In vitro assay of six UGT isoforms in human liver...
Transcript of In vitro assay of six UGT isoforms in human liver...
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In vitro assay of six UGT isoforms in human liver microsomes, using cocktails of probe
substrates and LC-MS/MS
Kyung-Ah Seo, Hyo-Ji Kim, Eun Sook Jeong, Nagi Abdalla, Chang-Soo Choi, Dong-Hyun
Kim, and Jae-Gook Shin
Department of Pharmacology and PharmacoGenomics Research Center, Inje University
College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D. -H.K, and J.-G.S)
Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.
Choi)
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Running title: In vitro cocktail analysis for the inhibition of six UGTs
Address correspondence to:
Jae-Gook Shin, M.D., Ph.D., Department of Pharmacology and PharmacoGenomics Research
Center, Inje University College of Medicine, #633-165 Gaegum-Dong, Busanjin-Gu, Busan
614-735, Korea. Tel.: +82-51-890-6720 Fax: +82-51-893-1232, E-mail: [email protected]
Dong Hyun Kim, Ph.D., Department of Pharmacology and PharmacoGenomics Research
Center, Inje University College of Medicine, #633-165 Gaegum-Dong, Busanjin-Gu, Busan
614-735, Korea. Tel.: +82-51-890-6411 Fax: +82-51-893-1232, E-mail: [email protected]
Number of text pages: 26
Number of Tables: 2
Number of Figures: 5
Number of References: 29
Number of words in the Abstract: 190
Number of words in the Introduction: 426
Number of words in the Discussion: 1154
ABBREVIATIONS:
P450, cytochrome P450; HLM, human liver microsome; LC-MS/MS, liquid
chromatography-tandem mass spectrometry; UGT, UDP-glucuronosyltransferase
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ABSTRACT
UDP-glucuronosyltransferase (UGT)-mediated drug-drug interactions are commonly
evaluated during drug development. We present a validated method for the simultaneous
evaluation of drug-mediated inhibition of six major UGT isoforms, developed in human liver
microsomes through the use of pooled specific UGT probe substrates (cocktail assay) and
rapid LC-MS/MS analysis. The six probe substrates used in this assay were estradiol
(UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-hydroxyindole
(UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). In a cocktail incubation,
UGT1A1, UGT1A9, and UGT 2B7 activities were substantially inhibited by other substrates.
This interference could be eliminated by dividing substrates into two incubations, one
containing estradiol, trifluoperazine, and 4-hydroxyindole, and the other containing
chenodeoxycholic acid, propofol, and naloxone. Incubation mixtures were pooled for the
simultaneous analysis of glucuronyl conjugates in a single LC-MS/MS run. The optimized
cocktail method was further validated against single-probe substrate assays, using compounds
known to inhibit UGTs. The degree of inhibition of UGT isoform activities by such known
inhibitors in this cocktail assay was not substantially different from that in single-probe
assays. This six-isoform cocktail assay may be very useful in assessing the UGT-based drug-
interaction potential of candidates in a drug-discovery setting.
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INTRODUCTION
Most pharmacokinetic drug-drug interactions occur at the metabolic level, and usually
involve changes in the activity of the major drug metabolizing enzymes. Identification of
these enzymes allows us to predict potential drug-drug interactions, which is critical for new
drug development. Although cytochrome P450 enzymes (CYP) are mainly responsible for
the initial oxidative metabolism of xenobiotic compounds, a considerable number of drugs
(approximately 15% of approved drugs on the market) are known to be metabolized by UDP-
glucuronosyltransferases (UGT), either directly or following initial oxidative metabolism
(Williams et al., 2004). Therefore, rapid and sensitive tools for in vitro evaluation of
compound-mediated inhibition of UGT isoform activities, along with those for CYPs, are
required for studies of drug-drug interactions in drug discovery.
Several in vitro CYP 'cocktail methods' have been developed, in which a mixture of
several CYP-selective substrates are included in a single human microsomal incubation, and
the metabolism of the substrates is determined by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) (Dixit et al., 2007, Pillai et al., 2013). Selective substrates,
antibodies, or inhibitors of UGT isoforms can be employed in metabolism studies with
human liver microsomes, and have been extremely useful in estimating the contribution of
each UGT isoform to metabolism of the compound of interest (e.g. a new chemical entity)
(Manevski et al., 2010). However most individual UGTs exhibit distinct, but overlapping
substrate selectivity, and differ in their regulation of expression, their genetic polymorphism,
and in other factors known to influence the activity of drug metabolizing enzymes in humans
(Lepine et al., 2004, Court, 2005, Itaaho et al., 2008). As a result, few selective substrates and
inhibitors useful for phenotyping UGTs have been identified to date (Donato et al., 2010).
