RESUMEN
A high throughput approach for the determination of in vitro metabolic stability and metabolic profiles of drug candidates has been developed. This approach comprises the combination of a Biomek FX liquid handling system with 96-channel pipetting capability and a custom-designed 96-well format on-line incubator with efficient thermal conductivity. This combination facilitates automated reagent preparation, sample incubation, and sample purification for microsome stability studies. The overall process is both fast and accurate and meets the challenges of high throughput screening for drug discovery. A custom designed, user-friendly computer program has been incorporated for large-scale data processing and report generation. Several applications are discussed that implement this strategy for rapid selection of compounds in early drug discovery.
Asunto(s)
Diseño de Fármacos , Animales , Cromatografía Liquida , Humanos , Espectrometría de Masas , Microsomas Hepáticos , Reproducibilidad de los ResultadosRESUMEN
Automation of plasma sample preparation for pharmacokinetic studies on VLA-4 antagonists has been achieved by using 96-well format solid-phase extraction operated by Beckman Coulter Biomek 2000 liquid handling system. A Biomek 2000 robot is used to perform fully automated plasma sample preparation tasks that include serial dilution of standard solutions, pipetting plasma samples, addition of standard and internal standard solutions, performing solid-phase extraction (SPE) on Waters OASIS 96-well plates. This automated sample preparation process takes less than 2 h for a typical pharmacokinetic study, including 51 samples, 24 standards, 9 quality controls, and 3-6 dose checks with minimal manual intervention. Extensive validation has been made to ensure the accuracy and reliability of this method. A two-stage vacuum pressure controller has been incorporated in the program to improve SPE efficiency. This automated SPE sample preparation approach combined with liquid chromatography coupled with the high sensitivity and selectivity of tandem mass spectrometry (LC/MS)/MS has been successfully applied on both individual and cassette dosing for pharmacokinetic screening of a large number of VLA-4 antagonists with a limit of quantitation in the range of 1-5 ng/ml. Consequently, a significant throughput increase has been achieved along with an elimination of tedious labor and its consequential tendency to produce errors.
Asunto(s)
Integrina alfa4beta1/antagonistas & inhibidores , Integrina alfa4beta1/sangre , Animales , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Masculino , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
The effect of dosing vehicle excipients such as PEG400, propylene glycol, Tween 80, and hydroxypropyl-beta-cyclodextrin on the accuracy of LC/MS measurements used in pharmacokinetic studies is examined. Using PEG400 as a probe compound, the concentration-time profile of the excipient in plasma from rats dosed both orally and intravenously is determined. These excipient plasma concentrations can result in a 2-5-fold increase in calculated plasma clearance values when the excipient interferes with the quantitation of the dosed compound. This can result in false rejection of a compound in a drug discovery screen. Several plasma purification methods and enhanced chromatographic selectivity are examined as ways to minimize or avoid excipient effects, particularly for very polar compounds. The combination of efficient sample purification and selective chromatography provides an effective way to diminish the significant interference effects of PEG400 and Tween 80. When appropriate, using negative ion mode MS or changing a dosing vehicle excipient, such as substituting propylene glycol for PEG400, provides an alternative approach for eliminating signal interference. The mechanism of excipient-related signal interference is discussed in relation to both competition of gas-phase proton-transfer reactions and high viscosity of dosing excipients.