Design and optimization of the cocktail assay for rapid assessment of the activity of UGT enzymes in human and rat liver microsomes

Abstract

Along with the prevalence of drug combination therapies, an increasing number of cases about drug-drug interactions (DDI) have been reported, which has drawn a lot of attention due to the potential toxicity and/or therapeutic failure. Pharmacokinetic interactions based on drug metabolic enzymes should be responsible for a great many of DDI. UDP-glucuronosyltransferases (UGT) as the main phase II metabolic enzymes are involved in the metabolism of many endogenous and exogenous substrates. Herein, we designed and optimized a validated cocktail method for the simultaneous evaluation of drug-mediated inhibition of the main five UGT isoforms using respective specific probe substrates (estradiol for UGT1A1, chenodeoxycholic acid for UGT1A3, serotonin for UGT1A6, propofol for UGT1A9/PROG and zidovudine for UGT2B7/AZTG) in human and rat liver microsomes by liquid chromatography–tandem mass spectrometry (LCMS/MS). Moreover, we investigated the risk of interactions among UGT probe substrates, and validated the cocktail method by known positive inhibitors of UGT isoforms. To minimize the substrates interaction, we developed two cocktail subgroups which were further optimized via exploring the experimental conditions. In particular, the cocktail inhibition assay for rapid assessment of in vitro rat UGTs was firstly reported and the values of Km in the liver microsomes from humans and rats were close to each other in the specific UGT subtype. In conclusion, this study has successfully established the cocktail approach to explore UGT activity, especially for UGT inhibition in a fast and efficient way.