Optimized assays for human UDP-glucuronosyltransferase (UGT) activities: Altered alamethicin concentration and utility to screen for UGT inhibitors

Article abstract of DOI:10.1124/dmd.111.043117

The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: β-estradiol- 3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5′-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 μg/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzyme-selective chemical inhibitors. Copyright

Full text of DOI:10.1124/dmd.111.043117

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2012/02/22/dmd.111.043117
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1521-009X/12/4005-1051–1065$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:1051–1065, 2012
Vol. 40, No. 5
43117/3766807
Optimized Assays for Human UDP-Glucuronosyltransferase (UGT)
Activities: Altered Alamethicin Concentration and Utility to Screen
□
S
for UGT Inhibitors
Robert L. Walsky,1 Jonathan N. Bauman, Karine Bourcier, Georgina Giddens,
Kimberly Lapham, Andre Negahban, Tim F. Ryder, R. Scott Obach, Ruth Hyland,
and Theunis C. Goosen
Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Groton, Connecticut (R.L.W., J.N.B., K.L., A.N.,
T.F.R., R.S.O., T.C.G.); and Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Sandwich, Kent, United
Kingdom (K.B., G.G., R.H.)
Received September 30, 2011; accepted February 22, 2012
ABSTRACT:
The measurement of the effect of new chemical entities on human binant UGT incubations. Apparent enzyme kinetic parameters, par-
UDP-glucuronosyltransferase (UGT) marker activities using in vitro ticularly for UGT1A1 and UGT1A4, were affected by nonspecific
experimentation represents an important experimental approach binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7
in drug development to guide clinical drug-interaction study de- increased significantly after addition of 2% bovine serum albumin,
signs or support claims that no in vivo interaction will occur.
with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven
Selective high-performance liquid chromatography-tandem mass potential UGT and cytochrome P450 inhibitors were evaluated as
spectrometry functional assays of authentic glucuronides for five UGT inhibitors, resulting in observation of nonselective UGT inhi-
major hepatic UGT probe substrates were developed: ␤-estradiol- bition by chrysin, mefenamic acid, silibinin, tangeretin, ketocona-
3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), zole, itraconazole, ritonavir, and verapamil. The pan-cytochrome
5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-gluc- P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT ac-
uronide (UGT1A9), and zidovudine-5ꢀ-glucuronide (UGT2B7). High tivities and may be useful in reaction phenotyping of mixed UGT
analytical sensitivity permitted characterization of enzyme kinetic and cytochrome P450 substrates. These methods should prove
parameters at low human liver microsomal and recombinant UGT useful in the routine assessments of the potential for new drug
protein concentration (0.025 mg/ml), which led to a new recom- candidates to elicit pharmacokinetic drug interactions via inhibi-
mended optimal universal alamethicin activation concentration of tion of human UGT activities and the identification of UGT enzyme-
10 ␮g/ml for microsomes. Alamethicin was not required for recom- selective chemical inhibitors.
Introduction
This work was presented in part as follows: Walsky RL, Bauman JN, Bourcier
Drug-drug interactions (DDIs) due to the inhibition of UDP-gluc-
uronosyltransferases (UGTs), both as perpetrator and victim, should
be considered when developing new chemical entities and are impor-
tant in drug discovery research or evaluation of patient safety, as noted
by regulatory agencies (Bjornsson et al., 2003; Zhang et al., 2010). In
general, the risk of significant clinical DDIs due to the inhibition of
UGTs during drug coadministration is low compared with inhibition
of cytochrome P450 (P450) activities (Williams et al., 2004; Kiang et
al., 2005). Likewise, few clinically relevant examples that require
dose adjustment for a poor UGT-metabolizer genotype exist, except
for clinical reports on UGT1A1 substrates (Toffoli et al., 2006;
Williams et al., 2008; Court, 2010). Nevertheless, a thorough evalu-
K, Giddens G, Lapham K, Negahban A, Ryder TF, Obach RS, Hyland R, and
Goosen TC (2011) Optimized assays for human UDP-glucuronosyltransferase
(UGT) activities: altered alamethicin concentration and utility to identify UGT
inhibitors. 17th North American International Society for the Study of Xenobiotics
Meeting; 2011 Oct 16–20; Atlanta, GA. International Society for the Study of
Xenobiotics, Washington, DC.
1 Current affiliation: Department of Drug Metabolism and Pharmacokinetics,
AstraZeneca Pharmaceuticals LP, Waltham, Massachusetts.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
http://dx.doi.org/10.1124/dmd.111.043117.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: DDI, drug-drug interaction; UGT, UDP-glucuronosyltransferase; P450, cytochrome P450; HLM, human liver microsomes;
LC-MS/MS, liquid chromatography equipped with tandem mass spectrometry; ES, ␤-estradiol; ES3-G, ␤-estradiol-3-glucuronide; TFP, trifluoper-
azine; 5HTOL, 5-hydroxytryptophol; PRO, propofol; AZT, 3Ј-azido-3Ј-deoxythymidine or zidovudine; CLint, intrinsic clearance; BSA, bovine serum
albumin; AZT-G, AZT-5Ј-glucuronide; PRO-G, propofol-O-glucuronide; TFP-G, trifluoperazine-N-glucuronide; UDPGA, UDP-glucuronic acid;
DMSO, dimethyl sulfoxide; 5HTOL-G, 5-hydroxytryptophol-O-glucuronide; HPLC, high-performance liquid chromatography; rUGT, recombinant
UGT; IS, internal standard; aSICCO, artificial signal insertion for calculation of concentration observed; COSY, correlation spectroscopy; HMBC,
heteronuclear multiple bond coherence; CLmax, maximal clearance.
1051

1052
WALSKY ET AL.
ation throughout the discovery and development of new drugs is 2010a). In addition to serotonin (5-hydroxytryptamine), the metabo-
required to inform particular groups of the potential for a significant lite 5-hydroxytryptophol (5HTOL) has been identified with LC/UV
DDI liability. Accordingly, the development of reliable and robust quantification as a marker substrate for UGT1A6 (Krishnaswamy et
assay conditions and analytical methods for measuring glucuronida- al., 2004). Literature reports have shown convincing evidence of
tion in vitro, which could be used to obtain relative activity factors propofol (PRO) selectivity for UGT1A9 activity (Soars et al., 2004;
(Venkatakrishnan et al., 2000) and to develop appropriate tools that Court, 2005), and 3Ј-azido-3Ј-deoxythymidine or zidovudine (AZT) is
are useful in identifying UGT enzyme-selective chemical inhibitors, an established UGT2B7 probe substrate (Court et al., 2003).
are required to advance UGT reaction-phenotyping techniques (Min-
ers et al., 2010a; Parkinson et al., 2010).
Several experimental variables affect in vitro UGT enzyme activity
and ultimately intrinsic clearance (CLint) estimates, including buffer
UGT1A1, -1A3, -1A4, -1A6, -1A9, -2B7, and -2B15 play a signif- type, pH, ionic strength, latency, organic solvent, glucuronide stabil-
icant role in hepatic drug and xenobiotic metabolism (Miners et al., ity, atypical kinetics, and the albumin effect (Easterbrook et al., 2001;
2010a) of the 19 human UGTs currently identified (Mackenzie et al., Fisher et al., 2001; Boase and Miners, 2002; Soars et al., 2003;
2005; Miners et al., 2010b). The triad of UGT reaction-phenotyping Uchaipichat et al., 2004; Engtrakul et al., 2005; Rowland et al., 2007,
techniques are not equally advanced (Court, 2004). In particular, a 2008). The latter results from long-chain unsaturated fatty acids being
limited number of UGT-selective probe substrates are available to released from membranes during the course of an incubation and acts
correlate in vitro-glucuronidation activities of the major UGTs ex- as a potent inhibitor of UGT1A9, UGT2B7, and microsomal gluc-
pressed in human liver with those of drugs or new chemical entities in uronidation activity, which results in overestimation of the Km value
human liver microsomes (HLM) or hepatocytes, or to evaluate UGT (Tsoutsikos et al., 2004; Rowland et al., 2007, 2008). Inhibitory fatty
contribution in DDIs through either enzyme inhibition or induction acids are sequestered by addition of bovine serum albumin (BSA), and
(Court, 2005; Miners et al., 2010a). During the course of these studies, altered incubation conditions (in the presence and absence of BSA)
we developed selective liquid chromatography equipped with tandem should be evaluated to select appropriate substrate concentrations
mass spectrometry (LC-MS/MS) functional assays of authentic gluc- (ՅKm) when used in an inhibitor screening assay. The objectives of
uronides for five major hepatic UGT probe substrates (Fig. 1). Selec- this study are to develop improved analytical methods (Donato et al.,
tive UGT1A1 substrates include bilirubin and etoposide, but, due to 2010) for measuring the in vitro activities of five major human UGT
challenging substrate assays, ␤-estradiol (ES) is mostly used to char- enzymes that use authentic analytical glucuronide standards (Fig. 1)
acterize UGT1A1 kinetics, with the understanding that other hepatic analogous to those used for P450 (Walsky and Obach, 2004). We also
UGTs (e.g., UGT1A3) may contribute to ␤-estradiol-3-glucuronide evaluated these methods in high-throughput UGT inhibitor screening
(ES3-G) formation (Le´pine et al., 2004; Ita¨aho et al., 2008). Triflu- of several potential inhibitors of these enzymes. Various attributes of
operazine (TFP) is selective for measurement of UGT1A4 activity incubation conditions frequently used to measure UGT activities (e.g.,
(Uchaipichat et al., 2006a), and other potentially selective substrates albumin, buffer, saccharolactone, and alamethicin) were explored to
have been characterized (e.g., 1-hydroxymidazolam) (Miners et al., define optimal conditions for each enzyme at low protein concentra-
FIG. 1. Major reactions catalyzed by human
UDP-glucuronosyltransferase enzymes. Inter-
nal standards for LC-MS/MS analytical meth-
ods shown for each reaction.

