RESUMO
Weight gain and diabetes have been reported during treatment with atypical antipsychotic drugs (AAPDs). Patients treated with the glucocorticoid receptor antagonist (GRA) and the progesterone receptor antagonist (PRA) mifepristone [estra-4,9-dien-3-one, 11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)-(11ß,17ß)-(9CI)] experienced significant reduction in the weight gain observed when patients were treated with olanzapine or risperidone. To understand the pharmacology responsible for this finding, we discovered LLY-2707 [N-(5-(tert-butyl)-3-(2-fluoro-5-methylpyridin-4-yl)-2-methyl-1H-indol-7-yl)methanesulfonamide], a novel and selective GRA, and evaluated its utility in preclinical models of AAPD-associated weight gain and diabetes. In vitro, LLY-2707 was a highly selective and potent GRA. GR occupancy in vivo was assessed using ex vivo binding where LLY-2707 inhibited [(3)H]dexamethasone binding to the liver. Modest but statistically significant decreases in brain ex vivo binding were observed with high doses of CORT-108297 [(R)-4α-(ethoxymethyl)-1-(4-fluorophenyl)-6-((4-(trifluoromethyl)phenyl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinoline] and LLY-2707, but mifepristone inhibited at all doses. Central activity of the GRAs was confirmed by their ability to suppress amphetamine-induced increases in locomotor activity. The increases in the body weight of female rats treated with olanzapine (2 mg/kg PO) over 14 days were reduced in a dose-dependent manner by coadministration of LLY-2707. Similar decreases, although less robust, in body weight were seen with mifepristone and CORT-108297. In addition, sGRAs prevented the glucose excursion after intragastric olanzapine infusions consistent with a direct effect on the hyperglycemia observed during treatment with AAPDs. At doses effectively preventing weight gain, LLY-2707 did not substantially interfere with the dopamine D2 receptor occupancy by olanzapine. Therefore, GRA coadministration may provide a novel treatment modality to prevent the weight gain and diabetes observed during treatment with AAPDs.
Assuntos
Antipsicóticos/toxicidade , Indóis/farmacologia , Receptores de Glucocorticoides/antagonistas & inibidores , Sulfonamidas/farmacologia , Aumento de Peso/efeitos dos fármacos , Redução de Peso/efeitos dos fármacos , Animais , Compostos Aza/química , Compostos Aza/farmacologia , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Indóis/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mifepristona/química , Mifepristona/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/fisiologia , Sulfonamidas/química , Aumento de Peso/fisiologia , Redução de Peso/fisiologiaRESUMO
Orphan G protein-coupled receptors (oGPCRs) are a class of integral membrane proteins for which endogenous ligands or transmitters have not yet been discovered. Transgenic animal technologies have uncovered potential roles for many of these oGPCRs, providing new targets for the treatment of various diseases. Understanding signaling pathways of oGPCRs and validating these receptors as potential drug targets requires the identification of chemical probe compounds to be used in place of endogenous ligands to interrogate these receptors. A novel chemical probe identification platform was created in which GPCR-focused libraries were screened against sets of oGPCR targets, with a goal of discovering fit-for-purpose chemical probes for the more druggable members of the set. Application of the platform to a set of oGPCRs resulted in the discovery of the first reported small molecule agonists for GPR39, a receptor implicated in the regulation of insulin secretion and preservation of beta cells in the pancreas. Compound 1 stimulated intracellular calcium mobilization in recombinant and native cells in a GPR39-specific manner but did not potentiate glucose-stimulated insulin secretion in human islet preparations.
RESUMO
This article summarizes the proceedings of a symposium held at the conference on "Alcoholism and Stress: A Framework for Future Treatment Strategies" in Volterra, Italy, May 6-9, 2008. Chaired by Markus Heilig and Roberto Ciccocioppo, this symposium offered a forum for the presentation of recent data linking neuropetidergic neurotransmission to the regulation of different alcohol-related behaviors in animals and in humans. Dr. Donald Gehlert described the development of a new corticotrophin-releasing factor receptor 1 antagonist and showed its efficacy in reducing alcohol consumption and stress-induced relapse in different animal models of alcohol abuse. Dr. Andrey Ryabinin reviewed recent findings in his laboratory, indicating a role of the urocortin 1 receptor system in the regulation of alcohol intake. Dr. Annika Thorsell showed data supporting the significance of the neuropeptide Y receptor system in the modulation of behaviors associated with a history of ethanol intoxication. Dr. Roberto Ciccocioppo focused his presentation on the nociceptin/orphanin FQ (N/OFQ) receptors as treatment targets for alcoholism. Finally, Dr. Markus Heilig showed recent preclinical and clinical evidence suggesting that neurokinin 1 antagonism may represent a promising new treatment for alcoholism. Collectively, these investigators highlighted the significance of neuropeptidergic neurotransmission in the regulation of neurobiological mechanisms of alcohol addiction. Data also revealed the importance of these systems as treatment targets for the development of new medication for alcoholism.
