RESUMO
The administration of antiretrovirals (ARVs) for HIV pre-exposure prophylaxis (PrEP) is highly efficacious and may benefit from new long-acting (LA) drug delivery approaches. This paper describes a subcutaneous, reservoir-style implant for the LA delivery of tenofovir alafenamide (TAF) and documents the preclinical assessment of implant safety and pharmacokinetics (PK) in New Zealand White (NZW) rabbits (3 groups of n = 5), beagle dogs (2 groups of n = 6), and rhesus macaques (2 groups of n = 3). Placebo implants were placed in rabbits (n = 10) and dogs (n = 12). Implant parameters, including selection of the TAF form, choice of excipient, and PCL formulation were tuned to achieve targeted concentrations of the active anabolite of TAF, tenofovir diphosphate (TFV-DP), within peripheral blood mononuclear cells (PBMCs) and mucosal tissues relevant to HIV transmission. Sustained concentrations of TFV-DP in PBMCs over 100 fmol/106 cells were achieved in all animal species indicating that the implants effectively delivered TAF for 3-6 months. Unlike placebo implants without TAF, all active implants resulted in local adverse events (AEs) proximal to the implant ranging in severity from mild to moderate and included dermal inflammation and necrosis across all species. Despite these AEs, the implant performed as designed and achieved a constant drug release profile, supporting the continued development of this drug delivery platform.
RESUMO
OBJECTIVES: To advance the initiative of ending the global epidemic, long-lasting HIV protection is needed through sustained release of antiretroviral drugs for months to years. We investigated in macaques the safety and efficacy of biodegradable polycaprolactone implants releasing tenofovir alafenamide for HIV pre-exposure prophylaxis (PrEP). METHODS: Implants were administered subcutaneously in the arm using a contraceptive trocar. Efficacy against vaginal simian-HIV (SHIV) infection was investigated in six pigtailed macaques that received two tenofovir alafenamide implants (0.35â mg/day), one in each arm, for a total release rate of tenofovir alafenamide at 0.7â mg/day. Macaques were exposed to SHIV twice weekly for 6â weeks. Statistical analyses were used to compare outcome with eight untreated controls. Histological assessments were performed on skin biopsies collected near implantation sites. RESULTS: Median (range) tenofovir diphosphate level in PBMCs was 1519 (1068-1898)â fmol/106 cells. All macaques with tenofovir alafenamide implants were protected against vaginal SHIV infection. In contrast, 7/8 controls were infected after a median of 4 SHIV exposures (Pâ=â0.0047). Histological assessment of tissues near tenofovir alafenamide implant sites showed inflammation and necrosis in 5/6 animals, which were not evident by visual inspection. CONCLUSIONS: We demonstrated complete protection against vaginal SHIV infection with two implants releasing a total of 0.7â mg of tenofovir alafenamide per day. We also identified tenofovir diphosphate concentrations in PBMCs associated with complete vaginal protection. Consistent with previous findings, we observed adverse local toxicity and necrosis near the tenofovir alafenamide implant site. Improved tenofovir alafenamide implants that are safe and maintain high efficacy have the potential to provide long-lasting protection against vaginal HIV infection.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Vírus da Imunodeficiência Símia , Animais , Feminino , Infecções por HIV/prevenção & controle , Infecções por HIV/tratamento farmacológico , Tenofovir/efeitos adversos , Fármacos Anti-HIV/uso terapêutico , Macaca , Implantes Absorvíveis , HIV , Necrose/tratamento farmacológico , Emtricitabina/uso terapêutico , Alanina/uso terapêuticoRESUMO
The aim of the study was to assess the quality of life (QOL) and the physical activity of liver transplant recipients compared with the general population. The case-controlled pilot study was accomplished through the administration of 2 questionnaires: 36-item Medical Outcomes Study, Short-Form General Health Survey (SF-36) for quality of life (10 scores) and International Physical Activity Questionnaire (IPAQ) to estimate the physical activity (metabolic equivalent score). Fifty-four patients who underwent liver transplantation using the piggyback technique and 108 controls from the general population at the orthopedic ambulatories were enrolled between 2002 and 2009. Participants had a mean age of 55 years (range, 41-73). The multivariate analysis showed significant differences for some scales of the SF-36: liver transplant recipients displayed lower values for "Mental Composite Score" (P = .043), "physical activity" (P = .001), "role limitations due to physical health" (P = .006), "role limitations due to the emotional state" (P = .006), and "mental health" (P = .010). The metabolic equivalent positively associated with all examined SF-36 scales. The present study focused on the QOL and physical activity of liver transplant recipients, demonstrating that transplant recipients scored lower than the general population. Liver transplantation may allow full recovery of health status, but the physical and social problems persist in some patients. Interventions aimed at improving rehabilitation programs, regular psychosocial support, and follow-up in all phases of treatment may give patients a more satisfying lifestyle after transplantation.