UGT INHIBITION ASSAYS
1053
Other reagents and solvents used were from standard suppliers and were of reagent
or high-performance liquid chromatography (HPLC) grade, with all purities as
defined by the manufacturer.
Human liver microsomes were prepared from a mixed gender pool of 50
donors (provided by BD Biosciences, Woburn, MA). Recombinant
UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 Supersomes were
heterologously expressed from human cDNA in a baculovirus expression
system; protein concentrations and initial activity assessments were pro-
vided by BD Biosciences.
tion. In the course of these studies, the more precise identification of
the glucuronide metabolite of 5-hydroxytryptophol was made to fa-
cilitate the development of the assay for UGT1A6. The findings are
presented here, and these assay methods should be useful to investi-
gators who are engaged in research on these enzymes and the xeno-
biotics they metabolize.
Materials and Methods
Materials. Substrates, metabolite standards, internal standards (IS), and
other materials were from the following sources: AZT-5Ј-glucuronide (AZT-
G), [13C6]AZT-5Ј-glucuronide, 5-hydroxytryptophol, propofol-O-glucuronide
(PRO-G), trifluoperazine-N-glucuronide, and [D3]trifluoperazine-N-glucuro-
nide (Cerilliant, Austin, TX); magnesium chloride, potassium phosphate, Tris
(base), and Tris-HCl (Mallinckrodt Baker, Inc., Phillipsburg, NJ); ammonium
formate and formic acid, (Fluka, Buchs, Switzerland); alamethicin, AZT,
bovine serum albumin (crude BSA), diclofenac, ␤-estradiol, ␤-estradiol-3-
glucuronide, ␤-estradiol-17-glucuronide, propofol, D-saccharic acid 1,4-lac-
tone (saccharolactone), and uridine-diphosphate-glucuronic acid trisodium salt
(UDPGA) (Sigma-Aldrich, St. Louis, MO); and 1-naphthyl-glucuronide (Se-
quoia Research Products, Ltd., Pangbourne, UK). Inhibitors were obtained
from Sigma-Aldrich or Sequoia Research Products. Pure (100%) stable-labeled
dimethyl sulfoxide (DMSO)-d6 and methanol-d4 were obtained from Cam-
bridge Isotope Laboratories, Inc. (Andover, MA). 5-Hydroxytryptophol-O-
glucuronide (5HTOL-G) was obtained by biosynthesis as detailed under 5-Hy-
General UGT Assay Incubation Conditions. Specific aspects of the
incubation condition for each assay are defined in Table 1. In general, for the
final optimized method, a premix containing HLM or expressed UGT
enzyme (0.025 mg/ml) were mixed with 100 mM Tris-HCl buffer (pH 7.5
at 37°C), MgCl2 (5 mM), substrate, and alamethicin (10 ␮g/ml), with or
without 2% BSA. The premix was placed on ice for 15 min to allow
alamethicin pore formation, and then aliquots of this mixture (0.09 ml)
were delivered to each well of a polypropylene 96-well polymerase chain
reaction plate (maintained at 37°C) that contained the inhibitor or control
solvent (DMSO), as applicable. Final solvent concentrations were 1% (v/v)
or less. Incubations were commenced with the addition of UDPGA (5 mM)
to a final incubation volume of 0.1 ml and incubated at 37°C for the period
defined in Table 1. Incubations were typically terminated by addition of
acidified organic solvent (acetonitrile) that contained the internal standard.
The terminated incubation mixtures were centrifuged and either directly
droxytryptophol-O-glucuronide Isolation, Quantitation, and Structural Elucidation. injected, evaporated under nitrogen and reconstituted, or filtered before
TABLE 1
Incubation conditions and analytical parameters for human UDP-glucuronosyltransferase assays
Assay
UGT1A1
UGT1A4
UGT1A6
UGT1A9
UGT2B7
Incubation Conditions
Human liver microsomes
Protein conc., mg/ml
Incubation time, min
Recombinant UGT
Protein conc., mg/ml
Incubation time, min
Analytical conditions
Analyte
0.025
60
0.025
30
0.025
20
0.025
30
0.025
60
0.025
45
0.025
45
0.025
90
0.025
30
0.025
60
ES3-G
TFP-G
͓D3͔TFP-G
5HTOL-G
Diclofenac
PRO-G
Diclofenac
AZT-G
13C6͔AZT-G
Internal standard:
identity
Internal standard
conc., ␮M
1-Naphthyl-O-
glucuronide
5.0
͓
3.0
0.24
0.42
1.0
Injection volume, ␮l
Flow, ml/min
20
0.4
20
0.75
10
1.0
20
0.6
10
0.5
Gradient program, %B 29(0)3 29(2)3 98(4.1) 10(0)3 2(0.1)3 80(2.0) 20(0)3 95(1.6)3 95(1.8) 90(0)3 90(1.2)3 97(4.4) 2(0)3 2(0.5)3 35(2.6)
(min)
a
Mass spectrometer
Mass spectrometer
conditions
Sciex (1)
Sciex (1)
Sciex (2)
Sciex (2)
Waters (3)
Detection mode
Ion spray/capillary
voltage
Negative
Positive
Negative
Negative
Negative
Ϫ4.50 kV
5.00 kV
Ϫ4.20 kV
Ϫ3.00 kV
Ϫ3.00 kV
Declustering/cone
voltage
Source/desolvation
temperature
Ϫ55 eV
75 eV
500°C
30 eV
700°C
40 eV
650°C
Ϫ80 eV
400°C
325°C
Collision energy
Analyte m/z transition
Internal standard m/z
transition
Ϫ30 eV
4473113
3193113
45 eV
5843408
5873411
Ϫ35 eV
3523176
2943249
Ϫ30 eV
3533177
2943249
Ϫ26 eV
4423125
4483125
Rt: analyte, min
Rt: internal standard,
min
1.3
1.1
1.2
1.1
1.4
1.1
1.3
1.1
2.0
2.0
Standard curve range,
nM
20–2500
17Ϫ4300
5.0Ϫ2000
56Ϫ28,000
50Ϫ5000
Accuracy, %b,c
Precision, %b,d
89.3
8.6
101
11
112
12
101
7.5
101
5.1
Rt, retention time.
a Analytical quantification was conducted by LC-MS/MS using three systems, as described under Materials and Methods.
b Determined at 2nd lowest analytical standard concentration.
c Accuracy (%) ϭ 100 ϫ ͓analyte͔measured/͓analyte͔
.
nominal
d Precision (%) ϭ 100 ϫ S.D. ͓analyte͔measured/͓analyte͔
.
measured

1054
WALSKY ET AL.
transferring into a receiver 96-well, microtiter plate for LC-MS/MS anal-
ysis, as described for each assay below.
Assay Incubation Conditions. Stock solutions of ES (100 mM), ES3-G (1 mM),
and ␣-naphthylglucuronide (0.5 mM), as internal standard, were prepared in
Optimization of UGT Assay Incubation Conditions. Linearity of product DMSO. Reactions (0.1 ml) were terminated at the indicated times (Table 1) by
formation with respect to time and protein concentration were conducted with addition of 0.05 ml of 47:50:3 acetonitrile/water/formic acid containing
HLM and recombinant UGT (rUGT) for each assay. Multiple time-course ␣-naphthylglucuronide (5 ␮M final), then centrifuged at 500g for 10 min, and
experiments were conducted to evaluate the effects of the following: 100 mM
Tris-HCl versus 100 mM phosphate buffers (pH 7.5), MgCl2 concentration (0, ES3-G standard curve samples (0.02–2.5 ␮M final concentration) were serially
1, 5, and 10 mM), and use of 5 mM saccharolactone. Incubations (1.0 ml) were diluted (1:100) in incubation matrix and treated identically to samples. The
conducted as described under General UGT Assay Incubation Conditions at LC-MS/MS method used a binary gradient using water/0.1% formic acid
four HLM or rUGT protein concentrations (0.01, 0.025, 0.05, 0.1 mg/ml) for (mobile phase A) and acetonitrile/0.1% formic acid (mobile phase B). A
the supernatant was transferred to 96-well plates for LC-MS/MS analysis.
each specific UGT assay at substrate concentrations approximating S50 or Km. mobile phase composition of 29% B was held for 2.0 min, then ramped to 90%
Aliquots (0.1 ml, n ϭ 2) were typically collected over a 90-min time course, B over 2.1 min, returned to 29% B over 0.1 min, and held. ES and ES3-G were
terminated, and analyzed for metabolite formation as described for each assay
below.
analytically separated on a Phenomenex Gemini 5␮ C18 2.0 ϫ 50 mm
(Phenomenex, Torrance, CA) column. Under these HPLC conditions ES3-G,
The optimal alamethicin concentration in incubation (Table 2) was determined ␤-estradiol-17-glucuronide, and the IS (␣-naphthylglucuronide) had elution
using pooled HLM and rUGT for UGT1A1, UGT1A4, and UGT2B7 since initial times of 1.3, 1.9, and 1.1 min, respectively.
incubations for UGT2B7 performed with published alamethicin concentrations (50
␮g/mg protein) shown optimal for pore-formation (Fisher et al., 2000) were not
Trifluoperazine N-Glucuronide (UGT1A4) Assay. Unless otherwise in-
dicated, TFP (1–550 ␮M final concentration) was incubated as described under
linear for AZT-G formation with respect to protein. Alamethicin, which was General UGT Assay Incubation Conditions. Stock solutions of TFP (55 mM)
dissolved in MeOH/water (50:50) as a 100ϫ stock, was evaluated with four pooled were prepared in DMSO. TFP-G (0.858 mM), and [D3]TFP-G (0.03 mM), as
HLM or rUGT protein concentrations (0.01, 0.05, 0.2, 0.5 mg/ml), and each was
assessed without and with alamethicin (1–25 ␮g/ml, n ϭ 3) pretreatment at the ml) were terminated at the indicated time (Table 1) by addition of 0.01 ml of
appropriate substrate S50 or Km. 17:80:3 acetonitrile/water/formic acid that contained [D3]trifluoperazine-N-
internal standard, were prepared in water/acetonitrile (50:50). Reactions (0.1
Determination of Nonspecific Protein Binding. The fraction of unbound glucuronide ([D3]TFP-G, 3 ␮M final) as IS and centrifuged at 500g for 10 min,
substrate in incubation matrices (fu,inc) was determined by equilibrium dialysis and the supernatant was transferred to 96-well plates for LC-MS/MS analysis
using the rapid equilibrium dialysis method (Waters et al., 2008). Substrates as described below. TFP-G standard curve samples (0.017–8.6 ␮M final
were spiked into the human liver microsomal sample (0.025 mg/ml, with and
without 2% BSA; 2.0-ml incubation volume) at a concentration close to the Km
or S50, and then dialyzed against incubation mixture on a shaker (450 rpm)
within a humidified CO2 (5%) incubator at 37°C in the absence of protein
concentration) were serially diluted (1:100) in incubation mixture and treated
identically to samples. The LC-MS/MS method used a binary gradient using
water/0.1% formic acid (mobile phase A) and acetonitrile/0.1% formic acid
(mobile phase B). A mobile phase composition of 10% B was held for 0.1 min,
(HLM or BSA) for 4 h. Aliquots (0.22 ml) were removed to assess recovery, then ramped to 80% B over 1.9 min (2.0 min total), returned to 10% B over
and, at the end of the incubation, protein was precipitated with acetonitrile 0.1 min, and held (3.0 min total). Analytes were separated on a Phenome-
(0.18 ml containing 5% DMSO and IS) and analyzed by LC-MS/MS as nex Synergi max-RP 4␮ 2 ϫ 30 mm column. Under these HPLC condi-
described for each assay below. tions, TFP-G and the IS ([D3]TFP-G) had elution times of 1.2 and 1.1 min,
UGT Enzyme Kinetic and Inhibition Experiments. Substrate saturation respectively.
experiments were conducted to generate rate data for determining the appro-
5-Hydroxytryptophol-O-glucuronide (UGT1A6) Assay. Unless other-
priate enzyme kinetic fit (see Data Analysis), and the apparent substrate Km wise indicated, 5HTOL (20–10,000 ␮M final) was incubated as described
and Vmax values were calculated for each assay in pooled HLM and rUGT under General UGT Assay Incubation Conditions. Stock solutions of 5HTOL,
enzymes (Tables 3 and 4). The experiments were performed using at least eight
substrate concentrations that spanned the anticipated Km (n ϭ 3) with and DMSO-d6, and acetonitrile, respectively. 5HTOL-G concentrations were de-
without addition of 2% BSA (w/v). Incubations were typically performed in a termined by quantitative NMR using the artificial signal insertion for calcu-
polypropylene 96-well polymerase chain reaction plate, which was incubated lation of concentration observed (aSICCO) method described by Walker et al.
5HTOL-G (6.6 mM), and diclofenac (0.32 ␮M) were prepared in water,
using a thermostatically controlled heater block or a shaking water bath at
37°C, as described above. In brief, aliquots (0.09 ml) of pooled HLM or rUGT
enzyme premix were prewarmed at 37°C for 5 min before initiation with 0.01
ml of UDPGA (5 mM). The samples were incubated for the times indicated in
Table 1, terminated by the addition of an internal standard as described for
each assay, and analyzed to quantify the specific glucuronide products by
LC-MS/MS, as detailed for each assay below.
(2011). Reactions (0.1 ml) were terminated at the indicated times (Table 1) by
the addition of 0.3 ml of acetonitrile, which contained diclofenac (0.24 ␮M) as
an internal standard, and centrifuged at 500g for 10 min. The supernatant (0.3
ml) was transferred to 96-well plates, evaporated to dryness under nitrogen at
40°C, and reconstituted in 5:95 acetonitrile/water (0.1 ml) containing 0.1%
formic acid for LC-MS/MS analysis as described below. 5HTOL-G standard
curve samples (0.01–20 ␮M) were serially diluted (1:10) in incubation matrix
␤-Estradiol-3-Glucuronide (UGT1A1) Assay. Unless otherwise indi- and treated identically to samples. The LC-MS/MS method used a binary
cated, ES (3–1000 ␮M final) was incubated as described under General UGT gradient using 95:5 water/acetonitrile containing 0.1% formic acid (mobile
TABLE 2
Optimal alamethicin concentration for activation of ␤-estradiol (UGT1A1) and trifluoperazine (UGT1A4) glucuronidation in human liver microsomes
Alamethicin Concentrationa
␤-Estradiol-3-glucuronide
␮M
Trifluoperazine N-Glucuronide
␮M
␮g/ml
␮g/mg HLM protein
HLM conc., mg/ml
0.01–0.5
0
1.0
1.71
2.92
5.0
8.55
14.6
25
0.01
0
100
171
292
500
855
1460
2500
0.05
0
20
34.2
58.4
100
171
292
500
0.2
0
5.0
8.55
14.6
25
42.8
73
125
0.5
0
2.0
3.42
5.84
0.01
0.20
0.17
0.18
0.19
0.28
0.32
0.32
0.27
0.05
0.79
0.77
0.79
0.88
1.3
1.5
1.4
1.2
0.2
2.6
2.2
2.3
2.2
3.4
3.4
3.7
3.3
0.5
3.5
3.3
3.3
3.4
4.1
4.6
4.7
4.5
0.01
0.28
0.29
0.28
0.31
0.44
0.52
0.51
0.47
0.05
1.3
1.4
1.4
1.5
2.2
2.6
2.6
2.5
0.2
3.4
3.5
3.5
3.5
4.6
5.2
5.2
5.3
0.5
4.8
4.8
4.6
4.7
5.5
6.4
6.6
6.8
10
17.1
29.2
50
a HLM were incubated without and with alamethicin at a constant concentration ranging 1 to 25 ␮g/ml at 4 HLM protein concentrations ranging 0.01 to 0.5 mg/ml, as described under Materials
and Methods. Based on the HLM concentration in incubation, the corresponding alamethicin concentration expressed as ␮g/mg HLM protein ranges 100 to 2500 ␮g/mg for 0.01 mg/ml HLM and
2 to 50 ␮g/mg for 0.5 mg/ml HLM.

