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Vol. 30, Issue 11, 1266-1273, November 2002
Department of Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (J.A.W., B.J.R., V.E.C., K.C., S.A.W.); and Department of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana (D.R.J., S.D.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Previous results demonstrating homotropic activation of human
UDP-glucuronosyltransferase (UGT) 1A1-catalyzed
estradiol-3-glucuronidation led us to investigate the effects of 16 compounds on estradiol glucuronidation by human liver microsomes (HLM).
In confirmation of previous work using alamethicin-treated HLM pooled
from four livers, UGT1A1-catalyzed estradiol-3-glucuronidation
demonstrated homotropic activation kinetics (S50 = 22 µM, Hill coefficient, n = 1.9) whereas
estradiol-17-glucuronidation (catalyzed by other UGT enzymes) followed
Michaelis-Menten kinetics (Km = 7 µM). Modulatory effects of the following compounds were investigated:
bilirubin, eight flavonoids, 17
-ethynylestradiol (17-EE),
estriol, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), anthraflavic acid, retinoic
acid, morphine, and ibuprofen. Although the classic UGT1A1 substrate
bilirubin was a weak competitive inhibitor of
estradiol-3-glucuronidation, the estrogens and anthraflavic acid
activated or inhibited estradiol-3-glucuronidation dependent on
substrate and effector concentrations. For example, at substrate
concentrations of 5 and 10 µM, estradiol-3-glucuronidation activity
was stimulated by as much as 80% by low concentrations of 17-EE but
was unaltered by flavanone. However, at higher substrate concentrations
(25-100 µM) estradiol-3-glucuronidation was inhibited by about 55%
by both compounds. Anthraflavic acid and PhIP were also stimulators of
estradiol 3-glucuronidation at low substrate concentrations. The most
potent inhibitor of estradiol 3-glucuronidation was the flavonoid
tangeretin. The UGT2B7 substrates morphine and ibuprofen had no effect
on estradiol 3-glucuronidation, whereas retinoic acid was slightly
inhibitory. Estradiol-17-glucuronidation was inhibited by 17-EE,
estriol, and naringenin but was not activated by any compound. This
study demonstrates that the interactions of substrates and inhibitors
at the active site of UGT1A1 are complex, yielding both activation and
competitive inhibition kinetics.
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Introduction |
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Introduction
Materials and Methods
Results
Discussion
References
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Glucuronidation
catalyzed by the UDP-glucuronosyltransferases
(UGT2) is a major pathway of metabolism of
endogenous steroids, bile acids, drugs, carcinogens, and environmental
pollutants (Tephly and Green, 2000
). Based on evolutionary divergence,
two families of UGT enzymes have been identified, UGT1 and UGT2. The
UGT1A gene is located on chromosome 2 and encodes for all
members of the UGT1A subfamily by differential splicing of the gene
product (Ritter et al., 1992). Within the UGT1A subfamily, the
catalytic activity of UGT1A1 has been relatively well studied.
Important physiological roles of UGT1A1 include glucuronidation of the
toxic heme breakdown product bilirubin, as well as the glucuronidation of catechol estrogens, and flavonoids (Senafi et al., 1994). UGT1A1 also glucuronidates anthraqinones (Senafi et al., 1994), the oral contraceptive 17-ethynylestradiol (17-EE) (Ebner et al., 1993), and oripavine opioids such as buprenorphine (Senafi et al., 1994). In
addition, UGT1A1-catalyzed estradiol-3-glucuronidation by microsomes from small intestine exceed rates found with liver microsomes (Fisher
et al., 2000a). Functional polymorphisms in the UGT1A1 gene decrease
bilirubin glucuronidation activity and depending on the nature of the
polymorphism can lead to the toxicity associated with Gilbert syndrome,
or the more severe Crigler-Najar syndrome (Mackenzie et al., 2000).
