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1.
Bioorg Med Chem ; 28(2): 115247, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31843461

RESUMO

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.


Assuntos
Aminopterina/farmacologia , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Metotrexato/farmacologia , Pró-Fármacos/farmacologia , Aminopterina/administração & dosagem , Aminopterina/química , Animais , Antirreumáticos/administração & dosagem , Antirreumáticos/química , Apoptose/efeitos dos fármacos , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Injeções Intraperitoneais , Metotrexato/administração & dosagem , Metotrexato/química , Estrutura Molecular , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
2.
Nat Metab ; 1(8): 830-843, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-32694768

RESUMO

Human and rodent brown adipose tissues (BAT) appear morphologically and molecularly different. Here we compare human BAT with both classical brown and brite/beige adipose tissues of 'physiologically humanized' mice: middle-aged mice living under conditions approaching human thermal and nutritional conditions, that is, prolonged exposure to thermoneutral temperature (approximately 30 °C) and to an energy-rich (high-fat, high-sugar) diet. We find that the morphological, cellular and molecular characteristics (both marker and adipose-selective gene expression) of classical brown fat, but not of brite/beige fat, of these physiologically humanized mice are notably similar to human BAT. We also demonstrate, both in silico and experimentally, that in physiologically humanized mice only classical BAT possesses a high thermogenic potential. These observations suggest that classical rodent BAT is the tissue of choice for translational studies aimed at recruiting human BAT to counteract the development of obesity and its comorbidities.


Assuntos
Tecido Adiposo Marrom/fisiologia , Animais , Humanos , Camundongos , Termogênese
4.
J Biomed Mater Res B Appl Biomater ; 106(7): 2673-2680, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29424962

RESUMO

The extent of metal release from implant materials that are irradiated during radiotherapy may be influenced by irradiation-formed radicals. The influence of gamma irradiation, with a total dose of relevance for radiotherapy (e.g., for cancer treatments) on the extent of metal release from biomedical stainless steel AISI 316L and a cobalt-chromium alloy (CoCrMo) was investigated in physiological relevant solutions (phosphate buffered saline with and without 10 g/L bovine serum albumin) at pH 7.3. Directly after irradiation, the released amounts of metals were significantly higher for irradiated CoCrMo as compared to nonirradiated CoCrMo, resulting in an increased surface passivation (enhanced passive conditions) that hindered further release. A similar effect was observed for 316L showing lower nickel release after 1 h of initially irradiated samples as compared to nonirradiated samples. However, the effect of irradiation (total dose of 16.5 Gy) on metal release and surface oxide composition and thickness was generally small. Most metals were released initially (within seconds) upon immersion from CoCrMo but not from 316L. Albumin induced an increased amount of released metals from AISI 316L but not from CoCrMo. Albumin was not found to aggregate to any greater extent either upon gamma irradiation or in the presence of trace metal ions, as determined using different light scattering techniques. Further studies should elucidate the effect of repeated friction and fractionated low irradiation doses on the short- and long term metal release process of biomedical materials. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2673-2680, 2018.


Assuntos
Raios gama , Radioterapia , Vitálio/química , Animais , Bovinos , Humanos , Soroalbumina Bovina/química
5.
Microbiol Res ; 199: 10-18, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28454705

RESUMO

The budding yeast S. cerevisiae is widely used as a eukaryotic model organism to elucidate the mechanism of action of low molecular weight compounds. This report describes the development of two high throughput screening methods based on cell viability either by monitoring the reduction of alamarBlue® (resazurin) or by direct optical measurement of cell growth. Both methods can be miniaturized to allow screening of large numbers of samples, and can be performed using S. cerevisiae in 384 and 1536-well format. The alamarBlue® approach achieves Z' values of >0.7 with signal to basal ratios of >6.5, and around 1.1 million low molecular weight compounds were screened, identifying approximately 25,000 primary hits. Dose response curves generated for a subset (1930) using both alamarBlue® and optical density methods showed significant overlap. In genome-wide haploinsufficiency profiling (HIP), 572 of these hits demonstrated a diverse mechanism of action, affecting >25% of all yeast strains.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Saccharomyces cerevisiae/química , Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Teóricos , Oxazinas/análise , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/química , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/crescimento & desenvolvimento , Xantenos/análise
6.
PLoS Genet ; 12(11): e1006374, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27855158

RESUMO

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.


Assuntos
Proteínas de Transporte/química , Criptococose/tratamento farmacológico , Cryptococcus neoformans/efeitos dos fármacos , Ergolinas/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Antifúngicos/farmacologia , Proteínas de Transporte/genética , Criptococose/microbiologia , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Ergolinas/química , Humanos , Testes de Sensibilidade Microbiana , Conformação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética
7.
AIDS Res Hum Retroviruses ; 28(12): 1766-74, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22364185

RESUMO

Multidrug-resistant (MDR) HIV-1 presents a challenge to the efficacy of antiretroviral therapy (ART). To examine mechanisms leading to MDR variants in infected individuals, we studied recombination between single viral genomes from the genital tract and plasma of a woman initiating ART. We determined HIV-1 RNA sequences and drug resistance profiles of 159 unique viral variants obtained before ART and semiannually for 4 years thereafter. Soon after initiating zidovudine, lamivudine, and nevirapine, resistant variants and intrapatient HIV-1 recombinants were detected in both compartments; the recombinants had inherited genetic material from both genital and plasma-derived viruses. Twenty-three unique recombinants were documented during 4 years of therapy, comprising ~22% of variants. Most recombinant genomes displayed similar breakpoints and clustered phylogenetically, suggesting evolution from common ancestors. Longitudinal analysis demonstrated that MDR recombinants were common and persistent, demonstrating that recombination, in addition to point mutation, can contribute to the evolution of MDR HIV-1 in viremic individuals.