UGT INHIBITION ASSAYS
1055
TABLE 3
Kinetic parameters for human UDP-glucuronosyltransferase activities in human liver microsomes and recombinant UGT enzymes
b
Enzymea
Incubation Conditionsa
Km or S50
␮M
Vmax
Ksi
n
CLint or CLmax
pmol ꢁ minϪ1 ꢁ mgϪ1
␮M
␮l ꢁ minϪ1 ꢁ mgϪ1
UGT1A1
HLM
HLM ϩ BSA
rUGT
rUGT ϩ BSA
HLM
HLM ϩ BSA
rUGT
rUGT ϩ BSA
HLM
HLM ϩ BSA
rUGT
rUGT ϩ BSA
HLM
HLM ϩ BSA
rUGT
11 Ϯ 0.8
170 Ϯ 20
13 Ϯ 0.8
120 Ϯ 4
42 Ϯ 10
67 Ϯ 10
15 Ϯ 4
140 Ϯ 60
420 Ϯ 50
330 Ϯ 30
570 Ϯ 50
490 Ϯ 200
98 Ϯ 30
46 Ϯ 4
200 Ϯ 70
63 Ϯ 10
610 Ϯ 30
150 Ϯ 10
900 Ϯ 30
320 Ϯ 10
820 Ϯ 20
1400 Ϯ 70
1300 Ϯ 20
1700 Ϯ 30
1500 Ϯ 300
870 Ϯ 50
970 Ϯ 200
5900 Ϯ 2000
66,000 Ϯ 3000
47,000 Ϯ 1100
4900 Ϯ 200
5400 Ϯ 1000
1400 Ϯ 300
780 Ϯ 10
N.A.
N.A.
N.A.
N.A.
64 Ϯ 20
N.A.
2.5 Ϯ 0.4
1.8 Ϯ 0.2
2.6 Ϯ 0.3
2.7 Ϯ 0.2
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
40
4.1
51
7.6
36
13
67
43
160
140
8.6
11
14
17
10
20
3.5
32
1.1
9.8
UGT1A4
UGT1A6
UGT1A9
UGT2B7
82 Ϯ 30
490 Ϯ 300
15,000 Ϯ 3000
41,000 Ϯ 9000
13,000 Ϯ 2000
17,000 Ϯ 10,000
690 Ϯ 200
N.A.
2000 Ϯ 500
1300 Ϯ 90
2100 Ϯ 30
4700 Ϯ 50
900 Ϯ 10
330 Ϯ 100
3200 Ϯ 1000
N.A.
rUGT ϩ BSA
HLM
HLM ϩ BSA
rUGT
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
rUGT ϩ BSA
3100 Ϯ 40
Km, apparent substrate concentration at half-maximal velocity; S50 apparent substrate concentration at half-maximal velocity for substrates exhibiting atypical kinetics; Vmax, maximal velocity;
Ksi, inhibition constant for substrates exhibiting substrate inhibition kinetics; n, Hill coefficient; CLint, intrinsic clearance; CLmax, maximal clearance; N.A., not applicable.
a HLM or rUGTs (0.025 mg/ml) were fully activated with alamethicin (10 ␮g/ml) and incubated with increasing substrate concentrations in 100 mM Tris-HCl buffer (pH ϭ 7.5) containing 5
mM MgCl2, 5 mM UDPGA, with or without 2% BSA, as described under Materials and Methods. Values were not corrected for nonspecific binding in incubation and represent mean Ϯ S.E.M.
for three to four experiments.
b CLint applies to all UGTs except UGT1A1, whereas CLmax was calculated because ES3-G formation displays atypical kinetics, as described under Materials and Methods.
phase A) and 95:5 acetonitrile/water containing 0.1% formic acid (mobile water/acetonitrile containing 0.01% formic acid (mobile phase A) and aceto-
phase B). A mobile phase composition of 20% B was ramped to 95% B over
1.6 min and maintained for 0.2 min (1.8 min total), returned to 10% B over 0.1
nitrile/0.01% formic acid (mobile phase B). A mobile phase composition of
90% B was held for 1.2 min, then ramped to 97% B over 1.2 min (4.2 min
min, and held (2.5 min total). Analytes were separated on a Waters XTerra MS total), returned to 90% B over 0.1 min, and held (5.0 min total). Analytes were
3.5␮ C18 2.1 ϫ 100 mm (Waters, Milford, MA) column. Under these condi-
tions, 5HTOL-G and the IS (diclofenac) had elution times of 1.4 and 1.1 min,
respectively.
separated with a Waters SunFire 3.5␮ C18 2.1 ϫ 30 mm column. Under these
HPLC conditions, PRO-G and the IS (diclofenac) had elution times of 1.3 and
1.1 min, respectively. PRO quantitation for nonspecific protein binding was
Propofol-O-glucuronide (UGT1A9) Assay. Unless otherwise indicated, performed using the following: an AB Sciex (AB Sciex LLC, Foster City, CA)
PRO (1–1000 ␮M final) was incubated as described under General UGT Assay API5500 QTrap mass spectrometer equipped with an electrospray source
Incubation Conditions. Stock solutions of PRO (100 mM) and PRO-G (1.1 (electrospray ionization) in negative detection mode; two Shimadzu LC-20AD
mM) were prepared in methanol/water (50:50) and diclofenac (0.629 ␮M), as pumps, a CBM20A controller, and DGU-20A solvent degasser (Shimadzu,
internal standard, in acetonitrile. Reactions (0.2 ml) were terminated at the
indicated times (Table 1) by the addition of 0.4 ml of acetonitrile containing
0.63 ␮M diclofenac (0.42 ␮M final concentration) as IS, centrifuged at 500g
Columbia, MD); a LEAP CTC HTS PAL autosampler (CTC Analytics, Carr-
boro, NC); and a Valco 2-position switching valve (Valco, Houston, TX).
Separation was on a Kinetex 2.6␮ C18 50 ϫ 2 mm column and the LC-MS/MS
for 10 min, and the supernatant was transferred to 96-well plates for LC- method used a binary gradient using water/0.01% formic acid (mobile phase
MS/MS analysis as described below. PRO-G standard curve samples (0.14–28 A) and acetonitrile (mobile phase B). A mobile phase composition of 5% B
␮M final) were serially diluted (1:40) in incubation matrix and treated iden- was held for 0.3 min, then ramped to 95% B over 2 min, held at 95% B for 0.3
tically to samples. The LC-MS/MS method used a binary gradient using 95:5 min, returned to 5% B over 0.1 min, and held (3.0 min total).
TABLE 4
Unbound kinetic parameters for human UDP-glucuronosyltransferase activities in human liver microsomes
a
b
Enzyme
Incubation Conditionsa
fu,inc
Km,u or S50,u
␮M
Vmax
Ksi,u
CLint,u or CLmax,u
pmol/ ꢁ minϪ1 ꢁ mgϪ1
␮M
␮l ꢁ minϪ1 ꢁ mgϪ1
UGT1A1
UGT1A4
UGT1A6
UGT1A9
UGT2B7
HLM
HLM ϩ BSA
HLM
HLM ϩ BSA
HLM
HLM ϩ BSA
HLM
HLM ϩ BSA
HLM
HLM ϩ BSA
0.14
0.039
0.28
0.061
0.93
0.90
1.0
0.17
0.69
0.69
1.4
6.6
11
820
1400
1500
870
66,000
47,000
1400
780
N.A.
N.A.
290
110
130
210
170
160
14
100
5.1
47
18
4.1
N.A.
14000
37,000
690
N.A.
N.A.
N.A.
390
300
98
7.8
420
100
2100
4700
f
u,inc, mean unbound fraction in incubation determined at substrate concentrations close to Km or S50 for ES (170 ␮M), TFP (67 ␮M), AZT (373 ␮M), or a concentration range for 5HTOL (3,
30, 300 ␮M) and PRO (4, 40 ␮M); Km,u, unbound apparent substrate concentration at half-maximal velocity; S50,u, unbound apparent substrate concentration at half-maximal velocity for substrates
exhibiting atypical kinetics; Vmax, maximal velocity; Ksi,u, unbound inhibition constant for substrates exhibiting substrate inhibition kinetics; CLint,u, unbound intrinsic clearance; CLmax,u, unbound
maximal clearance; N.A., not applicable.
a HLMs or rUGTs (0.025 mg/ml) were fully activated with alamethicin (10 ␮g/ml) and incubated with increasing substrate concentrations in 100 mM Tris-HCl buffer (pH ϭ 7.5) containing 5
mM MgCl2, 5 mM UDPGA, with or without 2% BSA, as described under Materials and Methods. Values represent mean Ϯ S.E.M. for three to four experiments.
b CLint,u applies to all UGTs except UGT1A1 where CLmax,u was calculated because ES3-G formation displays atypical kinetics as described under Materials and Methods.

