1521-009X/43/7/1137–1146$25.00 http://dx.doi.org/10.1124/dmd.114.063016DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1137–1146, July 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Simultaneous Screening of Activities of Five Cytochrome P450 andFour Uridine 59-Diphospho-glucuronosyltransferase Enzymes inHuman Liver Microsomes Using Cocktail Incubation and Liquid

Chromatography–Tandem Mass Spectrometry s

Boram Lee, Hyeon-Kyeong Ji, Taeho Lee, and Kwang-Hyeon Liu

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea

Received January 4, 2015; accepted April 22, 2015

ABSTRACT

CytochromeP450 (P450) and uridine 59-diphospho-glucuronosyltransferase(UGT) are major metabolizing enzymes in the biotransformation ofmost drugs. Altered P450 and UGT activities are a potential cause ofadverse drug-drug interaction. A method for the simultaneousevaluation of the activities of five P450s (CYP1A2, CYP2C9, CYP2C19,CYP2D6, and CYP3A) and four UGTs (UGT1A1, UGT1A4, UGT1A9, andUGT2B7) was developed using in vitro cocktail incubation and tandemmass spectrometry. The nine probe substrates used in this assaywere 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). Thisnew method involves incubation of two cocktail doses and singlecassette analysis. The two cocktail doses and the concentration ofeach probe substrate in vitro were determined to minimize mutual

drug interactions among substrates. Cocktail A comprised phenac-etin, diclofenac, S-mephenytoin, dextromethorphan, and midazolam,whereas cocktail B comprised SN-38, trifluoperazine, mycophenolicacid, and naloxone. In the incubation study of these cocktails, thereaction mixtures were pooled and simultaneously analyzed usingliquid chromatography–tandem mass spectrometry. The method wasvalidated by comparing inhibition data obtained from the incubation ofeach probe substrate alone with data from the cocktail method. TheIC50 values obtained in both cocktail and individual incubations werein agreement with values previously reported in the literature. Thiscocktail method offers a rapid and robust way to simultaneouslyevaluate phase I and II enzyme inhibition profiles of many newchemical entities. This new method will also be useful in the drugdiscovery process and for advancing the mechanistic understandingof drug interactions.

Introduction

Drug-drug interaction (DDI) can cause intense clinical complications,either by increasing the toxicity or by weakening the therapeutic efficacyof drugs. DDIs are one of the major reasons for drug withdrawal fromthe market (Huang et al., 2008). Therefore, early detection of DDIs isa critical factor of drug discovery that has led to the development of newscreening methods for determining drug interactions. The presentguidelines on pharmacokinetics and DDIs from the U.S. Food and DrugAdministration and European Medicines Agency noted that phase I andphase II metabolizing enzymes are of clinical relevance.Cytochrome P450 (P450) and uridine 59-diphospho-glucuronosyltransferase

(UGT) enzymes are the representative phase I and II enzymes,respectively, which play important roles in the metabolism of most

drugs. In practice, P450s are the enzymes involved in the bio-transformation of about 75% of all drugs metabolized by phase Ienzymes (Guengerich, 2008). UGT enzymes are involved in thebiotransformation of about 20–30% of all drugs (Meech et al., 2012;Stingl et al., 2014). Several in vitro screening methods for thesimultaneous evaluation of potential P450-mediated DDIs have beendeveloped, which include a mixture of P450 probe substrates ina cocktail incubation. The resultant P450-mediated metabolites aredetermined by liquid chromatography–tandem mass spectrometry(LC-MS/MS) (Kim et al., 2005; Na et al., 2011; Spaggiari et al.,2014). Recently, three screening methods for UGT enzyme activitiesusing cocktail incubation and tandem mass spectrometry have alsobeen reported (Gagez et al., 2012; Joo et al., 2014; Seo et al., 2014).A screening method for the simultaneous evaluation of P450 and UGTenzyme activities would accelerate the evaluation of the DDI potentialof new chemical entities during drug development. To date, however,no such method has been developed.In this study, we report a new screening method that allows the

simultaneous evaluation of the activities of five human hepatic P450 andfour UGT enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A,UGT1A1, UGT1A4, UGT1A9, and UGT2B7). Their established probesubstrates are used for the screening of potential inhibitory interactions

This study was supported by grants from the Korea Health Technology R&DProject, Ministry of Health & Welfare, Republic of Korea [Grant No. A111345], and theNational Research Foundation of Korea, Ministry of Science, ICT and Future Planning[NRF-2014M3A9D9069714] and Ministry of Education [NRF-2013R1A1A2008442],Republic of Korea.

dx.doi.org/10.1124/dmd.114.063016.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: DDI, drug-drug interaction; HLM, human liver microsome; IS, internal standard; LC-MS/MS, liquid chromatography–tandemmass spectrometry; P450, cytochrome P450; QC, quality control; SN-38, 7-ethyl-10-hydroxy-camptothecin; SRM, selected reaction monitoring;UDPGA, uridine 59-diphospho-glucuronic acid; UGT, UDP-glucuronosyltransferase.

