Metabolism of 1′- and 4-hydroxymidazolam by glucuronide conjugation is largely mediated by UDP-glucuronosyltransferases 1A4, 2B4, and 2B7

Article abstract of DOI:10.1124/dmd.110.035295

Midazolam undergoes oxidative hydroxylation by CYP3A to its metabolites, which are excreted mainly as glucuronidated conjugates into the urine. In this study, we examined the glucuronidation of hydroxymidazolam in human liver microsomes (HLMs) and characterized the UDP-glucuronosyltransferases (UGTs) involved in 1′- and 4-hydroxymidazolam glucuronidation. Among the 12 UGT isoforms tested, the O- and N-glucuronidation of 1′-hydroxymidazolam was mediated by UGT2B4/2B7 and 1A4, respectively. In contrast, the glucuronidation of 4-hydroxymidazolam was mediated by UGT1A4. Consistent with these observations, the UGT1A4 inhibitor hecogenin and the UGT2B7 substrate diclofenac potently inhibited the N- and Oglucuronidation of 1′-hydroxymidazolam in HLMs, respectively. A correlation analysis of UGT enzymatic activity and the formation rate of glucuronide metabolites from 1′- and 4-hydroxymidazolam in 25 HLMs showed that hydroxymidazolam glucuronidation is correlated with UGT1A4-mediated lamotrigine glucuronidation and UGT2B7-mediated diclofenac glucuronidation activity. Taken together, these findings indicate that UGT1A4, 2B4, and 2B7 are major isoforms responsible for glucuronide conjugate formation from 1′- and 4-hydroxymidazolam, which are the two major oxidative metabolites of midazolam. Copyright

Full text of DOI:10.1124/dmd.110.035295

0090-9556/10/3811-2007–2013$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:2007–2013, 2010
Vol. 38, No. 11
35295/3635725
Printed in U.S.A.
Metabolism of 1؅- and 4-Hydroxymidazolam by Glucuronide
Conjugation Is Largely Mediated by UDP-Glucuronosyltransferases
1A4, 2B4, and 2B7
Kyung-Ah Seo, Soo Kyung Bae, Young-Kil Choi, Chang Soo Choi, Kwang-Hyeon Liu,
and Jae-Gook Shin
Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea
(K.-A.S., S.K.B., K.-H.L., J.-G.S.); and Departments of Clinical Pharmacology (S.K.B., J.-G.S.) and Surgery (Y.-K.C., C.S.C.),
Inje University Busan Paik Hospital, Busan, Korea
Received July 5, 2010; accepted August 16, 2010
ABSTRACT:
Midazolam undergoes oxidative hydroxylation by CYP3A to its UGT2B7 substrate diclofenac potently inhibited the N- and O-
metabolites, which are excreted mainly as glucuronidated con- glucuronidation of 1؅-hydroxymidazolam in HLMs, respectively.
jugates into the urine. In this study, we examined the gluc- A correlation analysis of UGT enzymatic activity and the forma-
uronidation of hydroxymidazolam in human liver microsomes tion rate of glucuronide metabolites from 1؅- and 4-hydroxy-
(HLMs) and characterized the UDP-glucuronosyltransferases
midazolam in 25 HLMs showed that hydroxymidazolam gluc-
(UGTs) involved in 1؅- and 4-hydroxymidazolam glucuronidation. uronidation is correlated with UGT1A4-mediated lamotrigine
Among the 12 UGT isoforms tested, the O- and N-glucuronida- glucuronidation and UGT2B7-mediated diclofenac glucuronida-
tion of 1؅-hydroxymidazolam was mediated by UGT2B4/2B7 and tion activity. Taken together, these findings indicate that
1A4, respectively. In contrast, the glucuronidation of 4-hy-
UGT1A4, 2B4, and 2B7 are major isoforms responsible for gluc-
droxymidazolam was mediated by UGT1A4. Consistent with uronide conjugate formation from 1؅- and 4-hydroxymidazolam,
these observations, the UGT1A4 inhibitor hecogenin and the which are the two major oxidative metabolites of midazolam.