Assuntos
Transplante de Fígado , Atividade Motora , Qualidade de Vida , Adulto , Idoso , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Nível de Saúde , Humanos , Itália , Estilo de Vida , Modelos Lineares , Transplante de Fígado/efeitos adversos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Satisfação do Paciente , Projetos Piloto , Medição de Risco , Fatores de Risco , Inquéritos e Questionários , Fatores de Tempo , Resultado do TratamentoRESUMO
A growing body of evidence suggests that the alpha7 neuronal nicotinic receptor (NNR) subtype is an important target for the development of novel therapies to treat schizophrenia, offering the possibility to address not only the positive but also the cognitive and negative symptoms associated with the disease. In order to probe the relationship of alpha7 function to relevant behavioral correlates we employed TC-5619, a novel selective agonist for the alpha7 NNR subtype. TC-5619 binds with very high affinity to the alpha7 subtype and is a potent full agonist. TC-5619 has little or no activity at other nicotinic receptors, including the alpha4beta2, ganglionic (alpha3beta4) and muscle subtypes. The transgenic th(tk-)/th(tk-) mouse model that reflects many of the developmental, anatomical, and multi-transmitter biochemical aspects of schizophrenia was used to assess the antipsychotic effects of TC-5619. In these mice TC-5619 acted both alone and synergistically with the antipsychotic clozapine to correct impaired pre-pulse inhibition (PPI) and social behavior which model positive and negative symptoms, respectively. Antipsychotic and cognitive effects of TC-5619 were also assessed in rats. Similar to the results in the transgenic mice, TC-5619 significantly reversed apomorphine-induced PPI deficits. In a novel object recognition paradigm in rats TC-5619 demonstrated long-lasting enhancement of memory over a wide dose range. These results suggest that alpha7-selective agonists such as TC-5619, either alone or in combination with antipsychotics, could offer a new approach to treating the constellation of symptoms associated with schizophrenia, including cognitive dysfunction.
Assuntos
Comportamento Animal/efeitos dos fármacos , Benzofuranos/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Neurônios/metabolismo , Agonistas Nicotínicos/uso terapêutico , Quinuclidinas/uso terapêutico , Receptores Nicotínicos/fisiologia , Esquizofrenia/tratamento farmacológico , Psicologia do Esquizofrênico , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Benzofuranos/farmacologia , Clozapina/farmacologia , Clozapina/uso terapêutico , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/psicologia , Comportamento Exploratório/efeitos dos fármacos , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Agonistas Nicotínicos/farmacologia , Regiões Promotoras Genéticas , Quinuclidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Reflexo de Sobressalto/efeitos dos fármacos , Esquizofrenia/metabolismo , Comportamento Social , Tirosina 3-Mono-Oxigenase/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.
Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP2D6/química , Avaliação Pré-Clínica de Medicamentos/métodos , Contagem de Cintilação/métodos , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Químicos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredutases O-Desmetilantes/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/metabolismoRESUMO
A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.
