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Development of immobilized enzyme reactors based on human recombinant cytochrome P450 enzymes for phase I drug metabolism studies

Bartolini, Manuela
Andrisano, Vincenza
Published in Journal of Chromatography. A. 2008, vol. 1206, no. 1, p. 2-10
Abstract Two cytochrome P450 (CYP)-based immobilized enzyme reactors (IMERs) were developed to perform automated on-line phase I drug metabolism studies. For this purpose, biotinylated recombinant CYP2D6 or CYP3A4 reconstituted systems were anchored to the surface of two monolithic mini-columns (2 mm x 6 mm I.D.), which had been covalently grafted with NeutrAvidin. After optimization of immobilization conditions, the obtained IMERs were integrated on-line into a LC hyphenated to an electrospray ionization MS/MS system. Studies with probe substrates and a known competitive inhibitor were performed, showing the potential of CYP-based IMERs in drug metabolism. In the optimized conditions, ca. 15 experiments were carried out with each bioreactor.
Keywords AvidinBioreactorsBiotinylationChromatography, Liquid/instrumentation/methodsCytochrome P-450 CYP2D6/antagonists & inhibitors/metabolismCytochrome P-450 CYP3A/antagonists & inhibitors/metabolismDrug Evaluation, Preclinical/methodsEnzyme Inhibitors/pharmacologyEnzyme StabilityEnzymes, Immobilized/metabolismHumansKineticsMicrosomes, Liver/chemistryRecombinant Proteins/metabolismSpectrometry, Mass, Electrospray Ionization/instrumentation/methods
Stable URL https://archive-ouverte.unige.ch/unige:1233
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PMID: 18556005

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Deposited on : 2009-03-27

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