Boram Lee, Hyeon Kyeong Ji, Taeho Lee and Kwang-Hyeon Liu...2015/04/22 · Boram Lee, Hyeon Kyeong...

DMD #63016

1

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

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

College of Pharmacy and Research Institute of Pharmaceutical Sciences,

Kyungpook National University, Daegu 702-701, Republic of Korea

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on April 22, 2015 as DOI: 10.1124/dmd.114.063016

at ASPE

T Journals on June 11, 2021

dmd.aspetjournals.org

Dow

nloaded from

http://dmd.aspetjournals.org/

DMD #63016

2

Running title: Simultaneous Evaluation of P450 and UGT Enzyme Activities

Address correspondence to: Professor. Kwang-Heyon Liu, College of Pharmacy and

Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-

ro, Buk-gu, Daegu 702-701, Korea.

Tel: +82 53 950 8567; Fax: +82 53 950 8557; E-mail: [email protected]

Number of Text Pages: 15

Number of Tables: 5

Number of Figures: 5

Number of References: 53

Number of Words

In the Abstract: 259

In the Introduction: 414

In the Discussion: 1505

ABBREVIATIONS:

HLM, human liver microsomes; P450, cytochrome P450; UDPGA, uridine 5′-diphospho-

glucuronic acid; UGT, UDP-glucuronosyltransferase.


at ASPE



Dow

nloaded from


DMD #63016

3

ABSTRACT Cytochrome P450 (P450) and uridine 5′-diphosphoglucuronosyltransferase (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 (DDI). 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 (CYP3A4), 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, while 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 value 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.


at ASPE



Dow

nloaded from


DMD #63016

4

Introduction

Drug–drug interaction (DDI) can cause intense clinical complications, either by increasing

the toxicity or by weakening the therapeutic efficacy of drugs. DDIs are one of the major

reasons for drug withdrawal from the market (Huang et al., 2008). Therefore, the early

detection of DDIs is a critical factor of drug discovery that has led to the development of new

screening methods for determining drug interactions. The present guidelines on

pharmacokinetics and DDIs from the United States Food and Drug Administration (FDA) and

European Medicines Agency (EMA) noted that phase I and phase II metabolizing enzymes

are of clinical relevance.

Cytochrome P450 (P450) and uridine 5′-diphosphoglucuronosyltransferase (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

biotransformation of about 75% of all drugs metabolized by phase I enzymes (Guengerich,

2008). UGT enzymes are involved in the biotransformation of about 20 to 30% of all drugs

(Meech et al., 2012; Stingl et al., 2014). Several in vitro screening methods for the

simultaneous evaluation of potential P450-mediated DDIs have been developed, which

include a mixture of P450 probe substrates in a cocktail incubation. The resultant P450-

mediated metabolites are determined 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 activities using cocktail incubation and tandem mass

spectrometry also have been reported (Gagez et al., 2012; Joo et al., 2014; Seo et al., 2014).

A screening method for the simultaneous evaluation of P450 and UGT enzyme activities

would accelerate the evaluation of the DDI potential of new chemical entities during drug

development. To date, however, no such method has been developed.


at ASPE



Dow

nloaded from


DMD #63016

5

In this study, we report a new screening method that allows the simultaneous evaluation of

the activities of five human hepatic P450 and four UGT enzymes (CYP1A2, CYP2C9,

CYP2C19, CYP2D6, CYP3A, UGT1A1, UGT1A4, UGT1A9, and UGT2B7). Their

established probe substrates are used for the screening of potential inhibitory interactions of

test compounds. We explored the optimal incubation conditions to avoid potential

interactions between the cocktail compounds. Furthermore, we developed an analytical

method for the simultaneous determination of five P450-specific substrate metabolites and

four UGT-specific glucuronide metabolites using LC-MS/MS. To validate our newly

developed method, the IC50 values of known P450 and UGT inhibitors from the new

screening method were directly compared with the IC50 values from the incubation of

individual substrates.


at ASPE



Dow

nloaded from


DMD #63016

6

Materials and Methods

Chemicals and Reagents. Acetaminophen, alamethicin, α-naphthoflavone, β-

nicotinamide adenine dinucleotide phosphate (NADP+), dextromethorphan, hecogenin,

glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), ketoconazole,

magnolol, mefenamic acid, miconazole, mycophenolic acid, naloxone, naloxone 3-

glucuronide, niflumic acid, phenacetin, quinidine, D-saccharic acid 1,4-lactone monohydrate,

sulfaphenazole, trifluoperazine, trizma base, troleandomycin, and UDP-glucuronic acid

(UDPGA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SN-38 was provided

by Santa Cruz Biotechnology (Dallas, TX, USA) and atazanavir, S-benzylnirvanol, bilirubin,

dextrorphan, diclofenac, furafylline, 4-hydroxydiclofenac, 4′-hydroxymephenytoin, 1′-

hydroxymidazolam, ketoconazole, S-mephenytoin, midazolam, mycophenolic acid-β-D-

glucuronide, SN-38 glucuronide, and omeprazole were obtained from Toronto Research

Chemicals (Toronto, ON, Canada). Solvents were LC-MS grade (Fisher Scientific Co.,

Pittsburgh, PA, USA). Pooled human liver microsomes (HLMs, Catalog No. H2630, Mixed

Gender) were purchased from XenoTech (Lenexa, KS, USA).