1056
WALSKY ET AL.
AZT-5ꢀ-glucuronide (UGT2B7) Assay. Unless otherwise indicated, AZT 10ADvp controller and DGU-14 solvent degasser (Shimadzu), a LEAP CTC
(180–4500 ␮M final concentration) was incubated as described under General HTS PAL autosampler with a multisolvent peristaltic self-washing system
UGT Assay Incubation Conditions. Stock solutions of AZT (1100 mM),
using 5:95 water/acetonitrile and 95:5 acetonitrile/water (both containing 0.1%
AZT-G (0.5 mM), and [13C6]AZT-G (0.5 mM), as internal standard, were formic acid) as the wash solvents (CTC Analytics), and a LabPro switching
prepared in acetonitrile/water (50:50). Reactions (0.1 ml) were terminated at valve (Reodyne LLC, Rohnert Park, CA). In all three systems, flow was
the indicated times (Table 1) by the addition of 0.01 ml of 5:92:3 acetonitrile/
diverted from the mass spectrometer to waste for the first 0.5 min of the
water/formic acid containing 10 ␮M [13C6]AZT-G (1 ␮M final concentration) gradient to remove nonvolatile salts.
as IS, filtered on a Millipore (Millipore Corporation, Billerica, MA) Multi-
Quantitative and qualitative NMR was performed using a Bruker (Billerica,
screen-HA 0.45 ␮m mixed cellulose ester 96-well membrane vacuum filtration MA) Avance 600 MHz system controlled by TOPSPIN V2.0, equipped with a
module, and the filtrate was transferred to 96-well plates for LC-MS/MS
5-mm TCI cryoprobe. Semipreparative HPLC was performed using a Shi-
analysis as described below. AZT-G standard curve samples (0.05–5 ␮M final madzu SiL-HTC autosampler, two LC-20AD solvent pumps, an SPD-M20A
concentration) were serially diluted (1:100) in incubation matrix and treated
identically to samples. The LC-MS/MS method used a binary gradient using 5
mM ammonium formate/0.05% formic acid (mobile phase A) and 95:5 ace-
tonitrile/methanol containing 0.05% formic acid (mobile phase B). A mobile
phase composition of 2% B was held for 0.5 min, then ramped to 35% B over
2.1 min (2.6 min total), returned to 2% B over 0.1 min, and held (4.0 min).
diode array detector, and a FRC-10A fraction collector.
5-Hydroxytryptophol-O-glucuronide Isolation, Quantitation, and
Structural Elucidation. A pure sample of 5HTOL-G was prepared by incu-
bating 5HTOL (200 ␮M) with pooled HLM (1.0 mg/ml), UDPGA (1.0 mM),
alamethicin (10 ␮g/ml), and MgCl2 (5 mM) in 100 mM potassium phosphate
buffer (pH 7.5) containing 1% BSA (4–10-ml incubations). Incubations were
Analytes were separated on a Phenomenex Luna 5␮ C18(2) 3.0 ϫ 30 mm conducted in a shaking water bath at 37°C for 3 h, protein was precipitated by
column. Under these HPLC conditions, AZT-G and the IS ([13C6]AZT-G) had addition of acetonitrile (30 ml), and supernatants were combined and concen-
elution times of 2.0 min.
UGT Inhibition Assays. In experiments where inhibition of glucuronide tuted in 10:90 acetonitrile/water (2 ml) and purified using a semipreparative
formation was investigated (Table 5), 50 and 500 ␮M inhibitors (final con- HPLC system in five injections (0.4 ml) using a binary gradient of 5 mM
trated using an evaporative centrifuge set to 37°C. The residue was reconsti-
centration) in DMSO (or solvent control) were added to the 96-well plate (n ϭ ammonium acetate/0.1% formic acid (mobile phase A) and acetonitrile (mobile
2) before addition of the rUGT enzyme mixture and initiated with UDPGA (5 phase B). A mobile phase composition of 10% B was held for 10 min, then
mM) as described above. For IC50 determination, HLM or rUGT were incu- ramped to 30% B over 30 min. Separation was performed on a Phenomenex
bated with increasing inhibitor concentrations (0.1–100 ␮M) as described Gemini 5␮ C18 10 ϫ 250 mm (Phenomenex), semipreparative HPLC column
above. UGT substrate concentrations were at or below Km; ES (10 or 100 ␮M with a flow rate of 4 ml/min. Aliquots (0.025 ml) of fractions at retention times
with BSA), TFP (40 ␮M HLM or 67 ␮M with BSA; 10 ␮M rUGT or 140 of UV peaks believed to be the metabolite and the parent were taken for
␮M with BSA), 5HTOL (350 ␮M), PRO (100 ␮M HLM, 200 ␮M rUGT, or HPLC/MS/UV analysis for verification. Fractions containing 5HTOL-G were
40 ␮M with BSA), and AZT (842 ␮M HLM, 1080 ␮M rUGT, 374 ␮M HLM combined and concentrated to dryness using an evaporative centrifuge set to
with BS, or 596 ␮M rUGT with BSA).
37°C (Genevac, Valley Cottage, NY). Under these HPLC conditions,
Instrumentation. Analytical quantification was conducted by LC-MS/MS 5HTOL-G had an elution time of 13.4 min. The isolate was reconstituted in
using three systems as identified in Table 1: the LC-MS/MS systems com-
prised an 1) AB Sciex LLC 4000 QTrap mass spectrometer equipped with an
DMSO-d6 (200 ␮l) and placed in 3-mm diameter tubes before NMR analysis.
One-dimensional spectra were recorded using a sweep width of 12,000 Hz and
electrospray source, two Shimadzu LC-20AD pumps, a CBM20A controller a total recycle time of 7.2 s. The resulting time-averaged free-induction decays
and DGU-20A solvent degasser, a LEAP CTC HTS PAL autosampler using
20:80 acetonitrile/water and 80:20 acetonitrile/water (both containing 0.1%
were transformed using an exponential line broadening of 1.0 Hz to enhance
signal to noise. All spectra were referenced using residual DMSO-d6 (1H ␦ ϭ
formic acid) as the wash solvents (CTC Analytics), and a Valco 2-position 2.5 ppm and 13C ␦ ϭ 39.5 relative to tetramethylsilane, ␦ ϭ 0.00). Phasing,
switching valve; 2) an AB Sciex LLC 4000 triple quadrupole mass spectrom-
eter equipped with an electrospray source, two Jasco X-LCT 3080PU pumps,
and a LC-NetII ADC controller (Jasco Analytical Instruments, Easton, MD), a
LEAP CTC HTS PAL autosampler using 5:95 water/acetonitrile and methanol
(both containing 0.1% formic acid) as the wash solvents (CTC Analytics), and
baseline correction, and integration were all performed manually. If needed,
the BIAS and SLOPE functions for the integral calculation were adjusted
manually. The correlation spectroscopy (COSY), multiplicity-edited heteronu-
clear single quantum coherence, and heteronuclear multiple bond coherence
(HMBC) data were recorded using the standard pulse sequence provided by
a Valco 2-position switching valve; and 3) a Waters Micromass Quattro Ultima Bruker. Two-dimensional experiments were typically acquired using a 1K ϫ
triple quadrupole mass spectrometer equipped with an electrospray ionization 128 data with 16 dummy scans and a spectral width of 8000 Hz in the f2
source (Micromass, Beverly, MA), two LC-10ADvp pumps with a SCL- dimension. The data were zero-filled to a size of 1K ϫ 1K. A relaxation delay
TABLE 5
Inhibition of human UDP-glucuronosyltransferase activities in recombinant UGT enzymes
Recombinant UGTs (0.025 mg/ml) were incubated with alamethicin (10 ␮g/ml), 50 or 500 ␮M inhibitor (or DMSO solvent control) in 100 mM Tris-HCl buffer (pH ϭ 7.5) containing 5
mM MgCl2, 5 mM UDPGA, without 2% BSA as described under Materials and Methods. Percentage of activity remaining (relative to solvent control) represents the mean of two experiments.
UGT substrate concentrations were at or below the respective Km; 10 ␮M ES, 10 ␮M TFP, 350 ␮M 5HTOL, 200 ␮M PRO, 842 ␮M AZT.
Percentage Activity Remaining
rUGT1A6
Inhibitor
rUGT1A1
rUGT1A4
rUGT1A9
rUGT2B7
500 ␮M
50 ␮M
500 ␮M
50 ␮M
500 ␮M
50 ␮M
500 ␮M
50 ␮M
500 ␮M
50 ␮M
UGT inhibitors
Chrysin
Diflunisal
Mefenamic acid
Silibinin
Tangeretin
Valproic acid
Cytochrome P450 inhibitors
1-Aminobenzotriazole
Itraconazole
Ketoconazole
Ritonavir
2
62
63
6
14
97
2
9
10
1
25
104
60
103
89
22
71
14
96
53
6
64
107
56
90
76
100
22
100
41
20
32
48
69
89
37
13
3
126
6
21
2
2
1
5
35
96
7
74
76
92
55
68
1
16
55
83
107
94
70
102
11
17
18
35
102
6
1
4
9
99
13
15
5
98
10
3
1
39
100
89
80
100
63
100
85
86
100
43
99
84
110
73
80
58
10
44
16
124
91
12
64
79
111
87
0
46
22
Verapamil
72
70