Introduction
Individual UGTs exhibit distinct, but overlapping, substrate
selectivities and differ in their regulation of expression, genetic
polymorphisms, and other factors known to influence the activity
of drug-metabolizing enzymes in humans (Miners et al., 2002,
2004). Despite recognition of the importance of glucuronidation as
a clearance and detoxification mechanism for drugs, environmental
chemicals, and endogenous compounds, relatively few selective sub-
strates and inhibitors useful for the phenotyping of glucuronidated com-
pounds have been identified (Court, 2005).
Midazolam is a potent benzodiazepine with sedative activities. It is
metabolized to 1Ј- and 4-hydroxymidazolam by CYP3A4 and
CYP3A5 (Kronbach et al., 1989) and is further metabolized to gluc-
uronide conjugates by UGT (Fig. 1). Glucuronide conjugates of
1Ј-hydroxymidazolam account for 60 to 70% of the midazolam dose
(Heizmann and Ziegler, 1981). Zhu et al. (2008) recently identified an
N-linked glucuronide conjugate of 1Ј-hydroxymidazolam by incubation
with human liver microsomes (HLMs). Although the O- and N-gluc-
uronidation of 1Ј-hydroxymidazolam is catalyzed by UGT2B4/2B7 and
1A4, respectively (Zhu et al., 2008), the specific hepatic UGT
isoforms involved in 4-hydroxymidazolam glucuronidation have
not been identified.
Glucuronidation is an important detoxification and elimination
pathway for endogenous and exogenous compounds, and it ac-
counts for ϳ35% of all phase II drug metabolism (Evans and
Relling, 1999). UDP-glucuronosyltransferases (UGTs) mediate
glucuronidation. In humans, the UGT superfamily is composed of
two families (UGT1 and UGT2) and three subfamilies (UGT1A,
2A, and 2B) (Mackenzie et al., 2005). The lone UGT1A locus on
chromosome 2q37 encodes nine functional proteins: UGT1A1,
1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, and 1A10. The nomencla-
ture of UGT1A is based on the relative position of the first variable
exon with respect to four common exons in the unspliced mRNA.
The UGT2A subfamily has multiple loci located on chromosome
4q13 and comprises the following functional proteins: UGT2A1,
2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28 (Guillemette,
2003).
This study was supported by the Korean Health 21 R&D Project, Ministry of
Health, Welfare, and Family Affairs [Grant A030001]; and by the Ministry of
Education, Science, and Engineering [Korean Science and Engineering Founda-
tion Grant R13-2007-023-00000-0].
The objective of this study was to identify and kinetically
characterize and compare the UGT isoforms responsible for 1Ј-
and 4-hydroxymidazolam glucuronidation in vitro. Our data will
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.035295.
ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; HLM, human liver microsome; UDPGA, uridine diphosphoglucuronic acid; LC, liquid
chromatography; MS/MS, tandem mass spectrometry.
2007

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SEO ET AL.
FIG. 1. Proposed pathway of midazolam metabolism in HLMs.
droxymidazolam, an API 3000 LC-MS/MS system (Applied Biosystems,
Foster City, CA), coupled with an 1100 series high-performance liquid
chromatography system (Agilent Technologies, Santa Clara, CA) was used.
Separation was performed on a Polar-RP column (2 mm i.d. ϫ 250 mm,
4-␮m particle size; Phenomenex, Torrance, CA) with a mobile phase
consisting of water (A) and acetonitrile (B) containing 0.1% formic acid.
The analytes were eluted at a flow rate of 0.2 ml/min using a linear
gradient. Solvent B started at 30%, then increased as follows: 30% (1 min),
50% (6–8 min), and 30% (9 min). The TurboIonSpray interface was
operated in the positive ion mode at 5500 V and 500°C. The operating
conditions were as follows: nebulizing gas flow, 7 psi; curtain gas flow, 12
psi; and collision energy, 35 eV. Quadrupoles Q1 and Q3 were set on unit
resolution. Multiple-reaction-monitoring mode using specific precursor/
product ion transition was used for quantification. Detection of the ions was
performed by monitoring the transitions of m/z 518 3 324 and 518 3 342
for the hydroxymidazolam glucuronides and 339 3 163 for 7-hydroxycou-
marin glucuronide (internal standard). Peak areas for all compounds were
automatically integrated using Analyst software (version 1.2; Applied
Biosystems).