Microsomal Incubations. All microsomal incubations were done under linear incubation

time and protein concentration conditions for the formation of metabolites. Incubations

contained either each substrate cocktail set (A set: phenacetin, diclofenac, S-mephenytoin,

dextromethorphan, 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) (Easterbrook et al., 2001; Uchaipichat et al., 2004). The

incubation mixtures (final volume, 100 μL) for the cocktail A set contained 0.25 mg/mL

microsomal protein, 0.1 M potassium phosphate buffer (pH 7.4), and various P450 enzyme-


at ASPE



Dow

nloaded from


DMD #63016

7

specific substrates or a substrate cocktail A set. After a 5-min pre-incubation at 37ºC, the

reactions were initiated by adding a NADPH-generating system containing 3.3 mM G6P, 1.3

mM β-NADP+, 3.3 mM MgCl2, and 500 unit/mL G6PDH, and further incubated for 15 min at

37ºC in a thermoshaker (Kim et al., 2005). The incubation mixtures (final volume, 100 μL)

for the cocktail B set consisted of 0.25 mg/mL microsomal protein, 25 μg/mL alamethicin,

0.1 M Tris-HCl (pH 7.4), 10 mM MgCl2, and various UGT enzyme-specific substrates or a

substrate cocktail B set (Joo et al., 2014). After pre-incubation on ice for 15 min, the

reactions were initiated by the addition of 5 mM UDPGA, and incubated for 1 h at 37°C. All

reactions were terminated by the addition of 50 μL cold acetonitrile containing 5 ng/mL

terfenadine (internal standard, IS) to the mixtures, and centrifuging at 10,000 g for 5 min at

4 °C. The supernatants of the individual incubation samples and pooled cocktail reaction

samples (A set/B set, 1/1) were analyzed by LC-MS/MS.

LC-MS/MS Analysis. The samples were analyzed using a Thermo Vantage triple

quadrupole mass spectrometer coupled with a high-performance liquid chromatography

(HPLC) system (Thermo Fisher Scientific, San Jose, CA, USA). Analytes separation was

performed on a Phenomenex Kinetex XB-C18 HPLC column (2.6 μm, 100 Å, 100 mm ×

2.10 mm). The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1%

formic acid in water (B) that formed the following gradient: 0 min (0% A), 0 to 1 min (40%

A), 1 to 5 min (50% A), 5 to 5.1 min (0% A), and 5.1 to 8 min (0% A) (Joo et al., 2014).

Quantitation was performed by selected reaction monitoring (SRM) of the [M+H]+ (or [M–

H]–) ion and the related product ion for each metabolite, using an IS to establish peak area

ratios. The SRM transitions and collision energies determined for each metabolite and IS are

listed in Table 1.


at ASPE



Dow

nloaded from


DMD #63016

8

Method Validation. Four inter- and intra-day validations were performed to validate the

LC-MS/MS method for the simultaneous quantification of the five P450 and four UGT probe

metabolites in microsomal incubates. Calibration standards were prepared at six different

concentrations from 20 to 5,000 nM (acetaminophen, 4-hydroxydiclofenac, 4’-

hydroxymephenytoin, trifluoperazine N-glucuronide, and mycophenolic acid glucuronide) or

2 to 500 nM (dextrorphan, 1’-hydroxymidazolam, SN-38 glucuronide, and naloxone 3-

glucuronide), in a blank microsomal mixture. Quality control (QC) samples were prepared

separately at two different concentrations (5 and 20 nM for dextrorphan, 1’-

hydroxymidazolam, SN-38 glucuronide, and naloxone 3-glucuronide; 50 and 200 nM for

other metabolites) for assays. Inter- and intra-day precision and accuracy were determined by

analyzing replicates of the QC samples (n=4).

Comparison of the Cocktail and Individual Substrates for Inhibition Screening.

Known inhibitors of specific P450 and UGT enzymes (α-naphthoflavone for CYP1A2;

sulfaphenazole for CYP2C9; S-benzylnirvanol for CYP2C19; quinidine for CYP2D6;

ketoconazole for CYP3A4; atazanavir for UGT1A1; hecogenin for UGT1A4; niflumic acid

UGT1A9; and mefenamic acid for UGT2B7) were incubated with each substrate cocktail set

and with the individual substrates alone and the results were compared. The incubations were

performed (as described above) with various inhibitor concentrations and all incubations

were performed in triplicate. Furafylline, paroxetine and troleandomycin were pre-incubated

for 10 min at 37 °C with HLMs and an NADPH-generating system (as described above). The

reactions were initiated by the addition of the individual or cocktail substrate. With the

exception of the addition of P450- or UGT-isoform-specific inhibitors, all other incubation

conditions were as described above.


at ASPE



Dow

nloaded from


DMD #63016

9

Data Analysis. The P450- or UGT-isoform-mediated activities in the presence of

inhibitors were expressed as percentages of the corresponding control values. The percentage

of inhibition was calculated by the ratio of the amounts of metabolites formed, in the

presence and absence of the specific inhibitor. To calculate the enzyme inhibition IC50 values,

the relevant data were fitted to an inhibitory effect model [i.e. v = Emax × (1- [I]/(IC50 + [I]))]

using the WinNonlin (Pharsight, Mountain View, CA, USA).


at ASPE



Dow

nloaded from


DMD #63016

10

Results

Substrate Selection and Optimization of Microsomal Incubations. The structures of the

P450 and UGT probe substrates, their metabolites, and the internal standard used in the

assays are shown in Fig. 1. In this study, we selected a P450-isoform specific probe substrate

based on the preferred and accepted P450 substrates for assessing activity in vitro (Tucker et

al., 2001; Na et al., 2011). The UGT-isoform specific substrates were selected on the basis of

their specificity and previously published data (Hanioka et al., 2001; Di Marco et al., 2005;

Picard et al., 2005; Uchaipichat et al., 2006; Joo et al., 2014). The probe substrates for the

P450 and UGT isoforms were as follows: phenacetin, CYP1A2; diclofenac, CYP2C9; S-

mephenytoin, CYP2C19; dextromethorphan, CYP2D6; midazolam, CYP3A; SN-38,

UGT1A1; trifluoperazine, UGT1A4; mycophenolic acid, UGT1A9; and naloxone, UGT2B7.