UGT INHIBITION ASSAYS
1057
of 1.5 s was used between transients. Quantitation of the prepared 5HTOL-G
sample was performed using the aSICCO method, as described previously
(Walker et al., 2011).
Data Analysis. Standard curve fitting was accomplished with AB Sciex
Analyst software (version 1.4.2; AB Sciex LLC) or MassLynx QuanLynx
software (version 4.1; Micromass) as described above and indicated in Table
1. Data were typically fit to quadratic curves using 1/x2 weighting, and
standard curves were run for each experiment.
assays developed, and this applies to the protein concentration of
0.025 mg/ml used in this study.
Tris versus Phosphate Buffers. Activity comparisons in human
liver microsomes and recombinant UGT enzymes for UGT1A1,
UGT1A4, UGT1A6, UGT1A9, and UGT2B7 substrates were con-
ducted in 100 mM Tris-HCl (pH 7.5 at 37°C) and 100 mM potassium
phosphate (pH 7.5) buffers to evaluate which provided the greatest
overall glucuronide metabolite formation. Incubations in Tris buffer
with HLM showed marked increases in metabolite formation for
Substrate concentration [S] and velocity (V) data were fitted to the appro-
priate enzyme kinetic model by nonlinear least-squares regression analysis
(SigmaPlot version 12; Systat Software, Inc., San Jose, CA) to derive the UGT1A4 (2-fold) and UGT1A9 (ϳ1.5-fold), whereas the other UGTs
apparent enzyme kinetic parameters Vmax (maximal velocity) and Km or S50
(substrate concentration at half-maximal velocity). The Michaelis-Menten
model (eq. 1), the substrate inhibition model (eq. 2), and the substrate activa-
tion model (eq. 3), which incorporates the Hill coefficient (n), were used:
activities were relatively unaffected (Fig. 2). In rUGT incubations,
Tris buffer resulted in increased product formation for all UGTs
studied, except UGT1A6. Based on these results, a 100 mM Tris-HCl
buffer was selected for all subsequent experiments.
MgCl2 Concentration. To determine a common magnesium chlo-
ride concentration for use in all UGT assays, glucuronide metabolite
formation was measured in HLM and rUGT enzymes for all UGT
assays at 0, 1, 5, and 10 mM MgCl2. The effect of MgCl2 concen-
tration on UGT activities in HLM and recombinant UGT enzymes are
shown in Fig. 2. The inclusion of MgCl2 in HLM resulted in increased
glucuronidation activity, ranging from 2- to 4-fold for UGT1A1,
UGT1A4, UGT1A6, and UGT2B7, and up to 8-fold for UGT1A9.
Similar trends with more modest activation were observed in rUGTs
with a 10 mM MgCl2 concentration, resulting in decreased activity for
rUGT1A4. Based on these findings, a 5 mM MgCl2 concentration was
selected as general UGT incubation condition.
Inclusion of Saccharolactone. The effect of saccharolactone, an
inhibitor of ␤-glucuronidase, was assessed at 5 mM to determine
whether its use was required with HLM, and/or rUGTs for all assays
developed. The fold-change in metabolite formation in the presence
and absence of saccharolactone for incubations with HLM and rUGTs
are shown in Fig. 2. Saccharolactone inclusion did not significantly
impact the activities of UGT1A1, UGT1A6, and UGT2B7, whereas
decreases in metabolite formation were observed for UGT1A4 and
UGT1A9. Based on these results, saccharolactone was excluded from
the optimized UGT incubation conditions.
V ϭ Vmax ϫ S/͑Km ϩ S͒
(1)
(2)
(3)
V ϭ Vmax ϫ S/͑Km ϩ S ϫ ͑1 ϩ S/Ksi͒͒
n
V ϭ Vmax ϫ Sn/͑S50 ϩ Sn͒
where Vmax is the maximal velocity, Km or S50 is the substrate concentration at
half-maximal velocity, n is an exponent indicative of the degree of curve
sigmoidicity, and Ksi is an inhibition constant. The best fit was based on a
number of criteria, including visual inspection of the data plots (Michaelis-
Menten and Eadie-Hofstee), distribution of the residuals, size of the sum of the
squared residuals, and the S.E. of the estimates. Selection of models other than
Michaelis-Menten was based on the F-test (P Ͻ 0.05) and the Akaike Infor-
mation Criterion (AIC). The CLint was calculated as the Vmax/Km for Michae-
lis-Menten and substrate inhibition kinetics, and unbound intrinsic clearance
(CLint,u) was corrected for the fraction of unbound substrate in incubation
(fu,inc) as CLint/fu,inc. Because Km and S50 are not equivalent, the maximum
clearance (CLmax) is suggested as an appropriate alternate clearance parameter
for substrates exhibiting substrate activation kinetics or positive cooperativity
(Houston and Kenworthy, 2000; Uchaipichat et al., 2004) and was calculated
from (eq. 4):
Vmax
͑n Ϫ 1͒
CLmax ϭ
ϫ
(4)
1/n
S50
n͑n Ϫ 1͒
Alamethicin Concentration in UGT Incubations. After observ-
ing a lack of linearity between AZT-G formation and protein concen-
tration in HLM, which had been pretreated with alamethicin at the
previously reported optimal concentration for activation (50 ␮g/mg
HLM) (Fisher et al., 2000), the use of alamethicin at low protein
concentrations was evaluated further. Four HLM protein concentra-
IC50 estimates for inhibition of glucuronidation were determined by non-
linear curve fitting with SigmaPlot (version 12; Systat Software, Inc.) and were
defined as the concentration of inhibitor required to inhibit control glucuroni-
dation reactions by 50%.
Results
General UGT Assay Incubation Conditions. In this report, we tions were assessed at eight alamethicin concentrations using the
describe optimized LC-MS/MS analytical methods for five human optimized incubation conditions for UGT2B7 (Fig. 3), UGT1A1, and
UDP-glucuronosyltransferase assays. Although efforts were made to UGT1A4 (Table 2). Similar results were observed for all three en-
control any potential analytical method variability, a potentially zymes studied. In general, we observed initial activation of UGT
greater source of variability resides with the incubation method. activity at a universal alamethicin concentration of 5 ␮g/ml and
Linear conditions for each assay were established by conducting the apparent maximal activation at 8.55 ␮g/ml. The corresponding alame-
incubations at four protein concentrations and measuring the forma- thicin concentrations for 8.55 ␮g/ml expressed as microgram per
tion of glucuronide metabolite over time. The incubation times were milligram HLM are 855, 171, 42.8, and 17.1 ␮g/mg for HLM con-
selected such that all reactions were linear with time (Ͻ10% substrate centrations of 0.01, 0.05, 0.2, and 0.5 mg/ml HLM, respectively
consumption), and the lowest protein concentration was selected such (Table 2). Therefore, maximal activation would be observed with 50
that the amount of glucuronide metabolite formed were within the ␮g alamethicin/mg HLM only when protein concentrations exceed
dynamic range of the analytical assays (Table 1).
0.17 mg/ml (8.55 ␮g/ml divided by 50 ␮g/mg). Accordingly, it is the
Optimization of UGT Incubation Conditions. Because experi- alamethicin solution concentration (microgram per milliter) that is
ments were performed at significantly lower protein concentrations relevant to increasing metabolite formation at the microsomal protein
(0.025 mg/ml) than typically described previously (0.25–2.5 mg/ml) concentrations examined (0.01–0.5 mg/ml), irrespective of pro-
(Fisher et al., 2000; Court, 2004; Donato et al., 2010), and considering tein concentration in incubation. Based on these results, an alamethi-
that enzyme kinetic parameters are affected by incubation conditions cin concentration of 10 ␮g/ml is suggested as optimal for activation of
(Boase and Miners, 2002), a limited number of incubation conditions UGT activity regardless of the protein concentration used; therefore,
known to affect UGT enzyme activity were evaluated. The overall corresponding alamethicin concentrations required for activation at
goal was to select universal incubation conditions for all five UGT protein concentrations of 0.01, 0.05, 0.2, and 0.5 mg/ml are 1000, 200,

1058
WALSKY ET AL.
FIG. 2. Effect of incubation conditions on ma-
jor human hepatic UGT activities in pooled
human liver microsomes (A–C), and recombi-
nantly expressed UGT enzymes (D–F). Values
expressed as fold increase above activity in
presence of phosphate buffer (A and D), ab-
sence of MgCl2 (B and E), or absence of sac-
charolactone (C and F).
50, and 20 ␮g/mg HLM, which indicates that a significantly higher as a single peak at 5.3 min, suggesting a single isolated component.
alamethicin/protein ratio is required for UGT activation at low protein There are three biologically plausible positions for glucuronidation of
concentrations. At microsomal protein concentrations below 0.2 mg/ 5HTOL; 1) the C5 (phenolic) hydroxyl of the indole, 2) the aliphatic
ml, no activation is realized at an alamethicin concentration of 50 hydroxyl, and 3) the amine group of the indole (Fig. 4). The 1H-
␮g/mg (see Fig. 3B). AZT-G formation (UGT2B7) with HLM is spectrum of the isolate, when dissolved in DMSO-d6, contains two
plotted as log alamethicin concentration in microgram per milliliter resonances at ␦ 10.78 and 10.67 that together integrate to a single
versus metabolite formed for each protein concentration (Fig. 3A). hydrogen. The COSY spectrum contains cross-peaks, which indicates
The same data were plotted as the log of alamethicin concentration in that these resonances are coupled to resonances at ␦ 7.18 and 7.11
microgram per milligram microsomal protein versus metabolite (Fig. 4). When the sample is diluted in methanol-d4, the ␦ 10.78 and
formed for each protein concentration to illustrate the lack of effect at 10.67 resonances are absent from both the 1H and COSY spectrum
low microsomal protein concentrations (Fig. 3B). An alamethicin (Supplemental Fig. 1). Based on these data, the ␦ 10.78 and 10.67 are
solution concentration of at least 10 ␮g/ml (400 ␮g alamethicin/mg assigned as the NH of the indole and eliminates this site as a potential
HLM at 0.025 mg/ml HLM) would result in optimal increases in site for glucuronidation.
metabolite formation in all three UGT assays and was used for all
The HMBC spectrum of the isolated sample diluted in methanol-d4
optimized incubations. Similar experiments were conducted with contains cross-peaks that indicate long-range coupling from the ano-
rUGT2B7 and showed no benefit from alamethicin addition at con- meric proton (␦ 4.89 ppm) to a carbon with a chemical shift of 152.7
centrations up to 25 ␮g/ml (data not shown).
ppm. The HMBC data also contains cross-peaks from two aromatic
Structural Interpretation of 5-Hydroxytryptophol Glucuro- proton resonances at 7.33 and 7.25 ppm to the carbon at 152.7 ppm
nide. HPLC analysis of the isolated fraction of purified 5HTOL-G (Supplemental Fig. 2). This strongly indicates that the 5HTOL is
indicated a single UV peak at a retention time of 13.4 min, using the glucuronidated at the C5 (phenolic) position of the indole. The HMBC
semipreparative HPLC system. Using an analytical column (Phe- spectrum (diluted in methanol-d4) contains no evidence for any cou-
nomenex HydroRP 4␮ C18, 4.6 ϫ 150 mm), the isolate also appeared pling of the anomeric proton to any aliphatic carbon. These combined

