DMD #30676
1
Investigation into UDP-glucuronosyltransferase enzyme kinetics of
imidazole and triazole containing antifungal drugs in Human Liver
Microsomes and Recombinant UGT Enzymes
Authors: Karine Bourcier Ruth Hyland Sarah Kempshall Russell Jones Jacqueline Maximilien Nicola Irvine Barry Jones Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich, UK. (K.B., R.H., S.K., R.J., J.M., B.J.), Department of Pharmacy and Pharmacology, University of Bath, UK (N.I.).
DMD Fast Forward. Published on March 19, 2010 as doi:10.1124/dmd.109.030676
Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
2
Running Title: Azole glucuronidation in HLM and rUGT
Address for correspondence:
Karine Bourcier
Pharmacokinetics Dynamics and Metabolism
Pfizer Global R&D
Ramsgate Road
Sandwich, Kent CT13 9NJ
UK
Tel: +44 1304 643646
Fax: +44 1304 651987
E-mail: [email protected]
Number of pages: 25
Number of Tables: 5
Number of Figures: 4
Number of References: 32
Word Count: Abstract: 245
Introduction: 690
Discussion: 1325
Abbreviations:
UGT, UDP-glucuronosyltransferase; rUGT, recombinant UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; HLM, human liver microsomes; QTOF, quadrupole time of flight; LC, liquid chromatography; MS, mass spectrometry; HPLC, high pressure liquid chromatography
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
3
Abstract
Imidazoles and triazoles represent major classes of antifungal azole derivatives. With
respect to UDP-glucuronosyltransferase (UGT) enzymes the drug metabolism focus
for these has mainly concentrated on their inhibitory effects with little known about
azoles as substrates for UGTs. N-glucuronide metabolites of the imidazole
antifungals, tioconazole and croconazole, have been reported but there are currently
no reports of N-glucuronidation of triazole antifungal agents. In this study, evidence
for glucuronidation of azole containing compounds was studied in human liver
microsomes (HLM). Where a glucuronide metabolite was identified azoles were
incubated in twelve recombinant UGT (rUGT) enzymes and enzyme kinetics
determined for the UGT with the most intense glucuronide peak. Six imidazole
antifungals, three triazoles and the benzodiazepine alprazolam (triazole) were
evaluated in this study. All investigated compounds were identified as substrates of
UGT. UGT1A4 was the main enzyme involved in the metabolism of all compounds
except for fluconazole which was mainly metabolised by UGT2B7, likely mediating
its O-glucuronide metabolism. UGT1A3 was also found to be involved in the
metabolism of all imidazoles but not triazoles. In both HLM and rUGT KM values
were lower for imidazoles (14.8 – 144 µM) than for triazoles (158-3037 µM), with the
exception of itraconazole (8.4 µM). All the imidazoles studied inhibited their own
metabolism at high substrate concentrations. In terms of UGT1A4 metabolism,
itraconazole showed kinetic features characteristic of imidazole rather than triazole
anifungals. This behaviour is attributed to the similar physicochemical properties of
itraconazole to imidazoles in terms of clog P.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
4
Introduction
Glucuronide conjugation is catalysed by a family of UDP-glucuronosyltransferase
(UGT) enzymes and occurs at nucleophilic functional groups containing oxygen (e.g.
hydroxyl or carboxylic acid), nitrogen (e.g. amines), sulphur (e.g. thiols) and more
rarely carbon (King et al., 2000). It is therefore not surprising to find that a myriad of
compounds, including drugs from all therapeutic classes, dietary chemicals,
environmental pollutants and endogenous substances such as bilirubin and bile acids,
are cleared by glucuronidation (Miners et al., 2004). UGTs catalyse nucleophilic
attack by the acceptor group of a substrate at the C1 position on the acid ring of UDP-
glucuronic acid (UDPGA cofactor) resulting in the formation of UDP and a ß-D-
glucuronide product (King et al., 2000). UGT proteins are located in the endoplasmic
reticulum with the active site situated on the luminal side and in the nuclear envelope
membranes (Tephly and Burchell, 1990). Glucuronidation may occur in most tissues
and in common with the cytochrome P450 family of enzymes UGTs abound in the
liver. However, some UGT enzymes, like 1A7, 1A8, 1A10, 2A1 and 2B17 are only
expressed in extra-hepatic tissues (Tukey and Strassburg, 2000). The most commonly
described pathway is O-glucuronidation which may have overshadowed N-
glucuronidation as discussed during the 1996 ASPET N-Glucuronidation of
Xenobiotics Symposium (Franklin, 1998). In an evaluation of the importance of local
chemical structure for metabolism by UGT enzymes most UGT isoforms were found
to have a preference for oxygen over nitrogen glucuronidation, with the converse for
UGT1A4 (Sorich et al., 2006).
