Simultaneous Screening of Activities of Five Cytochrome P450 and Four Uridine 5′-Diphospho-glucuronosyltransferase Enzymes in Human Liver Microsomes Using Cocktail Incubation and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major metabolizing enzymes in the biotransformation of most drugs. Altered P450 and UGT activities are a potential cause of adverse drug-drug interaction. A method for the simultaneous evaluation of the activities of five P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) and four UGTs (UGT1A1, UGT1A4, UGT1A9, and UGT2B7) was developed using in vitro cocktail incubation and tandem mass spectrometry. The nine probe substrates used in this assay were phenacetin (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), 7-ethyl-10-hydroxy-camptothecin (SN-38) (UGT1A1), trifluoperazine (UGT1A4), mycophenolic acid (UGT1A9), and naloxone (UGT2B7). This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses and the concentration of each probe substrate in vitro were determined to minimize mutual drug interactions among substrates. Cocktail A comprised phenacetin, diclofenac, S-mephenytoin, dextromethorphan, and midazolam, whereas cocktail B comprised SN-38, trifluoperazine, mycophenolic acid, and naloxone. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography-tandem mass spectrometry. The method was validated by comparing inhibition data obtained from the incubation of each probe substrate alone with data from the cocktail method. The IC50 values obtained in both cocktail and individual incubations were in agreement with values previously reported in the literature. This cocktail method offers a rapid and robust way to simultaneously evaluate phase I and II enzyme inhibition profiles of many new chemical entities. This new method will also be useful in the drug discovery process and for advancing the mechanistic understanding of drug interactions.

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

“…Moreover, the range of obtained IC 50 values was much narrower than those previously reported, such as 0.6–1.3 (Liu et al, ), 0.68–2.61 (Dahlinger et al, ), 0.87–1.82 (Li et al, ), 0.6–1.8 (Kozakai et al, ), 0.67–1.4 (Kim et al, ) and 0.79–1.29 (Peng et al, ), indicating that this method is more accurate than previous cocktail assays. In addition, the measured IC 50 values were consistent with previously reported values (Chen et al, ; Donato et al, ; Eagling, Tjia, & Back, ; Kim et al, ; Kozakai et al, ; Lafite et al, ; Lee et al, a, ; Liu et al, , 2015; Perloff et al, ; Phuc et al, ; Stresser et al, ; Walsky & Obach, ; Walsky et al, b; Wu et al, ), demonstrating that the IC 50 values of NCEs against P450 isoform‐mediated metabolism could be determined precisely using a cocktail assay instead of individual substrate incubations, which would save a tremendous amount of time in DDI studies for NCEs.…”

“…The structure of the 12 P450 probe substrates, their metabolites and internal standard used in the assays are shown in Figure . The substrates of the cocktail doses established for the incubation study (Table ) were selected based on the recommendations of the US FDA (2016) and our previously published data (Lee et al, a; Phuc et al, ). In the case of CYP3A, three substrates were selected due to the existence of multiple binding sites within the CYP3A active site (Kenworthy et al, ; Lee, Shon, & Liu, ).…”

Simultaneous evaluation of substrate‐dependent CYP3A inhibition using a CYP3A probe substrates cocktail

“…However, it showed a lower detection signal than testosterone in the proposed LC‐MS/MS method. The cocktail incubation technique has been used in order to screen simultaneously the activities of several enzymes such as multiple cytochrome P450s or uridine diphosphate glucuronosyltransferases (UGTs) . It is important to optimize substrate concentrations in substrate cocktail incubations to minimize interactions between substrates.…”

Induction of Cytochrome P450 Involved in the Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomes In Vivo

“…Probe drugs that are mainly or exclusively metabolized by an individual P450 enzyme can be used to determine the activity of individual P450s in vivo. Recently, relative P450 activity has been determined by quantitative analysis of substrates in a multiple-probe cocktail assay, providing information on the activity of multiple P450 isoforms in a single experiment (Wang et al, 2013;Lee et al, 2015). According to the enzyme selectivity and specificity of probe substrates and criteria for selecting index substrates recommended by the Food and Drug Administration [(FDA); https://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf], we developed a P450 activity cocktail assay using model substrates for the seven important P450s including CYP1A2 (phenacetin), CYP2B1 (bupropion) (Nishiya et al, 2009), CYP2C7 (amodiaquine), CYP2C11 (omeprazole), CYP2D2 (dextromethorphan) (Walsky and Obach, 2004), CYP2C6 (diclofenac), and CYP3A1/2 (midazolam) (Turpeinen et al, 2009).…”