Glucuronidation of Hydroxymidazolam in HLMs or Recombinant
UGT Isoforms. The optimal conditions for microsomal incubation were
determined in the linear range for the formation of glucuronide metabolites
from hydroxymidazolam. In all experiments, 1Ј- and 4-hydroxymidazolam
were dissolved and serially diluted with methanol to the required concen-
trations. The final concentration of organic solvent did not exceed 1% in
the final incubation mixtures. Typical incubation mixtures (total volume,
125 ␮l), containing either 0.25 mg of HLMs or recombinant UGT (0.1 mg),
1Ј- or 4-hydroxymidazolam (1–200 ␮M), and 1.5 mg/ml alamethicin, were
reconstituted in 0.5 M Tris-HCl buffer (pH 7.5) and preincubated on ice for
15 min. The reaction was initiated by adding UDPGA (5 mM) and run for
allow a better understanding of the factors affecting midazolam
pharmacokinetics and drug interactions.
Materials and Methods
Chemicals and Reagents. Midazolam, 1Ј-hydroxymidazolam, and 4-hy-
droxymidazolam were purchased from Ultrafine Chemical Co. (Manches-
ter, UK). Alamethicin (from Trichoderma viride), uridine diphosphogluc-
uronic acid (UDPGA), saccharic acid-1, 4-lactone, diclofenac, hecogenin,
and lamotrigine were obtained from Sigma-Aldrich (St. Louis, MO). The
solvents were of high-performance liquid chromatography grade (Thermo
Fisher Scientific, Waltham, MA); all other chemicals were of the highest
quality available. Recombinant human UGT isoforms (UGT1A1, 1A3,
1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) and pooled
and 10 single-donor HLMs were purchased from BD Gentest (Woburn,
MA). The vendor supplied information regarding the protein concentration.
Fifteen single-donor HLMs were obtained from the Inje PharmacoGenom-
ics Research Center Tissue Bank (Busan, Korea).
In Vitro Metabolism of Hydroxymidazolam by HLMs. For the micro-
somal phase II metabolism of hydroxymidazolam, 0.25 mg of pooled
HLMs (H161; BD Gentest), 1Ј- or 4-hydroxymidazolam (100 ␮M), and 1.5
mg/ml alamethicin were reconstituted in 0.5 M Tris-HCl buffer (pH 7.5)
and preincubated on ice for 15 min. The reaction was initiated by addition
of UDPGA (5 mM) and run for 60 min at 37°C in a shaking water bath. In
this study, a 60-min incubation time was selected because the reaction
showed linearity until 60 min from the preliminary experiments. The
reaction was terminated by the addition of 100 ␮l of acetonitrile on ice and
then centrifuged at 10,000g for 5 min at 4°C. Aliquots of the supernatant
were analyzed by liquid chromatography/tandem mass spectrometry (LC-
MS/MS) for the identification of glucuronide conjugate metabolites of 1Ј-
and 4-hydroxymidazolam.
Analysis of the Glucuronide Conjugates of Hydroxymidazolam by LC- 60 min at 37°C in a shaking water bath. The reaction was terminated by the
MS/MS. For the identification of the glucuronide metabolites of hy- addition of 100 ␮l of acetonitrile on ice, and the mixture was centrifuged

UGT ISOFORMS INVOLVED IN 1Ј- AND 4-HYDROXYMIDAZOLAM
2009
at 10,000g for 5 min at 4°C. Aliquots of the supernatant were used for
LC-MS/MS.
Chemical Inhibition Studies with HLMs. The inhibitory effects of the
known UGT1A4 inhibitor hecogenin (final concentration, 10 ␮M) or the
UGT2B7 substrate diclofenac (50 ␮M) on the formation of 1Ј- and 4-hy-
droxymidazolam glucuronides were evaluated to determine the UGT isoforms
involved in this metabolic pathway. Hecogenin was dissolved in dimethyl
sulfoxide and diclofenac was dissolved in methanol. The final concentration of
the organic solvents in the reaction mixture was Ͻ1% (v/v). The formation
ratios of 1Ј- and 4-hydroxymidazolam glucuronides from 1Ј- and 4-hy-
droxymidazolam were determined from reaction mixtures incubated in the
presence or absence of hecogenin or diclofenac. Except for the addition of
hecogenin or diclofenac, all other incubation conditions were similar to those
described previously.
