In vitro assay of six UGT isoforms in human liver ...dmd.aspetjournals.org/content/dmd/early/2014/08/14/dmd.114.058818.full.pdf · DMD #58818 In vitro assay of six UGT isoforms in

DMD #58818

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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)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 13, 2014 as DOI: 10.1124/dmd.114.058818

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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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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)

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 August 13, 2014 as D

OI: 10.1124/dm

d.114.058818 at ASPET Journals on December 30, 2019 dmd.aspetjournals.org Downloaded from


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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