benzazepinas/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Aprendizagem por Discriminação/fisiologia , Relação Dose-Resposta a Droga , Antagonistas dos Receptores Histamínicos H3/administração & dosagem , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Neurogênese/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Testes Neuropsicológicos , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Percepção Olfatória/efeitos dos fármacos , Percepção Olfatória/fisiologia , Ratos , Ratos Wistar , Escopolamina , Fatores de Tempo
12.
Eur J Neurosci ; 29(12): 2363-74, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19490084

RESUMO

Histaminergic neurons of the hypothalamic tuberomammillary nuclei (TMN) send projections to the whole brain. Early anatomical studies described histaminergic neurons as a homogeneous cell group, but recent evidence indicates that histaminergic neurons are heterogeneous and organized into distinct circuits. We addressed this issue using the double-probe microdialysis in freely moving rats to investigate if two compounds acting directly onto histaminergic neurons to augment cell firing [thioperamide and bicuculline, histamine H(3)- and gamma-aminobutyric acid (GABA)(A)-receptor (R) antagonists, respectively] may discriminate groups of histaminergic neurons impinging on different brain regions. Intra-hypothalamic perfusion of either drug increased histamine release from the TMN and cortex, but not from the striatum. Thioperamide, but not bicuculline, increased histamine release from the nucleus basalis magnocellularis (NBM), bicuculline but not thioperamide increased histamine release from the nucleus accumbens (NAcc). Intra-hypothalamic perfusion with thioperamide increased the time spent in wakefulness. To explore the local effects of H(3)-R blockade in the histaminergic projection areas, each rat was implanted with a single probe to simultaneously administer thioperamide and monitor local changes in histamine release. Thioperamide increased histamine release from the NBM and cortex significantly, but not from the NAcc or striatum. The presence of H(3)-Rs on histaminergic neurons was assessed using double-immunofluorescence with anti-histidine decarboxylase antibodies to identify histaminergic cells and anti-H(3)-R antibodies. Confocal analysis revealed that all histaminergic somata were immunopositive for the H(3)-R. This is the first evidence that histaminergic neurons are organized into functionally distinct circuits that influence different brain regions, and display selective control mechanisms.


Assuntos
Vias Eferentes/metabolismo , Histamina/metabolismo , Região Hipotalâmica Lateral/metabolismo , Neurônios/metabolismo , Receptores Histamínicos H3/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Núcleo Basal de Meynert/metabolismo , Córtex Cerebral/metabolismo , Vias Eferentes/citologia , Vias Eferentes/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Imunofluorescência , Antagonistas GABAérgicos/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/efeitos dos fármacos , Masculino , Microdiálise , Neurônios/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Histamínicos H3/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Vigília/efeitos dos fármacos , Vigília/fisiologia
13.
J Neurochem ; 110(3): 966-75, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19549006

RESUMO

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b - anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.


Assuntos
Modelos Animais de Doenças , Dopamina/fisiologia , Microglia/metabolismo , Oxidopamina , Doença de Parkinson Secundária/metabolismo , Substância Negra/patologia , Animais , Masculino , Microglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Fatores de Tempo
14.
Exp Neurol ; 216(2): 459-70, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19320004

RESUMO

Chronic inflammation is known to occur in the brains of Alzheimer's Disease (AD) patients, including the presence of activated microglia close to amyloid plaques. We utilised real time autoradiography and immunohistochemistry to investigate microglial activation and the potential anti-inflammatory effects of PPARgamma agonists in the Thy-1 APP695swe/Thy-1 PS-1.M146V (TASTPM) overexpressing transgenic mouse model of AD. An age dependent increase in specific [3H](R)-PK11195 binding to peripheral benzodiazepine receptors (PBR)/translocator protein (18 kDa) (TSPO) was observed in the cortex of TASTPM mice compared to wild type mice, indicative of microglial activation. This was consistent with immunohistochemical data showing age-dependent increases in CD68 immunoreactivity co-localised with amyloid beta (Abeta) deposits. In 10 month old TASTPM mice, pioglitazone (20 mg/kg) and ciglitazone (50 mg/kg) significantly reduced [3H](R)-PK11195 and [3H]DPA-713 binding in cortex and hippocampus, indicative of reduced microglial activation. In AD brain, significant [3H](R)-PK11195 and [3H]DPA-713 binding was observed across all stages of the disease. These results support the use of PBR/TSPO autoradiography in TASTPM mice as a functional readout of microglial activation to assess anti-inflammatory drugs prior to evaluation in AD patients.


