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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 (LC MS/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 K m 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. [ABSTRACT FROM AUTHOR]

• The inhibition risk of shikonin on UGT enzymes was firstly investigated. • Shikonin significantly inhibited UGT1A1, UGT1A3, UGT1A9/PROG, and UGT2B7/AZTG activities in human and rat liver microsomes. • Shikonin exhibited no time-dependent inhibition on UGT activities. • Shikonin displayed competitive inhibition on UGT isoforms. Shikonin, a natural red colorant, is widely used for food garnishment and cosmetic ingredient in the world. Shikonin also possesses a variety of pharmacological actions, including anti-inflammation and anti-cancer activities. However, little is known about its effects on the UDP-glucuronosyltransferases (UGT) activity. Therefore, the aim of this study was to evaluate the effect of shikonin on the UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 activities via the human and rat liver microsomal assay and cocktail approach. The results showed shikonin inhibited human and rat liver microsomal UGT activity only in a dose-dependent manner. The further enzyme kinetic studies demonstrated that shikonin was not only a competitive inhibitor of human UGT1A1, UGT1A9, and UGT2B7, but also presented competitive inhibition on rat UGT1A1 and AZTG reactions. In conclusion, shikonin as a reversible inhibitor of UGT enzyme has a high-risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions. [ABSTRACT FROM AUTHOR]