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Some UGT substrates have been used as probe drugs without proper validation, which can
lead to biased study results (Hanioka et al., 2001). Recently, a cocktail method using multiple
UGT substrates has been developed for determining UGT activity in vitro (e.g. in human
liver microsomes) (Gagez et al., 2012), but no validated method for measuring the inhibitory
potential of a given compound on the major UGT enzymes has yet been reported.
The purpose of the present study was to develop a new cocktail method for simultaneous
evaluation of the activities of six major human liver microsomal UGT isoforms (UGT1A1,
1A3, 1A4, 1A6, 1A9, and 2B7). We evaluated the specificity and sensitivity of each probe
substrate and validated those substrates with specific UGT inhibitors. We explored the
optimal experimental conditions to avoid potential interactions among the cocktail drugs, and
developed an analytical method for cocktail experiments using LC-MS/MS.
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Materials and Methods
Chemicals and reagents
Alamethicin (from Trichoderma viride), uridine 5’- diphosphoglucuronic acid
(UDPGA), 1-napthol, β-estradiol, bilirubin, chenodeoxycholic acid, fluconazole, hecogenin,
lithocholic acid, naloxone, niflumic acid, propofol, trifluoperazine, troglitazone, and β-
estradiol-3-β-D-glucuronide were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4-Hydroxyindole and propofol glucuronide were obtained from Toronto Research Chemicals
(North York, ON, Canada). Recombinant human UGT isoforms (UGTs 1A1, 1A3, 1A4,
1A6, 1A9, 2B4, 2B7, 2B15, and 2B17) and pooled human liver microsomes (HLMs)
were purchased from BD Gentest Co. (Woburn, MA, USA). HPLC-grade acetonitrile
and methanol were purchased from J. T. Baker (Phillipsburg, NJ, USA). All other
chemicals were the highest analytical grade commercially available.
Microsomal incubations
The incubation mixtures consisted of 0.25 mg/ml of pooled human liver microsomes,
25 μg/ml alamethicin, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and substrates (various
UGT enzyme-specific substrates or a substrate cocktail set), in a total volume of 125 μl.
After pre-incubation on ice for 15 min, reactions were initiated by the addition of 5
mM UDPGA, and incubated for 1 h at 37°C in a shaking water bath. The reactions were
terminated by the addition of 125 μl acetonitrile containing estrone glucuronide (2 μM,
internal standard) and centrifuged at 10,000 g for 5 min at 4°C. An aliquot of the
supernatant was injected into LC-MS/MS for the determination of glucuronide
conjugates.
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Selectivity screening of known UGT isoform substrates
Incubation mixtures, containing 0.25 mg of recombinant human UGTs, substrates, and 25
μg/mL of alamethicin, were reconstituted in 50 mM Tris-HCl (pH 7.5) and pre-incubated on
ice for 15 min. The selective substrates were estradiol (10 μM) for UGT1A1,
chenodeoxycholic acid (5 μM) for UGT1A3, trifluoperazine (10 μM) for UGT1A4, 4-
hydroxyindole (10 μM) for UGT1A6, propofol (50 μM) for UGT1A9, and naloxone (250 μM)
for UGT2B7. The concentration of each probe substrate was initially chosen near its Km
value reported elsewhere (Supplemental Table 1). Under these conditions, drug interactions
among substrates were observed in cocktail incubation and their concentrations were reduced
to 1/2-1/4 of their Km values to avoid such interactions. The final volume of the organic
solvents in each incubation mixture was 1% (v/v). Reactions were initiated by adding 5 mM
UDPGA, and were incubated for 1 h at 37°C in a shaking water bath. Reactions were
terminated by the addition of 125 μl acetonitrile containing estrone glucuronide (2 μM,
internal standard) and centrifuged at 10,000g for 5 min at 4°C. Aliquots of the supernatants
were analyzed by LC-MS/MS for the identification of the glucuronide metabolites.