Assuntos
Mapeamento Encefálico , Córtex Cerebral/efeitos dos fármacos , PPAR gama/agonistas , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Acetamidas/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Autorradiografia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Humanos , Isoquinolinas/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Fragmentos de Peptídeos/metabolismo , Pioglitazona , Presenilina-1/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Pirazóis/metabolismo , Pirimidinas/metabolismo , Tiazolidinedionas/farmacologia , Fatores de Tempo
15.
Eur J Pharmacol ; 604(1-3): 1-11, 2009 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-19100256

RESUMO

The human 5-hydroxytryptamine (5-HT(4)) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT(4a) (r5-HT(4a)), 5-HT(4b) (r5-HT(4b)) and 5-HT(4e) (r5-HT(4e)). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT(4c1)) and present the first comprehensive analysis of 5-HT(4) splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT(4) splice variant specific antibodies. In transfected cells, r5-HT(4c1) receptor exhibited similar binding properties to r5-HT(4a) and r5-HT(4b). Functional studies showed that 5-HT(4) agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (+/-)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT(4c1), but full agonists at r5-HT(4a) and r5-HT(4b). Moreover, in contrast to r5-HT(4a) and r5-HT(4b), r5-HT(4c1) was not constitutively active. TaqMan mRNA analysis showed that r5-HT(4a) expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT(4b) and r5-HT(4c1). mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT(4a) and r5-HT(4b) specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT(4b) immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT(4) splice variant function.


Assuntos
Processamento Alternativo , Anticorpos Monoclonais/farmacologia , Receptores 5-HT4 de Serotonina/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , AMP Cíclico/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores 5-HT4 de Serotonina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Agonistas do Receptor de Serotonina/farmacologia , Transfecção
16.
Psychopharmacology (Berl) ; 201(4): 483-94, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18762914

RESUMO

OBJECTIVES: To test the novel nonimidazole histamine H3 receptor antagonist 5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazapin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide (GSK207040) in a series of behavioral and neurochemical paradigms designed to evaluate its antipsychotic potential. MATERIALS AND METHODS: Acute orally administered GSK207040 was investigated for its capacity to reverse a 24-h-induced deficit in novel object recognition memory, deficits in prepulse inhibition (PPI) induced by isolation rearing, and hyperlocomotor activity induced by amphetamine. The acute neurochemical effects of GSK207040 were explored by analyzing rat anterior cingulate cortex microdialysates for levels of dopamine, noradrenaline, and acetylcholine and by c-fos immunohistochemistry. The potential for interaction with the antipsychotic dopamine D2 receptor antagonist haloperidol was explored behaviorally (spontaneous locomotor activity and catalepsy), biochemically (plasma prolactin), and via ex vivo receptor occupancy determinations. RESULTS: GSK207040 significantly enhanced object recognition memory (3 mg/kg) and attenuated isolation rearing-induced deficits in PPI (1.0 and 3.2 mg/kg) but did not reverse amphetamine-induced increases in locomotor activity. There was no evidence of an interaction of GSK207040 with haloperidol. GSK207040 (3.2 mg/kg) raised extracellular concentrations of dopamine, noradrenaline, and acetylcholine in the anterior cingulate cortex and c-fos expression in the core of the nucleus accumbens was increased at doses of 3.2 and 10.0 mg/kg. CONCLUSIONS: The behavioral and neurochemical profile of GSK207040 supports the potential of histamine H3 receptor antagonism to treat the cognitive and sensory gating deficits of schizophrenia. However, the failure of GSK207040 to reverse amphetamine-induced locomotor hyperactivity suggests that the therapeutic utility of histamine H(3) receptor antagonism versus positive symptoms is less likely, at least following acute administration.