The optimum microsomal incubation time was 15 min and 1 hr for phase I (cocktail A set)

and phase II (cocktail B set), respectively, with 0.25 mg/mL microsomal protein. The

concentration of each probe substrate was optimized to avoid interactions between them: 100

μM for phenacetin, 10 μM for diclofenac, 100 μM for S-mephenytoin, 5 μM for

dextromethorphan, 5 μM for midazolam, 0.5 μM for SN-38, 0.5 μM for trifluoperazine, 0.2

μM for mycophenolic acid, and 1 μM for naloxone (Table 1). These concentrations were

lower than their respective Km values.

Development of a Simultaneous Analytical Method using LC-MS/MS. We developed a

method for simultaneously analyzing metabolites of five P450 and four UGT probe substrates,

using LC-MS/MS. Among the nine metabolites, only mycophenolic acid glucuronide was

detected in the negative mode because of its poor ionization property in the positive mode.

The remaining eight metabolites and the IS were monitored in the positive mode. The SRM


at ASPE



Dow

nloaded from


DMD #63016

11

transitions and optimal MS/MS collision energy are described in Table 1. The representative

chromatograms for the five P450 and four UGT probe metabolites and the IS in microsomal

incubation mixtures are presented in Fig. 2. As shown in Fig. 2, all the metabolites were

eluted in less than 6.5 min and separated into their individual SRM channels. The retention

times for acetaminophen, 4-hydroxydiclofenac, 4′-hydroxymephenytoin, dextrorphan, 1′-

hydroxymidazolam, SN-38 glucuronide, trifluoperazine N-glucuronide, mycophenolic acid

glucuronide, naloxone 3-glucuronide, and terfenadine were approximately 3.3, 6.2, 3.9, 3.4,

3.9, 3.6, 4.8, 4.0, 3.0, and 5.8 min, respectively. For acetaminophen, three peaks were

observed in the microsomal incubation. Similar results have also been observed in other

reported studies (Joo and Liu, 2013; Pillai et al., 2013; Song et al., 2013). The peak with a

retention time of 3.3 min was identified as acetaminophen by comparing it with the retention

time of an authentic standard.

Cocktail Dose Selection. The inhibition potential of each P450 or UGT substrate was

evaluated by comparing the reaction of each metabolite in the single substrate incubations, to

the response of the same metabolite formed in incubations with the two-substrate cocktail set.

The change in each P450 and UGT enzyme activity level was less than 15% in each cocktail

set, compared with the individual incubation (Fig. 3). The relative standard deviations (RSDs)

ranged from 2.4–10.3% (n = 3) for the results of the cocktail incubation procedure.

Method Validation. The inter- and intra-day precision and accuracy of the method were

determined by analyzing four QC replicates. The method accuracy was expressed as the

percentage of the metabolite concentration measured in each sample relative to the known

amount of metabolites added (Joo et al., 2014). Calibration curves produced good correlation


at ASPE



Dow

nloaded from


DMD #63016

12

coefficients for nine metabolites in the mixture (r > 0.999). The inter-day and intra-day

accuracy and precision data for the five P450 and four UGT probe metabolites in human liver

microsomal incubates are summarized in Table 2 and 3. Overall, the inter- and intra-day

accuracy 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 determining P450 and UGT inhibition, was validated

using known selective inhibitors of the isoforms (α-naphthoflavone, CYP1A2;

sulfaphenazole, CYP2C9; S-benzylnirvanol, CYP2C19; quinidine, CYP2D6; ketoconazole,

CYP3A; atazanavir, UGT1A1; hecogenin, UGT1A4; niflumic acid, UGT1A9; and

mefenamic acid, UGT2B7) with their corresponding substrates. The IC50 value of each P450-

and UGT-isoform selective inhibitor was estimated in both the individual and cocktail

incubations (Table 2). The inhibition curves (Fig. 4) show there was no substantial difference

between the individual inhibitor profiles for the two different incubation methods (single vs.

cocktail).


at ASPE



Dow

nloaded from


DMD #63016

13

Discussion

Pharmacodynamic interactions between drugs alter the response to one or both drugs while

also affecting their plasma concentrations (Singh, 2006). Therefore, the risk of metabolism-

based DDIs is always a potential problem to consider during the drug development process.

For this reason, in vitro drug interaction studies using HLMs are used for the early estimation

and prediction of the in vivo drug interaction potential of new candidate drugs (Baranczewski

et al., 2006). Several LC-MS/MS-based P450 (Dierks et al., 2001; Kim et al., 2005; Smith et

al., 2007; Zambon et al., 2010; Na et al., 2011; Pillai et al., 2013) or UGT (Gagez et al., 2012;

Joo et al., 2014; Seo et al., 2014) inhibition methods for the evaluation of in vitro drug

interaction potential have been reported. To date, however, a simultaneous evaluation method

for both P450 and UGT enzyme activities has not been developed. The aim of this study was

to develop and validate a cocktail assay to simultaneously evaluate the activity of hepatic

P450 and UGT isoforms 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 and Relling, 1999). Among the

numerous P450 and UGT enzymes identified to date, five P450 (CYP1A2, CYP2C9,

CYP2C19, CYP2D6, and CYP3A) and four UGT (UGT1A1, UGT1A4, UGT1A9, and

UGT2B7) enzymes have been shown to play an important role in the metabolism of marketed

drugs. P450 enzymes biotransform about 75% of all drugs, and the above mentioned five

P450 human isoforms biotransform about 95% of marketed drugs (Baj-Rossi et al., 2011). In

the case of UGT enzymes, UGT2B7 was suggested to be responsible for the hepatic

glucuronidation of 40% of drugs, while UGT1A1, 1A4, and 1A9 contribute to additional 47%

(Williams et al., 2004; Burchell et al., 2005). Based on these data, we attempted to develop a


at ASPE



Dow

nloaded from


DMD #63016

14

method for the simultaneous evaluation of the activities of five P450 (P450s 1A2, 2C9, 2C19,

2D6, and 3A) and four UGT (UGTs 1A1, 1A4, 1A9, and 2B7) enzyme, which are primarily

responsible for drug metabolism.

