Simultaneous determination of in vitro CYP activities in human microsomes using hops as a CYP trial inhibitor: A method development study
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Cytochrome P450 (CYP) constitutes a superfamily of hemeproteins that plays a key role in the metabolism of drugs and other xenobiotics. Xenobiotic interactions are a major reason for druginduced toxicity and multiple drug therapy and/or the concomitant use of herbal medicine is common, particularly in patients with several diseases or conditions. A popular assay to evaluate in vitro interactions is the cocktail method using pooled microsomes to simultaneous determine several CYP activities. Hops (Humulus lupulus) is a herb often recommended for sleep deprivations and menopausal problems in women. The aim of this thesis was to develop an in vitro cocktail method based on Inje’s in vivo cocktail to investigate the inhibitory potential of natural remedies and other potential CYP inhibitors toward the CYP enzymes 1A2, 2C9, 2C19, 2D6 and 3A4. The final method was tested using hops as a CYP trial inhibitor. The conducted pilot study gave indications of substrate concentrations, HLM concentration and incubation time for the cocktail method, and the method was further optimized. Addition of MgCl2, ethanol inhibition and substrate specificity was also investigated. The following conditions were chosen; 20 µM phenacetin, 2 µM losartan, 2 µM omeprazole, 10 µM dextromethorphan and 10 µM midazolam, incubated with 0.4 mg proteins/mL HLM for 20 minutes. Maximum ethanol concentration was 1 %. Substrates and metabolites were analyzed by an adapted and validated LC-MSMS method. The inhibitory potential of two types of hops, dried hops and the dietary supplement Hops Flowers, were investigated. All five CYP enzymes were inhibited by hops, but to a different extent, evaluated by calculated IC50-values. Ethanol extracted hops had lower IC50-values compared to water extracted hops. The order of inhibition was as following: CYP2C9 was the most affected, then 3A4, 2C19 or 1A2 and 2D6 being the least affected. In this thesis a cocktail method was developed, validated and showed to be functional. An LCMSMS method was adapted and validated. In vitro «cocktail» inhibition studies are cheap, effective and relatively easy to perform. Even though extrapolation of in vitro data to humans have many limitations, in vitro cocktail inhibition studies are useful to get increased knowledge of CYP interactions between herbal and conventional medicines.