A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliablein vitroinhibitory drug–drug interaction evaluation

Abstract

1. A comprehensive method for the simultaneous characterization of xenobiotic compound inhibition of nine major CYP enzymes in human liver microsomes was established by using 16 CYP-catalyzed reactions of 14 probe substrates with three cocktail incubation sets and a single LC/MS/MS analysis. 2. The three cocktail subgroups were developed to minimize the effects of organic solvents, polyunsaturated fatty acids and mutual substrate interactions: Group I was composed of tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), testosterone (CYP3A4), dextromethorphan (CYP2D6); Group II was composed of nifedipine (CYP3A4), midazolam (CYP3A4), coumarin (CYP2A6), bupropion (CYP2B6), diclofenac (CYP2C9); Group III was composed of phenacetin (CYP1A2), chlorzoxazone (CYP2E1), omeprazole (CYP2C19 and CYP3A4), paclitaxel (CYP2C8), (+)-bufuralol (CYP2D6). In the case of CYP2C9, CYP2C19, CYP2D6 and CYP3A4, multiple probe substrates were used due to the phenomenon of multiple substrate-binding pockets and substrate-dependent inhibition. All probe metabolites were simultaneously analyzed with a polarity switching mode in a single LC/MS/MS run. 3. This method was validated against the single probe substrate assay using 12 well-characterized CYP inhibitors and two new entities (GT0918, MDV3100). The IC50 values of each inhibitor in the cocktail agreed well with that of the individual probe drug as well as with values reported in previous literatures.

Screening of ten cytochrome P450 enzyme activities with 12 probe substrates in human liver microsomes using cocktail incubation and liquid chromatography–tandem mass spectrometry

“…In this study, the same internal standard was used for the two cocktail sets although it is ideal to use different internal standards for two separate cocktail sets. Several studies also successfully used the same internal standard for cocktail sets (Kim et al, ; Lee & Kim, ; Peng et al, ).…”

“…In vitro P450 enzyme inhibition assays have been used routinely to assess the P450‐mediated DDI potential of these nine enzymes. Several cocktail assays (also known as N‐in‐one assays) that use a mixture of P450 probe substrates added to liver microsomal incubation (Peng et al, ) have been developed for the high‐throughput screening (HTS) of the P450 inhibition of the nine major human P450 isoforms simultaneously using liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Dinger et al, ; Kim et al, , ; Li, Huang, Nikolic, & van Breemen, ; Sevior et al, ; Tolonen, Petsalo, Turpeinen, Uusitalo, & Pelkonen, ). Several earlier reported cocktail approaches used the N‐in‐one assay (Abass & Pelkonen, ; Dahlinger et al, ; Kozakai et al, ; Li et al, ; Liu et al, ; Sevior et al, ; Tolonen et al, ).…”

“…Several earlier reported cocktail approaches used the N‐in‐one assay (Abass & Pelkonen, ; Dahlinger et al, ; Kozakai et al, ; Li et al, ; Liu et al, ; Sevior et al, ; Tolonen et al, ). However, in recent years, the ‘N‐in‐two’ or ‘N‐in‐three’ cocktail approach has also been used widely to minimize DDI among the cocktail substrates (Di, Kerns, Li, & Carter, ; Dinger et al, ; Dinger, Meyer, & Maurer, ; Kim et al, ; Lee & Kim, ; Peng et al, ).…”

Cell-based in vitro models in environmental toxicology: a review

“…It has been reported that in the EROD assay, the S9 supplement might have an additional direct inhibitory effect on the resorufin fluorescence, and should be used with caution in this approach [50]. Like in the EROD assay, cytochrome activity can be measured using other fluorescent substrates, and specific assays are available for different enzyme subfamilies and polypeptides (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4) [51,52]. Moreover, liquid-chromatography/mass-spectroscopy (LC-MS/MS) can be used to detect and quantify specific cytochrome/substrate reactions (CYP2B6/bupropion, CYP2C8/paclitaxel, CYP2C9/tolbutamide, CYP2C19/Smephenytoin, CYP2D6/dextromethorphan, CYP3A4/ midazolam, CYP3A4/testosterone) [53].…”

The effect of ZnO nanoparticles on liver function in rats

“…38 The quality and quantity of Measurements of cYP 1a2, 2c11, and 3a2 activity CYP450 1A2, CYP450 2C11, and CYP450 3A2 activity were assessed as described. [39][40][41] All microsomal incubations were conducted for 60 minutes at 37°C in a final volume of 500 μL. Each incubation mixture contained pooled microsomes (1.0 mg protein/mL) and an nicotinamide adenine dinucleotide phosphate-regenerating system consisting of MgCl 2 (10 mM), glucose 6-phosphate (10 mM), NADP + (1.0 mM), and 6-phosphate dehydrogenase (2.0 U/mL).…”