LC-MS/MS analysis of glucuronide metabolites of selective substrates
LC-MS/MS analysis was performed on an API 4000 LC-MS/MS system (Applied
Biosystems, Foster City, CA, USA), coupled with an Agilent 1100 series HPLC system
(Agilent, Wilmington, DE, USA). The separation was performed on a Synergi RP 80A
column (2 x 150 mm, 4 μm, Phenomenex, Torrance, CA) using a mobile phase of 0.1%
formic acid and acetonitrile (60:40, v/v). The flow rate was 0.2 ml/min. Electrospray
ionization was performed in positive and negative ion modes with nitrogen as the
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nebulizing, turbo, and curtain gases with the optimum values set at 50, 50, and 30
(arbitrary units). The Turbo ion spray interface was operated in the positive ion mode
at 4500 V and at -4500 V in the negative ion mode. Multiple reaction monitoring
(MRM) mode, using specific precursor/product ion transition, was employed for
quantification. Detection of the positive ions was performed by monitoring the
transitions of m/z 584.5 → 408.5 for trifluoperazine glucuronide, 310.0 → 134.0 for 4-
hydroxyindole glucuronide, and 504.0 → 310.0 for naloxone-3-glucuronide. Detection
of the negative ions was performed by monitoring the transitions of m/z 447.0 →
271.0 for estradiol-3-glucuronide, 567.5 → 391.5 for chenodeoxycholic acid
glucuronide, 353.0 → 177.0 for propofol glucuronide, and 445.0 → 269.0 for the
internal standard estrone glucuronide. Peak areas for all compounds were
automatically integrated using the Analyst software (version 1.4, Applied Biosystems).
Concentrations of glucuronides that lacked reference compounds were estimated as
molar equivalents, with respect to the calibration curve of the respective parent probe.
Chemical inhibition
The inhibitory effects of known UGT isoform-selective inhibitors on the formation of probe-
drug glucuronides were evaluated to identify the feasibility of the cocktail method for
screening the inhibitory effects of test compounds. Inhibitors used in this study were as
follows: bilirubin (50 μM) for UGT1A1, lithocholic acid (10 μM) for UGT1A3, hecogenin (5
μM) for UGT1A4, troglitazone (100 μM) for UGT1A6, niflumic acid (5 μM) for UGT1A9,
and fluconazole (2.5 mM) for UGT2B4 and 2B7. The formation rates of probe-drug
glucuronides were determined from reaction mixtures incubated in the presence or absence of
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inhibitors. With the exception of the addition of UGT-isoform-specific inhibitors, all other
incubation conditions were as described above.
Data analysis
In microsomal incubation studies, the apparent kinetic parameters of biotransformation (Km
and Vmax) were determined by fitting a one-enzyme Michaelis–Menten (V = Vmax[S]/Km+[S]),
a substrate inhibition (V=Vmax[S]/(Km+[S]*(1+[S]/Ksi))), or a Hill equation (V = Vmax[S]n/
S50n+[S]n). The calculated parameters included the maximum rate of formation (Vmax),
substrate concentration at half-maximal rate (apparent Km or S50), and the intrinsic clearance
(CLint = Vmax/apparent Km or S50). UGT-mediated activities in the presence of inhibitors
were expressed as a percentage of the corresponding control values. A sigmoid curve
was fitted to the data, and the enzyme inhibition parameter (IC50) was calculated using
a nonlinear least squares regression analysis of the plot of percent control activity
versus concentration of the test inhibitor. Calculations were performed using the
WinNonlin software (Pharsight, Mountain View, CA, USA). The percentages of
inhibition were calculated by the ratio of the amounts of metabolites formed, with and
without the specific inhibitor.
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Results
Glucuronidation of UGT isoform-selective substrates
Our initial efforts were focused on the selection of six UGT isoform-specific substrates
suitable for cocktail incubations. It is generally known that UGT isoforms show broad
substrate specificity. The probe substrates for each UGT isoform used in the cocktail assay
were selected on the basis of previous reports and on our preliminary screening results: β-
estradiol for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, 4-
hydroxyindole for UGT1A6, propofol for UGT1A9, and naloxone for UGT2B4/7. A
simultaneous analytical method using LC-MS/MS for six UGT isoform-specific probe
metabolites and an internal standard was developed for the cocktail assay of UGT activity in
human liver microsomes. The MRM transitions and optimized collision-induced dissociation
conditions are described in Table 1. The specificity of the tandem mass spectrometer allowed
a fast LC gradient to be employed. The representative chromatograms for six probe
metabolites in microsomal incubation mixtures are presented in Fig. 1. There was no
interference from other substrates or metabolites at any of the retention times of interest for
any metabolite MRM channel. In the case of β-estradiol, two glucuronides were observed in
the microsomal incubation; one at a retention time of 3.23 was β-estradiol-3-glucuronide and
the other at 3.82 was β-estradiol-17-glucuronide. The formation of β-estradiol-3-glucuronide
is mediated by UGT1A1 whereas the formation of β-estradiol-17-glucuronide is mainly
catalyzed by UGT2B7 (Alkharfy and Frye, 2002).