Correlation Experiments. For the correlation analysis, the comparative
metabolic rates of hydroxymidazolam in 25 different HLMs were investi-
gated by the incubation of 1Ј- and 4-hydroxymidazolam (50 ␮M) with 0.25
mg/ml microsomal protein for 60 min. The activity of each UGT isoform
was determined by LC-MS/MS as described previously for lamotrigine
(Rowland et al., 2006) and diclofenac glucuronidation (King et al., 2001).
The correlation coefficients between the formation rates of glucuronide
metabolites of 1Ј- and 4-hydroxymidazolam and the activity of each UGT
isoform in the various HLMs were calculated by Spearman’s correlation
coefficient analysis (SAS version 8.01; SAS Institute Inc., Cary, NC). p Ͻ
0.05 was considered to be statistically significant.
Data Analysis. All results are expressed as the mean Ϯ S.D. of estimates
obtained from three different HLMs in triplicate experiments. Because of
the absence of authentic standards for the hydroxymidazolam glucuronides,
the conjugates were semiquantified using a hydroxymidazolam calibration
curve. The apparent kinetic parameters for the biotransformation of 1Ј- and
4-hydroxymidazolam (Km and Vmax) were determined by fitting an one-enzyme
Michaelis-Menten equation or a Hill equation (V ϭ Vmax ⅐ [S]n/[Km ϩ (S)n]). The
calculated parameters include the maximum rate of formation (Vmax), the Michae-
lis-Menten constant (apparent Km), the intrinsic clearance (CLint ϭ Vmax/apparent
Km), and Hill coefficient (n). All calculations were performed using WinNonlin
software (Pharsight, Mountain View, CA). The percent inhibition was calculated
from the ratio of the amount of metabolite formed with and without the specific
inhibitor.
FIG. 2. Multiple-reaction-monitoring (MRM) chromatogram of glucuronide metab-
olites obtained by LC-MS/MS using HLM incubates of 1Ј- (A) and 4-hydroxy-
midazolam (B) in the presence of UDPGA.
lam glucuronides after the incubation of 1Ј- or 4-hydroxymidazolam
with the UGT isoforms are shown in Fig. 3. UGT1A4 and 2B4/2B7
metabolized 1Ј-hydroxymidazolam to 1Ј-hydroxymidazolam N- and
O-glucuronide, respectively, more efficiently than any other UGT
(Fig. 3, A and B), whereas UGT1A4 metabolized 4-hydroxymidazo-
lam to its glucuronide metabolites with little contribution from
UGT1A3, 1A6, and 2B7 (Fig. 3, C and D). Hydroxymidazolam
glucuronide formation from 1Ј- and 4-hydroxymidazolam (50 ␮M)
was also studied using a UGT isoform inhibitor or substrate (Fig. 4).
1Ј-Hydroxymidazolam N-glucuronidation was inhibited by the
UGT1A4 inhibitor hecogenin (IC50 ϭ 1.5 ␮M), whereas 1Ј-hy-
droxymidazolam O-glucuronidation was inhibited by the UGT2B7
substrate diclofenac (IC50 ϭ 2.0 ␮M). The formation of 4-hydroxy-
midazolam glucuronide I and II was also inhibited by hecogenin
(IC50 ϭ 0.8 ␮M) and diclofenac (IC50 ϭ 46.6 ␮M), respectively
(Table 1). The formation rates of 1Ј-hydroxymidazolam N-glucuro-
nide and 4-hydroxymidazolam glucuronide I were significantly cor-
related with the activity of UGT1A4, whereas that of 1Ј-hydroxymid-
azolam O-glucuronide was correlated with the activity of UGT2B7
and 1A4 (Fig. 5).