Assuntos
Fármacos Anti-HIV/sangue , Cromatografia Líquida/métodos , Inibidores da Protease de HIV/sangue , Indanos/sangue , Indinavir/sangue , Espectrometria de Massas/métodos , Piperazinas/sangue , Fármacos Anti-HIV/farmacocinética , Automação , Inibidores da Protease de HIV/farmacocinética , Humanos , Indanos/farmacocinética , Indinavir/farmacocinética , Piperazinas/farmacocinética , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
TC-2559 [(E)-N-Methyl-4-[3-(5-ethoxypyridin)yl]-3-buten-1-amine] is a novel nicotinic agonist markedly more selective than recently reported novel nicotinic receptor ligands (selectivity ratio for central nervous system (CNS) to peripheral nervous system (PNS)>4000). TC-2559 competes effectively with [3H]-nicotine binding (K(i)=5 nM) but not with [125I]-bungarotoxin (>50,000 nM). Dopamine release from striatal synaptosomes and ion flux from thalamic synaptosomes indicate that TC-2559 is potent and efficacious in the activation of CNS receptors and significantly reduced glutamate-induced neurotoxicity in vitro. TC-2559 has no detectable effects on muscle and ganglion-type nicotinic acetylcholine receptors at concentrations up to 1 mM. TC-2559 significantly attenuates scopolamine-induced cognitive deficits in a step-through passive avoidance task. Acute and repeated oral dosing of TC-2559 enhances performance in a radial arm maze task. In contrast to the effects of equimolar concentrations of (-) nicotine, TC-2559 does not induce hypothermia and locomotor activity is not enhanced following repeated daily administration of 14 days. TC-2559 has a markedly enhanced CNS-PNS selectivity ratio and an intra-CNS selectivity as evidenced by the improved cognition without increased locomotor activity. The in vitro and in vivo studies in the present study suggest that TC-2559 has the desired profile to be further evaluated as a potential therapeutic agent for neurodegenerative diseases.
Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/efeitos dos fármacos , Piridinas/farmacologia , Receptores Colinérgicos/efeitos dos fármacos , Amnésia/induzido quimicamente , Amnésia/prevenção & controle , Animais , Temperatura Corporal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Dopamina/metabolismo , Ligantes , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Antagonistas Muscarínicos , Fármacos Neuroprotetores/farmacologia , Células PC12 , Ratos , Ratos Sprague-Dawley , Radioisótopos de Rubídio , Escopolamina , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Many proteins contain targeting signals within their sequences that specify their delivery to particular organelles. The peroxisomal targeting signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes. The PTS1 sequence closely approximates Ser-Lys-Leu-COO-. PEX5, the receptor for PTS1, interacts with the signal via a series of tetratricopeptide repeats (TPRs) within its C-terminal half. Here we report the crystal structure of a fragment of human PEX5 that includes all seven predicted TPR motifs in complex with a pentapeptide containing a PTS1 sequence. Two clusters of three TPRs almost completely surround the peptide, while a hinge region, previously identified as TPR4, forms a distinct structure that enables the two sets of TPRs to form a single binding site. This structure reveals the molecular basis for PTS1 recognition and demonstrates a novel mode of TPR-peptide interaction.
Assuntos
Peroxissomos/química , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia em Gel , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Sequências Repetitivas de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: Intrasphincteric injection of botulinum toxin (Botx) has been proposed as treatment for oesophageal achalasia. However, the predictors of response and optimal dose remain unclear. AIMS: To compare the effect of different doses of Botx and to identify predictors of response. PATIENTS/METHODS: A total of 118 achalasic patients were randomised to receive one of three doses of Botx in a single injection: 50 U (n=40), 100 U (n=38), and 200 U (n=40). Of those who received 100 U, responsive patients were reinjected with an identical dose after 30 days. Clinical and manometric assessments were performed at baseline, 30 days after the initial injection of botulinum toxin, and at the end of follow up (mean 12 months; range 7-24 months). RESULTS: Thirty days after the initial injection, 82% of patients were considered responders without a clear dose related effect. At the end of follow up however, relapse of symptoms was evident in 19% of patients who received two injections of 100 U compared with 47% and 43% in the 50 U and 200 U groups, respectively. Using Kaplan-Meier analysis, patients in the 100x2 U group were more likely to remain in remission at any time (p<0.04), with 68% (95% CI 59-83) still in remission at 24 months. In a multiple adjusted model, response to Botx was independently predicted by the occurrence of vigorous achalasia (odds ratio 3.3) and the 100x2 U regimen (odds ratio 3.2). CONCLUSIONS: Two injections of 100 U of Botx 30 days apart appeared to be the most effective therapeutic schedule. The presence of vigorous achalasia was the principal determinant of the response to Botx.