Assuntos
Alcoolismo/etiologia , Hormônio Liberador da Corticotropina/fisiologia , Neuropeptídeo Y/fisiologia , Estresse Psicológico/complicações , Alcoolismo/tratamento farmacológico , Animais , Ansiedade/etiologia , Humanos , Antagonistas dos Receptores de Neurocinina-1 , Peptídeos Opioides/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Urocortinas/fisiologia , NociceptinaRESUMO
Resilience to mental and physical stress is a key determinant for the survival and functioning of mammals. Although the importance of stress resilience has been recognized, the underlying neural mediators have not yet been identified. Neuropeptide Y (NPY) is a peptide known for its anti-anxiety-like effects mediated via the amygdala. The results of our current study demonstrate, for the first time that repeated administration of NPY directly into the basolateral nucleus of the amygdala (BLA) produces selective stress-resilient behavioral responses to an acute restraint challenge as measured in the social interaction test, but has no effect on hypothalamic-adrenal-pituitary axis activity or stress-induced hyperthermia. More importantly, the resilient behaviors observed in the NPY-treated animals were present for up to 8 weeks. Antagonizing the activity of calcineurin, a protein phosphatase involved in neuronal remodeling and present in NPY receptor containing neurons within the BLA, blocked the development of long-term, but not the acute increases in social interaction responses induced by NPY administration. This suggests that the NPY-induced long-term behavioral resilience to restraint stress may occur via mechanisms involving neuronal plasticity. These studies suggest one putative physiologic mechanism underlying stress resilience and could identify novel targets for development of therapies that can augment the ability to cope with stress.
Assuntos
Febre/fisiopatologia , Sistema Hipotálamo-Hipofisário/fisiologia , Neuropeptídeo Y/administração & dosagem , Sistema Hipófise-Suprarrenal/fisiologia , Comportamento Social , Estresse Psicológico/prevenção & controle , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiologia , Animais , Febre/tratamento farmacológico , Febre/psicologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Masculino , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Ratos , Ratos Wistar , Estresse Psicológico/fisiopatologia , Estresse Psicológico/psicologia , TempoRESUMO
Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green fluorescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.
Assuntos
Receptores de Neuropeptídeo Y/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Adenilil Ciclases/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Macaca mulatta , Mesocricetus , Microscopia Confocal , Neuropeptídeo Y/química , Neuropeptídeo Y/farmacologia , Fragmentos de Peptídeos/farmacologia , Ensaio Radioligante , Receptores de Neuropeptídeo Y/química , Receptores de Neuropeptídeo Y/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
We describe a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist with advantageous properties for clinical development, and its in vivo activity in preclinical alcoholism models. 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) inhibited 125I-sauvagine binding to rat pituitary membranes and cloned human CRF1 with subnanomolar affinities, with no detectable activity at the CRF2 receptor or other common drug targets. After oral administration to rats, MTIP inhibited 125I-sauvagine binding to rat cerebellar membranes ex vivo with an ED50 of approximately 1.3 mg/kg and an oral bioavailability of 91.1%. Compared with R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine) and CP154526 (N-butyl-N-ethyl-4,9-dimethyl-7-(2,4,6-trimethylphenyl)-3,5,7-triazabicyclo[4.3.0]nona-2,4,8,10-tetraen-2-amine), MTIP had a markedly reduced volume of distribution and clearance. Neither open-field activity nor baseline exploration of an elevated plus-maze was affected by MTIP (1-10 mg/kg). In contrast, MTIP dose-dependently reversed anxiogenic effects of withdrawal from a 3 g/kg alcohol dose. Similarly, MTIP blocked excessive alcohol self-administration in Wistar rats with a history of dependence, and in a genetic model of high alcohol preference, the msP rat, at doses that had no effect in nondependent Wistar rats. Also, MTIP blocked reinstatement of stress-induced alcohol seeking both in postdependent and in genetically selected msP animals, again at doses that were ineffective in nondependent Wistar rats. Based on these findings, MTIP is a promising candidate for treatment of alcohol dependence.