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of test compounds. We explored the optimal incubation conditions toavoid potential interactions between the cocktail compounds. Further-more, we developed an analytical method for the simultaneousdetermination of five P450-specific substrate metabolites and fourUGT-specific glucuronide metabolites using LC-MS/MS. To validateour newly developed method, the IC50 values of known P450 and UGT

inhibitors from the new screening method were directly compared withthe IC50 values from the incubation of individual substrates.

Materials and Methods

Chemicals and Reagents. Acetaminophen, alamethicin, a-naphthoflavone,NADP+, dextromethorphan, hecogenin, glucose-6-phosphate, glucose-6-phosphate

TABLE 1

SRM conditions for the major metabolites of the nine substrates used in all assays

Enzyme Substrate Conc. Metabolite Transition Polarity Collision Energy

mM m/z eV

CYP1A2 Phenacetin 100 Acetaminophen 152 . 110 ESI+ 25CYP2C9 Diclofenac 10 4-Hydroxydiclofenac 312 . 231 ESI+ 23CYP2C19 S-mephenytoin 100 49-Hydroxymephenytion 235 . 150 ESI+ 27CYP2D6 Dextromethorphan 5 Dextrorphan 258 . 157 ESI+ 50CYP3A Midazolam 5 19-Hydroxymidazolam 342 . 203 ESI+ 25UGT1A1 SN-38 0.5 SN-38-glucuronide 569 . 393.4 ESI+ 30UGT1A4 Trifluoperazine 0.5 Trifluoperazine N-glucuronide 584.5 . 408.5 ESI+ 30UGT1A9 Mycophenolic acid 0.2 Mycophenolic acid-glucuronide 495 . 319 ESI2 25UGT2B7 Naloxone 1 Naloxone 3-glucuronide 504 . 310 ESI+ 30IS Terfenadine — 472 . 436 ESI+ 25

Conc., concentration; ESI2, electrospray ionization (negative mode); ESI+, electrospray ionization (positive mode).

Fig. 1. Structures of P450 and UGT isoform–specific probe substrates, their metabolites, and IS.

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dehydrogenase, ketoconazole, magnolol, mefenamic acid, miconazole, mycophe-nolic acid, naloxone, naloxone 3-glucuronide, niflumic acid, phenacetin, quini-dine, D-saccharic acid 1,4-lactone monohydrate, sulfaphenazole, trifluoperazine,Trizma base, troleandomycin, and UDP-glucuronic acid (UDPGA) were pur-chased from Sigma-Aldrich (St. Louis, MO). 7-Ethyl-10-hydroxy-camptothecin(SN-38) was provided by Santa Cruz Biotechnology (Dallas, TX), andatazanavir, S-benzylnirvanol, bilirubin, dextrorphan, diclofenac, furafyl-line, 4-hydroxydiclofenac, 49-hydroxymephenytoin, 19-hydroxymidazolam,ketoconazole, S-mephenytoin, midazolam, mycophenolic acid-b-D-glucuronide,SN-38 glucuronide, and omeprazole were obtained from Toronto Re-search Chemicals (Toronto, ON, Canada). Solvents were LC-MS grade(Fisher Scientific Co., Pittsburgh, PA). Pooled human liver microsomes(HLMs, catalog no. H2630, mixed gender) were purchased from XenoTech(Lenexa, KS).

Microsomal Incubations. All microsomal incubations were done underlinear incubation time and protein concentration conditions for theformation of metabolites. Incubations contained either each substratecocktail set (A set: phenacetin, diclofenac, S-mephenytoin, dextromethor-phan, and midazolam; B set: SN-38, trifluoperazine, mycophenolic acid,and naloxone) or an individual substrate. The final concentration of

