In vitro LC-MS cocktail assays to simultaneously determine human cytochrome P450 activities.
Using liquid chromatography-mass spectrometry (LC-MS), two sensitive cocktail assays were developed, one to simultaneously determine activities of the cytochrome P450 enzymes, CYP1A2 (phenacitin), 2B6 (bupropion), 2C8 (amodiaquine) and 2C19 (omperazole), and the other to determine simultaneously activities of CYP3A4/5 (testosterone), 2C9 (tolbutamide) and 2D6 (dextromethorphan). These cocktail assays are sensitive, require only a small amount of microsomal protein, employ selective and high turnover CYP substrates and do not require post-incubation extraction. In each of these cocktails, no interactions were observed between the substrates. Combining the two cocktails into a single cocktail resulted in significant inhibition of CYP2D6 by amiodiaquine. These assays were used successfully to determine induction of CYP enzymes in microsomes isolated from human hepatocytes treated (72 h) with or without the prototypic inducer, rifampin.
Dixit
Vaishali
V
Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195, USA.
Hariparsad
Niresh
N
Desai
Pankaj
P
Unadkat
Jashvant D
JD
D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000008:administration & dosage / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System