The selectivity of each UGT substrate was evaluated using cDNA-expressed human UGT
isoforms (Fig. 2). The concentration of each substrate was optimized to avoid interactions
among probe substrates. The formation rate of β-estradiol-3-glucuronide by UGT1A1 was
11-fold greater than that by UGT1A3. Conversely, the formation of chenodeoxycholic acid
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glucuronide by UGT1A3 was 14-fold greater than that by UGT1A1. UGT2B7 showed
minimal activity on chenodeoxycholic acid. 4-Hydroxyindole was glucuronidated mainly by
UGT1A6, with minor activity by UGT1A9. Kinetic analysis also demonstrated that UGT1A1,
1A3 and 1A6 could play major roles in the glucuronidation of estradiol, chenodeoxycholic
acid and 4-hydroxyindole, respectively (Supplemental Fig. 1-3, Supplemental Table 2). The
glucuronidation of trifluoperazine, propofol, and naloxone was almost exclusively catalyzed
by UGT1A4, UGT1A9, and UGT2B7, respectively. Our results indicate that the UGT
isoform-selective targets used in this experiment are appropriate substrates, representing the
corresponding UGT isoform activities, when incubated in a cocktail.
Comparison of UGT isoform activities between individual and cocktail incubations
Potential interactions among UGT substrates were evaluated during simultaneous incubations
with human liver microsomes. The simultaneous incubation of six substrates with human
liver microsomes showed glucuronidation activities different from those obtained with single
individual incubations (Fig. 3A). The formation of estradiol-3-glucuronide,
chenodeoxycholic acids, propofol glucuronide, and naloxone-3-glucuronide was inhibited by
greater than 30% when 6 substrates were co-incubated with microsomes. When pairs of
substrates were incubated, an interaction between estradiol and propofol was observed. In the
presence of estradiol, propofol glucuronidation catalyzed by UGT1A9 was reduced to
approximately 50% of basal activity; Inhibition of UGT1A9 activity was independent on the
concentration of propofol. When estradiol was replaced with the UGT1A1-selective substrate
SN-38 (Hanioka et al., 2001), SN-38 glucuronidation was inhibited by both trifluoperazine
and naloxone. Naloxone glucuronidation was also inhibited by other UGT isoform-selective
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substrates. Replacement of naloxone with the UGT2B7-selective substrate efavirenz (Bae et
al., 2011) inhibited the glucuronidation of trifluoperazine and propofol. When zidovudine
was added as an UGT2B7-selective substrate (Barbier et al., 2000) to cocktail incubations,
the glucuronidation of the drug was inhibited by estradiol (Supplemental Fig. 4). These
results collectively indicated that simultaneous incubation of all six UGT isoform-selective
substrates with human liver microsomes caused interactions among substrates that resulted in
the inhibition of at least one or two UGT isoforms. Therefore, two cocktails of substrates
were prepared for the microsomal incubation step. Cocktail A included estradiol,
trifluoperazine, and 4-hydroxyindole, and cocktail B contained chenodeoxycholic acid,
propofol, and naloxone. These mixtures were pooled after incubation and analyzed together
by LC-MS/MS to reduce total assay time. As shown in Fig. 3B, each UGT isoform’s activity
was not substantially inhibited by other substrates within the cocktail sets except UGT1A3
(percent inhibition < 20%). UGT1A3 activity was enhanced 1.3-fold over single-substrate
incubations.
Assay validation using UGT isoform-selective inhibitors
The utility of this cocktail incubation as a screening tool for UGT inhibition was evaluated
using known UGT inhibitors. The IC50 value of each UGT isoform-selective inhibitor was
determined in both individual and cocktail incubations. As shown in Fig. 4, the inhibition
profile of each inhibitor was not substantially different between the two incubation methods,
with the exception of that of lithocholic acid, a UGT1A3 inhibitor. The IC50 values measured
by the different approaches are summarized in Table 2. The IC50 value of lithocholic acid for
the formation of chenodeoxycholic acid glucuronide in single incubations was 2.6-fold lower
than in cocktail incubations (Table 2). The effects of isoform-selective inhibitors on other
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UGT isoforms were also evaluated in cocktail incubations (Fig. 5). Bilirubin, an UGT1A1-
selective inhibitor (Williams et al., 2002) resulted in greater inhibition of UGT1A1 activity
compared to those of the activities of UGT1A4 or 1A6. Hecogenin, lithocholic acid, and
niflumic acid demonstrated selective inhibition of UGT1A4, 1A3, and 1A9 activities,
respectively, without affecting other isoform activities measured in cocktail incubations.