UGT INHIBITION ASSAYS
1059
FIG. 3. Optimal alamethicin concentration for activation of AZT glucuronidation in human liver microsomes. A, AZT-G formation shown at four concentrations (0.01–0.5
mg/ml) of pooled HLM versus alamethicin concentration expressed as microgram of alamethicin per milliliter of incubate. B, the same data were also plotted expressing
alamethicin concentration as microgram of alamethicin per milligram of microsomal protein. Dashed lines signify the current recommended optimal alamethicin activation
concentration (10 ␮g/ml) (A) and the previously used alamethicin activation concentration (50 ␮g/mg) (B). Corresponding data for ␤-estradiol and trifluoperazine
glucuronidation are shown in Table 2.
data suggest a single site of glucuronidation of 5HTOL in human liver free) with the addition of 2% BSA. These values reflect significant
microsomes at the phenolic oxygen (Fig. 4). These experiments con- microsomal binding, even at low protein concentrations, and are in
firmed that of the three likely glucuronidation sites, the analogous agreement with reported ES binding to BSA (9–11% free with 0.5%
serotonin 5-hydroxyl moiety was glucuronidated (Krishnaswamy et BSA) (Rowland et al., 2009) and high plasma protein binding (ϳ2%
al., 2003).
free). TFP was moderately bound to HLM (28% free) and highly
Nonspecific Binding. The fraction of unbound substrate in incu- bound with 2% BSA (6.1% free), in agreement with previously
bation (fu,inc) is shown in Table 4 and was determined at substrate reported microsomal binding at the concentration tested (Uchaipichat
concentrations close to Km or S50 for ES (170 ␮M), TFP (67 ␮M), et al., 2006a). The results of previous studies indicated a degree of
AZT (373 ␮M), or a concentration range for 5HTOL (3, 30, or 300 saturable protein binding for TFP in HLM between a concentration
␮M) and PRO (4 or 40 ␮M). Substrate recovery for all compounds range of 10 to 200 ␮M, with fu,inc increasing from 0.21 to 0.59
met acceptance criteria and ranged from 73 to 134%. ES is moderately (Uchaipichat et al., 2006a). In this study, unbound TFP enzyme
bound (14% free) to HLM at 0.025 mg/ml and is highly bound (3.9% kinetic parameters (Table 4) did not account for the possibility of
FIG. 4. Structure of 5-hydroxytryptophol-O-
glucuronide showing NMR HMBC coupling at
positions A, B, and C, which confirm glucu-
ronidation at the phenolic 5-hydroxytryptophol
position.

1060
WALSKY ET AL.
saturable binding. 5HTOL was poorly bound to HLM (93% free) and BSA binding (Ն90% free) was reported by other authors (Court et
HLM with BSA (90% free), and mean fu,inc is reported in Table 4. al., 2003; Rowland et al., 2007, 2009).
The fu,inc in HLM at 3, 30, or 300 ␮M were 0.85, 0.98, and 0.95,
␤-Estradiol-3-glucuronide Glucuronidation (UGT1A1). An LC-
respectively, and for HLM with BSA 0.79, 0.94, and 0.97, respec- MS/MS assay for ␤-estradiol-3-glucuronosyltransferase, a probe sub-
tively. PRO at 4 and 40 ␮M was not bound to HLM (100% free) strate for UGT1A1 activity (Williams et al., 2002), was adopted and
and moderately bound with BSA (17% free), in agreement with optimized for use at low protein concentrations. The resulting ES3-G
previous HLM (70% free at 0.5 mg/ml) and BSA-binding studies product formation displayed atypical kinetics (curved Eadie-Hofstee
(ϳ20–60% free) (Rowland et al., 2008, 2009). AZT exhibited plot) and best fit the Hill equation, which is consistent with previous
moderate binding to HLM and HLM with BSA (69% free), in literature (Fisher et al., 2000). The mean Hill coefficient value (n),
agreement with microsomal (60% free) and BSA binding (49% which gives an indication of the degree of sigmoidicity of the curve,
free) reported by Kilford et al. (2009), and reported AZT plasma ranged from 1.8 to 2.7 for all incubation conditions (Table 3; Fig. 5),
protein binding of 62 to 66% free (product label) or 72 to 82% free which is indicative of homotropic activation or positive cooperativity.
(Luzier and Morse, 1993). A lower degree of AZT microsomal or The mean HLM and rUGT S50 values of 11 and 13 ␮M, respectively,
FIG. 5. Enzyme kinetics of ␤-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), and 5-hydroxytryptophol-O-glucuronide (UGT1A6) formation
in pooled HLM (A, C, and E) or recombinant UGTs (B, D, and F) in the absence and presence of 2% BSA. ES3-G formation displayed atypical kinetics, as elucidated
by the insert at low substrate concentrations in B. TFP-G and 5HTOL-G formation displayed substrate inhibition kinetics, except TFP-G formation with HLM in presence
of 2% BSA, which displayed Michaelis-Menten kinetics. Enzyme kinetic parameters and incubation conditions are summarized in Tables 3 and 4.

UGT INHIBITION ASSAYS
1061
is in agreement with apparent S50 values previously reported for HLM values in HLM were 42 and 11 ␮M, respectively, in agreement with
ranging from 17 to 61 ␮M (Fisher et al., 2000; Alkharfy and Frye, Km values reported in HLM that ranged from 35 to 84 ␮M or 4.7 to
2002; Williams et al., 2002; Soars et al., 2003; Ita¨aho et al., 2008) and 7.6 ␮M when corrected for nonspecific binding (Uchaipichat et al.,
8.7 to 23 ␮M for rUGT1A1 (Soars et al., 2003; Le´pine et al., 2004; 2006a). In rUGT1A4, total and unbound Km values were 15 and 4.1
Fujiwara et al., 2007; Zhou et al., 2011).
␮M, respectively, in agreement with reported rUGT1A4 values (bind-
In the presence of 2% BSA, total S50 increased (9–15-fold) with ing corrected) of 20 to 44 (4.1) ␮M (Uchaipichat et al., 2006a;
minor increases (1.3–1.7-fold) in Vmax, resulting in decreased appar- Fujiwara et al., 2007; Kubota et al., 2007; Kerdpin et al., 2009). When
ent CLmax (Table 3). However, when corrected for unbound kinetics corrected for nonspecific binding, HLM unbound CLint increased
(Table 4), S50 values were significantly lower (ϳ8–26-fold) than total slightly (1.6-fold) due to a relatively higher degree of BSA binding
S50 and more comparable between HLM and rUGT (1.4–6.6 ␮M) due (Table 4). Although unbound HLM Km was comparable with previous
to a high degree of microsomal and BSA binding (Table 4). The reports, Vmax was higher with a pronounced effect of BSA on
resultant HLM unbound maximal clearance (CLmax,u) decreased rUGT1A4 Vmax, comparable with observations with lamotrigine as
slightly with BSA addition, but it was relatively similar (within UGT1A4 substrate (Rowland et al., 2006).
2.6-fold) to values obtained in the absence of BSA.
5-Hydroxytryptophol-O-glucuronidation (UGT1A6). A 5-hy-
Trifluoperazine N-Glucuronidation (UGT1A4). A trifluopera- droxytryptophol-O-glucuronosyltransferase LC-MS/MS assay for
zine-N-glucuronosyltransferase LC-MS/MS assay, which has been UGT1A6 was developed and optimized. The 5HTOL-G formation
shown to be selective for measurement of UGT1A4 activity (Uchai- displayed weak substrate inhibition kinetics (Table 3; Fig. 5). The
pichat et al., 2006a) as previously quantified by LC/UV based on mean Km values in HLM and rUGT were 420 and 570 ␮M, respec-
aglycone absorbance (Uchaipichat et al., 2006b), was developed. The tively. Values cited for Km in HLM have ranged from 134 to 156 ␮M,
structural identity of TFP-G isolated from in vitro HLM incubations and the value for rUGT1A6 was 135 ␮M (Krishnaswamy et al., 2004).
were in agreement with the TFP-G analytical standard compared by In the presence of 2% BSA, unbound kinetic parameters (Table 4) did
NMR spectroscopy (data not shown). The TFP-G product formation not change significantly, due to a low degree of nonspecific binding.
data in HLM and rUGT exhibited substrate inhibition kinetics (Table
Propofol-O-glucuronidation (UGT1A9). A propofol-O-gluc-
3; Fig. 5), whereas HLM in the presence of BSA displayed Michaelis- uronosyltransferase LC/MS-MS assay was developed to evaluate
Menten kinetics. Substrate concentrations higher than 200 ␮M failed UGT1A9 activities in HLM and rUGT. The PRO-G product formation
to remain in solution in the absence of BSA, accordingly the 300 and data displayed substrate inhibition kinetics except for data collected
550 ␮M TFP concentrations were not used for determining apparent using HLM in the presence of 2% BSA, which displayed Michaelis-
enzyme kinetic parameters; however, solubility did not appear to be Menten kinetics (Table 3; Fig. 6). The optimized method yielded mean
affected in the presence of BSA. The mean total and unbound Km Km values of 98 and 200 ␮M for HLM and rUGT1A9, respectively.
FIG. 6. Enzyme kinetics of propofol-O-glucuronide and AZT-5Ј-glucuronide formation in pooled HLM (A and C) or recombinant UGTs (B and D) in the absence and
presence of 2% BSA. PRO-G formation displayed substrate inhibition kinetics, except with HLM in the presence of 2% BSA, which displayed Michaelis-Menten kinetics.
AZT-G formation displayed Michaelis-Menten kinetics. Enzyme kinetic parameters and incubation conditions are summarized in Tables 2 and 3.