The optimum incubation conditions for the assessment of enzyme activities are different

between the P450 and UGT enzymes. The microsomal incubation of P450 enzymes was

conducted in phosphate buffer, 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 β-glucuronidase inhibitor

(Oleson and Court, 2008) using a longer incubation time (1 hr). In addition, UDPGA also has

inhibitory potential against CYP2C9 and CYP2C19 activity to some degree (Yan and

Caldwell, 2003). Therefore, we used two separate microsomal incubations with P450 and

UGT probe substrates. The substrates were divided into two cocktail groups (cocktail A for

five P450 probe substrates and cocktail B for four UGT probe substrates). These two cocktail

mixtures were pooled after incubation and analyzed together using 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 the metabolism of a single drug. Therefore, in this

study we chose specific probe substrates for the nine major P450 and UGT enzymes in the

human liver based on previous reports (Table 1). Phenacetin, dextromethorphan, and

midazolam are the most frequently employed probe substrate for in vitro activity assessment

of CYP1A2, CYP2D6, and CYP3A enzymes, respectively, using the cocktail approach, and

are also 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; Otten et al., 2011; Pillai et al., 2013) and omeprazole (Testino and


at ASPE



Dow

nloaded from


DMD #63016

15

Patonay, 2003; He et al., 2007; Tolonen et al., 2007; Song et al., 2013) are used as CYP2C19

substrates in cocktail incubation studies. In our preliminary study, omeprazole (20 μM)

inhibited CYP3A-mediated midazolam hydroxylase activity (40% of control) and CYP1A2-

mediated phenacetin O-deethylation activity (>20% of control) (Supplemental Figure 1).

Diclofenac (Dierks et al., 2001; Smith et al., 2007; Pillai et al., 2013) and tolbutamide (Bu et

al., 2001; Testino and Patonay, 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 CYP2C9 probe substrates. In this

study, diclofenac was selected as the CYP2C9 probe substrate, because it showed higher

detection intensity than tolbutamide in the proposed LC-MS/MS method. Based our

preliminary findings, phenacetin, diclofenac, S-mephenytoin, dextromethorphan, 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 as probe substrates for

UGT1A1, UGT1A4, UGT1A9, and UGT2B7, respectively, based on previously reported data

(Uchaipichat et al., 2004; Di Marco et al., 2005; Picard et al., 2005) and our recently

published data (Joo et al., 2014).

Following the selection of the P450 and UGT probe substrates for the cocktail incubation

study, the potential metabolic interactions among the five P450 metabolites and four UGT

metabolites of the selected probe substrates were evaluated. The simultaneous incubation of

five or four substrates may lead to interactions. As shown in Fig. 3, P450 and UGT enzyme

activities in the cocktail incubations were in accordance with the results from individual

incubations, suggesting that drug interactions among probe substrates in the present cocktail

set were negligible.

P450 and UGT-isoform selective inhibitors represent the most powerful tools available for


at ASPE



Dow

nloaded from


DMD #63016

16

metabolizing enzyme phenotyping. The well-known P450 and UGT inhibitors include α-

naphthoflavone for CYP1A2 (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), ketoconazole for CYP3A (Khojasteh et al., 2011),

hecogenin for UGT1A4 (Uchaipichat et al., 2006), and niflumic acid for UGT1A9 (Miners et

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 α-

naphthoflavone, sulfaphenazole, S-benzylnirvanol, quinidine, hecogenin, and niflumic acid

strongly and selectively inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, UGT1A4, and

UGT1A9, respectively (Table 3 and Fig. 5). Liu et al. (2011) also reported that selective P450

inhibitors such as α-naphthoflavone, sulfaphenazole, and quinidine have negligible inhibitory

potential against UGT isoforms. Ketoconazole also selectively inhibits CYP3A with an IC50

value of 0.03 μM, which is at least 100-fold more selective compared to its effects against all

other P450s and UGTs tested (Fig. 5E) (Ren et al., 2013). The inhibitory potential of

ketoconazole on UGT1A1-mediated SN-38 glucuronidation (IC50 = 4.8 μM) concurred with

previous findings in which ketoconazole had inhibitory potential on UGT1A1-catalyzed β-

estradiol glucuronidation (IC50 = 4.1 μM) (Liu et al., 2011). Hecogenin and niflumic acid are

UGT1A4 and UGT1A9 selective inhibitor, respectively, and they have negligible inhibitory

effect on the P450 isoforms tested (IC50 > 20 μM, Table 3). Atazanavir strongly inhibited

UGT1A1 activity with an IC50 value of 0.4 μM, however it also inhibited CYP3A activity

(IC50 = 1.8 μM, Fig. 5F). Mefenamic acid inhibited UGT2B7 activity (IC50 = 5.0 μM), but

also weakly inhibited CYP1A2 and CYP2C9 activity with IC50 values of 8.3 and 12 μM,

respectively (Fig. 5I).

We also evaluated the IC50 values of the characterized inhibitors for each P450 and UGT


at ASPE



Dow

nloaded from


DMD #63016

17

enzymes using both the individual and the cocktail substrates (Fig. 4). The IC50 value of each

cocktail set using this approach was comparable to those of the individual substrates and were

in agreement with those previously reported (Table 2). This confirms that the IC50 values of

P450 and UGT inhibitors can be accurately determined using the cocktail assay instead of

individual substrate incubations, which would save considerable time in the screening process

for new chemical entities. One exception observed was in the inhibition of CYP2C19 by S-

benzylnirvanol, which showed a slight difference compared with previously published values.

The published IC50 value of 0.41 μM for S-benzylnirvanol against CYP2C19-mediated S-

mephenytoin hydroxylaiton activity (Walsky and Obach, 2003), was lower than our present

data (1.2 μM, Table 2). Such a difference is not unusual and could be due to the use of

different microsomal incubation conditions (Dierks et al., 2001). Nonetheless, our results

demonstrate that the IC50 values of inhibitors against the nine enzymes can be determined

accurately by cocktail incubation and cassette analysis thereby reducing the time required.