Fluconazole inhibited UGT2B7 activity in a concentration-dependent manner up to 10 mM,
although UGT1A3 and 1A9 activities were also inhibited by 40% and 21%, respectively at 10
mM fluconazole. Troglitazone is reported to be a UGT1A6 inhibitor (Ito et al., 2001).
However, this compound inhibited the activity of UGT1A1 and UGT1A4 to a greater extent
than UGT1A6 in cocktail incubations. This was also observed in individual incubations with
estradiol and trifluoperazine, suggesting that the inhibition observed in cocktail incubations
was not due to substrate interactions. Troglitazone caused greater inhibition of UGT1A6
activities when incubated with recombinant UGT1A6 instead of microsomes (Supplemental
Fig 5).
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Discussion
During the early stages of drug development, knowledge of the metabolic characteristics of
new drug candidates is very important in the selection of lead compounds and in minimizing
failures during clinical studies due to major kinetic problems such as drug-drug interactions.
For this reason, several in vitro methods have been developed and are being utilized to study
drug metabolism and metabolic interactions in the early phases of drug discovery and
development (Pelkonen et al., 2005). The aim of this study was to develop a simple and rapid
cocktail assay to simultaneously monitor the activity of hepatic UGT isoforms in human liver
microsomes.
The probe substrates for six human hepatic UGT isoforms were selected from the literature
and from our own preliminary screening of their specificity for each isoform. The specificity
of each substrate was evaluated using cDNA-expressed UGTs. It is well known that UGTs
exhibit partially distinct but frequently overlapping substrate specificities, which make it
difficult to identify a selective substrate for each UGT isoform (Lepine et al., 2004). In
addition, substrates selective for one UGT isoform often modulate the activities of other
isoforms. Therefore, considerable efforts have been made to choose probe substrates
relatively specific for single UGT isoforms that do not interfere with other isoform activities.
We found that trifluoperazine, propofol, and naloxone were almost exclusively
glucuronidated by UGT1A4, 1A9, and 2B7, respectively, and these results are consistent with
data reported elsewhere (Uchaipichat et al., 2006, Court, 2005, Di Marco et al., 2005). The
formation of estradiol-3-glucuronide is mediated mainly by UGT1A1, whereas estradiol-17-
glucuronide is generated by UGT2B7 (Alkharfy and Frye, 2002). Chenodeoxycholic acid is
reported to be glucuronidated by UGT1A3 (Trottier et al., 2006). We also found that
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UGT1A1 was the major enzyme catalyzing the formation of estradiol-3-glucuronide.
Although UGT1A3 also contributed to the glucuronidation of estradiol, its rate of formation
was ~9% of that seen with UGT1A1. On the other hand, chenodeoxycholic acid was mainly
glucuronidated by UGT1A3, with UGT1A1 catalyzing glucuronide formation at only 7% of
the UGT1A3 rate. Recently, Fallon et al. (Fallon et al., 2013b) reported that the average
protein level of UGT1A1 is 4.5-fold higher than that of UGT1A3 in human liver microsomes
(36.2 vs. 8.0 pmol/mg protein). As reported in the same study, BD supersomes expressed 2.6-
fold more recombinant UGT1A1 than UGT1A3 (Fallon et al., 2013a). When intrinsic
clearance values obtained from kinetic analyses (Supplemental Table 1, Supplemental Fig. 1)
and relative ratios of expression are considered, the contributions of UGT1A3 to estradiol-3-
glucuronide formation and UGT1A1 to chenodeoxycholic acid glucuronide formation in
human liver microsomes were estimated to be 8.2% of UGT1A1 and 7.9% of UGT1A3,
respectively. The relative contributions of UGT1A6 and 1A9 to the formation of 4-
hydroxylindole was estimated to be 81.3 and 18.7%, respectively when calculated in the
same way. These results collectively indicate that the substrates selected for the present study
were suitable as probe substrates for each UGT isoform.