Assuntos
Antidiscinéticos/administração & dosagem , Toxinas Botulínicas/administração & dosagem , Acalasia Esofágica/terapia , Adolescente , Adulto , Idoso , Relação Dose-Resposta a Droga , Feminino , Humanos , Injeções Intramusculares , Masculino , Manometria , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Resultado do TratamentoRESUMO
The three-dimensional structure of a protein can greatly illuminate the relationship between its sequence and its function. However, in the absence of a set of experimentally derived coordinates, one often seeks a model of the protein of interest to guide future study. We describe the combined utilization of orthologous sequence information along with knowledge of the related structural fold to model the interaction between PEX5 and its ligand, the peroxisomal targeting signal-1 (PTS1). With this model, we are able to identify residues within PEX5 that appear to be important for peptide recognition, as well as explain some of the sequence requirements of the PTS1. Specifically, our model highlights four asparagine residues as important for ligand backbone atom recognition, which, along with previously observed examples, suggests this as a general mechanism for the binding of extended polypeptides.
Assuntos
Receptores Citoplasmáticos e Nucleares/química , Sequência de Aminoácidos , Proteínas Fúngicas/química , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
BACKGROUND: Although band ligation is now recommended for prevention of rebleeding from oesophageal varices in cirrhosis, sclerotherapy is still widely used. Patients submitted to chronic sclerotherapy undergo several endoscopies and experience a large number of serious complications. However, long-term outcome is poorly defined. AIMS: To assess the clinical course and prognostic indicators of patients undergoing chronic sclerotherapy for prevention of variceal rebleeding as a basis for future evaluation of long-term band ligation outcome. METHODS: Prospective cohort study; prognostic analysis by the Cox proportional hazards model. RESULTS: A total of 218 consecutive cirrhotic patients (37 Child class A, 154 B, 27 C) were enrolled in the study Varices were obliterated in 139 (64%) patients in a mean of 5 (+/-2.6) sessions and recurred in 58/139 (41.7%) within one year. A total of 132 (60%) patients experienced 283 rebleeding episodes and 73 (33%) died. Bleeding from oesophageal ulcers was the most serious complication causing 14% of all rebleeding episodes. Significant prognostic indicators of sclerotherapy outcome were: Child-Pugh class for variceal obliteration; gastric varices and platelet count for recurrence of varices; failure to obliterate varices, variceal size and gastric varices for rebleeding; blood urea nitrogen and failure to obliterate varices for death. Presence of gastric varices was the only prognostic indicator for death in the 79 patients not achieving variceal obliteration. A mean of 10 endoscopies and of 6 hospital admissions were needed per each patient with an estimated cost of US dollars 7154 per patient during the first two years of therapy. CONCLUSIONS: Sclerotherapy is a very demanding and costly treatment, and is associated with frequent and serious side-effects. The probability of treatment failure is significantly higher in Child C patients with gastric varices. Alternative treatments should be considered for these patients.