Assuntos
Alcoolismo/tratamento farmacológico , Encéfalo/metabolismo , Piridazinas/farmacocinética , Piridazinas/uso terapêutico , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Tiazóis/farmacocinética , Tiazóis/uso terapêutico , Administração Oral , Consumo de Bebidas Alcoólicas/psicologia , Alcoolismo/genética , Alcoolismo/psicologia , Proteínas de Anfíbios , Animais , Ansiedade/psicologia , Comportamento Animal/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Etanol/administração & dosagem , Masculino , Hormônios Peptídicos , Peptídeos/antagonistas & inibidores , Peptídeos/metabolismo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Piridazinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Autoadministração , Estresse Fisiológico/psicologia , Síndrome de Abstinência a Substâncias/psicologia , Tiazóis/administração & dosagemRESUMO
Preclinical brain receptor occupancy measures have heretofore been conducted by quantifying the brain distribution of a radiolabeled tracer ligand using either scintillation spectroscopy or tomographic imaging. For smaller animals like rodents, the majority of studies employ tissue dissection and scintillation spectroscopy. These measurements can also be accomplished using liquid chromatography coupled to mass spectral detection to measure the brain distribution of tracer molecules, obviating the need for radioligands. In order to validate mass spectroscopy-based receptor occupancy methods, we examined dopamine D2 receptor dose-occupancy curves for a number of antipsychotic drugs in parallel experiments using either mass spectroscopy or radioligand-based approaches. Oral dose-occupancy curves were generated for 8 antipsychotic compounds in parallel experiments using either radiolabeled or unlabeled raclopride tracer. When curves generated by these two methods were compared and ED(50) values determined, remarkably similar data were obtained. Occupancy ED(50) values were (mg/kg): chlorpromazine, 5.1 and 2.7; clozapine, 41 and 40; haloperidol, 0.2 and 0.3; olanzapine, 2.1 and 2.2; risperidone, 0.1 and 0.4; spiperone, 0.5 and 0.4; thioridazine 9.2 and 9.5; and ziprasidone 1.4 and 2.1 (unlabeled and radiolabeled raclopride tracer, respectively). The observation that in vivo application of both techniques led to comparable data adds to the validation state of the mass spectroscopy-based approach to receptor occupancy assays.
Assuntos
Antipsicóticos/metabolismo , Antagonistas de Dopamina , Racloprida , Receptores de Dopamina D2/metabolismo , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Antagonistas de Dopamina/farmacocinética , Relação Dose-Resposta a Droga , Masculino , Espectrometria de Massas , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Racloprida/farmacocinética , Compostos Radiofarmacêuticos , Ratos , Ratos Sprague-DawleyRESUMO
Repeated exposure to stressful conditions is linked to the etiology of affective disorders. The melanin-concentrating hormone-1 receptor (MCHR1) may be a novel mechanism that is involved in the modulation of stress responses and affective states. The role of MCHR1 in neuroendocrine, behavioral, and neurochemical stress, and anxiety-related responses was examined by monitoring the effects of melanin-concentrating hormone (MCH) and the selective MCHR1 antagonist, GW3430, in inbred C57Bl/6NTac and MCHR1-knockout (KO) and wild-type (WT) mice. Intracerebroventricular injection of MCH increased plasma corticosterone, and produced anxiety-related responses in the elevated plus maze. The selective MCHR1 antagonist, GW3430, blocked the neuroendocrine and behavioral effects of MCH and produced anxiolytic-like effects by itself in animal models of anxiety. Moreover, KO mice had an anxiolytic-like phenotype in behavioral models of anxiety, and GW3430 had anxiolytic-like effects in WT, but not KO mice. Lastly, stressor-evoked acetylcholine release within the prefrontal cortex of inbred and WT mice, but not KO mice, was blocked by GW3430. We show that MCH elicits anxiety-like responses and that the effects of a selective MCHR1 antagonist and the phenotype of KO mice are consistent with anxiolytic-like action. Distinct behavioral, physiological, and neurochemical stress, and anxiety-related responses were selectively modulated by the MCHR1, and these actions may involve corticolimbic regulation of stress responsivity and anxiety.