Estradiol is glucuronidated at the 3-position by UGT1A1 (Senafi et al.,
1994; Fisher et al., 2000a) and at the 17-position by several UGT
enzymes, including UGT2B7 (Gall et al., 1999). The kinetics of
estradiol-3-glucuronidation by UGT1A1 in human liver microsomes
demonstrate homotropic activation (autoactivation kinetics) whereas
estradiol-17-glucuronidation follows Michaelis-Menten kinetics (Fisher
et al., 2000a,b). The mechanism of homotropic activation of
UGT1A1-catalyzed estradiol-3-glucuronidation is unknown. One
possibility is the existence of the enzyme in multimeric form, so that
the binding of one substrate molecule to one subunit may increase the
affinity of the other subunit(s) for another substrate molecule. A
limited number of studies have provided experimental evidence to
support the existence of UGT1A1 (Bruni and Chang, 1999) and
UGT2B1 (Meech and Mackenzie, 1997) as multimeric enzymes. Since
UGT1A1-catalyzed estradiol-3-glucuronidation undergoes homotropic
activation, the possibility also exists that UGT1A1 substrates or other
compounds may act as heterotropic activators. Therefore, 16 structurally diverse compounds, including representatives of five
distinct classes of compounds known to be UGT1A1 substrates were
examined for their potential as activators and/or inhibitors of
UGT1A1-catalyzed estradiol-3-glucuronidation over a wide range of
effector and substrate concentrations. The known UGT1A1 substrates examined for modulatory effects on this activity were bilirubin, anthraflavic acid, 17 -EE (Tephly and Green, 2000), the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) (Malfatti and Felton, 2001), and the flavonoids naringenin and
quercetin (Senafi et al., 1994). Four additional flavonoids, flavone,
flavanone, chrysin, silymarin, and the two polymethoxyflavonoids,
nobiletin and tangeretin, were also examined. The UGT2B7
substrates retinoic acid (Carrier et al., 2000), morphine (Coffman et
al., 1997), and ibuprofen (Jin et al., 1993) were examined as examples
of non-UGT1A1 substrates. Estriol (16-hydroxy estradiol), which is
not a substrate of UGT1A1 but is structurally related to 17-EE, was
included in the study to help determine whether the modulating effects
of these compounds on estradiol-3-glucuronidation were unique to UGT1A1
substrates. In parallel, the effects of each of these compounds on
estradiol-17-glucuronidation were also determined.
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Materials and Methods |
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Materials and Methods
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Chemicals. Unless stated all chemicals were purchased from the Sigma-Aldrich (St. Louis, MO). Tangeretin was obtained from Indofine Chemical Co. (Somerville, NJ). Nobiletin from orange oil was a gift from Dr. William Widmer, Florida Department of Citrus, Lakeland, FL. PhIP was purchased from ICN Biomedicals Inc. (Costa Mesa, CA). Morphine was obtained from Sigma/RBI (Natick, MA).
Liver Specimens.
Human liver samples were obtained from the liver transplant unit at the
Medical College of Wisconsin under a protocol approved by the Committee
for the Conduct of Human Research. Microsomes from a mixture of four
human liver samples (B, H, M, and P) were prepared by differential
centrifugation as described previously (van der Hoeven and Coon, 1974)
and mixed for use in these assays. The livers were chosen because they
had average levels of estradiol-3-glucuronidation (UGT1A1) activities
(Fisher et al., 2000a).
Assay for Modulation of Estradiol Glucuronidation Activity. The primary focus of the study was to investigate modulation of UGT1A1 activity using estradiol-3-glucuronidation as a probe. Preliminary experiments (data not shown) were performed to ensure estradiol glucuronidation experiments were carried out under initial rate conditions. Preliminary range finding experiments (data not shown) were also performed at an estradiol concentration of 25 µM (near its S50 value, substrate concentration at half-maximal activity) to generate information on the potency of each compound as a modulator (at 5, 10, 30, 50, and 100 µM modulator) of estradiol-3-glucuronidation. Under these conditions, the 16 compounds either resulted in inhibition of estradiol 3-glucuronidation or had no effect. The results obtained in the preliminary studies were used to generate the appropriate ranges of modulator concentrations for subsequent experiments. Structures of each of the compounds are shown in Table 1.