Enzyme Kinetic Analysis. Kinetic analyses of 1Ј- and 4-hy-
droxymidazolam metabolite formation were performed using three
different HLM preparations. Under the experimental conditions
used, the metabolism of 1Ј- and 4-hydroxymidazolam by the HLMs
was best fitted by a Hill equation (Fig. 6). The fitting of the data
to the Michaelis-Menten two-enzyme model did not significantly
improve the regression, compared with fitting of the data to a
one-enzyme model. The kinetic parameters estimated from the
three HLMs are shown in Table 2. The sums of the formation
(CLint) of both metabolites were 346.2 and 90.3 ␮l/(min ⅐ mg
Results
Identification of Glucuronide Metabolites of Hydroxymid-
azolam in HLMs. After the incubation of 1Ј-hydroxymidazolam
with HLMs in the presence of UDPGA, two metabolites were pro-
filed. These metabolites were confirmed as glucuronide conjugates by
full-scan analysis, which showed the addition of 176 Da (glucuronic
acid) to the parent (m/z 518). These two metabolites were tentatively
identified as 1Ј-hydroxymidazolam O- and N-glucuronide based on
the MS/MS fragmentation pattern. A fragment ion at m/z 324 (loss of
glucuronic acid and a water molecule) was the base peak observed
with 1Ј-hydroxymidazolam O-glucuronide, whereas that at m/z 342
(loss of glucuronic acid) was the major fragment ion for 1Ј-hy-
droxymidazolam N-glucuronide (Fig. 2). These results are consistent
with those from previous reports (Zhu et al., 2008). When 4-hy-
droxymidazolam was incubated with the HLMs, two glucuronide
conjugates (m/z 518) were detected. On the basis of the structures of
the 1Ј-hydroxymidazolam O- and N-glucuronides, which were re-
ported by Zhu et al. (2008), it is reasonable to assume that glucuronic
acid was conjugated to 4-hydroxymidazolam at the same N and OH
positions. These two metabolites were designated as 4-hydroxymid-
azolam glucuronide I and II because we could not distinguish 4-hy-
droxymidazolam O-glucuronide from the N-glucuronide.
Identification of the UGT Isoforms Involved in the Metabolism
of Hydroxymidazolams. Two concentrations of 1Ј- and 4-hy- protein) for 1Ј- and 4-hydroxymidazolam, respectively (Table 2).
droxymidazolam (20 and 100 ␮M) were incubated with a panel of
Next, we examined the enzyme kinetic parameters for the for-
recombinant UGT isoforms. The formation rates of hydroxymidazo- mation of glucuronide conjugates from each hydroxymidazolam

2010
SEO ET AL.
FIG. 3. Representative plots of the formation of 1Ј-hydroxymid-
azolam N-glucuronide (A) and O-glucuronide (B) from 1Ј-hy-
droxymidazolam, from 4-hydroxymidazolam glucuronides I (C)
and II (D) and from 4-hydroxymidazolam by recombinant human
UGT isoforms. IS, internal standard.
(0–200 ␮M) upon incubation with recombinant human UGT1A4, mediated N-glucuronidation of 1Ј-hydroxymidazolam showed a
2B4, and 2B7 (Fig. 7). Under the experimental conditions used, the lower Km and higher Vmax than did UGT2B4- and 2B7-catalyzed
metabolism of 1Ј- and 4-hydroxymidazolam by these UGT iso- O-glucuronidation, resulting in a higher CLint [29.5 versus 6.8
forms was also best described by a Hill equation. The UGT1A4- ␮l/(min ⅐ mg protein)] (Table 3). The in vitro intrinsic clearance for
FIG. 4. Effects of the UGT isoform-selective inhibitor hecogenin
(10 ␮M, UGT1A4) or the substrate diclofenac (50 ␮M, UGT2B7)
on the formation of glucuronide metabolites from 1Ј-hydroxymid-
azolam (A) and 4-hydroxymidazolam (B) by HLMs. Each bar
represents the mean of duplicate determinations.
TABLE 1
IC50 values for the inhibition of UGT-mediated hydroxymidazolam glucuronidation by the UGT isoform-selective inhibitor or substrate in pooled HLMs
IC50 Valuesa
UGT
Inhibitor
Isoform
1Ј-Hydroxymidazolam N-glucuronide
1Ј-Hydroxymidazolam O-glucuronide
␮M
4-Hydroxymidazolam Glucuronide I
4-Hydroxymidazolam Glucuronide II
1A4
2B7
Hecogenin
Diclofenac
1.5
802.8
Ͼ5
2.0
0.8
941.1
Ͼ5
46.6
a Averages of duplicate determinations.

