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

Abstract

Testing for potential drug interactions of new chemical entities is essential when developing a novel drug. In this study, an assay was designed to evaluate drug interactions with 10 major human cytochrome P450 (P450) enzymes incubated in liver microsomes, involving 12 probe substrates with two cocktail incubation sets used in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run. The P450 substrate composition in each cocktail set was optimized to minimize solvent effects and mutual drug interactions among substrates as follows: cocktail A was composed of phenacetin for CYP1A2, bupropion for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, and dextromethorphan for CYP2D6; cocktail B was composed of coumarin for CYP2A6, chlorzoxazone for CYP2E1, astemizole for CYP2J2, and midazolam, nifedipine, and testosterone for CYP3A. Multiple probe substrates were used for CYP3A owing to the multiple substrate-binding sites and substrate-dependent inhibition. After incubation in human liver microsomes, each incubation mixture was pooled and all probe metabolites were simultaneously analysed in a single LC-MS/MS run. Polarity switching was used to acquire the negative-ion mode for hydroxychlorzoxazone and positive-ion mode for the remaining analytes. The method was validated by comparing the inhibition data obtained from incubation of each individual probe substrate alone and with the substrate cocktails. The half-maximal inhibitory concentration values obtained from the cocktail and individual incubations were well correlated and in agreement with previously reported values. This new method will be useful in assessing the drug interaction potential of new chemical entities during new drug development. KEYWORDS drug interaction, high-throughput screening, IC 50 , liquid chromatography-tandem mass spectrometry, new chemical entities *These authors contributed equally to this work.

Lack of Correlation between In Vitro and In Vivo Studies on the Inhibitory Effects of (‒)-Sophoranone on CYP2C9 Is Attributable to Low Oral Absorption and Extensive Plasma Protein Binding of (‒)-Sophoranone

“…The inhibitory effects of SPN on the activities of nine CYP isozymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in human liver microsomes are shown in Figure 2, and the IC 50 values are listed in Table 2. The IC 50 values for the positive controls used in the reversible inhibition studies were in an acceptable degree of accuracy with published values [12,[19][20][21]. Of the P450 isoforms tested, SPN exerted the strongest inhibitory effect on CYP2C9-catalyzed tolbutamide hydroxylation, with an IC 50 value of 0.966 ± 0.149 µM ( Table 2).…”

“…All incubations were performed in triplicate, and the data are shown as the mean ± standard deviation. Incubation samples containing well-known CYP inhibitors for each isozyme (Table 2) in parallel were included to compare inhibitory effects, all of which appear on the US FDA list of recommended or accepted in vitro inhibitors [12,[19][20][21].…”

Inhibitory Effects of Schisandra Lignans on Cytochrome P450s and Uridine 5′-Diphospho-Glucuronosyl Transferases in Human Liver Microsomes

“…The inhibitory potential of six lignans on nine P450 activities was evaluated as previously described with slight modifications [30]. Lignans were first dissolved in methanol.…”

Strong and Selective Inhibitory Effects of the Biflavonoid Selamariscina A against CYP2C8 and CYP2C9 Enzyme Activities in Human Liver Microsomes

“…The inhibitory potential of the five biflavonoids on the metabolism of nine P450 probe substrates was evaluated using previously developed methods with minor modifications [23,24]. Biflavonoids were dissolved in methanol.…”