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-Napthylglucuronide (4 nmol) was added as internal standard, and the
tubes were kept on ice for 30 min. The mixtures were centrifuged and
supernatant transferred to vials for mass spectrometry analysis. The
effects of 17-EE (10 µM) on estradiol glucuronidation at 1.25, 2.5, 5, 10, 15, 20, 30, 50, 75, and 100 µM estradiol were also examined.
Analyses of glucuronide formation were performed on a Micromass
platform LCZ system (Micromass Ltd., Manchester, UK) equipped with a
Waters Alliance 2690 separations module and column oven (30°C), a
3-µm, 100 × 2 mm Prodigy ODS (3) high performance liquid chromatography column (Phenomenex, Torrance, CA) and a SecurityGuard column (Phenomenex). The mobile phase solution A was 10 mM ammonium acetate, and solution B was 90% acetonitrile/10% water/10% ammonium acetate. Initial conditions were 85% A/15% B pumped at 0.25 ml/min. A
linear gradient from 15 to 31% B between 0 and 8 min was used, followed by 1 min at 100% B, and a re-equilibration at 15% B. Analytes were detected as their [M 
H]
ions using negative ion electrospray ionization. The source block temperature was 125°C, desolvation temperature was 400°C, capillary voltage was 2.70 KV, and the cone voltage was 30V. The internal standard -napthylglucuronide, estradiol-3-glucuronide, and
estradiol-17-glucuronide were detected by single ion monitoring at
m/z 319, 447, and 447 and eluted at 5.6, 8.5 and
9 min, respectively. Standard curve correlation coefficients
(r2) were 
0.99.
Data Analysis.
Duplicate values for rate of estradiol glucuronide formation for each
substrate concentration were fit to equations describing hyperbolic
(Michaelis-Menten, eq. 1; Segel, 1975) or sigmoidal (Hill, eq.
2; Segel, 1975) relationships using WinNonlin software (Pharsight
Corporation, Mountain View, CA). The best fit of the data to a
relationship was determined according to established criteria (Ring et
al., 1994).
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Materials and Methods
Results
Discussion
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Modulation of Estradiol-3-glucuronidation. In the absence of added modulator, enzyme kinetics consistent with homotropic activation were observed for estradiol-3-glucuronidation over a concentration range of 5 to 100 µM in each of 16 experiments. The mean Vmax (± standard deviation) and S50 (± standard deviation) values for the 16 determinations were 433 ± 79 pmol/mg/min and 22 ± 5 µM. The mean Hill coefficient value (n), which gives an indication of the degree of sigmoidicity of the curve (n of 1 = Michaelis-Menten kinetics, no sigmoidicity) was 1.9 ± 0.6.
The structures of the 16 compounds examined are shown in Table 1. Figures 1 and 2 show the modulatory effect of each of the flavonoids and nonflavonoid compounds, respectively, over the indicated range of concentrations on estradiol-3-glucuronidation. Results are expressed as a percentage of control activity at estradiol concentrations of 5, 10, 25, 50, and 100 µM. Interestingly, several patterns of interaction between the modulators and UGT1A1-catalyzed estradiol-3-glucuronidation were observed. The rates of estradiol 3-glucuronidation at all estradiol concentrations examined were basically unaffected by the addition of increasing concentrations of the UGT2B7 substrates ibuprofen and morphine (Fig. 2). The pattern expected for a competitive inhibitor of estradiol 3-glucuronidation is a decrease in activity as the modulator concentration increases. In addition for competitive inhibition, as substrate concentration increases the observed loss of glucuronide formation in the presence of inhibitor should decrease. Such patterns were observed for the effects of tangeretin (Fig. 1) and bilirubin (Fig. 2). However, as the kinetics of estradiol 3-glucuronide formation fitted the sigmoidal kinetic model best, and not hyperbolic kinetics described by the Michaelis-Menten equation, it was not appropriate to fit the inhibitory effects data to classic models of inhibition to generate Ki values.