Assuntos
Varizes Esofágicas e Gástricas/complicações , Hemorragia Gastrointestinal/prevenção & controle , Cirrose Hepática/complicações , Escleroterapia/métodos , Idoso , Estudos de Coortes , Varizes Esofágicas e Gástricas/diagnóstico , Varizes Esofágicas e Gástricas/mortalidade , Esofagoscopia/métodos , Feminino , Seguimentos , Hemorragia Gastrointestinal/mortalidade , Humanos , Cirrose Hepática/diagnóstico , Cirrose Hepática/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Prevenção Secundária , Estatísticas não Paramétricas , Taxa de Sobrevida , Resultado do TratamentoRESUMO
The case of Poland syndrome reported incorporates a number of previously unrecorded features: hypoplasia of the left lung and agenesia of the epiglottis in addition to the standard symptoms. The case also presents certain features typical of the Moebius Syndrome.
Assuntos
Síndrome de Poland/patologia , Adulto , Epiglote/anormalidades , Epiglote/patologia , Evolução Fatal , Feminino , Humanos , Recém-Nascido , Pulmão/anormalidades , Pulmão/patologiaRESUMO
BACKGROUND: Intrasphincteric injection of botulinum toxin has been reported as a safe and effective alternative treatment in oesophageal achalasia, especially in high-risk and elderly patients. AIM: : To compare two formulations of botulinum toxin in the management of achalasia. PATIENTS AND METHODS: We randomly compared the efficacy and safety of 100 U of Botox (Allergan, Irvine, USA) and 250 U of Dysport (Ipsen, Milan, Italy), injected through a sclerotherapy needle at the level of the lower oesophageal sphincter, in 78 consecutive patients with achalasia. Symptom score, oesophageal manometry and 24 h pH-metry were recorded (before and 1 month after therapy). Symptom score was also obtained 6 months after treatment. RESULTS: One month after treatment, the effects of the toxin on symptoms and oesophageal tests were similar for both formulations. Lower oesophageal sphincter pressure decreased from 31 +/- 12 to 18 +/- 5 mmHg after Botox, and from 35 +/- 9 to 18 +/- 10 after Dysport. At the end of the follow-up period (6 months), symptom score decreased from 5 +/- 1.2 to 1.2 +/- 0.8 after Botox and from 5.2 +/- 1.5 to 1.5 +/- 1 after Dysport. Moreover, the percentages of patients who failed to respond to treatment (10% and 17.5%) and who relapsed during follow-up (12% and 24%) did not differ significantly. No patient complained of reflux symptoms after treatment, although abnormal acid exposure was documented in two subjects. CONCLUSIONS: Both formulations of botulinum toxin have comparable efficacy in the treatment of oesophageal achalasia, for up to 6 months of follow-up.
Assuntos
Toxinas Botulínicas Tipo A/administração & dosagem , Acalasia Esofágica/tratamento farmacológico , Fármacos Neuromusculares/administração & dosagem , Adulto , Idoso , Toxinas Botulínicas Tipo A/efeitos adversos , Química Farmacêutica , Relação Dose-Resposta a Droga , Método Duplo-Cego , Acalasia Esofágica/complicações , Feminino , Refluxo Gastroesofágico/etiologia , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Fármacos Neuromusculares/efeitos adversosRESUMO
Most biological information is contained within gene and genome sequences. However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities. We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches. CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae. Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes. CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes. More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes. Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins. Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine, a heretofore unrecognized role for peroxisomes. These results expand our understanding of peroxisome content and function, demonstrate the utility of CoSMoS for context-sensitive motif scanning, and point to the benefits of improved in silico genome analysis.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genoma Fúngico , Microcorpos/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , SoftwareRESUMO
A drug discrimination procedure was used to characterize the ethanol-like effects of a variety of 5-HT1 agonists. Previous studies found that the degree of substitution of the 5-HT1B/2C agonist TFMPP (m-trifluoromethylphenylpiperazine) depended on the training dose of ethanol. The present studies extend this initial finding to four additional 5-HT agonists with different selectivity for 5-HT1A, 5-HT1B, or 5-HT2C receptors: CGS 12066B (7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo[1,2a]quinoxaline maleate), mCPP [1-(3-chlorophenyl)piperazine diHCl], RU 24969 [5-methoxy-3(1,2,3,4-tetrahydro-4-pyridinyl]-1H-indole succinate and 8-OH DPAT [(+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr]. Separate groups of rats were trained to discriminate 1.0 g/kg (n = 7), 1.5 g/kg (n = 6) or 2.0 g/kg (n = 8) ethanol from water. Following training, three to five doses of each 5-HT agonist were tested twice in each rat. The most selective 5-HT1B agonist tested, CGS 12066B (3-17 mg/kg; IP), completely substituted for the 1.0 g/kg ethanol, but not for 1.5 or 2.0 g/kg ethanol. Likewise, the 5-HT1B/2C agonist mCPP (0.56-1.7 mg/kg; IP) completely substituted only in the 1.0 g/kg ethanol training group. The 5-HT1A/1B agonist RU 24969 (0.1-3.0 mg/kg; IP) substituted for all training doses of ethanol, although in a lower proportion of the rats tested in the 2.0 g/kg ethanol training group. Finally, the 5-HT1A agonist 8-OH DPAT (0.1-1.0 mg/kg, IP) did not substitute completely for any ethanol training dose. The results consistently show that agonists with 5-HT1B activity produce discriminative stimulus effects similar to low and intermediate, but not high, ethanol training doses.
