boram lee, hyeon kyeong ji, taeho lee and kwang-hyeon liu...2015/04/22 · boram lee, hyeon kyeong...
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DMD #63016
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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
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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.
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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.
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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.
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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.
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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-
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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.
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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.
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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).
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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
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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
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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).
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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
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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
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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
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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
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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.
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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
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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.
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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.
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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.
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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
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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).
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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).
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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
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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
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Fig.1
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V (
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UG
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UG
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(A) (B)
Fig.3
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Fig. 5
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