UGT ISOFORMS INVOLVED IN 1Ј- AND 4-HYDROXYMIDAZOLAM
2011
FIG. 5. Simple regression analysis of lamotrigine or diclofenac
glucuronidation and the rates of 1Ј-hydroxymidazolam O-gluc-
uronidation (A) and N-glucuronidation (B) and 4-hydroxymidazo-
lam glucuronide I formation (C) in 25 different HLMs.
glucuronide conjugate formation from 1Ј-hydroxymidazolam by nin (10 ␮M, ϳ90%), a potent UGT1A4-selective inhibitor (Fig. 4)
UGT1A4 was similar to that for 4-hydroxymidazolam by UGT1A4
[29.5 versus 26.1 ␮l/(min ⅐ mg protein)].
(Uchaipichat et al., 2006), whereas those of 1Ј-hydroxymidazolam O-
glucuronide were inhibited by diclofenac (50 ␮M, ϳ95%), a selective
UGT2B7 substrate (King et al., 2001). We found a significant correlation
between 1Ј-hydroxymidazolam N-glucuronidation in HLMs (n ϭ 25) and
the glucuronidation activity of the UGT1A4 substrate lamotrigine (Row-
land et al., 2006), whereas 1Ј-hydroxymidazolam O-glucuronidation
was correlated with glucuronidation activity mediated by the
UGT2B7 substrates diclofenac (King et al., 2001) and lamotrigine.
The significant correlation observed between the activity of
UGT1A4 and 1Ј-hydroxymidazolam O-glucuronidation in the
panel of HLMs tested may not directly involve UGT1A4. Because
our inhibition and recombinant experiments do not support a
significant role for UGT1A4 in 1Ј-hydroxymidazolam O-
glucuronidation, the observed correlation may be derived from the
involvement of UGT2B7 in lamotrigine glucuronidation (Rowland
et al., 2006). These results are compatible with those from a
previous report by Zhu et al. (2008).
Discussion
The glucuronidation of midazolam and its oxidized metabolites
is an important part of midazolam metabolism in humans. It has
been reported that the amount of hydroxymidazolam O-
glucuronide excreted in the urine is quite variable (Heizmann and
Ziegler, 1981). This variability could be one factor responsible for
the large interindividual differences seen in midazolam metabo-
lism. In the present study, the glucuronidation of 1Ј- and 4-hy-
droxymidazolam in HLMs and the recombinant UGT isoforms
mediating this metabolism was thoroughly evaluated.
In this study, we reconfirmed that 1Ј-hydroxymidazolam is metabo-
lized to N- and O-glucuronide in the presence of UDPGA in HLMs and
that UGT1A4 and 2B4/2B7 are the major enzymes responsible for
1Ј-hydroxymidazolam N- and O-glucuronidation, respectively. Recom-
binant human UGT2B4/2B7 and 1A4 metabolized 1Ј-hydroxy-
midazolam to 1Ј-hydroxymidazolam O- and N-glucuronide, respectively,
whereas the other UGT isoforms did not (Fig. 3). The formation rates of
In addition, we demonstrated for the first time that 4-hydroxy-
midazolam is transformed to two glucuronide metabolites (I and II).
Formation of the major metabolite, 4-hydroxymidazolam glucuronide
1Ј-hydroxymidazolam N-glucuronide were strongly inhibited by hecoge- I, was catalyzed by UGT1A4 but not by the other UGT isoforms

2012
SEO ET AL.
FIG. 6. Representative Michaelis-Menten (left panels) and Eadie-Hofstee (right
panels) plots of the glucuronide conjugation of 1Ј- (A and B) and 4-hydroxymid-
azolam (C and D). HLMs were incubated with 0 to 200 ␮M 1Ј-hydroxymidazolam
or 4-hydroxymidazolam at 37°C for 60 min in the presence of UDPGA (5 mM). The
data points represent the averages obtained from three different HLMs.