methanol in the cocktail incubation conditions was 1.0% (v/v) (Easterbrooket al., 2001; Uchaipichat et al., 2004). The incubation mixtures (finalvolume: 100 ml) for the cocktail A set contained 0.25 mg/ml microsomalprotein, 0.1 M potassium phosphate buffer (pH 7.4), and various P450enzyme–specific substrates or a substrate cocktail A set. After a 5-minutepreincubation at 37�C, the reactions were initiated by adding a NADPH-generating system containing 3.3 mM glucose-6-phosphate, 1.3 mMb-NADP+, 3.3 mM MgCl2, and 500 unit/ml glucose-6-phosphatedehydrogenase, and further incubated for 15 minutes at 37�C in athermoshaker (Kim et al., 2005). The incubation mixtures (final volume:100 ml) for the cocktail B set consisted of 0.25 mg/ml microsomal protein,25 mg/ml alamethicin, 0.1 M Tris-HCl (pH 7.4), 10 mM MgCl2, andvarious UGT enzyme–specific substrates or a substrate cocktail B set (Jooet al., 2014). After preincubation on ice for 15 minutes, the reactions wereinitiated by the addition of 5 mM UDPGA and incubated for 1 hour at 37�C. Allreactions were terminated by the addition of 50 ml of cold acetonitrilecontaining 5 ng/ml terfenadine [internal standard (IS)] to the mixtures, and werecentrifuged at 10,000g for 5 minutes at 4�C. The supernatants of the individualincubation samples and pooled cocktail reaction samples (A set/B set, 1/1) wereanalyzed by LC-MS/MS.

Fig. 2. Representative selected reaction monitoring chromatograms of P450- and UGT-mediated metabolites of probe substrate and internal standard: acetaminophen (A),4-hydroxydiclofenac (B), 49-hydroxymephenytoin (C), dextrorphan (D), 19-hydroxymidazolam (E), SN-38 glucuronide (F), trifluoperazine N-glucuronide (G), mycophenolicacid glucuronide (H), naloxone 3-glucuronide (I), and terfenadine (internal standard) (J).

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LC-MS/MS Analysis. The samples were analyzed using a Thermo Vantagetriple quadrupole mass spectrometer coupled with a high-performance liquidchromatography system (Thermo Fisher Scientific, San Jose, CA). Analytesseparation was performed on a Phenomenex (Torrance, CA) Kinetex XB-C18high-performance liquid chromatography column (2.6 mm, 100 Å, 100� 2.10 mm).The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1%formic acid in water (B), which formed the following gradient: 0 minute (0% A),0–1 minute (40% A), 1–5 minutes (50% A), 5–5.1 minutes (0% A), and 5.1–8 min-utes (0% A) (Joo et al., 2014). Quantitation was performed by selected reac-tion monitoring (SRM) of the [M+H]+ (or [M–H]–) ion and the related production for each metabolite, using an IS to establish peak area ratios. The SRMtransitions and collision energies determined for each metabolite and IS arelisted in Table 1.

Method Validation. Four inter- and intraday validations were performedto validate the LC-MS/MS method for the simultaneous quantification of thefive P450 and four UGT probe metabolites in microsomal incubates.Calibration standards were prepared at six different concentrations from 20 to5000 nM (acetaminophen, 4-hydroxydiclofenac, 49-hydroxymephenytoin,trifluoperazine N-glucuronide, and mycophenolic acid glucuronide) or 2 to500 nM (dextrorphan, 19-hydroxymidazolam, SN-38 glucuronide, andnaloxone 3-glucuronide) in a blank microsomal mixture. Quality control(QC) samples were prepared separately at two different concentrations (5 and20 nM for dextrorphan, 19-hydroxymidazolam, SN-38 glucuronide, andnaloxone 3-glucuronide; 50 and 200 nM for other metabolites) for assays.Inter- and intraday precision and accuracy were determined by analyzingreplicates of the QC samples (n = 4).

Comparison of the Cocktail and Individual Substrates for InhibitionScreening. Known inhibitors of specific P450 and UGT enzymes (a-naphthoflavonefor CYP1A2, sulfaphenazole for CYP2C9, S-benzylnirvanol for CYP2C19,quinidine for CYP2D6, ketoconazole for CYP3A, atazanavir for UGT1A1,hecogenin for UGT1A4, niflumic acid for UGT1A9, and mefenamic acid forUGT2B7) were incubated with each substrate cocktail set and with theindividual substrates alone, and the results were compared. The incubationswere performed (as described earlier) with various inhibitor concentrations, andall incubations were performed in triplicate. Furafylline, paroxetine, andtroleandomycin were preincubated for 10 minutes at 37�C with HLMs and aNADPH-generating system (as described earlier). The reactions were initiatedby the addition of the individual or cocktail substrate. With the exception of theaddition of P450 or UGT isoform–specific inhibitors, all other incubationconditions were as described earlier.

Data Analysis. The P450 or UGT isoform–mediated activities in the presenceof inhibitors were expressed as percentages of the corresponding control values.The percentage of inhibition was calculated by the ratio of the amounts ofmetabolites formed, in the presence and absence of the specific inhibitor. Tocalculate the enzyme inhibition IC50 values, the relevant data were fitted to aninhibitory effect model [i.e., v = Emax � (12 [I]/(IC50 + [I]))] using WinNonlin(Pharsight, Mountain View, CA).