When relatively selective substrates for six hepatic UGTs (1A1, 1A3, 1A4, 1A6, 1A9,
and 2B7) were incubated in a cocktail assay, UGT1A1, 1A9, and 2B7 activities were
substantially inhibited relative to those seen in individual incubations (Fig. 3A). Substrates
affecting the activity of other UGT isoforms were initially identified by measuring activity in
pairwise incubations. Identified substrates were then replaced by others reported to be
isoform selective, as described in the results section. However, cross-interactions among
substrates could not be avoided, even after the replacement of estradiol with the UGT1A1-
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selective substrates SN-38 or etoposide, or exchanging naloxone for the UGT2B7 substrates
efavirenz or zidovudine. Therefore, we employed a single LC-MS/MS analysis of incubation
mixtures pooled from two separate microsomal incubations with substrates. Substrates were
divided into two groups; cocktail A included estradiol, trifluoperazine, and 4-hydroxyindole,
and cocktail B contained chenodeoxycholic acid, propofol, and naloxone. For five UGT
isoforms (but not for UGT1A3), activities in these sets were similar to those observed in
individual incubations. In group B incubations, propofol glucuronidation by UGT1A9 was
increased 30% over individual incubations (Fig 3B). This may be due to catalytic activation.
However, this activation did not change the IC50 value of niflumic acid, a known UGT1A9
inhibitor (Table 2).
Although the availability of selective UGT inhibitors is currently limited, they represent
the most powerful tool available for reaction phenotyping. The best-known UGT inhibitors
are hecogenin for UGT1A4 (Uchaipichat et al., 2006), niflumic acid for UGT1A9 (Mano et
al., 2006), and fluconazole for UGT2B7 (Miners et al., 2010, Donato et al., 2010). Bilirubin and
lithocholic acid are known to be substrates for UGT1A1 and UGT1A3, respectively. These
compounds are also used for inhibition studies for UGT1A1 (Soars et al., 2003, Alkharfy and
Frye, 2002) and UGT1A3 (Matern.S. et al., 1984, Verreault et al., 2006). Our results
demonstrated that hecogenin and niflumic acid resulted in strong and selective inhibition of
UGT1A4 and UGT1A9, respectively, as expected (Fig. 4). Fluconazole was a moderately
selective inhibitor; we found that it inhibited both UGT1A1 and 2B7. Bilirubin inhibited
UGT1A1 activity, but also weakly inhibited UGT1A4 activity. Troglitazone was chosen as a
UGT1A6 inhibitor based on a report that it inhibited recombinant UGT1A6-mediated 1-
naphthol glucuronidation with an IC50 of 28 μM (Hanioka et al., 2001). However,
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troglitazone did not inhibit 1-naphthol glucuronidation under our experimental conditions,
and had no effect on the glucuronidation of 4-hydroxyindole, another reaction mediated by
UGT1A6 in microsomal incubations (data not shown). Unexpectedly, UGT1A1 and 1A4
were inhibited by troglitazone, with IC50 values less than 10 μM. This discrepancy may be
due to use of enzymes from different sources. With our cDNA-expressed human UGT1A6,
troglitazone inhibited the glucuronidation of 4-hydroxyindole and 1-naphtol, consistent with
the results of (Hanioka et al., 2001). These results suggest that recombinant UGTs may not be
suitable for evaluating the inhibition potential of chemicals, particularly in the case of
UGT1A6.
We found that all inhibitors tested showed similar inhibition profiles with both individual
substrates and substrate cocktails (Fig. 4). The IC50 values of the selective UGT inhibitors
determined using the substrate cocktails were in good agreement with those determined using
individual substrates, and were comparable to those reported by other groups (Table 2). This
suggests that the inhibitory potential of test compounds can be accurately determined using
our cocktail assay, rather than individual substrate incubations.
In conclusion, a method was developed for high-throughput inhibition screening of the
major human hepatic UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9,
and UGT2B7) using in vitro substrate cocktails and LC-MS/MS analysis. Probe substrates
were selected after evaluation of isoform selectivity to minimize possible interference by
other UGT isoforms. Six substrates divided into two cocktails for incubation and pooled for
analysis in a single run allowed us to evaluate the activity of six UGT isoforms without cross-
interference. With known UGT isoform-selective inhibitors, this cocktail assay produced
similar inhibition profiles to those obtained from single-substrate incubations, suggesting that
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this assay can be a useful tool for rapid screening of UGT inhibition and for the prediction of
clinical drug interactions.
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Authorship Contributions
Participated in research design: D.-H. Kim, and Shin
Conducted experiments: Seo, H.-J. Kim, Jeong, and N. Abdalla
Performed data analysis: Seo, H.-J. Kim, Jeong, C.-S. Choi, D.-H. Kim, and Shin
Wrote or contributed to the writing of the manuscript: Seo, D.-H. Kim, and Shin
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(2002) Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed
estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to
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Footnotes
This work was supported by the National Research Foundation of Korea grant funded by the
Korean Government [R13-2007-023-00000-0].
