An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes

Abstract

An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP) enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively) as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam) in the incubates were quantified using LC-MS/MS methods either by an internal standard (IS) calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC₅₀ values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.

Keywords: Cocktail-probe; Cytochrome P450; Drug screenning; Inhibition assessment; LC–MS/MS.

Fig. 1

Chromatograms of metabolites and IS in blank matrix observed in detection channels of acetaminophen, N-desethylamodiquine, 4-hydroxydiclofenac, 4-hydroxymephenytoin, dextrophan, 1-hydroxymidazolam, and verapamil. No significant interfere peaks were observed at the same retention time in the chromatograms of marker metabolites.

Fig. 2

Typical chromatographic profiling of acetaminophen, N-desethylamodiquine, 4-hydroxydiclofenac, 4-hydroxymephenytoin, dextrophan, 1-hydroxymidazolam, and verapamil; two significant peaks were observed in the channel of acetaminophen and the peak with earlier retention time was identified as acetaminophen (see Fig. 3).

Fig. 3

Chromatograms of acetaminophen and phenacetin detected in two detection channels. (A) Chromatogram of incubation sample observed in acetaminophen channel (152.1/110.3). Two peaks with the retention time of 2.04 min and 2.45 min were observed. (B) Chromatogram of acetaminophen standard observed in acetaminophen detection channel with the retention time 2.04 min, indicating that the peak with earlier retention time in A was the target. (C) Chromatogram of phenacetin standard observed in phenacetin channel. A peak was observed at the retention time of 2.45 min. (D) Chromatogram of phenacetin standard observed in acetaminophen transmit channel (180.0/138.2). A peak was observed at the retention time of 2.45 min, demonstrating the cross channel interference between phenacetin and acetaminophen.

Fig. 4

DMSO effect with different concentrations on the activities of CYPs in 6-substrate assay.

Fig. 5

Semi-log graphs obtained using individual or cocktail CYP probe substrates.