Results

Substrate Selection and Optimization of Microsomal Incuba-tions. The structures of the P450 and UGT probe substrates, theirmetabolites, and the internal standard used in the assays are shown inFig. 1. In this study, we selected a P450 isoform–specific probesubstrate based on the preferred and accepted P450 substrates forassessing activity in vitro (Tucker et al., 2001; Na et al., 2011). TheUGT isoform–specific substrates were selected on the basis of theirspecificity and previously published data (Hanioka et al., 2001; DiMarco et al., 2005; Picard et al., 2005; Uchaipichat et al., 2006; Jooet al., 2014). The probe substrates for the P450 and UGT isoforms wereas follows: phenacetin, CYP1A2; diclofenac, CYP2C9; S-mephenytoin,CYP2C19; dextromethorphan, CYP2D6; midazolam, CYP3A; SN-38,UGT1A1; trifluoperazine, UGT1A4; mycophenolic acid, UGT1A9; andnaloxone, UGT2B7. The optimum microsomal incubation times were15 minutes and 1 hour for phase I (cocktail A set) and phase II (cocktail Bset), respectively, with 0.25 mg/ml microsomal protein. The concentrationof each probe substrate was optimized to avoid interactions betweenthem: 100 mM for phenacetin, 10 mM for diclofenac, 100 mM forS-mephenytoin, 5 mM for dextromethorphan, 5 mM for midazolam,0.5 mM for SN-38, 0.5 mM for trifluoperazine, 0.2 mM for mycophenolicacid, and 1 mM for naloxone (Table 1). These concentrations were lowerthan their respective Km values.Development of a Simultaneous Analytical Method Using

LC-MS/MS. We developed a method for simultaneously analyzingmetabolites of five P450 and four UGT probe substrates usingLC-MS/MS. Among the nine metabolites, only mycophenolic acidglucuronide was detected in the negative mode because of its poorionization property in the positive mode. The remaining eightmetabolites and the IS were monitored in the positive mode. TheSRM transitions and optimal MS/MS collision energy are described inTable 1. The representative chromatograms for the five P450 and fourUGT probe metabolites and the IS in microsomal incubation mixturesare presented in Fig. 2. As shown in Fig. 2, all of the metabolites wereeluted in less than 6.5 minutes and separated into their individual SRMchannels. The retention times for acetaminophen, 4-hydroxydiclofenac,49-hydroxymephenytoin, dextrorphan, 19-hydroxymidazolam, SN-38glucuronide, trifluoperazine N-glucuronide, mycophenolic acid glucu-ronide, naloxone 3-glucuronide, and terfenadine were approximately3.3, 6.2, 3.9, 3.4, 3.9, 3.6, 4.8, 4.0, 3.0, and 5.8 minutes, respectively.For acetaminophen, three peaks were observed in the microsomalincubation. Similar results have also been observed in other reportedstudies (Joo and Liu, 2013; Pillai et al., 2013; Song et al., 2013). The

Fig. 3. Comparison of the results from the cocktailincubation with the individual incubations for each offive P450 (A) and four UGT (B) enzyme activities.Incubations were performed with the individual sub-strate (j) and each substrate cocktail set (u) in pooledHLMs. The activities are the means of triplicateincubations.

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peak with a retention time of 3.3 minutes was identified as acet-aminophen by comparing it with the retention time of an authenticstandard.

Cocktail Dose Selection. The inhibition potential of each P450 orUGT substrate was evaluated by comparing the reaction of eachmetabolite in the single-substrate incubations to the response of thesame metabolite formed in incubations with the two-substratecocktail set. The change in each P450 and UGT enzyme activitylevel was less than 15% in each cocktail set, compared with theindividual incubation (Fig. 3). The relative standard deviationsranged from 2.4 to 10.3% (n = 3) for the results of the cocktailincubation procedure.

Method Validation. The inter- and intraday precision and accuracyof the method were determined by analyzing four QC replicates. Themethod accuracy was expressed as the percentage of the metaboliteconcentration measured in each sample relative to the known amount ofmetabolites added (Joo et al., 2014). Calibration curves produced goodcorrelation coefficients for nine metabolites in the mixture (r . 0.999).The interday and intraday accuracy and precision data for the five P450and four UGT probe metabolites in human liver microsomal incubatesare summarized in Tables 2 and 3. Overall, the inter- and intradayaccuracy was 88.2–110.9% with a precision of less than 15.3%.

Validation of the Screening Method Using Selective Inhibitors.The newly developed screening method, used as a tool for determiningP450 and UGT inhibition, was validated using known selectiveinhibitors of the isoforms (a-naphthoflavone, CYP1A2; sulfaphenazole,CYP2C9; S-benzylnirvanol, CYP2C19; quinidine, CYP2D6; ketocona-zole, CYP3A; atazanavir, UGT1A1; hecogenin, UGT1A4; niflumicacid, UGT1A9; and mefenamic acid, UGT2B7) with their correspond-ing substrates. The IC50 value of each P450 and UGT isoform–selectiveinhibitor was estimated in both individual and cocktail incubations(Table 4). The inhibition curves (Fig. 4) show there was no substantialdifference between the individual inhibitor profiles for the two differentincubation methods (single versus cocktail).