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-EE was observed in two separate experiments designed to test for
this phenomenon (Figs. 2 and 3). Figure 2
shows that at a substrate concentration of 5 µM,
estradiol-3-glucuronidation was stimulated to 180% of the untreated
value by 10 µM 17-EE. However, at this substrate concentration, as
the 17-EE concentration increased, stimulation of
estradiol-3-glucuronidation was attenuated so that by 50 µM 17-EE,
estradiol-3-glucuronide formation was 125% of the control activity and
by 75 µM 17-EE, approximately 20% inhibition was observed. An
Eadie-Hofstee plot of this data (Fig. 3) clearly shows that increasing
concentrations of 17-EE (10 and 50 µM in this example) shift the
shape of the line from a "curve" (observed in the absence of
17-EE), which is typical of homotropic activation to a more linear
relationship observed for Michaelis-Menten behavior. To more fully
evaluate the effect of 17-EE on estradiol glucuronidation, 10 µM
17-EE was added to a wide range of estradiol concentrations, and its
effects on estradiol 3-glucuronidation and estradiol 17-glucuronidation
were examined (Fig. 4). The results
demonstrate that 10 µM 17-EE was shown to stimulate
estradiol-3-glucuronidation by as much as 180% of control values at
the low estradiol concentrations of 1.25 to 10 µM (below the
S50 value). This confirmed the observation of
heterotropic activation at low 17-EE and of substrate concentrations in the previously described experiment (Fig. 2) and that stimulation decreased with increasing substrate concentration. However, at higher
effector concentrations, estradiol 3-glucuronidation formation was
inhibited by 17-EE (Fig. 2). Interestingly, as would be expected for
competitive inhibition by 17-EE, estradiol 17-glucuronidation was
decreased by 10 µM 17-EE at lower substrate concentrations (Fig.
4). In addition, as shown in Fig. 6, estradiol 17-glucuronide formation
was inhibited by 17-EE in a concentration-dependent manner with the
greatest inhibition occurring at the lowest substrate concentration.
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-EE, PhIP, and anthraflavic acid.
Flavanone, naringenin, ibuprofen, and estriol had no effect on
estradiol 3-glucuronidation at a substrate concentration of 5 µM.
Information on the effects of morphine and silymarin at 5 µM were not
available due to technical difficulties with the assays. For silymarin,
estradiol 3-glucuronidation was 150% of control activity at 25 µM
estradiol and 10 µM silymarin (Fig. 1). There is no other data point
above 120% of control activity in this graph (Fig. 1). It is therefore
not appropriate to classify this compound as a stimulator (heterotropic
activator), since the data at 5 µM estradiol was incomplete for this
compound, and the apparent "stimulation" arising from a sporadic
single data point does not seem to follow a trend with regard to
estradiol concentration, as observed for 17-EE, for example
(described above and shown in Fig. 2).
Subtle differences in flavonoid structure had significant effects on
the ability of the flavonoid to modulate estradiol 3-glucuronidation (Table 1). The ring nomenclature for flavonoids is shown on flavone. A
hydroxyl group at position 3 is indicative of a flavonol (e.g., quercetin, Table 1), whereas saturation of the carbon atoms between position 2 and 3 is indicative of a flavonone (Table 1). The most
potent inhibitors of estradiol-3-glucuronidation at a concentration of
5 µM estradiol were the flavones, tangeretin
(4',5,6,7,8-pentamethoxyflavone, IC50 = 1 µM)
and chrysin (5,7-dihydroxyflavone, IC50 < 10 µM). The presence of a methoxy group at the 3' position appears to significantly reduce the inhibitory potency of flavones [e.g., nobiletin (3', 4',5,6,7,8-hexamethoxyflavone,
IC50 > 10 µM) compared with tangeretin].