1062
WALSKY ET AL.
Previously reported Km values obtained in HLM ranged from 64 to 280 500 ␮M) for their ability to inhibit the five hepatic UGTs studied, to
␮M (Soars et al., 2003; Shimizu et al., 2007; Rowland et al., 2008) and evaluate a high-throughput UGT inhibition screening scenario
from 28 to 111 ␮M for rUGT1A9 (Soars et al., 2003; Rowland et al., (Table 5). Activities were compared with solvent control. Chrysin
2008; Takahashi et al., 2008; Fujiwara et al., 2010). In the presence of 2% significantly inhibited UGT1A1, UGT1A6, UGT1A9, and UGT2B7;
BSA, total Km values for HLM and rUGT1A9 were 46 and 63 ␮M for diflunisal appeared to be selective as UGT1A9 inhibitor; mefenamic
HLM and rUGT1A9, respectively. Previously reported Km values in the acid mostly inhibited UGT1A9 and UGT2B7; silibinin was most
presence of BSA were 15.5 and 7.2 ␮M for HLM and rUGT, respectively potent as UGT1A1 inhibitor; and tangeretin inhibited UGT1A1 and
(Rowland et al., 2008). As shown in Table 4, the addition of BSA resulted UGT1A9. Valproic acid exhibited minimal UGT inhibition at 500
in significant increases in unbound CLint (7.1-fold).
␮M. The P450 inhibitor 1-aminobenzotriazole did not inhibit UGTs
AZT-5ꢀ-glucuronidation (UGT2B7). An AZT-5Ј-glucuronosyl- up to 500 ␮M, whereas the other P450 inhibitors tested appeared to be
transferase assay with LC-MS/MS detection (Engtrakul et al., 2005) nonselective inhibitors of UGT1A1 and UGT1A4 (itraconazole);
was optimized to evaluate UGT2B7 activities in HLM and rUGT as UGT1A1, UGT1A4, and UGT2B7 (ketoconazole and ritonavir); and
an established UGT2B7 probe substrate (Court et al., 2003). The weak UGT1A1 inhibition by verapamil at 50 ␮M with nonselective
AZT-G product formation data displayed Michaelis-Menten kinetics inhibition at the higher concentration tested.
(Table 3; Fig. 6). Statistical evaluation (F-test) indicated a statistically
IC50 experiments (Table 6) with chrysin confirmed potent inhibi-
better enzyme kinetic fit (p Ͻ 0.05) with a substrate inhibition model tion of UGT1A1 with less than 10-fold selectivity over UGT1A6,
for incubation conditions, except for rUGT in the presence of BSA. UGT1A9, and UGT2B7. Itraconazole is a potent inhibitor (IC50 Ͻ 1
However, visual inspection of the fit did not justify use of a more ␮M) of UGT1A1 and UGT1A4, whereas activities of UGT1A6,
complex model, and conclusive evidence would require incubation UGT1A9, and UGT2B7 were unaffected up to 100 ␮M. The addition
with substrate concentrations Ͼ4500 ␮M. The mean Km values for of BSA typically increased IC50 values, probably due to an increase in
HLM (840 ␮M), HLM with BSA (160 ␮M), and rUGT (1100 ␮M) nonspecific inhibitor binding (not specifically measured in these stud-
estimated with the substrate inhibition model were comparable with ies), reflecting the importance of correcting IC50 or Ki values for
those reported in Table 3, where the mean total Km values for HLM nonspecific binding when attempting DDI predictions.
and rUGT2B7 were 610 and 900 ␮M, respectively. Values are in
Discussion
agreement with published Km values in HLM ranging from 518 to
1600 ␮M, and for rUGT2B7 values of 478 to 770 ␮M have been
Over the past couple of decades, major advances were made in the
reported (Court et al., 2003; Engtrakul et al., 2005; Uchaipichat et al., characterization of UGT enzyme substrate and inhibitor selectivities,
2006b; Peterkin et al., 2007; Rowland et al., 2009). In the presence of highlighting the importance of glucuronidation in drug metabolism, in
2% BSA, mean Km values for HLM and rUGT2B7 were 150 and 320 vitro-in vivo extrapolation of drug clearance, and prediction of DDIs
␮M, respectively. Previously reported Km values in the presence of (Miners et al., 2010a). The UGT reaction-phenotyping techniques
BSA were 69 to 105 ␮M and 40 to 70 ␮M for HLM and rUGT, have progressed significantly with the identification and characteriza-
respectively (Uchaipichat et al., 2006b; Rowland et al., 2007, 2009). tion of selective probe substrates, availability of recombinant UGTs,
Corrections for unbound concentrations in microsomes resulted in and optimization of in vitro incubation conditions required to measure
slight decreases in Km, whereas addition of BSA decreased Km (Table drug glucuronidation. However, a serious shortcoming in the UGT
4). Unbound HLM Km (100 ␮M) with BSA was comparable with reaction-phenotyping process is the lack of isoform-selective inhibi-
globulin-free BSA (188 ␮M) and reported values in the presence of tors that could be used in liver microsomal incubations, analogous to
crude BSA (87 ␮M) (Rowland et al., 2007). The decreases in unbound those available for P450 enzymes (Zhang et al., 2007). Selective UGT
Km (4.2-fold) and increases in Vmax (2.2-fold) in the presence of BSA inhibitor probes are limited to hecogenin (UGT1A4), niflumic acid
resulted in a significantly increased unbound HLM CLint (9.2-fold) (UGT1A9), and fluconazole (UGT2B7) (Miners et al., 2010a). Thus,
(Table 4).
the confidence in UGT reaction phenotyping could benefit signifi-
UGT Inhibition. Eleven previously reported UGT inhibitors and cantly from the identification of a greater number of enzyme-selective
known P450 inhibitors were screened at two concentrations (50 and inhibitors for UGTs that contribute to hepatic drug metabolism. Ac-
TABLE 6
Inhibition of human UDP-glucuronosyltransferase activities in human liver microsomes and recombinant UGT enzymes
b
IC50
Inhibitor and Incubation Conditionsa
UGT1A1
UGT1A4
UGT1A6
UGT1A9
UGT2B7
␮M
Chrysin
HLM
HLM ϩ BSA
4.6
24
6.1
13
38
Ͼ100
43
Ͼ100c,d
Ͼ100c
22
43
26
15
Ͼ100c
23
rUGT
Ͼ100c
Ͼ100c
rUGT ϩ BSA
Itraconazole
HLM
HLM ϩ BSA
rUGT
Ͼ100c
Ͼ100c
Ͼ100c
0.42
1.5
0.97
0.71
0.70
1.06
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
Ͼ100c
36
rUGT ϩ BSA
Ͼ100c
a HLMs or rUGT (0.025 mg/ml) were fully activated with alamethicin (10 ␮g/ml) and incubated with increasing inhibitor concentrations (0.1–100 ␮M) in 100 mM Tris-HCl (pH ϭ 7.5) buffer
containing 5 mM MgCl2, 5 mM UDPGA, with or without 2% BSA, as described under Materials and Methods. UGT substrate concentrations were at or below Km; ES (10 ␮M or 100 ␮M with
BSA), TFP (40 ␮M HLM or 67 ␮M with BSA; 10 ␮M rUGT or 140 ␮M with BSA), 5HTOL (350 ␮M), PRO (100 ␮M HLM, 200 ␮M rUGT, or 40 ␮M with BSA), AZT (842 ␮M HLM, 1080
␮M rUGT, 374 ␮M HLM with BSA, or 596 ␮M rUGT with BSA).
b IC50 values represent mean from two experiments and are not corrected for nonspecific binding.
c Negligible or low degree of inhibition (IC50 Ͼ 100 ␮M).
d Low degree of inhibition, 40% activity remaining at 100 ␮M chrysin.

UGT INHIBITION ASSAYS
1063
cordingly, highly selective, robust, and sensitive LC-MS/MS analyt- with as little as 17.1 ␮g/mg HLM at high HLM protein concentration,
ical techniques would assist high-throughput screening efforts to whereas lower HLM protein incubations may require as much as 855
increase future success in this endeavor.
␮g alamethicin/mg protein. Hence, to standardize, it is more appro-
Glucuronidation reactions have been described to be impacted priate to express optimal alamethicin activation concentrations as
significantly by in vitro incubation conditions such as buffer type and microgram per milliliter in the incubation, especially when using
ionic strength, latency, glucuronide stability, atypical kinetics, and the HLM protein concentrations Ͻ0.2 mg/ml (Fig. 3). The exact reason
albumin effect (refer to Introduction). Some standardization occurred for this phenomenon is not clear, but it is apparent that a critical
over time, but universally accepted incubation conditions are not alamethicin concentration in solution is required for optimal activa-
generally available with phosphate and Tris buffers used interchange- tion, independent of protein concentration. The current recommenda-
ably for in vitro UGT reactions; however, some authors noted highest tion is to use a standard alamethicin activation concentration of at
AZT glucuronidation activity with a physiologically relevant carbon- least 10 ␮g/ml incubate for microsomal protein concentrations be-
ate buffer or Williams E medium (Engtrakul et al., 2005). In this tween 0.01 and 0.5 mg/ml. In contrast, although alamethicin is often
study, Tris buffer provided greater activity for UGT1A4 and UGT1A9 included in incubations of insect cell baculoviral-expressed UGTs, no
in HLM, whereas all rUGT activities, except rUGT1A6, were in- activation was observed in this study, which suggests little benefit and
creased relative to phosphate buffer. Similar findings were observed is consistent with similar reports (Kaivosaari et al., 2008).
for acetaminophen glucuronidation with rUGT1A9 (Mutlib et al.,
Enzyme kinetic parameters for UGT1A9 and UGT2B7 were most
2006) and negligible impact of Tris on UGT1A1 (ES) (Soars et al., significantly affected by the addition of 2% BSA, whereas UGT1A1,
2003) or UGT2B7 (AZT) (Boase and Miners, 2002; Engtrakul et al., UGT1A4, and UGT1A6 were affected to a lesser degree. In general,
2005) activities. Boase and Miners (2002) demonstrated a trend of total Km or S50 increased for UGT1A1 and UGT1A4, remained
higher apparent Vmax for AZT glucuronidation in Tris compared with unchanged for UGT1A6, and decreased for UGT1A9 and UGT2B7,
phosphate buffers and, interestingly, a significant decrease in CLint at whereas apparent Vmax remained relatively unaffected (Ͻ2-fold
higher phosphate ionic strength (20–100 mM), primarily because of change). When corrected for nonspecific binding in the incubation,
an increase in apparent Km. The mechanistic effect or impact of the unbound CLint in the presence of BSA increased most signifi-
phosphate on UGT activity is not completely clear, although altered cantly for UGT1A9 and UGT2B7 and remained relatively unaffected
substrate selectivity and activity have been reported due to UGT for other UGTs investigated. Previous studies indicated that BSA
phosphorylation (Basu et al., 2005).
decreased PRO Km without affecting Vmax in HLM and rUGT, and
Divalent metal ions increase UGT activity, and MgCl2 (2–10 mM) similar observations were found for AZT in HLM (Rowland et al.,
is typically included in in vitro UGT incubations, with some using 2008, 2009). A more recent study indicated decreases in Km, but
MgCl2 concentrations closer the endoplasmic reticulum interior (1 significant increases in Vmax for entacapone (UGT1A9), and only
mM) or as high as 50 mM (Fisher et al., 2001). Inclusion of MgCl2 decreases in the Km for AZT in HLM and rUGT (Manevski et al.,
increased UGT activity by ϳ2- to 8-fold in HLM and to a lesser extent 2011). These findings indicate more complex mechanisms, other
in rUGTs with 5 mM generally resulting in maximal stimulation of that competitive inhibition by fatty acids may be involved in the
glucuronidation. A final MgCl2 concentration of 5 mM for the opti- inhibition of enzyme activity released from membranes. It has been
mized incubation conditions is in agreement with general practice postulated that the albumin effect is affected by enzyme source or
(4–5 mM) (Boase and Miners, 2002; Court, 2004). Saccharolactone, different fatty acid compositions released from membranes (Row-
an inhibitor of endogenous ␤-glucuronidase-catalyzed hydrolysis of land et al., 2008, 2009), which could result in differences in
the glucuronide conjugate, is often added to UGT incubations, and in apparent kinetic parameter estimates for HLM or rUGT; however,
some cases it is required to preserve glucuronide stability (Bauman et comparisons between the human embryonic kidney (HEK293) and
al., 2005). In this study, saccharolactone did not increase glucuronide Spodoptera frugiperda (Sf9) expression systems does not explain
formation, and it occasionally decreased glucuronidation (e.g., the observations for entacapone (Manevski et al., 2011). It should
UGT1A4 and UGT1A9). These findings are in agreement with reports also be noted that in vitro CLint could differ depending on micro-
indicating limited saccharolactone benefit or an inhibitory effect (Kai- somal enzyme source due to significant interindividual variation of
vosaari et al., 2008; Oleson and Court, 2008).
AZT glucuronidation rates (Peterkin et al., 2007), and apparent
Alamethicin, a pore-forming peptide, is currently the preferred Vmax estimates were determined without an authentic glucuronide
agent to activate microsomal UGT activity, presumably by increasing standard (Rowland et al., 2007).
access of substrate and cofactor to the luminal orientation of UGT
The S50 for UGT1A1-catalyzed ES glucuronidation reported in this
proteins (Fisher et al., 2001). A standard alamethicin concentration of study (1.4–6.6 ␮M) is significantly lower than previous observations
50 ␮g/mg microsomal protein is routinely used based on optimization for apparent ES S50 (17–61 ␮M) (Fisher et al., 2000; Ita¨aho et al.,
(50–100 ␮g alamethicin/mg HLM) with microsomal protein concen- 2008) because of considerable microsomal binding even when using
trations ranging from 0.5 to 2.5 mg/ml (Fisher et al., 2000) and other a low protein concentration in incubation, reiterating the importance
supportive studies (Kaivosaari et al., 2008). Activation in hepatocytes of correcting for nonspecific binding in kinetic parameter estimates.
required higher alamethicin concentrations (Ն200 ␮g/ml) (Ba´nhegyi Although glucuronidation is often considered to be a low-affinity
et al., 1993). Development of LC-MS/MS analytical methods allows process (Williams et al., 2004), high micromolar affinity for ES and
use of lower protein concentrations in incubation, and initial optimi- TFP (4.1–11 ␮M) was observed, similar to other reports for UGT1A
zation (0.01–0.1 mg/ml HLM) indicated a lack of linearity in gluc- substrates (Goosen et al., 2007; Liu et al., 2010).
uronide product formation using standard alamethicin activation con-
Inhibition of UGT activities was confirmed with potential UGT
ditions (50 ␮g/mg). Further evaluation of optimal alamethicin and characterized P450 inhibitors, which indicated nonselective
concentration (2–2500 ␮g/mg HLM) indicated dependence of the inhibition by chrysin, mefenamic acid, silibinin, and tangeretin.
critical alamethicin activation concentration on microsomal protein Diflunisal may be a potent and selective UGT1A9 inhibitor, but
concentration and loss of activation at low protein concentrations requires it further characterization because weak UGT2B7 inhibi-
(Table 2; Fig. 3), even with alamethicin levels as high as 292 ␮g/mg tion (IC50 ϭ 370 ␮M) was reported previously (Knights et al.,
microsomal protein. Accordingly, maximal activation could occur 2009). Nonselective UGT inhibition was apparent with ketocona-

