RESUMO
BACKGROUND: The Gyrolab™ workstation benefits from fully automated transfer of reagents and samples originating from a storage microplate onto a compact disc containing solid-phase microstructures composed of a 15 nl streptavidin-derivitized bead bed. RESULTS: This paper describes the development, full validation and use of the method in a regulated environment to measure a humanized bispecific monoclonal antibody-domain antibody (GSK-A) molecule using the Gyrolab immunoassay system in cynomolgus nonhuman primate plasma ranging from 5 to 250 µg/ml. The method was subsequently used in support of the TK portion of a regulated preclinical study in monkeys. CONCLUSION: The Gyrolab immunoassay system proved to be a viable alternative to traditional immunoassays and was used to support a regulated preclinical TK study. The speed of analysis that the Gyrolab provides was beneficial in meeting timelines to complete this project as multiple assays and repeat sample analysis could be completed in the same day.
Assuntos
Automação , Imunoensaio/métodos , Animais , Macaca fascicularis , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The cardioprotective effects of glucagon-like peptide-1 (GLP-1) and analogs have been previously reported. We tested the hypothesis that albiglutide, a novel long half-life analog of GLP-1, may protect the heart against I/R injury by increasing carbohydrate utilization and improving cardiac energetic efficiency. METHODS/PRINCIPAL FINDINGS: Sprague-Dawley rats were treated with albiglutide and subjected to 30 min myocardial ischemia followed by 24 h reperfusion. Left ventricle infarct size, hemodynamics, function and energetics were determined. In addition, cardiac glucose disposal, carbohydrate metabolism and metabolic gene expression were assessed. Albiglutide significantly reduced infarct size and concomitantly improved post-ischemic hemodynamics, cardiac function and energetic parameters. Albiglutide markedly increased both in vivo and ex vivo cardiac glucose uptake while reducing lactate efflux. Analysis of metabolic substrate utilization directly in the heart showed that albiglutide increased the relative carbohydrate versus fat oxidation which in part was due to an increase in both glucose and lactate oxidation. Metabolic gene expression analysis indicated upregulation of key glucose metabolism genes in the non-ischemic myocardium by albiglutide. CONCLUSION/SIGNIFICANCE: Albiglutide reduced myocardial infarct size and improved cardiac function and energetics following myocardial I/R injury. The observed benefits were associated with enhanced myocardial glucose uptake and a shift toward a more energetically favorable substrate metabolism by increasing both glucose and lactate oxidation. These findings suggest that albiglutide may have direct therapeutic potential for improving cardiac energetics and function.
Assuntos
Cardiotônicos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Peptídeo 1 Semelhante ao Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Coração , Testes de Função Cardíaca , Hemodinâmica/efeitos dos fármacos , Técnicas In Vitro , Insulina/sangue , Ácido Láctico/sangue , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Domain antibodies (dAbs; â¼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb. RESULTS: A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody. This method was used to analyze pharmacokinetic studies early on in drug development. Furthermore, an immunoassay was developed and the pharmacokinetic samples were reanalyzed. CONCLUSION: The two assays show good correlation (r(2) = 0.92), giving confidence in using either method for quantification of the dAb.