Discussion

Pharmacodynamic interactions between drugs alter the response toone or both drugs while also affecting their plasma concentrations(Singh, 2006). Therefore, the risk of metabolism-based DDIs is alwaysa potential problem to consider during the drug development process.For this reason, in vitro drug interaction studies using HLMs are usedfor the early estimation and prediction of the in vivo drug interactionpotential of new candidate drugs (Baranczewski et al., 2006). SeveralLC-MS/MS–based P450 (Dierks et al., 2001; Kim et al., 2005; Smith

TABLE 2

Intraday (n = 4) validation data for the simultaneous determination of five P450- and four UGT-specific probemetabolites in human liver microsomal incubations using the LC-MS/MS method

Intraday accuracy and precision were determined at two different concentration levels (n = 4).

Metabolites50 nM 200 nM

Conc. Founda Accuracy RSD Conc. Founda Accuracy RSD

nM % % nM % %

Acetaminophen 49.3 98.6 8.1 202.1 101.0 5.54-Hydroxydiclofenac 45.8 91.7 6.4 182.0 91.0 11.749-Hydroxymephenytoin 51.5 103.0 13.4 202.9 101.5 14.1Dextrorphanb 4.41 88.2 15.3 20.0 99.9 3.919-Hydroxymidazolamb 4.85 97.1 7.2 19.2 95.8 12.1SN-38 glucuronideb 5.30 106.0 10.8 19.1 95.7 6.4Trifluoperazine N-glucuronide 45.4 90.8 1.9 179.9 89.9 8.5Mycophenolic acid glucuronide 47.2 94.4 6.3 182.5 91.2 6.8Naloxone 3-glucuronideb 5.55 110.9 3.8 19.4 94.6 3.7

Conc., concentration; RSD, relative standard deviation.aEach value represents the mean of four replicates.bDextrorphan, 19-hydroxymidazolam, SN-38 glucuronide, and naloxone 3-glucuronide concentrations were 5 and 20 nM.

TABLE 3

Interday (n = 4) validation data for the simultaneous determination of five P450- and four UGT-specific probemetabolites in human liver microsomal incubations using the LC-MS/MS method

Interday accuracy and precision were determined at two different concentration levels (n = 4).

Metabolites50 nM 200 nM

Conc. Founda Accuracy RSD Conc. Founda Accuracy RSD

nM % % nM % %

Acetaminophen 52.0 104.1 4.9 202.0 101.0 3.04-Hydroxydiclofenac 50.1 100.1 9.6 198.5 99.2 8.449-Hydroxymephenytoin 51.3 102.7 3.4 203.8 101.9 2.3Dextrorphanb 4.61 92.1 8.5 20.6 103.0 7.119-Hydroxymidazolamb 5.35 107.0 5.7 20.2 100.7 8.7SN-38 glucuronideb 4.81 96.2 9.9 19.9 99.3 5.4Trifluoperazine N-glucuronide 50.7 101.5 3.3 201.4 100.7 5.3Mycophenolic acid glucuronide 46.0 92.9 8.0 188.8 94.4 4.2Naloxone 3-glucuronideb 5.46 109.2 4.9 20.2 101.0 3.2

Conc., concentration; RSD, relative standard deviation.aEach value represents the mean of four replicates.bDextrorphan, 19-hydroxymidazolam, SN-38 glucuronide, and naloxone 3-glucuronide concentrations were 5 and 20 nM.

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et al., 2007; Zambon et al., 2010; Na et al., 2011; Pillai et al., 2013) orUGT (Gagez et al., 2012; Joo et al., 2014; Seo et al., 2014) inhibitionmethods for the evaluation of in vitro drug interaction potential havebeen reported. To date, however, a simultaneous evaluation methodfor both P450 and UGT enzyme activities has not been developed. Theaim of this study was to develop and validate a cocktail assay tosimultaneously evaluate the activity of hepatic P450 and UGTisoforms in HLMs using LC-MS/MS.P450 and UGT are representative phase I and II enzymes responsible

for the biotransformation of a wide variety of drugs (Evans andRelling, 1999). Among the numerous P450 and UGT enzymesidentified to date, five P450 (CYP1A2, CYP2C9, CYP2C19,CYP2D6, and CYP3A) and four UGT (UGT1A1, UGT1A4,UGT1A9, and UGT2B7) enzymes have been shown to play animportant role in the metabolism of marketed drugs. P450 enzymesbiotransform about 75% of all drugs, and the aforementioned fiveP450 human isoforms biotransform about 95% of marketed drugs