Flavone, flavanone, silymarin, naringenin, and quercetin were
considerably less potent than tangeretin or nobiletin in inhibiting
estradiol 3-glucuronidation at a concentration of 5 µM estradiol (see
Fig. 1 and Table 1).
Modulation of Estradiol-17-glucuronidation.
Figures 5 and
6 show the effect of each of the
flavonoids and nonflavonoid compounds, respectively over the indicated
range of concentrations on estradiol 17-glucuronidation expressed as a
percentage of control activity at each of the 5 estradiol
concentrations examined. Hyperbolic Michaelis-Menten kinetics for the
formation of estradiol 17-glucuronide were observed in each experiment
yielding average Vmax and
Km values (± standard deviations) for
the 16 determinations of, respectively, 111 ± 26 pmols/mg/min and
7 ± 4 µM. 17-EE was a competitive inhibitor of
estradiol-17-glucuronidation (Ki = 20 µM) at the modulator
concentrations tested. Estriol and naringenin were found to be
noncompetitive inhibitors of estradiol 17-glucuronide and less potent
inhibitors (Ki >50 µM) than 17-EE. Interestingly, unlike the
behavior observed for estradiol 3-glucuronidation, none of the examined
compounds demonstrated a pattern of modulation consistent with
stimulation of estradiol 17-glucuronidation (Figs. 5 and 6), although
sporadic data points above 120% of control activity were observed in
some cases (e.g., at 25 µM estradiol and 10 µM silymarin, and at
100 µM estradiol and 50 µM flavone).
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The results presented in this study demonstrate that, depending on
effector and substrate concentrations, UGT1A1-catalyzed estradiol-3-glucuronidation activity is differentially sensitive to the
effects of additional UGT1A1 substrates and other compounds. For those
compounds (modulators) that stimulated estradiol 3-glucuronidation at
estradiol concentrations below its S50 value, the
greatest stimulation was observed at the lowest modulator
concentrations (Figs. 1 and 2). This was confirmed by a separate,
second experimental design using 17-EE as modulator (Fig. 4). At
higher concentrations of both modulator and substrate, no effect or
inhibition was observed (Figs. 1 and 2). Although this is the first
observation of heterotropic activation for a UGT enzyme, the in vitro
observation of crossover from stimulation to inhibition as modulator
concentration increases has also been observed for other enzymes
involved in endo- and xenobiotic metabolism [e.g., cytochrome P450
(Ekins et al., 1998; Maenpaa et al., 1998; Stresser et al., 2000)].
The demonstration of heterotropic activation of UGT1A1-catalyzed
estradiol-3-glucuronidation in the current study is consistent with the
previous studies (Fisher et al., 2000a,b) indicating homotropic
activation. Interestingly, homotropic activation kinetics for estradiol
3-glucuronidation activity have also been demonstrated using microsomes
expressing recombinant UGT1A1 in our laboratory, indicating that this
is a true behavior of the enzyme (unpublished observations).
Several compounds in the study were observed to be inhibitors of estradiol 3-glucuronidation by UGT1A1. Flavonoids were the most potent inhibitors with tangeretin being the most potent inhibitor of estradiol 3-glucuronidation. Unlike naringenin and the other hydroxylated flavonoids, which provide hydroxy groups for glucuronidation and are therefore substrates for UGT1A1, tangeretin has methoxy groups at various positions. It therefore appears that the most potent inhibitor determined in this study of UGT1A1-catalyzed estradiol-3-glucuronidation, tangeretin, is not a substrate of the enzyme.