FIG. 7. Representative Michaelis-Menten plots for glucuronide conjugate forma-
tion from 1Ј-hydroxymidazolam (A and B) and 4-hydroxymidazolam (C) by re-
combinant UGT isoforms 1A4, 2B4, and 2B7. The recombinant UGT isoforms
(0.25 mg/ml) were incubated with 0 to 100 ␮M 1Ј- or 4-hydroxymidazolam at 37°C
for 60 min in the presence of UDPGA (5 mM). Each data point represents the
average of triplicate incubations.
(Fig. 3C). The other isoforms, including UGT1A3, 1A6, 2B4, and
2B7, contributed to the formation of the minor metabolite 4-hy-
droxymidazolam glucuronide II (Fig. 3D). We also found that the
formation rates of 4-hydroxymidazolam glucuronide I were strongly
inhibited by hecogenin (10 ␮M, ϳ95%) (Fig. 4). In addition, diclofe-
nac, a well known UGT2B7 substrate, inhibited 4-hydroxymidazolam
glucuronide II formation (50 ␮M, ϳ50%). Our data significantly
correlated 4-hydroxymidazolam glucuronide I formation in HLMs
with UGT1A4-mediated lamotrigine N-glucuronidation (Fig. 5).
Taken together, these results suggest that 4-hydroxymidazolam gluc-
uronide I is the major glucuronide metabolite of 4-hydroxymidazolam
and was formed by UGT1A4.
showed convex relationships (Fig. 6B), indicating positive cooperat-
ivity (n Ͼ 1), whereas 4-hydroxymidazolam glucuronide I and II
formation showed convex (n ϭ 1.2) and concave (n ϭ 0.5) relation-
ships (Fig. 6D), respectively. The intrinsic clearance for 1Ј-hy-
droxymidazolam O-glucuronidation was roughly 3-fold higher than
that for N-glucuronidation (Table 2). A similar kinetic profile was
reported by Zhu et al. (2008), although the difference in intrinsic
clearance for metabolite formation was higher than observed in this
study. This may be accounted for by differences between the assay
conditions in their study (100 mM phosphate buffer, 2 mM MgCl2, 0.5
mg/ml HLMs, and 2 mM UDPGA) and in the present study. A
different kinetic profile for nicotine N-glucuronidation has also been
shown to be dependent on the incubation conditions (Ghosheh et al.,
2001; Nakajima et al., 2002). In the current study, 4-hydroxymidazo-
lam glucuronide I formation was a major metabolic pathway for
4-hydroxymidazolam glucuronidation [the CLint values for 4-hy-
droxymidazolm glucuronide I and II formation were 87.0 and 3.3
␮l/(min ⅐ mg protein), respectively]. In contrast to 4-hydroxymidazo-
lam, 1Ј-hydroxymidazolam showed high enzyme affinity (low Km
values), resulting in relatively high metabolic clearance (Table 2).
We observed large interindividual variability in 1Ј-hydroxymid-
azolam O-glucuronidation (roughly 9-fold), N-glucuronidation (ϳ20
fold), and 4-hydroxymidazolam glucuronide I formation (approxi-
mately 13-fold) in the HLMs (n ϭ 25). Large interindividual differ-
ences in the percentage of glucuronide conjugates excreted in the
urine may be due to interindividual differences in the formation of
these metabolites (i.e., differences in the catalytic properties of the
UGT isoforms, rather than in the process of excretion) (Nakajima et
al., 2002). There are genetic polymorphisms in certain UGT isoforms,
including UGT1A1, 1A4, 1A6, 2B4, 2B7, and 2B15 (Tukey and
Strassburg, 2000). Polymorphisms in the UGT1A4, 2B4, and 2B7
The glucuronidation of 1Ј- and 4-hydroxymidazolam in HLMs was
characterized by Hill kinetics, suggesting the involvement of a single
enzyme or more than one enzyme with similar affinities (Fig. 6).
Eadie-Hofstee plots of 1Ј-hydroxymidazolam glucuronide formation
TABLE 2
Mean enzyme kinetic parameters for glucuronide conjugate formation from
1Ј- and 4-hydroxymidazolam in HLMs
The values are the means of estimates from three different HLM preparations.