(Baj-Rossi et al., 2011). In the case of UGT enzymes, UGT2B7 wassuggested to be responsible for the hepatic glucuronidation of 40% ofdrugs, whereas UGT1A1, 1A4, and 1A9 contribute to an additional47% (Williams et al., 2004; Burchell et al., 2005). Based on these data,we attempted to develop a method for the simultaneous evaluation ofthe activities of five P450 (P450s 1A2, 2C9, 2C19, 2D6, and 3A) andfour UGT (UGTs 1A1, 1A4, 1A9, and 2B7) enzymes, which areprimarily responsible for drug metabolism.The optimum incubation conditions for the assessment of enzyme

activities are different between the P450 and UGT enzymes. Themicrosomal incubation of P450 enzymes was conducted in phosphatebuffer, whereas UGT enzymes were incubated in Tris-HCl buffer.UGT enzyme activity was assessed in the presence of alamethicin,a membrane permeabilizing agent (Fisher et al., 2000), and saccharolactone,a b-glucuronidase inhibitor (Oleson and Court, 2008), using a longerincubation time (1 hour). In addition, UDPGA also has inhibitorypotential against CYP2C9 and CYP2C19 activity to some degree (Yan

Fig. 4. Inhibition curves determined using individual substrates and the substrate cocktails. Each P450- and UGT-selective inhibitor was incubated in a separate experimentwith cocktail substrates (d) or an individual substrate set (s). The activity is expressed as the percentage of the remaining activity compared with a control sample containingno inhibitor. Inhibition of phenacetin O-deethylation by a-naphthoflavone (A), diclofenac 4-hydroxylation by sulfaphenazole (B), S-mephenytoin 49-hydroxylation byS-benzylnirvanol (C), dextromethorphan O-demethylation by quinidine (D), midazolam 19-hydroxylation by ketoconazole (E), SN-38 glucuronidation by atazanavir (F),trifluoperazine N-glucuronidation by hecogenin (G), mycophenolic acid glucuronidation by niflumic acid (H), and naloxone 3-glucuronidation by mefenamic acid (I).

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and Caldwell, 2003). Therefore, we used two separate microsomalincubations with P450 and UGT probe substrates. The substrates weredivided into two cocktail groups (cocktail A for five P450 probesubstrates and cocktail B for four UGT probe substrates). These twococktail mixtures were pooled after incubation and analyzed togetherusing LC-MS/MS to reduce the assay time.The selection of specific probe substrates for each P450 and UGT

enzyme is important because multiple enzymes are involved in themetabolism of a single drug. Therefore, in this study, we chosespecific probe substrates for the nine major P450 and UGT enzymes inthe human liver based on previous reports (Table 1). Phenacetin,dextromethorphan, and midazolam are the most frequently used probesubstrates for in vitro activity assessment of CYP1A2, CYP2D6, andCYP3A enzymes, respectively, using the cocktail approach, and arealso the preferred probes for regulatory authorities (Kim et al., 2005;Otten et al., 2011; Pillai et al., 2013). S-mephenytoin (Dierks et al.,2001; Kim et al., 2005; Smith et al., 2007; Zambon et al., 2010; Ottenet al., 2011; Pillai et al., 2013) and omeprazole (Testino and Patonay,

2003; He et al., 2007; Tolonen et al., 2007; Song et al., 2013) are usedas CYP2C19 substrates in cocktail incubation studies. In ourpreliminary study, omeprazole (20 mM) inhibited CYP3A-mediatedmidazolam hydroxylase activity (40% of control) and CYP1A2-mediated phenacetin O-deethylation activity (.20% of control)(Supplemental Fig. 1). Diclofenac (Dierks et al., 2001; Smith et al.,2007; Pillai et al., 2013) and tolbutamide (Bu et al., 2001; Testino andPatonay, 2003; Kim et al., 2005; He et al., 2007; Tolonen et al., 2007;Otten et al., 2011; Joo and Liu, 2013) are generally used as CYP2C9probe substrates. In this study, diclofenac was selected as the CYP2C9probe substrate because it showed higher detection intensity thantolbutamide in the proposed LC-MS/MS method. Based on ourpreliminary findings, phenacetin, diclofenac, S-mephenytoin, dextro-methorphan, and midazolam were selected as the CYP1A2, CYP2C9,CYP2C19, CYP2D6, and CYP3A probe substrates, respectively(cocktail A set). For the cocktail B set (UGT enzymes), SN-38,trifluoperazine, mycophenolic acid, and naloxone were selected asprobe substrates for UGT1A1, UGT1A4, UGT1A9, and UGT2B7,

Fig. 5. Inhibitory effects of a-naphthoflavone (A), sulfaphenazole (B), S-benzylnirvanol (C), quinidine (D), ketoconazole (E), atazanavir (F), hecogenin (G), niflumic acid(H), and mefenamic acid (I) on phenacetin O-deethylation (d), diclofenac 4-hydroxylation (s), S-mephenytoin 49-hydroxylation (.), dextromethorphan O-demethylation(n), midazolam 19-hydroxylation (j), SN-38 glucuronidation (u), trifluoperazine N-glucuronidation (♦), mycophenolic acid glucuronidation ()), and naloxone3-glucuronidation (m) on incubation with pooled HLMs. Data are the means of triplicate experiments.