Assuntos
Antipsicóticos/farmacologia , Benzazepinas/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Pirazinas/farmacologia , Esquizofrenia/tratamento farmacológico , Administração Oral , Anfetamina/farmacologia , Animais , Antipsicóticos/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Benzazepinas/administração & dosagem , Relação Dose-Resposta a Droga , Antagonistas dos Receptores Histamínicos/administração & dosagem , Hipercinese/induzido quimicamente , Hipercinese/prevenção & controle , Masculino , Memória/efeitos dos fármacos , Pirazinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores Histamínicos H3/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Esquizofrenia/fisiopatologia , Isolamento Social/psicologia
18.
Neuroreport ; 19(6): 657-60, 2008 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-18382281

RESUMO

The possible neuroprotective role of a novel and highly selective cyclooxygenase-2 inhibitor GW637185X was studied in a model of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury of nigrostriatal dopaminergic (DA) neurons in the mouse. Stereological and microdensitometrical analysis of nigral tyrosine hydroxylase-immunoreactive cell bodies and striatal tyrosine hydroxylase-immunoreactive terminals, respectively, showed that GW637185X exerted a full protection against MPTP-induced degeneration of the nigro-striatal pathway. In contrast to earlier studies, these findings demonstrate that acute inhibition of cyclooxygenase-2 can result in a full neuroprotective effect not only on nigral DA cell bodies, but also on striatal DA terminals in the mouse MPTP model.


Assuntos
Encéfalo/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Intoxicação por MPTP/tratamento farmacológico , Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Dopamina/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
19.
Pain ; 138(1): 61-69, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18164820

RESUMO

Several studies have implicated a potential role for histamine H(3) receptors in pain processing, although the data are somewhat conflicting. In the present study we investigated the effects of the novel potent and highly selective H(3) receptor antagonists GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride) and GSK334429 (1-(1-methylethyl)-4-([1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl]carbonyl)hexahydro-1H-1,4-diazepine) in two rat models of neuropathic pain, namely the chronic constriction injury (CCI) model and the varicella-zoster virus (VZV) model. Both GSK189254 (0.3, 3 and/or 10mg/kg p.o.) and GSK334429 (1, 3 and 10mg/kg p.o.) significantly reversed the CCI-induced decrease in paw withdrawal threshold (PWT) measured using an analgesymeter and/or von Frey hairs. In addition, GSK189254 (3mg/kg p.o.) and GSK334429 (10mg/kg p.o.) both reversed the VZV-induced decrease in PWT using von Frey hairs. We also investigated the potential site of action of this analgesic effect of H(3) antagonists using autoradiography. Specific binding to H(3) receptors was demonstrated with [(3)H]-GSK189254 in the dorsal horn of the human and rat spinal cord, and in human dorsal root ganglion (DRG), consistent with the potential involvement of H(3) receptors in pain processing. In conclusion, we have shown for the first time that chronic oral administration of selective H(3) antagonists is effective in reversing neuropathic hypersensitivity in disease-related models, and that specific H(3) receptor binding sites are present in the human DRG and dorsal horn of the spinal cord. These data suggest that H(3) antagonists such as GSK189254 and GSK334429 may be useful for the treatment of neuropathic pain.


Assuntos
Azepinas/administração & dosagem , Benzazepinas/administração & dosagem , Modelos Animais de Doenças , Antagonistas dos Receptores Histamínicos H3/administração & dosagem , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Niacinamida/análogos & derivados , Medição da Dor/efeitos dos fármacos , Piridinas/administração & dosagem , Receptores Histamínicos H3/metabolismo , Medula Espinal/metabolismo , Animais , Herpes Zoster/complicações , Herpes Zoster/tratamento farmacológico , Herpes Zoster/metabolismo , Humanos , Masculino , Neuralgia/etiologia , Niacinamida/administração & dosagem , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/metabolismo , Ratos , Distribuição Tecidual
20.
J Pharmacol Exp Ther ; 321(3): 1032-45, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17327487

RESUMO

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.


Assuntos
Benzazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos/farmacologia , Niacinamida/análogos & derivados , Nootrópicos/farmacologia , Receptores Histamínicos H3/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Benzazepinas/metabolismo , Benzazepinas/farmacocinética , Ligação Competitiva , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Cães , Agonistas dos Receptores Histamínicos/metabolismo , Agonistas dos Receptores Histamínicos/farmacocinética , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/farmacocinética , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Neurotransmissores/metabolismo , Niacinamida/metabolismo , Niacinamida/farmacocinética , Niacinamida/farmacologia , Nootrópicos/metabolismo , Nootrópicos/farmacocinética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Histamínicos H3/análise , Sus scrofa
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