Kinetic Parameter
Hill
Coefficient (n)
CLint
(Vmax/Km)
Km
Vmax
␮M
pmol/(min ⅐ mg
␮l/(min ⅐ mg
protein)
protein)
1Ј-Hydroxymidazolam
N-Glucuronide
O-Glucuronide
9.1
12.7
790.0
3285.8
1.6
1.5
86.8
259.4
4-Hydroxymidazolam
Glucuronide I
Glucuronide II
41.5
976.9
3609.9
3210.5
1.2
0.5
87.0
3.3

UGT ISOFORMS INVOLVED IN 1Ј- AND 4-HYDROXYMIDAZOLAM
2013
TABLE 3
Mean enzyme kinetic parameters for glucuronide conjugate formation from 1Ј- and 4-hydroxymidazolam by recombinant human UGT isoforms
Each data set is the average of triplicate incubations.
Kinetic Parameters
UGT Isoform
Metabolite
Km
Vmax
Hill Coefficient (n)
CLint (Vmax/Km)
␮M
pmol/(min ⅐ mg protein)
␮l/(min ⅐ mg protein)
1Ј-Hydroxymidazolam
1A4
2B4
2B7
N-Glucuronide
O-Glucuronide
15.9
29.8
35.5
468.3
127.0
90.1
1.31
1.00
0.83
29.5
4.3
2.5
4-Hydroxymidazolam
1A4
Glucuronide I
40.8
1064.2
1.23
26.1
King C, Finley B, and Franklin R (2000) The glucuronidation of morphine by dog liver
microsomes: identification of morphine-6-O-glucuronide. Drug Metab Dispos 28:661–663.
King C, Tang W, Ngui J, Tephly T, and Braun M (2001) Characterization of rat and human
UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of diclofenac.
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and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 36:89–96.
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.
Miners JO, McKinnon RA, and Mackenzie PI (2002) Genetic polymorphisms of UDP-
glucuronosyltransferases and their functional significance. Toxicology 181–182:453–456.
Miners JO, Smith PA, Sorich MJ, McKinnon RA, and Mackenzie PI (2004) Predicting human
drug glucuronidation parameters: application of in vitro and in silico modeling approaches.
Annu Rev Pharmacol Toxicol 44:1–25.
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Rowland A, Elliot DJ, Williams JA, Mackenzie PI, Dickinson RG, and Miners JO (2006) In vitro
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isoforms might cause the interindividual differences seen in 1Ј- and
4-hydroxymidazolam glucuronidation in humans.
In conclusion, we thoroughly characterized the glucuronidation of
1Ј- and 4-hydroxymidazolam in HLMs. Using UGT isoform-specific
inhibitors, correlation analyses, and recombinant UGT isoforms, we
found that UGT1A4 and 2B4/2B7 were responsible for the formation
of 1Ј-hydroxymidazolam N- and O-glucuronide, respectively, from
1Ј-hydroxymidazolam. In addition, the glucuronidation of 4-hy-
droxymidazolam was mainly metabolized by UGT1A4. The available
data suggest that UGT enzymes exhibit overlapping substrate selec-
tivity (Burchell et al., 1995; King et al., 2000). Therefore, identifying
the substrate of each UGT isoform could be useful in UGT pheno-
typing. For this purpose, 4-hydroxymidazolam could be used in vitro
as a probe substrate for UGT1A4 as has been suggested for 1Ј-
hydroxymidazolam (Zhu et al., 2008).
Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, ex-
pression, and disease. Annu Rev Pharmacol Toxicol 40:581–616.
Uchaipichat V, Mackenzie PI, Elliot DJ, and Miners JO (2006) Selectivity of substrate (trifluo-
perazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbuta-
zone, quinidine, quinine, and sulfinpyrazone) “probes” for human UDP-glucuronosyltrans-
ferases. Drug Metab Dispos 34:449–456.
Zhu B, Bush D, Doss GA, Vincent S, Franklin RB, and Xu S (2008) Characterization of
1Ј-hydroxymidazolam glucuronidation in human liver microsomes. Drug Metab Dispos 36:
331–338.
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Address correspondence to: Dr. J.-G. Shin, Department of Pharmacology
and PharmacoGenomics Research Center, Inje University College of Medi-
cine, #633-165 Gaegum-Dong, Busanjin-Gu, Busan 614-735, Korea. E-mail:
phshinjg@inje.ac.kr
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