The Hill equation (see "Data Analysis" under Materials and
Methods section) is a useful mathematical tool for describing the degree of sigmoidicity of the substrate concentration/enzyme activity relationship. It was originally proposed to provide an indication of
the number of subunits in a multimeric enzyme that bound successive ligands in a cooperative manner. The Hill coefficient value for UGT1A1-catalyzed estradiol 3-glucuronidation in the current set of
experiments was on average 1.9. There is evidence for UGT1A1 existing
as a tetramer (Peters and Jansen, 1986) and another UGT, UGT2B1
existing as a dimer (Meech and Mackenzie, 1997). Therefore, it is
tempting to speculate that UGT1A1 behaves as a cooperative ligand-binding multisubunit enzyme in microsomes with regard to UGT1A1-catalyzed estradiol 3-glucuronidation, thus explaining the
activation (homotropic and heterotropic) of estradiol
3-glucuronidation.
There are alternative explanations for the cooperative behavior of
UGT1A1 in human liver microsomes. One potential explanation is that
allosteric effector sites exist on the UGT1A1 enzyme or that two
substrate molecules occupy the UGT1A1 active site. For example,
a kinetic model (Korzekwa et al., 1998) describing this binding and
originally proposed to explain activation at low substrate concentrations and inhibition by effector at high substrate
concentrations for CYP3A-catalyzed reactions (Ueng et al., 1997)
adequately explains our data (results not shown). Therefore, should
this proposal also hold for UGTs, these two sites may be within the
UGT1A1 active site or on separate subunits, as described above. There
is evidence of overlapping but distinct binding locations for 17-EE
and bilirubin in the UGT1A1 active site (Ciotti and Owens, 1996).
However, there is no information to date on whether two estrogen-type
molecules can simultaneously occupy the active site.
It may be argued that the observed heterotropic and homotropic
activation of estradiol 3-glucuronidation is an artifact of the in
vitro systems examined. The lumenal location of UGT enzymes in the
endoplasmic reticulum presents a particular problem for investigating
UGT-catalyzed enzyme reactions. Sonication and detergents have been
used to overcome this problem but ultimately result in disruption of
the natural microenvironment of the enzymes. The alamethicin treatment
(used in the current study) of microsomes is not thought to be as
disruptive but does result in the free transit of substrate, cofactors,
and products to and from the UGT enzyme (Fisher et al., 2000a). Thus
the assay conditions used in the current studies appear to provide an
optimal system for examining the behavior of the UGT enzymes.
Furthermore, the lack of homotropic or heterotropic activation of
estradiol 17-glucuronidation over the same effector and substrate
concentration ranges demonstrates that activation is not a generalized
artifact of the incubation conditions. It is therefore appropriate to
conclude that the observations of homotropic and heterotropic
activation for estradiol-3-glucuronidation are true behaviors of UGT1A1
at least in vitro. However, further studies will need to be performed
to evaluate whether this in vitro phenomenon also occurs in vivo.
Observed Km/S50
values for UGT enzymes are often much higher than the plasma
concentrations of substrates (Senafi et al., 1994). For example, plasma
concentrations of estradiol are in the nanomolar range (Berrino et al.,
2001), and the S50 value for estradiol binding to
UGT1A1 is 22 µM. If UGT1A1 behaves in vivo as it does in vitro, then
at this low substrate concentration many compounds will be stimulators
of estradiol 3-glucuronidation activity (Table 1). The findings of this
study also raise an interesting point as to the ability of
alamethicin-treated human liver microsomes to predict drug-drug
interactions that result from the alteration of the catalytic activity
of UGT1A1. It seems appropriate that for the most accurate prediction
of drug-drug interactions, near-therapeutic concentrations of substrate
(and inhibitor) should be used rather than the higher concentrations reflecting the substrate Km or
S50 value. This study design may provide a more
relevant observation of enzyme behavior, as a compound predicted to be
an inhibitor from an experiment using the substrate at
Km concentrations may actually be a
stimulator or have no effect at therapeutic concentrations in vivo.