In conclusion, we developed an LC-MS/MS method for the simultaneous determination of

five P450 and four UGT enzyme activities. Nine substrates were divided into two cocktail

sets for incubation and pooled for LC-MS/MS analysis in a single run. This process allowed

us to simultaneously evaluate the activity of five P450 and four UGT enzyme activities, using

well-known P450- and UGT-isoform specific inhibitors. In addition, this cocktail method

involving multiple substrates, provided inhibition profiles similar to those obtained from

single substrate incubations. Therefore, these results suggest that this newly developed assay

can be a useful tool for robust and rapid screening, which accelerates the prediction of the

P450 and UGT inhibitory potential of new chemical entities.


at ASPE



Dow

nloaded from


DMD #63016

18

Authorship Contributions Participated 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


at ASPE



Dow

nloaded from


DMD #63016

19

References

Baj-Rossi C, De Micheli G, and Carrara S (2011) P450-Based Nano-Bio-Sensors for Personalized Medicine. INTECH Open Access Publisher.

Baranczewski P, Stanczak A, Sundberg K, Svensson R, Wallin A, Jansson J, Garberg P, and Postlind H (2006) Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development. Pharmacol Rep 58:453-472.

Bu HZ, Magis L, Knuth K, and Teitelbaum P (2001) High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1. Rapid Commun Mass Spectrom 15:741-748.

Burchell B, Lockley DJ, Staines A, Uesawa Y, and Coughtrie MW (2005) Substrate specificity of human hepatic udp-glucuronosyltransferases. Methods Enzymol 400:46-57.

Di Marco A, D'Antoni M, Attaccalite S, Carotenuto P, and Laufer R (2005) Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well radiometric assay. Drug Metab Dispos 33:812-819.

Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H, and Ball SE (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29:23-29.

Easterbrook J, Lu C, Sakai Y, and Li AP (2001) Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes. Drug Metab Dispos 29:141-144.

Evans WE and Relling MV (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286:487-491.

Fisher MB, Campanale K, Ackermann BL, VandenBranden M, and Wrighton SA (2000) In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab Dispos 28:560-566.

Gagez AL, Rouguieg-Malki K, Sauvage FL, Marquet P, and Picard N (2012) Simultaneous evaluation of six human glucuronidation activities in liver microsomes using liquid chromatography-tandem mass spectrometry. Anal Biochem 427:52-59.

Gao ZW, Shi XJ, Yu C, Li SJ, and Zhong MK (2007) Simultaneous determination of the inhibitory potency of compounds on the activity of five cytochrome P-450 enzymes using a cocktail probe substrates method. Yao Xue Xue Bao 42:589-594.

Guengerich FP (2008) Cytochrome P450 and chemical toxicology. Chem Res Toxicol 21:70-83.

Hanioka N, Ozawa S, Jinno H, Ando M, Saito Y, and Sawada J (2001) Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin. Xenobiotica 31:687-699.

He F, Bi HC, Xie ZY, Zuo Z, Li JK, Li X, Zhao LZ, Chen X, and Huang M (2007) Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry: application to high-throughput inhibition screening of terpenoids. Rapid Commun Mass Spectrom 21:635-643.

Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ, Chung S, Colangelo P, Frucht D, Green MD, Hepp P,


at ASPE



Dow

nloaded from


DMD #63016

20

Karnaukhova E, Ko HS, Lee JI, Marroum PJ, Norden JM, Qiu W, Rahman A, Sobel S, Stifano T, Thummel K, Wei XX, Yasuda S, Zheng JH, Zhao H, and Lesko LJ (2008) New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 48:662-670.

Joo J, Lee B, Lee T, and Liu K-H (2014) Screening of six UGT enzyme activities in human liver microsomes using liquid chromatography/triple quadrupole mass spectrometry. Rapid Commun Mass Spectrom 28:2405-2414.

Joo JM and Liu KH (2013) Inhibitory effect of honokiol and magnolol on cytochrome P450 enzyme activities in human liver microsomes. Mass Spectrom Lett 4: 34-37.

Khojasteh SC, Prabhu S, Kenny JR, Halladay JS, and Lu AY (2011) Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity. Eur J Drug Metab Pharmacokinet 36:1-16.

Kim MJ, Kim H, Cha IJ, Park JS, Shon JH, Liu KH, and Shin JG (2005) High-throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 19:2651-2658.

Knights KM, Winner LK, Elliot DJ, Bowalgaha K, and Miners JO (2009) Aldosterone glucuronidation by human liver and kidney microsomes and recombinant UDP-glucuronosyltransferases: inhibition by NSAIDs. Br J Clin Pharmacol 68:402-412.

Liu Y, She M, Wu Z, and Dai R (2011) The inhibition study of human UDP-glucuronosyltransferases with cytochrome P450 selective substrates and inhibitors. J Enzyme Inhib Med Chem 26:386-393.

Mano Y, Usui T, and Kamimura H (2006) In vitro inhibitory effects of non-steroidal anti-inflammatory drugs on 4-methylumbelliferone glucuronidation in recombinant human UDP-glucuronosyltransferase 1A9--potent inhibition by niflumic acid. Biopharm Drug Dispos 27:1-6.

Mano Y, Usui T, and Kamimura H (2007) Inhibitory potential of nonsteroidal anti-inflammatory drugs on UDP-glucuronosyltransferase 2B7 in human liver microsomes. Eur J Clin Pharmacol 63:211-216.

Meech R, Miners JO, Lewis BC, and Mackenzie PI (2012) The glycosidation of xenobiotics and endogenous compounds: versatility and redundancy in the UDP glycosyltransferase superfamily. Pharmacol Ther 134:200-218.