Assuntos
Depressores do Sistema Nervoso Central/administração & dosagem , Discriminação Psicológica , Etanol/administração & dosagem , Agonistas do Receptor de Serotonina/farmacologia , Animais , Condicionamento Operante , Indóis/farmacologia , Masculino , Piperazinas/farmacologia , Quinoxalinas/farmacologia , RatosRESUMO
The forced swimming test (FST) predicts the efficacy of clinically effective antidepressants. In the present study, using the FST we examined the antidepressant potential of three novel tropane analogs: 8-methyl-2beta-propanoyl-3beta-(4-(1-methylethyl)phenyl)-8-azabicy clo[3.2.1] (WF-31) and 2beta-propanoyl-3beta-(4-(1-methylethyl)phenyl)-8-azabicyclo[3.2.1 ]octane (WF-50), selective inhibitors of serotonin uptake, and 8-methyl-2beta-propanoyl-3beta-(4-(1-methylphenyl)-8-azabicyclo[3. 2.1] octane (PTT, WF-11), a selective inhibitor of dopamine uptake. Fluoxetine and GBR 12909 were used as controls for selective inhibitors of serotonin and dopamine, respectively. Drugs were administered three times in a 24-hr period between pretest and test sessions. Intraperitoneal administration of WF-31 (0.1-10.0 mg/kg), WF-50 (0.3-10.0 mg/kg) and fluoxetine (0.3-10.0 mg/kg) dose-dependently decreased immobility while increasing swimming. In contrast, WF-11 (0.3-3.0 mg/kg) dose-dependently decreased immobility and increased both swimming and climbing, whereas GBR 12909 (3.0-30.0 mg/kg) decreased immobility, increased climbing but did not affect swimming. In a separate experiment, WF-11 (1.0 mg/kg) increased locomotor activity, whereas a higher dose of WF-11 (3.0 mg/kg) and GBR-12909 (30.0 mg/kg) produced stereotypic behaviors, suggesting that the effects in the FST may have been attributable to increases in general activity. However, the effects of WF-11 on swimming in the FST indicate that WF-11 produces antidepressant-like effects in addition to motor stimulation. These results confirm previous results that behavioral patterns manifested in the FST are characteristic of specific monoamine uptake inhibitors. In addition, these results demonstrate that WF-31 and WF-50 produce behavioral patterns similar to fluoxetine in the FST without accompanying decreases in motor activity, suggesting a potential antidepressant action. Based on comparisons with fluoxetine, the data suggest WF-31 and WF-50 may be therapeutically useful as potential antidepressant medications.