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Figure legends
Fig. 1. Multiple reaction monitoring (MRM) chromatograms from the analysis of the major
metabolites of UGT substrates.
Fig. 2. Representative plots of the formation of estradiol-3-glucuronide from estradiol (A),
chenodeoxycholic acid (CDCA) glucuronide from chenodeoxycholic acid (B), trifluoperazine
(TFP) glucuronide from trifluoperazine (C), 4-hydroxyindole glucuronide from 4-
hydroxyindole (D), propofol glucuronide from propofol (E), and naloxone-3-glucuronide
from naloxone (F), by cDNA-expressed human UGT isoforms. Activities shown are means
of duplicate determinations from a single experiment.
Fig. 3. Effects of cocktail incubation on UGT isoform activities in human liver microsomes;
(A) six substrates were incubated together and (B) six substrates were divided into two
groups prior to incubation. Each bar represents the relative percentage of the activity assessed
by individual incubation with estradiol for UGT1A1 (10 μM), chenodeoxycholic acid for
UGT1A3 (5 μM), trifluoperazine for UGT1A4 (10 μM), 4-hydroxyindole for UGT1A6 (10
μM), propofol for UGT1A9 (50 μM), and naloxone for UGT2B7 (250 μM). Each activity
shown is the mean of triplicate experiments. Each bar represents the mean + SD of triplicate
determinations from a single experiment.
Fig. 4. Effects of various inhibitors on UGT isoform activity in human liver microsomes, in
individual and cocktail incubations. UGT isoform-selective inhibitors used were bilirubin (A,
50 μM), lithocholic acid (B, 10 μM), hecogenin (C, 5 μM), troglitazone (D, 100 μM),
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niflumic acid (E, 5 μM) and fluconazole (F, 2.5 mM). Each bar represents the mean + SD of
triplicate determinations from a single experiment.
Fig. 5. The effects of UGT isoform-selective inhibitors on other UGT isoform activities in
human liver microsomes in substrate cocktails. Cocktail A contained estradiol,
trifluoperazine, and 4-hydroxyindole, and cocktail B consisted of chenodeoxycholic acid,
propofol, and naloxone. Data represent the means ± SD of triplicate determinations from a
single experiment.
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Table 1
MRM parameters for the major metabolites of six UGT probe substrates.
UGT isoform
Metabolite Transition
(m/z) Polarity
CE (eV)
UGT1A1 β-Estradiol-3-glucuronide 447.0>271.0 ES- 50
UGT1A3 Chenodeoxycholic acid
glucuronide 567.5>391.5 ES- 50
UGT1A4 Trifluoperazine glucuronide 584.5>408.5 ES+ 35
UGT1A6 4-Hydroxyindole glucuronide 310.0>134.0 ES+ 20
UGT1A9 Propofol glucuronide 353.0>177.0 ES- 35
UGT2B7 Naloxone-3-glucuronide 504.0>310.0 ES+ 30
IS Estrone glucuronide 445.0>269.0 ES- 38
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Table 2
IC50 values obtained in this study using individual substrate and substrate cocktails, and published IC50 values for six UGT inhibitors.
UGT
isoform Substrate Inhibitor
IC50 (µM) Reported IC50
(µM) References
Individual
substrate
Cocktail
substrate
1A1 β-estradiol Bilirubin 22.5 31.5 4.0 - 30 (Williams et al., 2002,
Tachibana et al., 2005)
1A3 Chenodeoxycholic acid Lithocholic acid 4.8 12.3 - -
1A4 Trifluoperazine Hecogenin 2.2 3.2 1.5 - 15 (Uchaipichat et al., 2006,
Edavana et al., 2013)
1A6 4-Hydroxyindole Troglitazone 195.5 185.8 28 (Ito et al., 2001)
1A9 Propofol Niflumic acid 0.3 0.2 0.034 - 0.4 (Mano et al., 2006, Miners et