Miners JO, Bowalgaha K, Elliot DJ, Baranczewski P, and Knights KM (2011) Characterization of niflumic acid as a selective inhibitor of human liver microsomal UDP-glucuronosyltransferase 1A9: application to the reaction phenotyping of acetaminophen glucuronidation. Drug Metab Dispos 39:644-652.

Na DH, Ji HY, Park EJ, Kim MS, Liu KH, and Lee HS (2011) Evaluation of metabolism-mediated herb-drug interactions. Arch Pharm Res 34:1829-1842.

Oleson L and Court MH (2008) Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases. J Pharm Pharmacol 60:1175-1182.

Otten JN, Hingorani GP, Hartley DP, Kragerud SD, and Franklin RB (2011) An in vitro, high throughput, seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS. Drug Metab Lett 5:17-24.

Perloff ES, Mason AK, Dehal SS, Blanchard AP, Morgan L, Ho T, Dandeneau A, Crocker RM, Chandler CM, Boily N, Crespi CL, and Stresser DM (2009) Validation of cytochrome P450 time-dependent inhibition assays: a two-time point IC50 shift approach facilitates kinact assay design. Xenobiotica 39:99-112.


at ASPE



Dow

nloaded from


DMD #63016

21

Picard N, Ratanasavanh D, Premaud A, Le Meur Y, and Marquet P (2005) Identification of the UDP-glucuronosyltransferase isoforms involved in mycophenolic acid phase II metabolism. Drug Metab Dispos 33:139-146.

Pillai VC, Strom SC, Caritis SN, and Venkataramanan R (2013) A sensitive and specific CYP cocktail assay for the simultaneous assessment of human cytochrome P450 activities in primary cultures of human hepatocytes using LC-MS/MS. J Pharm Biomed Anal 74:126-132.

Ren S, Zeng J, Mei Y, Zhang JZ, Yan SF, Fei J, and Chen L (2013) Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors. Drug Metab Dispos 41:60-71.

Seo KA, Kim HJ, Jeong ES, Abdalla N, Choi CS, Kim DH, and Shin JG (2014) In Vitro Assay of Six UDP-Glucuronosyltransferase Isoforms in Human Liver Microsomes, Using Cocktails of Probe Substrates and Liquid Chromatography-Tandem Mass Spectrometry. Drug Metab Dispos 42:1803-1810.

Singh SS (2006) Preclinical pharmacokinetics: an approach towards safer and efficacious drugs. Curr Drug Metab 7:165-182.

Smith D, Sadagopan N, Zientek M, Reddy A, and Cohen L (2007) Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery. J Chromatogr B 850:455-463.

Song M, Hong M, Choi HG, Jahng Y, Lee SH, and Lee S (2013) Effects of Mollugin on Hepatic Cytochrome P450 in Male ICR Mice as Determined by Liquid Chromatography/Tandem Mass Spectrometry. Mass Spectrom Lett 3:104-107.

Spaggiari D, Geiser L, Daali Y, and Rudaz S (2014) A cocktail approach for assessing the in vitro activity of human cytochrome P450s: An overview of current methodologies. J Pharm Biomed Anal 101: 221-237.

Stingl JC, Bartels H, Viviani R, Lehmann ML, and Brockmoller J (2014) Relevance of UDP-glucuronosyltransferase polymorphisms for drug dosing: A quantitative systematic review. Pharmacol Ther 141:92-116.

Suzuki H, Kneller MB, Haining RL, Trager WF, and Rettie AE (2002) (+)-N-3-Benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital: new potent and selective in vitro inhibitors of CYP2C19. Drug Metab Dispos 30:235-239.

Testino SA, Jr. and Patonay G (2003) High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 30:1459-1467.

Tolonen A, Petsalo A, Turpeinen M, Uusitalo J, and Pelkonen O (2007) In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti-CYP antibodies. J Mass Spectrom 42:960-966.

Tucker GT, Houston JB, and Huang SM (2001) Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential--towards a consensus. Br J Clin Pharmacol 52:107-117.

Uchaipichat V, Mackenzie PI, Elliot DJ, and Miners JO (2006) Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) "probes" for human udp-glucuronosyltransferases. Drug Metab Dispos 34:449-456.

Uchaipichat V, Mackenzie PI, Guo XH, Gardner-Stephen D, Galetin A, Houston JB, and Miners JO (2004) Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic


at ASPE



Dow

nloaded from


DMD #63016

22

solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos 32:413-423.

Vietri M, Pietrabissa A, Mosca F, and Pacifici GM (2000) Mycophenolic acid glucuronidation and its inhibition by non-steroidal anti-inflammatory drugs in human liver and kidney. Eur J Clin Pharmacol 56:659-664.

von Moltke LL, Greenblatt DJ, Duan SX, Schmider J, Kudchadker L, Fogelman SM, Harmatz JS, and Shader RI (1996) Phenacetin O-deethylation by human liver microsomes in vitro: inhibition by chemical probes, SSRI antidepressants, nefazodone and venlafaxine. Psychopharmacology 128:398-407.

Walsky RL and Obach RS (2003) Verification of the selectivity of (+)N-3-benzylnirvanol as a CYP2C19 inhibitor. Drug Metab Dispos 31:343.

Walsky RL and Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32:647-660.

Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, and Ball SE (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32:1201-1208.

Yan Z and Caldwell GW (2003) Metabolic assessment in liver microsomes by co-activating cytochrome P450s and UDP-glycosyltransferases. Eur J Drug Metab Pharmacokinet 28:223-232.

Zambon S, Fontana S, and Kajbaf M (2010) Evaluation of cytochrome P450 inhibition assays using human liver microsomes by a cassette analysis /LC-MS/MS. Drug Metab Lett 4:120-128.

Zhang D, Chando TJ, Everett DW, Patten CJ, Dehal SS, and Humphreys WG (2005) In vitro inhibition of UDP glucuronosyltransferases by atazanavir and other HIV protease inhibitors and the relationship of this property to in vivo bilirubin glucuronidation. Drug Metab Dispos 33:1729-1739.