Assuntos
Antidepressivos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Glicoproteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Tropanos/farmacologia , Animais , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina , Comportamento Estereotipado/efeitos dos fármacos , NataçãoRESUMO
Previous drug discrimination studies have elucidated the importance of gamma-aminobutyric acidA (GABAA), N-methyl-D-aspartate (NMDA) glutamate, and serotonin (5-HT) receptor systems in mediating the discriminative stimulus effects of ethanol. The present study used a three-choice operant drug discrimination procedure in an attempt to determine if salient GABAergic effects could be separated from other stimulus effects of 2.0 g/kg ethanol. Adult male Long-Evans rats (n = 7) were trained to discriminate pentobarbital (10.0 mg/kg; intragastrically (i.g.) from ethanol (2.0 g/kg; i.g.) from water (4.7 ml; i.g.) using food reinforcement. Stimulus substitution tests were conducted following the administration of allopregnanolone (1.0-17.0 mg/kg; intraperitoneally (i.p.)), diazepam (0.1-7.3 mg/kg; i.p.), midazolam (0.0056-17.0 mg/kg; i.p.), dizocilpine (0.01-0.56 mg/kg; i.p.), phencyclidine (1.0-5.6 mg/kg; i.p.), CGS 12066B (3-30 mg/kg; i.p.), RU 24969 (0.1-5.6 mg/kg; i.p.) and morphine (1 or 3.0 mg/kg; i.p.). Within the group, allopregnanolone and midazolam completely substituted (> 80%), and diazepam partly substituted (67%) for the discriminative stimulus effects of pentobarbital. Dizocilpine and phencyclidine partly substituted (58 and 57%, respectively) for ethanol without substantial pentobarbital-appropriate responding. RU 24969, CGS 12066B and morphine did not result in complete substitution for either ethanol or pentobarbital, although RU 24969 resulted in partial (68%) pentobarbital substitution. The ability to train the present three-choice discrimination in rats indicates that the discriminative stimulus effects of 10.0 mg/kg pentobarbital were separable from those of 2.0 g/kg ethanol. The results suggest that the pharmacological effects of ethanol, which can control behavior, may seemingly be modified by training conditions (two-versus three-choice discrimination procedures), to the extent that a receptor system prominently linked to the behavioral activity of ethanol (i.e. GABAA) appears no longer to be involved in the interoceptive effects of the drug.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Aprendizagem por Discriminação , Etanol/farmacologia , Hipnóticos e Sedativos/farmacologia , Pentobarbital/farmacologia , Água/farmacologia , Animais , Diazepam/farmacologia , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Moduladores GABAérgicos/farmacologia , Indóis/farmacologia , Masculino , Midazolam/farmacologia , Morfina/farmacologia , N-Metilaspartato/antagonistas & inibidores , Entorpecentes/farmacologia , Fenciclidina/farmacologia , Pregnanolona/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Long-Evans , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
The aim of the present study was to investigate whether ethanol training affects the ability of Ro 15-4513 to block the discriminative stimulus effects of ethanol dose differentially. Three different groups of rats were trained to discriminate 1.0 g/kg ethanol (n = 8), 1.5 g/kg ethanol (n = 7) or 2.0 g/kg ethanol (n = 8) from water in a two-lever, food-reinforced procedure. Ethanol and water were administered by gavage 20 min before the onset of the session. When the discrimination performance was stable, rats were pretreated with Ro 15-4513 (1-17 mg/kg; i.p.) 5 min before the administration of ethanol. Ro 15-4513 attenuated the discriminative stimulus effects of 1.0 and 1.5 g/kg ethanol but not 2.0 g/kg ethanol in each of the ethanol training groups. Overall, blockade of the discriminative stimulus effects of 1.0 and 1.5 g/kg ethanol by 5.6 mg/kg Ro 15-4513 occurred without significantly altering response rates or blood ethanol concentrations. A decrease in blood ethanol concentration was, however, found with 17 mg/kg Ro 15-4513 in combination with 2.0 g/kg ethanol. These results suggest that the benzodiazepine partial inverse agonist, Ro 15-4513, can attenuate the discriminative stimulus effects associated with low to moderate doses of ethanol (1.0-1.5 g/kg).