Simultaneous Screening of Activities of Five Cytochrome P450 and Four Uridine 5'-Diphospho-glucuronosyltransferase Enzymes in Human Liver Microsomes Using Cocktail Incubation and Liquid Chromatography-Tandem Mass Spectrometry.

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Lee B, Ji HK, Lee T, Liu KH

Drug Metab Dispos. 2015 Jul;43(7):1137-46. doi: 10.1124/dmd.114.063016. Epub 2015 Apr 22.

PubMed ID

25904760 [ View in PubMed

]

Abstract

Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major metabolizing enzymes in the biotransformation of most drugs. Altered P450 and UGT activities are a potential cause of adverse drug-drug interaction. A method for the simultaneous evaluation of the activities of five P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) and four UGTs (UGT1A1, UGT1A4, UGT1A9, and UGT2B7) was developed using in vitro cocktail incubation and tandem mass spectrometry. The nine probe substrates used in this assay were phenacetin (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), 7-ethyl-10-hydroxy-camptothecin (SN-38) (UGT1A1), trifluoperazine (UGT1A4), mycophenolic acid (UGT1A9), and naloxone (UGT2B7). This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses and the concentration of each probe substrate in vitro were determined to minimize mutual drug interactions among substrates. Cocktail A comprised phenacetin, diclofenac, S-mephenytoin, dextromethorphan, and midazolam, whereas cocktail B comprised SN-38, trifluoperazine, mycophenolic acid, and naloxone. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography-tandem mass spectrometry. The method was validated by comparing inhibition data obtained from the incubation of each probe substrate alone with data from the cocktail method. The IC50 values obtained in both cocktail and individual incubations were in agreement with values previously reported in the literature. This cocktail method offers a rapid and robust way to simultaneously evaluate phase I and II enzyme inhibition profiles of many new chemical entities. This new method will also be useful in the drug discovery process and for advancing the mechanistic understanding of drug interactions.

DrugBank Data that Cites this Article

Drug Enzymes

Drug	Enzyme	Kind	Organism	Pharmacological Action	Actions
Mefenamic acid	Cytochrome P450 1A2	Protein	Humans	Unknown	Substrate Inhibitor	Details
Mefenamic acid	Cytochrome P450 2C9	Protein	Humans	Unknown	Substrate Inhibitor	Details
Mefenamic acid	UDP-glucuronosyltransferase 2B7	Protein	Humans	Unknown	Inhibitor	Details