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respectively, based on previously reported data (Uchaipichat et al.,2004; Di Marco et al., 2005; Picard et al., 2005) and our recentlypublished data (Joo et al., 2014).Following the selection of the P450 and UGT probe substrates for

the cocktail incubation study, the potential metabolic interactionsamong the five P450 metabolites and four UGT metabolites of theselected probe substrates were evaluated. The simultaneous incubationof five or four substrates may lead to interactions. As shown in Fig. 3,P450 and UGT enzyme activities in the cocktail incubations were inaccordance with the results from individual incubations, suggestingthat drug interactions among probe substrates in the present cocktailset were negligible.P450 and UGT isoform–selective inhibitors represent the most

powerful tools available for metabolizing enzyme phenotyping. Thewell known P450 and UGT inhibitors include a-naphthoflavone forCYP1A2 (von Moltke et al., 1996), sulfaphenazole for CYP2C9(Khojasteh et al., 2011), S-benzylnirvanol for CYP2C19 (Suzuki et al.,2002), quinidine for CYP2D6 (Khojasteh et al., 2011), ketoconazolefor CYP3A (Khojasteh et al., 2011), hecogenin for UGT1A4(Uchaipichat et al., 2006), and niflumic acid for UGT1A9 (Minerset al., 2011). Atazanavir (Zhang et al., 2005) and mefenamic acid(Mano et al., 2007) are known inhibitors of UGT1A1 and UGT2B7,respectively. Our results demonstrated that a-naphthoflavone, sulfa-phenazole, S-benzylnirvanol, quinidine, hecogenin, and niflumic acidstrongly and selectively inhibited CYP1A2, CYP2C9, CYP2C19,

CYP2D6, UGT1A4, and UGT1A9, respectively (Fig. 5; Table 5). Liuet al. (2011) also reported that selective P450 inhibitors such asa-naphthoflavone, sulfaphenazole, and quinidine have negligibleinhibitory potential against UGT isoforms. Ketoconazole alsoselectively inhibits CYP3A with an IC50 value of 0.03 mM, whichis at least 100-fold more selective compared with its effects against allother P450s and UGTs tested (Fig. 5E) (Ren et al., 2013). Theinhibitory potential of ketoconazole on UGT1A1-mediated SN-38glucuronidation (IC50 = 4.8 mM) concurred with previous findings inwhich ketoconazole had inhibitory potential on UGT1A1-catalyzedb-estradiol glucuronidation (IC50 = 4.1 mM) (Liu et al., 2011).Hecogenin and niflumic acid are UGT1A4 and UGT1A9 selectiveinhibitors, respectively, and they have a negligible inhibitory effect onthe P450 isoforms tested (IC50 . 20 mM; Table 5). Atazanavirstrongly inhibited UGT1A1 activity with an IC50 value of 0.4 mM;however, it also inhibited CYP3A activity (IC50 = 1.8 mM; Fig. 5F).Mefenamic acid inhibited UGT2B7 activity (IC50 = 5.0 mM), but alsoweakly inhibited CYP1A2 and CYP2C9 activity with IC50 values of8.3 and 12 mM, respectively (Fig. 5I).We also evaluated the IC50 values of the characterized inhibitors

for each P450 and UGT enzyme using both the individual and thecocktail substrates (Fig. 4). The IC50 value of each cocktail set usingthis approach was comparable to those of the individual substratesand was in agreement with those previously reported (Table 4). Thisconfirms that the IC50 values of P450 and UGT inhibitors can be

TABLE 4

Comparison of IC50 values estimated using the individual substrate and the substrate cocktails of well known P450- and UGT-selective inhibitors

Inhibitors of P450 and UGT enzymes were incubated with each substrate cocktail set and with individual substrate alone. IC50 values were calculated using a nonlinear least-squares regressionanalysis.