Although it has not been possible to identify all of the enzymes that
contribute to estradiol 17-glucuronidation in human liver microsomes,
UGT2B7 is known to be a contributor (Gall et al., 1999). In the current
study, three compounds were found to inhibit estradiol
17-glucuronidation. The competitive pattern of inhibition observed for
estradiol 17-glucuronidation in human liver microsomes suggests that
17-EE, estriol, and naringenin displace estrogen from the active
site(s) of enzymes responsible for estradiol 17-glucuronidation. As
already indicated, activation was not observed for this
biotransformation. Thus it appears that Michaelis-Menten relationships
between inhibitors and the substrate can be applied to the estradiol
17-glucuronidation results obtained in the current study.
In summary, evidence is provided for differential modulation, both
activation and inhibition of estradiol-3-glucuronidation by other
UGT1A1 substrates. For example, UGT1A1-catalyzed
estradiol-3-glucuronidation is stimulated by 17-EE at low substrate
concentrations. In contrast, bilirubin is a weak competitive-type
inhibitor. The most potent inhibitor of this activity at the lowest
estradiol concentration tested is the polymethoxyflavone, tangeretin
(IC50 = 1 µM at 5 µM estradiol
concentration). For compounds with similar structures to estrogens and
anthraflavic acid, the stimulatory effects on estradiol
3-glucuronidation appear to be dominant over the inhibitory effects at
low modulator concentrations, whereas at higher modulator concentrations, the inhibitory effects are dominant.
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Footnotes |
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Received June 4, 2002; accepted August 14, 2002.
1 Current address: Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Bldg. 25-235B, 2800 Plymouth Rd., Ann Arbor, MI 48105
Address correspondence to: Steven Wrighton, Department of Drug Metabolism, Drop Code 0710, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285. E-mail: wrighton_steven{at}lilly.com
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Abbreviations |
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Abbreviations used are:
UGT, UDP-glucuronosyltransferase;
17-EE, 17-ethynylestradiol;
PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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S. Takeda, Y. Kitajima, Y. Ishii, Y. Nishimura, P. I. Mackenzie, K. Oguri, and H. Yamada INHIBITION OF UDP-GLUCURONOSYLTRANSFERASE 2B7-CATALYZED MORPHINE GLUCURONIDATION BY KETOCONAZOLE: DUAL MECHANISMS INVOLVING A NOVEL NONCOMPETITIVE MODE Drug Metab. Dispos., August 1, 2006; 34(8): 1277 - 1282. [Abstract] [Full Text] [PDF]
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L. Xu, D. M. Krenitsky, A. M. Seacat, J. L. Butenhoff, T. R. Tephly, and M. W. Anders N-GLUCURONIDATION OF PERFLUOROOCTANESULFONAMIDE BY HUMAN, RAT, DOG, AND MONKEY LIVER MICROSOMES AND BY EXPRESSED RAT AND HUMAN UDP-GLUCURONOSYLTRANSFERASES Drug Metab. Dispos., August 1, 2006; 34(8): 1406 - 1410. [Abstract] [Full Text] [PDF]
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 E. Pfeiffer, C. R. Treiling, S. I. Hoehle, and M. Metzler Isoflavones modulate the glucuronidation of estradiol in human liver microsomes Carcinogenesis, December 1, 2005; 26(12): 2172 - 2178. [Abstract] [Full Text] [PDF]