Assuntos
Comportamento Animal/fisiologia , Química Encefálica/fisiologia , Sistemas Neurossecretores/metabolismo , Receptores de Somatostatina/fisiologia , Estresse Fisiológico , Acetilcolina/metabolismo , Hormônio Adrenocorticotrópico/sangue , Alprazolam/farmacologia , Animais , Ansiolíticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Corticosterona/sangue , Relação Dose-Resposta a Droga , Hormônios Hipotalâmicos/administração & dosagem , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Melaninas/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sistemas Neurossecretores/efeitos dos fármacos , Hormônios Hipofisários/administração & dosagem , Distribuição Aleatória , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/deficiência , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologia , Estresse Fisiológico/psicologiaRESUMO
BACKGROUND AND OBJECTIVES: Low level laser therapy (LLLT) offers promising symptomatic relief of osteoarthritic (OA) pain. We examined efficacy of active LLLT versus sham LLLT on finger joints and three superficial nerves. STUDY DESIGN/MATERIALS AND METHODS: OA-patients randomly assigned, received three treatments per week for 6 weeks of LLLT (n = 42) or sham LLLT (n = 46). RESULTS: Pain relief, morning stiffness, and functional status did not significantly improve for LLLT versus placebo. No significant differences were found in finger range of motion, except carpometacarpal opposition (P = 0.011), grip strength, and patient global assessment which improved for active LLLT participants (P = 0.041). CONCLUSIONS: LLLT is no better than placebo at reducing pain, morning stiffness, or improving functional status for OA-hand patients.
Assuntos
Mãos/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Osteoartrite/radioterapia , Idoso , Alumínio , Arsenicais , Feminino , Articulações dos Dedos/fisiopatologia , Articulações dos Dedos/efeitos da radiação , Gálio , Mãos/fisiopatologia , Força da Mão/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/fisiopatologia , Medição da Dor , Amplitude de Movimento Articular/fisiologia , Resultado do TratamentoRESUMO
Smallpox disease has been eradicated from the human population since 1979, but is again a concern because of its potential use as an agent of bioterrorism or biowarfare. World Health Organization-sanctioned repositories of infectious Variola virus are known to occur in both Russia and the United States, but many believe other undeclared and unregulated sources of the virus could exist. Thus, validation of improved methods for definitive identification of smallpox virus in diagnostic specimens is urgently needed. In this paper, we describe the discovery of suspected Variola infected human tissue, fixed and preserved for decades in largely unknown solutions, and the use of routine histology, electron microscopy, and ultimately DNA extraction and fluorogenic 5' nuclease (TaqMan) assays for its identification and confirmation.
Assuntos
Varíola/diagnóstico , Fixação de Tecidos , Vírus da Varíola/isolamento & purificação , Arquivos , Técnicas Bacteriológicas , DNA Viral/análise , DNA Viral/genética , Corantes Fluorescentes , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Pele/patologia , Pele/virologia , Varíola/virologia , Taq Polimerase/genética , Taq Polimerase/metabolismo , Vírus da Varíola/genética , Vírus da Varíola/ultraestruturaRESUMO
OBJECTIVES: To use multiplex polymerase chain reaction (PCR) to screen a large number of Escherichia coli clinical isolates for virulence factor genes and to evaluate the importance of several known factors in the etiology of urinary tract infection. METHODS: Eighty-six E. coli isolates from urine or vaginal or rectal swabs of patients with recurrent urinary tract infection were screened for P fimbria (pap), hemolysin (hly), aerobactin (aer), cytotoxic necrotizing factor 1 (cnf1), S fimbria (sfa), and afimbrial adhesion I (afaI) genes by multiplex PCR. The phenotype of the strains was determined for type 1 fimbriae and O antigen serotype. The infectivity of 11 strains with different combinations of virulence factors was tested using a mouse model of unobstructed urinary tract infection. RESULTS: Type 1 fimbriae were present in 81 of the 86 strains and was the only virulence factor in approximately one third of the isolates. Genes for hly, aer, cnf1, sfa, or pap were present in approximately one fourth of the strains; afaI was present less frequently. A positive type 1 fimbriae phenotype was common to all strains that induced a bladder infection in mice. CONCLUSIONS: Multiplex PCR methods can be effectively applied to studies that require genetic screening of numerous E. coli uropathogens. Where phenotypic information was available, it was consistent with genotypes identified by PCR. Infectivity studies showed that the presence of the type 1 fimbriae gene in an E. coli isolate was required to establish a bladder infection. Other genes that were not identified in this study may also be required in mice and humans.