N-glucuronidation may occur at various functional groups that include alkylamines,
arylamines, hydroxylamines, carbamates, ureas, thioureas and sulfonamides (Hawes,
1998). UGT1A4 is reported to be the major UGT involved in N-glucuronidation,
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
5
even though other UGT enzymes may also N-glucuronidate but to a lesser extent.
UGT1A3 and UGT1A4 were thought to be the only two enzymes able to
glucuronidate tertiary amines (Miners et al., 2004) however, more recently UGT2B10
has been shown to selectively glucuronidate medetomidine, a chiral imidazole, to the
N3-glucuronide of levomedetomidine (Kaivosaari et al., 2008). UGT2B10 has not
been extensively studied in the literature and may therefore be of greater importance
in N-glucuronidation reactions. Multiple examples of glucuronidation of tertiary
amines to quaternary ammonium glucuronides have been reported in the literature
with classes of compounds such as the tricyclic antidepressants (Lehman et al., 1983;
Breyer-Pfaff et al., 1997; Fischer and Breyer-Pfaff, 1997) and azole derivatives
(Vashishtha et al., 2001; Kaivosaari et al., 2008). Of the azole antifungals tioconazole
(Macrae et al., 1990), posaconazole (Ghosal et al., 2004) and croconazole (Takeuchi
et al., 1989) were the only three azole antifungal agents for which quaternary N-
glucuronidation evidence could be found in the literature and none are reported for
triazole antifungals.
However, a number of the azole antifungal agents have been shown to inhibit UGT
enzymes. Ketoconazole inhibits UGT 2B7-catalysed morphine glucuronidation
(Takeda et al., 2006), zidovudine glucuronidation (Sampol et al., 1995) and
recombinant UGT (rUGT) 1A1 and 1A9 catalysed 7-ethyl-10-hydroxycamptothecin
glucuronidation (Yong et al., 2005). The former researchers reported that although
ketoconazole is not known to be a substrate for UGT enzymes it appears to bind to the
substrate binding site of UGT2B7. Competitive inhibition of lorazepam
glucuronidation by fluconazole, miconazole and ketoconazole in vitro with rabbit
liver microsomes has also been observed (Sawamura et al., 2000). In addition the
imidazoles miconazole and ketoconazole were demonstrated to inhibit the
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
6
glucuronidation of zidovudine by human liver microsomes more potently than the
triazoles fluconazole and itraconazole (Sampol et al., 1995). In general however,
drug-drug interactions as a result of UGT inhibition are modest in magnitude
(Williams et al., 2004). For example, the in vitro inhibition of zidovudine metabolism
by fluconazole predicts an AUC ratio of 1.4 to 2.1 compared to the observed value of
1.9 (Uchaipichat et al., 2006b).
The aim of the current research was to investigate whether imidazole antifungals such
as ketoconazole and triazole antifungals like itraconazole form glucuronides
following incubations with human liver microsomes (HLM) and to identify the UGT
enzyme(s) involved (see figure 1 for structures of the imidazole and triazole
containing compounds investigated). The benzodiazepine alprazolam (triazole) was
also investigated for comparison with recently published data for midazolam (Hyland
et al., 2009).
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
7
Materials and Methods
Materials.
Fluconazole, tioconazole, ketoconazole, alprazolam, voriconazole were synthesised
by the Synthetic Services group, department of Chemistry, Pfizer Ltd (Sandwich).
All were of >98% purity. Econazole, miconazole, itraconazole, bifonazole,
sulconazole, dimethyl sulphoxide, alamethicin, D-saccharic acid 1,4-lactone
monohydrate (saccharolactone) and uridine 5’-diphosphoglucuronic acid (UDPGA)
were purchased from Sigma-Aldrich (Germany). Recombinant human UGT enzymes
and human liver microsomes were purchased from BD Biosciences (MA, USA). All
solvents were of HPLC grade and were purchased from Fisher Scientific (Germany).
Identification of Glucuronide Metabolites.