al., 2011)
2B7 Naloxone Fluconazole 5100 5100 1790 - 2500 (Mano et al., 2007, Donato et
al., 2010)
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Time (min)
0 2 4 6
Inte
nsi
ty,
cp
s
5.0e+3
1.0e+4
1.5e+4
Time (min)
0 2 4 6
Inte
nsi
ty,
cp
s
1.0e+4
2.0e+4
3.0e+4
Time (min)
0 2 4 6
Inte
nsi
ty,
cp
s
1.0e+4
2.0e+4
3.0e+4
4.0e+4
Time (min)
0 2 4 6
Inte
nsi
ty,
cp
s
5.0e+3
1.0e+4
1.5e+4
2.0e+4
Time (min)
0 2 4 6
Inte
nsi
ty,
cp
s
1.0e+5
2.0e+5
3.0e+5
4.0e+5
5.0e+5
Time (min)
0 2 4 6
Inte
nsi
ty,
cp
s
5.0e+3
1.0e+4
1.5e+4
2.0e+4
(A) Estradiol-3-glucuronide (B) Chenodeoxycholic acid glucuronide
(C) Trifluoperazine glucuronide (D) 4-Hydroxyindole glucuronide
(E) Propofol glucuronide (F) Naloxone-3-glucuronide
Fig.1
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Fig.2
Recombinant UGTs
1A1
1A3
1A4
1A6
1A9
2B42B7
2B152B17
% o
f U
GT
1A
1 a
cti
vit
y
0
20
40
60
80
100
120 -estradiol 10M
Recombinant UGTs
1A1
1A3
1A4
1A6
1A9
2B42B7
2B152B17
% o
f U
GT
1A
3 a
cti
vit
y
0
20
40
60
80
100
120CDCA 10M
Recombinant UGTs
1A1
1A3
1A4
1A6
1A9
2B42B7
2B152B17
% o
f U
GT
1A
4 a
cti
vit
y
0
20
40
60
80
100
120TFP 10
Recombinant UGTs
1A1
1A3
1A4
1A6
1A9
2B42B7
2B152B17
% o
f U
GT
1A
6 a
cti
vit
y
0
20
40
60
80
100
120 4-Hydroxyindole 100
Recombinant UGTs
1A1
1A3
1A4
1A6
1A9
2B42B7
2B152B17
% o
f U
GT
1A
9 a
cti
vit
y
0
20
40
60
80
100
120 Propofol 100
Recombinant UGTs
1A1
1A3
1A4
1A6
1A9
2B42B7
2B152B17
% o
f U
GT
2B
7 a
cti
vit
y
0
20
40
60
80
100
120 Naloxone 500
(B) (A) (C)
(E) (D) (F)
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Fig.3
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B7
Rela
tiv
e a
cti
vit
y (
%)
0
20
40
60
80
100
120
140
UGT1A1
UGT1A4
UGT1A6
UGT1A3
UGT1A9
UGT2B7
Rela
tiv
e a
cti
vit
y (
%)
0
20
40
60
80
100
120
140
Cocktail A Cocktail B
(B) (A)
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UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B7
% o
f co
ntr
ol
acti
vit
y
0
20
40
60
80
100
120
Individual
Cocktail
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B70
20
40
60
80
100
120
Individual
Cocktail
% o
f co
ntr
ol
acti
vit
y
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B70
20
40
60
80
100
120
Individual
Cocktail
% o
f co
ntr
ol
acti
vit
y
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B70
20
40
60
80
100
120
Individual
Cocktail
% o
f co
ntr
ol
acti
vit
y
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B70
20
40
60
80
100
120
Individual
Cocktail
% o
f co
ntr
ol
acti
vit
y
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B70
20
40
60
80
100
120
Individual
Cocktail
% o
f co
ntr
ol
acti
vit
y
(B) (A) (C)
(E) (D) (F)
Fig.4
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1 10 100
0
20
40
60
80
100
120
140
UGT1A1
UGT1A4
UGT1A6
Cocktail A
Bilirubin (M)
% o
f co
ntr
ol
acti
vit
y
1 10 100
0
20
40
60
80
100
120
140
UGT1A1
UGT1A4
UGT1A6
Cocktail A
Troglitazone (M)
% o
f co
ntr
ol
acti
vit
y
0.1 1 10
0
20
40
60
80
100
120
140
UGT1A1
UGT1A4
UGT1A6
Cocktail A
Hecogenin (M)%
of
co
ntr
ol
acti
vit
y
0.1 1 10
0
20
40
60
80
100
120
140
UGT1A3
UGT1A9
UGT2B7
Cocktail B
Niflumic acid (M)
% o
f co
ntr
ol
acti
vit
y
1 10
0
20
40
60
80
100
120
140
UGT1A3
UGT1A9
UGT2B7
Cocktail B
Lithocholic acid (M)
% o
f co
ntr
ol
acti
vit
y
1 10
0
20
40
60
80
100
120
140
UGT1A3
UGT1A9
UGT2B7
Cocktail B
Fluconazole (mM)%
of
co
ntr
ol
acti
vit
y
(B) (A) (C)
(E) (D) (F)
Fig.5
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