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D. Zhang, T. J. Chando, D. W. Everett, C. J. Patten, S. S. Dehal, and W. G. Humphreys IN VITRO INHIBITION OF UDP GLUCURONOSYLTRANSFERASES BY ATAZANAVIR AND OTHER HIV PROTEASE INHIBITORS AND THE RELATIONSHIP OF THIS PROPERTY TO IN VIVO BILIRUBIN GLUCURONIDATION Drug Metab. Dispos., November 1, 2005; 33(11): 1729 - 1739. [Abstract] [Full Text] [PDF]
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 S. E. Kostrubsky, J. F. Sinclair, S. C. Strom, S. Wood, E. Urda, D. B. Stolz, Y. H. Wen, S. Kulkarni, and A. Mutlib Phenobarbital and Phenytoin Increased Acetaminophen Hepatotoxicity Due to Inhibition of UDP-Glucuronosyltransferases in Cultured Human Hepatocytes Toxicol. Sci., September 1, 2005; 87(1): 146 - 155. [Abstract] [Full Text] [PDF]
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L. Luukkanen, J. Taskinen, M. Kurkela, R. Kostiainen, J. Hirvonen, and M. Finel KINETIC CHARACTERIZATION OF THE 1A SUBFAMILY OF RECOMBINANT HUMAN UDP-GLUCURONOSYLTRANSFERASES Drug Metab. Dispos., July 1, 2005; 33(7): 1017 - 1026. [Abstract] [Full Text] [PDF]
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M. Tachibana, M. Tanaka, Y. Masubuchi, and T. Horie ACYL GLUCURONIDATION OF FLUOROQUINOLONE ANTIBIOTICS BY THE UDP-GLUCURONOSYLTRANSFERASE 1A SUBFAMILY IN HUMAN LIVER MICROSOMES Drug Metab. Dispos., June 1, 2005; 33(6): 803 - 811. [Abstract] [Full Text] [PDF]
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H. Yamanaka, M. Nakajima, M. Katoh, A. Kanoh, O. Tamura, H. Ishibashi, and T. Yokoi TRANS-3'-HYDROXYCOTININE O- AND N-GLUCURONIDATIONS IN HUMAN LIVER MICROSOMES Drug Metab. Dispos., January 1, 2005; 33(1): 23 - 30. [Abstract] [Full Text] [PDF]
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J. A. Williams, R. Hyland, B. C. Jones, D. A. Smith, S. Hurst, T. C. Goosen, V. Peterkin, J. R. Koup, and S. E. Ball DRUG-DRUG INTERACTIONS FOR UDP-GLUCURONOSYLTRANSFERASE SUBSTRATES: A PHARMACOKINETIC EXPLANATION FOR TYPICALLY OBSERVED LOW EXPOSURE (AUCI/AUC) RATIOS Drug Metab. Dispos., November 1, 2004; 32(11): 1201 - 1208. [Abstract] [Full Text] [PDF]
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 N. K. Basu, S. Kubota, M. R. Meselhy, M. Ciotti, B. Chowdhury, M. Hartori, and I. S. Owens Gastrointestinally Distributed UDP-glucuronosyltransferase 1A10, Which Metabolizes Estrogens and Nonsteroidal Anti-inflammatory Drugs, Depends upon Phosphorylation J. Biol. Chem., July 2, 2004; 279(27): 28320 - 28329. [Abstract] [Full Text] [PDF]
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V. Uchaipichat, P. I. Mackenzie, X.-H. Guo, D. Gardner-Stephen, A. Galetin, J. B. Houston, and J. O. Miners HUMAN UDP-GLUCURONOSYLTRANSFERASES: ISOFORM SELECTIVITY AND KINETICS OF 4-METHYLUMBELLIFERONE AND 1-NAPHTHOL GLUCURONIDATION, EFFECTS OF ORGANIC SOLVENTS, AND INHIBITION BY DICLOFENAC AND PROBENECID Drug Metab. Dispos., April 1, 2004; 32(4): 413 - 423. [Abstract] [Full Text] < |
, 5 µM;
, 10 µM; 
, 25 µM; 
, 50 µM; 
, 100 µM. Mean control
activities ± standard deviation (in the absence of modulator)
from 16 experiments in pmol/mg/min were 43 ± 12 at 5 µM
estradiol, 94 ± 15 at 10 µM, 210 ± 54 at 25 µM,
349 ± 94 at 50 µM, and 369 ± 106 at 100 µM. The mean
S50 (± standard deviation) was 22 ± 5 µM. The mean
Hill coefficient, which gives an indication of the sigmoidicity of the
curve, was 1.9 ± 0.6.