Enzyme Substrates InhibitorsIC50 (mM 6 S.D.)a

ReferencesIndividual Substrate Cocktail Substrate Literature

CYP1A2 Phenacetin a-Naphthoflavone 0.014 6 0.003 0.013 6 0.004 0.01–0.27 (Gao et al., 2007; Zhang et al., 2008; Perloff et al., 2009)CYP2C9 Diclofenac Sulfaphenazole 0.70 6 0.08 0.68 6 0.08 0.3–1.5 (Dierks et al., 2001; Kim et al., 2005)CYP2C19 S-mephenytoin S-benzylnirvanol 1.02 6 0.13 1.20 6 0.06 0.4–0.41 (Walsky and Obach, 2003; Walsky and Obach, 2004)CYP2D6 Dextromethorphan Quinidine 0.11 6 0.01 0.13 6 0.01 0.02–0.68 (Dierks et al., 2001; Kim et al., 2005)CYP3A Midazolam Ketoconazole 0.038 6 0.001 0.032 6 0.0004 0.09–0.24 (Dierks et al., 2001; Kim et al., 2005)UGT1A1 SN-38 Atazanavir 0.37 6 0.11 0.38 6 0.13 0.4–2.5 (Zhang et al., 2005)UGT1A4 Trifluoperazine Hecogenin 0.89 6 0.21 0.86 6 0.11 0.64–1.5 (Uchaipichat et al., 2006)UGT1A9 Mycophenolic acid Niflumic acid 0.78 6 0.10 0.83 6 0.18 0.1–8 (Vietri et al., 2000; Mano et al., 2006; Miners et al., 2011)UGT2B7 Naloxone Mefenamic acid 4.18 6 1.01 5.04 6 1.34 0.3–370 (Mano et al., 2007; Knights et al., 2009)

aValues represent the mean (6S.D.) of experiments performed in triplicate.

TABLE 5

Effects of specific enzyme inhibitors on cytochrome P450 and uridine 59-diphospho-glucuronosyltransferase metabolicactivities in pooled human liver microsomes

Enzyme Inhibitors

IC50a

P450s UGTs

1A2 2C9 2C19 2D6 3A 1A1 1A4 1A9 2B7

mM mM mM mM mM mM mM mM mM

CYP1A2 a-Naphthoflavone 0.01 — — — — — — — —

CYP2C9 Sulfaphenazole — 0.7 — — — — — — —

CYP2C19 S-benzylnirvanol — — 1.2 — — — — — —

CYP2D6 Quinidine — — — 0.2 — — — — —

CYP3A Ketoconazole — — — — 0.03 4.8 — — —

UGT1A1 Atazanavir 15 20 10 13 1.8 0.4 15 — —

UGT1A4 Hecogenin — — — — — — 0.9 — —

UGT1A9 Niflumic acid 15 — — — — 20 — 0.8 —

UGT2B7 Mefenamic acid 8.3 12 — — — — — — 5.0

—, IC50 . 50 mΜ.aValues represent the mean of experiments performed in triplicate.

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accurately determined using the cocktail assay instead of individualsubstrate incubations, which would save considerable time in thescreening process for new chemical entities. One exception observedwas in the inhibition of CYP2C19 by S-benzylnirvanol, whichshowed a slight difference compared with previously publishedvalues. The published IC50 value of 0.41 mM for S-benzylnirvanolagainst CYP2C19-mediated S-mephenytoin hydroxylation activity(Walsky and Obach, 2003) was lower than our present data (1.2 mM;Table 4). Such a difference is not unusual and could be due to the useof different microsomal incubation conditions (Dierks et al., 2001).Nonetheless, our results demonstrate that the IC50 values ofinhibitors against the nine enzymes can be determined accuratelyby cocktail incubation and cassette analysis, thereby reducing thetime required.In conclusion, we developed a LC-MS/MS method for the si-

multaneous determination of five P450 and four UGT enzyme ac-tivities. Nine substrates were divided into two cocktail sets forincubation and pooled for LC-MS/MS analysis in a single run. Thisprocess allowed us to simultaneously evaluate the activity of five P450and four UGT enzyme activities, using well known P450 and UGTisoform–specific inhibitors. In addition, this cocktail method involvingmultiple substrates provided inhibition profiles similar to thoseobtained from single-substrate incubations. Therefore, these resultssuggest that this newly developed assay can be a useful tool for robustand rapid screening, which accelerates the prediction of the P450 andUGT inhibitory potential of new chemical entities.

Authorship ContributionsParticipated in research design: B. Lee, T. Lee, Liu.Conducted experiments: B. Lee, Ji, Liu.Contributed new reagents or analytic tools: B. Lee, Liu.Performed data analysis: B. Lee, T. Lee, Liu.Wrote or contributed to the writing of the manuscript: B. Lee, T. Lee, Liu.

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Address correspondence to: Kwang-Hyeon Liu, College of Pharmacy andResearch Institute of Pharmaceutical Sciences, Kyungpook National University,80 Daehak-ro, Buk-gu, Daegu 702-701, Korea. E-mail: dstlkh@knu.ac.kr

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