Zhang L, Wei MJ, Zhao CY, and Qi HM (2008) Determination of the inhibitory potential of 6 fluoroquinolones on CYP1A2 and CYP2C9 in human liver microsomes. Acta Pharmacol Sin 29:1507-1514.


at ASPE



Dow

nloaded from


DMD #63016

23

Footnotes

This study was supported by grants from the Korea Health Technology R&D Project,

Ministry of Health & Welfare, Republic of Korea [Grant No: A111345], and the National

Research Foundation of Korea, Ministry of Science, ICT and Future Planning [NRF-

2014M3A9D9069714] and Ministry of Education [NRF-2013R1A1A2008442], Republic of

Korea.


at ASPE



Dow

nloaded from


DMD #63016

24

Figure legends.

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

internal standard (IS).

Fig. 2. Representative selected reaction monitoring chromatograms of P450- and UGT-

mediated metabolites of probe substrate and internal standard: Acetaminophen (A), 4-

hydroxydiclofenac (B), 4′-hydroxymephenytoin (C), dextrorphan (D), 1′-hydroxymidazolam

(E), SN-38 glucuronide (F), trifluoperazine N-glucuronide (G), mycophenolic acid

glucuronide (H), naloxone 3-glucuronide (I), and terfenadine (internal standard, J).

Fig. 3. Comparison of the results from the cocktail incubation with the individual incubations

for each of five P450 (A) and four UGT (B) enzyme activities. Incubations were performed

with the individual substrate (■), and each substrate cocktail set (□) in pooled human liver

microsomes (HLMs). The activities are the means of triplicate incubations

Fig. 4. Inhibition curves determined using individual substrates and the substrate cocktails.

Each P450- and UGT-selective inhibitor was incubated in a separate experiment with cocktail

substrates (●) or an individual substrate set (○). The activity is expressed as the percentage of

the remaining activity compared with a control sample containing no inhibitor. Inhibition of

(A) phenacetin O-deethylation by α-naphthoflavone; (B) diclofenac 4-hydroxylation by

sulfaphenazole; (C) S-mephenytoin 4′-hydroxylation by S-benzylnirvanol; (D)

dextromethorphan O-demethylation by quinidine; (E) midazolam 1′-hydroxylation by

ketoconazole, (F) SN-38 glucuronidation by atazanavir; (G) trifluoperazine N-

glucuronidation by hecogenin; (H) mycophenolic acid glucuronidation by niflumic acid; and

(I) naloxone 3-glucuronidation by mefenamic acid.


at ASPE



Dow

nloaded from


DMD #63016

25

Fig. 5. Inhibitory effects of (A) α-naphthoflavone, (B) sulfaphenazole, (C) S-benzylnirvanol,

(D) quinidine, (E) ketoconazole, (F) atazanavir, (G) hecogenin, (H) niflumic acid, and (I)

mefenamic acid on phenacetin O-deethylation (●), diclofenac 4-hydroxylation (○), S-

mephenytoin 4′-hydroxylation (▼), dextromethorphan O-demethylation (△), midazolam 1′-

hydroxylation (■), SN-38 glucuronidation (□), trifluoperazine N-glucuronidation (◆),

mycophenolic acid glucuronidation (◇), and naloxone 3-glucuronidation (▲) on incubation

with pooled human liver microsomes (HLMs). Data are the means of triplicate experiments.


at ASPE



Dow

nloaded from


DMD #63016

26

Table 1. Selected reaction monitoring (SRM) conditions for the major metabolites of the nine substrates used in all assays.

Enzyme Substrate Conc.

(μM)

Metabolite Transition (m/z) Polarity Collision

energy (eV)

CYP1A2 Phenacetin 100 Acetaminophen 152 > 110 ESI+ 25

CYP2C9 Diclofenac 10 4-Hydroxydiclofenac 312 > 231 ESI+ 23

CYP2C19 S-Mephenytoin 100 4′-Hydroxymephenytion 235 > 150 ESI+ 27

CYP2D6 Dextromethorphan 5 Dextrorphan 258 > 157 ESI+ 50

CYP3A Midazolam 5 1′-Hydroxymidazolam 342 > 203 ESI+ 25

UGT1A1 SN-38 0.5 SN-38-glucuronide 569 > 393.4 ESI+ 30

UGT1A4 Trifluoperazine 0.5 Trifluoperazine

N-glucuronide

584.5 > 408.5 ESI+ 30

UGT1A9 Mycophenolic acid 0.2 Mycophenolic acid-

glucuronide

495 > 319 ESI- 25

UGT2B7 Naloxone 1 Naloxone 3-glucuronide 504 > 310 ESI+ 30

IS Terfenadine - 472 > 436 ESI+ 25

Conc., concentration; IS, internal standard

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on April 22, 2015 as D

OI: 10.1124/dm

d.114.063016 at ASPET Journals on June 11, 2021 dmd.aspetjournals.org Downloaded from


DMD #63016

27

Table 2. Intra-day (n=4) validation data for the simultaneous determination of five P450 and four UGT-specific probe metabolites in human liver microsomal incubations using the LC-MS/MS method.

Metabolites

50 nM 200 nM

Conc. found

(nM)b

Accuracy

(%)

RSD

(%)

Conc. found

(nM)b

Accuracy

(%)

RSD

(%)

Acetaminophen 49.3 98.6 8.1 202.1 101.0 5.5

4-Hydroxydicolfenac 45.8 91.7 6.4 182.0 91.0 11.7

4’-Hydroxymephenytoin 51.5 103.0 13.4 202.9 101.5 14.1

Dextrorphana 4.41 88.2 15.3 20.0 99.9 3.9

1’-Hydroxymidazolama 4.85 97.1 7.2 19.2 95.8 12.1

SN-38 glucuronidea 5.30 106.0 10.8 19.1 95.7 6.4

Trifluoperazine N-glucuronide 45.4 90.8 1.9 179.9 89.9 8.5

Mycophenolic acid glucuronide 47.2 94.4 6.3 182.5 91.2 6.8

Naloxone 3-glucuronidea 5.55 110.9 3.8 19.4 94.6 3.7

aDextrorphan, 1’-hydroxymidazolam, SN-38 glucuronide, and naloxone 3-glucuronide concentrations were 5 and 20 nM. bEach value represents the mean of four replicates Intra-day accuracy and precision were determined at two different concentration levels (n = 4).




