title: Phenotyping of CYP450 in human liver microsomes using the cocktail approach.
abstract: The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211, Geneva 4, Switzerland.
authors.forename: Dany
authors.initials: D
authors.lastname: Spaggiari
authors.forename: Laurent
authors.initials: L
authors.lastname: Geiser
authors.forename: Youssef
authors.initials: Y
authors.lastname: Daali
authors.forename: Serge
authors.initials: S
authors.lastname: Rudaz
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D055808:Drug Discovery; D006801:Humans; D007527:Isoenzymes; D007553:Isotope Labeling; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D010641:Phenotype; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System