1064
WALSKY ET AL.
Fisher MB, Campanale K, Ackermann BL, VandenBranden M, and Wrighton SA (2000) In vitro
glucuronidation using human liver microsomes and the pore-forming peptide alamethicin.
Drug Metab Dispos 28:560–566.
Fisher MB, Paine MF, Strelevitz TJ, and Wrighton SA (2001) The role of hepatic and extrahe-
patic UDP-glucuronosyltransferases in human drug metabolism. Drug Metab Rev 33:273–297.
Fujiwara R, Nakajima M, Oda S, Yamanaka H, Ikushiro S, Sakaki T, and Yokoi T (2010)
Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes.
J Pharm Sci 99:442–454.
zole, itraconazole, ritonavir, and verapamil, in agreement with
previous reports on ketoconazole (Liu et al., 2011; Zhou et al.,
2011). Of the P450 inhibitors investigated, minimal inhibition was
observed with the pan-P450 inhibitor 1-aminobenzotriazole, which
indicated that it may be useful when phenotyping mixed UGT and
P450 substrates and estimating fractional metabolism (fm) by P450
versus UGT (Kilford et al., 2009).
In summary, we describe optimized in vitro incubation and alame-
thicin activation conditions, LC-MS/MS analytical methods using
authentic glucuronide standards, and kinetic parameters for five he-
patic UGTs. These methods should prove useful in the routine assess-
ments of the potential for new drug candidates to elicit pharmacoki-
netic drug interactions via inhibition of human UGT activities. The
methods should also be advantageous when screening larger com-
pound libraries to identify UGT enzyme-selective chemical inhibitors.
Fujiwara R, Nakajima M, Yamanaka H, Katoh M, and Yokoi T (2007) Interactions between
human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug Metab
Dispos 35:1781–1787.
Goosen TC, Bauman JN, Davis JA, Yu C, Hurst SI, Williams JA, and Loi CM (2007)
Atorvastatin glucuronidation is minimally and nonselectively inhibited by the fibrates gemfi-
brozil, fenofibrate, and fenofibric acid. Drug Metab Dispos 35:1315–1324.
Houston JB and Kenworthy KE (2000) In vitro-in vivo scaling of CYP kinetic data not consistent
with the classical Michaelis-Menten model. Drug Metab Dispos 28:246–254.
Ita¨aho K, Mackenzie PI, Ikushiro S, Miners JO, and Finel M (2008) The configuration of the
17-hydroxy group variably influences the glucuronidation of beta-estradiol and epiestradiol by
human UDP-glucuronosyltransferases. Drug Metab Dispos 36:2307–2315.
Kaivosaari S, Toivonen P, Aitio O, Sipila¨ J, Koskinen M, Salonen JS, and Finel M (2008) Regio-
and stereospecific N-glucuronidation of medetomidine: the differences between UDP glucu-
ronosyltransferase (UGT) 1A4 and UGT2B10 account for the complex kinetics of human liver
microsomes. Drug Metab Dispos 36:1529–1537.
Kerdpin O, Mackenzie PI, Bowalgaha K, Finel M, and Miners JO (2009) Influence of N-terminal
domain histidine and proline residues on the substrate selectivities of human UDP-
glucuronosyltransferase 1A1, 1A6, 1A9, 2B7, and 2B10. Drug Metab Dispos 37:1948–1955.
Kiang TK, Ensom MH, and Chang TK (2005) UDP-glucuronosyltransferases and clinical
drug-drug interactions. Pharmacol Ther 106:97–132.
Kilford PJ, Stringer R, Sohal B, Houston JB, and Galetin A (2009) Prediction of drug clearance
by glucuronidation from in vitro data: use of combined cytochrome P450 and UDP-
glucuronosyltransferase cofactors in alamethicin-activated human liver microsomes. Drug
Metab Dispos 37:82–89.
Acknowledgments
We acknowledge Dr. Brian Ethell for helpful scientific discussions and
thank Howard Miller and Mark Snyder for experimental assistance with the
high-throughput experimental automation.
Authorship Contributions
Knights KM, Winner LK, Elliot DJ, Bowalgaha K, and Miners JO (2009) Aldosterone glucu-
ronidation by human liver and kidney microsomes and recombinant UDP-glucuronosyltrans-
ferases: inhibition by NSAIDs. Br J Clin Pharmacol 68:402–412.
Participated in research design: Walsky, Bauman, Lapham, Bourcier, Gid-
dens, Obach, Hyland, and Goosen.
Conducted experiments: Walsky, Bauman, Lapham, Bourcier, Giddens,
Negahban, Hyland, and Ryder.
Contributed new reagents or analytic tools: Walsky, Bauman, Lapham,
Bourcier, Giddens, Negahban, and Ryder.
Performed data analysis: Walsky, Bauman, Lapham, Bourcier, Giddens,
Negahban, Ryder, Hyland, and Goosen.
Krishnaswamy S, Duan SX, Von Moltke LL, Greenblatt DJ, Sudmeier JL, Bachovchin WW, and
Court MH (2003) Serotonin (5-hydroxytryptamine) glucuronidation in vitro: assay develop-
ment, human liver microsome activities and species differences. Xenobiotica 33:169–180.
Krishnaswamy S, Hao Q, Von Moltke LL, Greenblatt DJ, and Court MH (2004) Evaluation of
5-hydroxytryptophol and other endogenous serotonin (5-hydroxytryptamine) analogs as sub-
strates for UDP-glucuronosyltransferase 1A6. Drug Metab Dispos 32:862–869.
Kubota T, Lewis BC, Elliot DJ, Mackenzie PI, and Miners JO (2007) Critical roles of residues
36 and 40 in the phenol and tertiary amine aglycone substrate selectivities of UDP-
glucuronosyltransferases 1A3 and 1A4. Mol Pharmacol 72:1054–1062.
Le´pine J, Bernard O, Plante M, Teˆtu B, Pelletier G, Labrie F, Be´langer A, and Guillemette C
(2004) Specificity and regioselectivity of the conjugation of estradiol, estrone, and their
catecholestrogen and methoxyestrogen metabolites by human uridine diphospho-
glucuronosyltransferases expressed in endometrium. J Clin Endocrinol Metab 89:5222–5232.
Liu Y, Ramírez J, House L, and Ratain MJ (2010) Comparison of the drug-drug interactions
potential of erlotinib and gefitinib via inhibition of UDP-glucuronosyltransferases. Drug
Metab Dispos 38:32–39.
Liu Y, She M, Wu Z, and Dai R (2011) The inhibition study of human UDP-glucuronosyltrans-
ferases with cytochrome P450 selective substrates and inhibitors. J Enzyme Inhib Med Chem
26:386–393.
Luzier A and Morse GD (1993) Intravascular distribution of zidovudine: role of plasma proteins
and whole blood components. Antiviral Res 21:267–280.
Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, Owens
IS, and Nebert DW (2005) Nomenclature update for the mammalian UDP glycosyltransferase
(UGT) gene superfamily. Pharmacogenet Genomics 15:677–685.
Manevski N, Moreolo PS, Yli-Kauhaluoma J, and Finel M (2011) Bovine serum albumin
decreases Km values of human UDP-glucuronosyltransferases 1A9 and 2B7 and increases
Vmax values of UGT1A9. Drug Metab Dispos 39:2117–2129.
Wrote or contributed to the writing of the manuscript: Walsky, Bauman,
Lapham, Bourcier, Giddens, Negahban, Ryder, Obach, Hyland, and Goosen.
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Address correspondence to: Dr. Theunis C. Goosen, Department of Phar-
macokinetics, Dynamics, and Metabolism, Pfizer Worldwide Research and De-
velopment, Eastern Point Road, MS 8220-3525, Groton, CT 06340. E-mail:
theunis.goosen@pfizer.com