DM

D Fast Forw


OI: 10.1124/dm



DMD #63016

28

Table 3. Inter-day (n = 4) validation data for the simultaneous determination of five P450 and four UGT-specific probe metabolites in human liver microsomal incubations using LC-MS/MS method

Metabolites

50 nM 200 nM

Conc. found

(nM)b

Accuracy

(%)

RSD

(%)

Conc. found

(nM)b

Accuracy

(%)

RSD

(%)

Acetaminophen 52.0 104.1 4.9 202.0 101.0 3.0

4-Hydroxydicolfenac 50.1 100.1 9.6 198.5 99.2 8.4

4’-Hydroxymephenytoin 51.3 102.7 3.4 203.8 101.9 2.3

Dextrorphana 4.61 92.1 8.5 20.6 103.0 7.1

1’-Hydroxymidazolama 5.35 107.0 5.7 20.2 100.7 8.7

SN-38 glucuronidea 4.81 96.2 9.9 19.9 99.3 5.4

Trifluoperazine N-glucuronide 50.7 101.5 3.3 201.4 100.7 5.3

Mycophenolic acid glucuronide 46.0 92.9 8.0 188.8 94.4 4.2

Naloxone 3-glucuronidea 5.46 109.2 4.9 20.2 101.0 3.2

aDextrorphan, 1’-hydroxymidazolam, SN-38 glucuronide, and naloxone 3-glucuronide concentrations were 5 and 20 nM. bEach value represents the mean of four replicates Inter-day accuracy and precision were determined at two different concentration levels (n = 4).




DM

D Fast Forw


OI: 10.1124/dm



DMD #63016

29

Table 4. Comparison of IC50 values estimated using the individual substrate and the substrate cocktails of well-known P450- and UGT-selective inhibitors

Enzyme Substrates Inhibitors IC50 (μM±SD)a References

Individual substrate Cocktail substrate Literature

CYP1A2 Phenacetin α-Naphthoflavone 0.014 ± 0.003 0.013 ± 0.004 0.01–0.27 (Gao et al., 2007; Zhang et al., 2008; Perloff et al., 2009)

CYP2C9 Diclofenac Sulfaphenazole 0.70 ± 0.08 0.68 ± 0.08 0.3–1.5 (Dierks et al., 2001; Kim et al., 2005)

CYP2C19 S-Mephenytoin S-Benzylnirvanol 1.02 ± 0.13 1.20 ± 0.06 0.4–0.41 (Walsky and Obach, 2003; Walsky and Obach, 2004)

CYP2D6 Dextromethorphan Quinidine 0.11 ± 0.01 0.13 ± 0.01 0.02–0.68 (Dierks et al., 2001; Kim et al., 2005)

CYP3A Midazolam Ketoconazole 0.038 ± 0.001 0.032 ± 0.0004 0.09–0.24 (Dierks et al., 2001; Kim et al., 2005)

UGT1A1 SN-38 Atazanavir 0.37 ± 0.11 0.38 ± 0.13 0.4–2.5 (Zhang et al., 2005)

UGT1A4 Trifluoperazine Hecogenin 0.89 ± 0.21 0.86 ± 0.11 0.64–1.5 (Uchaipichat et al., 2006)

UGT1A9 Mycophenolic acid Niflumic acid 0.78 ± 0.10 0.83 ± 0.18 0.1–8 (Vietri et al., 2000; Mano et al., 2006; Miners et al., 2011)

UGT2B7 Naloxone Mefenamic acid 4.18 ± 1.01 5.04 ± 1.34 0.3–370 (Mano et al., 2007; Knights et al., 2009)

aValues represent the mean (±SD) of experiments performed in triplicate. Inhibitors of P450 and UGT enzymes were incubated with each substrates cocktail set and with individual substrate alone. IC50 values were calculated using a nonlinear least-squares regression analysis




DM

D Fast Forw


OI: 10.1124/dm



DMD #63016

30

Table 5. Effects of specific enzyme inhibitors on cytochrome P450 and uridine 5’-diphosphoglucuronosyltransferase metabolic activities in pooled human liver microsomes.

Enzyme Inhibitors

IC50 (μM) a

P450s UGTs

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

CYP1A2 α-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

aValues represent the mean of experiments performed in triplicate ‘-’ means IC50 > 50 μΜ




DM

D Fast Forw


OI: 10.1124/dm



Fig.1


at ASPE



Dow

nloaded from


Fig.2


at ASPE



Dow

nloaded from


V (

pm

ol/m

in/m

g p

rote

in)

0

100

200

300

400

CY

P1A

2

CY

P2C

9

CY

P2C

19

CY

P2D

6

CY

P3A

0

10

20

30

Individual

Cocktail (5+4)

UG

T1A

1

UG

T1A

4

UG

T1A

9

UG

T2B

7

(A) (B)

Fig.3


at ASPE



Dow

nloaded from


Fig. 4


at ASPE



Dow

nloaded from


Fig. 5


at ASPE



Dow

nloaded from

Boram Lee, Hyeon Kyeong Ji, Taeho Lee and Kwang-Hyeon Liu...2015/04/22 · Boram Lee, Hyeon Kyeong...

Documents

Transcript of Boram Lee, Hyeon Kyeong Ji, Taeho Lee and Kwang-Hyeon Liu...2015/04/22 · Boram Lee, Hyeon Kyeong...