Assuntos
Genitália Feminina/virologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Plasma/virologia , Recombinação Genética , Adulto , Fármacos Anti-HIV/administração & dosagem , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , Humanos , Lamivudina/administração & dosagem , Dados de Sequência Molecular , Nevirapina/administração & dosagem , RNA Viral/genética , Análise de Sequência de DNA , Zidovudina/administração & dosagem
8.
J Thorac Oncol ; 6(12): 2120-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21900840

RESUMO

INTRODUCTION: Pemetrexed is an established second-line therapy for non-small cell lung cancer (NSCLC). Everolimus has previously been shown to have some clinical activity when used as a single agent in NSCLC. The aim of this phase I study was to evaluate the safety and feasibility of combining pemetrexed with everolimus in patients with NSCLC who had disease progression after one previous treatment. METHODS: Patients with stage IIIb/IV NSCLC and one previous chemotherapy regimen were enrolled. A Bayesian dose-escalation model was used to determine the feasible doses of daily or weekly everolimus combined with pemetrexed (500 mg/m q3w). The primary end point was rate of cycle 1 dose-limiting toxicities (DLTs). Secondary end points included safety, relative dose intensity of pemetrexed, pharmacokinetics, and tumor response. RESULTS: Twenty-four patients received daily everolimus (2.5, 5, 7.5, or 10 mg) and 19 received weekly everolimus (30 or 50 mg) with pemetrexed. Cycle 1 DLTs in the daily regimen included febrile neutropenia, neutropenia, rash/pruritus, and thrombocytopenia; in the weekly regimen, DLTs included neutropenia and stomatitis. The most frequent grade 3/4 adverse events were neutropenia, dyspnea, and thrombocytopenia. Three partial responses were observed with everolimus 5 mg/d and two with 50 mg/wk. Pharmacokinetics did not suggest an influence of everolimus on pemetrexed parameters; pemetrexed resulted in a minor decrease in everolimus exposure with both daily and weekly regimens. CONCLUSIONS: Everolimus 5 mg/d or 50 mg/wk with the standard regimen of pemetrexed are feasible dosages in patients with stage IIIb/IV NSCLC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Teorema de Bayes , Erupção por Droga/etiologia , Dispneia/induzido quimicamente , Everolimo , Feminino , Glutamatos/administração & dosagem , Guanina/administração & dosagem , Guanina/análogos & derivados , Humanos , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Pemetrexede , Prurido/induzido quimicamente , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Estomatite/induzido quimicamente , Trombocitopenia/induzido quimicamente , Resultado do Tratamento
9.
J Pediatr Endocrinol Metab ; 19(11): 1353-7, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17220064

RESUMO

We describe a female infant who developed transient neonatal diabetes mellitus (TNDM) (MIM 601410). At birth she presented with growth retardation and macroglossia. Diabetes was diagnosed on the fourth day of life and it resolved after two months of insulin therapy. Genetic testing revealed the presence of paternal uniparental disomy of chromosome 6 (UPD6) including heterodisomy of 6q24. This is the first documented case of uniparental heterodisomy for chromosome 6.


Assuntos
Cromossomos Humanos Par 6 , Diabetes Mellitus/genética , Dissomia Uniparental/genética , Complicações do Diabetes/complicações , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/tratamento farmacológico , Feminino , Retardo do Crescimento Fetal/etiologia , Humanos , Hiperglicemia/etiologia , Hipoglicemiantes/uso terapêutico , Recém-Nascido , Insulina/uso terapêutico , Macroglossia/etiologia , Polimorfismo Genético , Resultado do Tratamento , Dissomia Uniparental/diagnóstico
10.
Mol Cell Biol ; 25(1): 488-98, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15601868

RESUMO

The Ccr4-Not complex is a conserved global regulator of gene expression, which serves as a regulatory platform that senses and/or transmits nutrient and stress signals to various downstream effectors. Presumed effectors of this complex in yeast are TFIID, a general transcription factor that associates with the core promoter, and Msn2, a key transcription factor that regulates expression of stress-responsive element (STRE)-controlled genes. Here we show that the constitutively high level of STRE-driven expression in ccr4-not mutants results from two independent effects. Accordingly, loss of Ccr4-Not function causes a dramatic Msn2-independent redistribution of TFIID on promoters with a particular bias for STRE-controlled over ribosomal protein gene promoters. In parallel, loss of Ccr4-Not complex function results in an alteration of the posttranslational modification status of Msn2, which depends on the type 1 protein phosphatase Glc7 and its newly identified subunit Bud14. Tests of epistasis as well as transcriptional analyses of Bud14-dependent transcription support a model in which the Ccr4-Not complex prevents activation of Msn2 via inhibition of the Bud14/Glc7 module in exponentially growing cells. Thus, increased activity of STRE genes in ccr4-not mutants may result from both altered general distribution of TFIID and unscheduled activation of Msn2.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Ribonucleases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Reagentes de Ligações Cruzadas/farmacologia , DNA/metabolismo , Regulação da Expressão Gênica , Genótipo , Glucose/metabolismo , Immunoblotting , Imunoprecipitação , Modelos Biológicos , Mutação , Hibridização de Ácido Nucleico , Fosfoproteínas Fosfatases/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Fosfatase 1 , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fator de Transcrição TFIID/química , Transcrição Genética , Técnicas do Sistema de Duplo-Híbrido
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