Human liver microsomes (pool of 60 donors) at 0.5 mg/mL, saccharolactone (5 mM),
alamethicin (50 µg/mg protein in incubation), MgCl2 (10 mM), Tris-HCl buffer pH
7.4 (50 mM) were mixed together and pre-incubated on ice for 15 minutes. The drug
solutions (100x DMSO stocks) were added to give final concentrations of 1, 10 and
50 µM and the incubation mix was pre-incubated on ice for a further 5 minutes.
Reaction mixtures were then warmed to 37 °C and the reaction was initiated by
addition of UDPGA (5 mM in incubation). Three volumes of ice-cold acetonitrile
were added to the reaction mixtures after 1 hour of incubation. The samples were
centrifuged at 3000 g for 1 hour at 4 °C and the supernatant was analysed by LC-MS
for identification of metabolites.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
8
Glucuronidation of Azole Containing Drugs in Human Liver Microsomes.
Enzyme kinetic experiments were performed in triplicate with human liver
microsomes. Incubation times and protein concentrations were optimised by
performing time courses for up to 120 minutes and protein courses up to 2 mg
protein/mL. Table 1 details the various optimum incubation times and protein
concentrations for each of the compounds studied. Incubations contained 50 mM
Tris-HCl pH 7.4 buffer, 10 mM MgCl2, 5 mM saccharolactone, alamethicin at 50
µg/mg protein, 5 mM UDPGA and azole substrate (50 nM to 1 mM, with a final
DMSO concentration of 1% v/v). Prior to substrate and UDPGA addition, the
reaction mixture was kept on ice for 15 minutes so that alamethicin, a pore-forming
peptide, had time to disrupt the microsomal membrane, since the UGT active site is
localised inside the endoplasmic reticulum (Fisher et al., 2000). Following pre-
incubation at 4 °C substrate was added and the reaction mixture warmed to 37 °C.
Incubations were initiated by addition of UDPGA and terminated with three volumes
of ice-cold acetonitrile containing 13.3 % of formic acid. Samples were centrifuged
at 3000 g for 45 minutes and the supernatant was analysed by LC-MSMS.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
9
UGT phenotyping.
In order to identify the UGT enzyme(s) responsible for glucuronidation of the azole
containing drugs being tested time courses under the assay conditions described above
were performed with the following recombinant UGT enzymes: UGT1A1, UGT1A3,
UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7,
UGT2B15 and UGT2B17 at 0.25 mg protein/mL in the incubation in quadruplicate.
Enzyme kinetic experiments were performed in triplicate, except where stated, with
the recombinant UGT enzyme identified in the UGT phenotyping experiment as being
the main enzyme involved. Experimental conditions were the same as described
above.
Enzyme Kinetics Analysis.
Reaction rates were calculated as peak area of glucuronide/mg protein/min as
metabolite standards were not available. The apparent kinetic parameters KM,
Relative Vmax and Ki (where substrate-inhibition was observed) were determined
using software GraFit 5.0. Where data fitted the Michaelis-Menten equation, the
following equation was applied:
v = (Vmax x [S]) / (KM + [S])
Where substrate-inhibition was observed the following equation (Houston and
Kenworthy, 2000) was applied:
v = (Vmax x [S]) / (KM + [S] + [S]2 / Ki)
Ki being the dissociation constant for the inhibitory substrate-enzyme-substrate
complex.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
10
Analytical Conditions.
For metabolite identification accurate mass determinations of parent compound and
glucuronide conjugates were carried out on a Waters QTOF Premier (Waters Ltd.,
Elstree, Herts, UK) operating in “V”mode using Leucine-Enkephalin (m/z 556.2771)
as lock spray internal mass calibrant. The instrument was operated under positive-ion
mode for full scanning acquisitions between m/z 100 and m/z 800 at 0.3 second per
scan. The LC system consisted of an 1100 binary pump (Agilent Tech. Inc., Palo
Alto, CA, U.S.A.), and a CTC PAL autosampler (Presearch Ltd, Basingstoke, Hants,
UK). Parent compound and glucuronide metabolites were resolved on a SunFireTM
3.5 µm C18 (100 x 2.1 mm ID) column (Waters Ltd., Elstree, Herts, UK) with a linear
gradient of acetonitrile (5-98 % over 8 min) in water, both containing 0.1 % (v/v)
formic acid, at a flow rate of 0.2 ml/min. Nitrogen was used as both the nebulising
and desolvation gas. The source and desolvation temperatures were 120ºC and 300°C
respectively; the capillary voltage was 3.2 kV, the sample cone voltage 25 V. MS/MS
analysis was carried out at a collision energy of 20 eV using Argon as collision gas.
Data was processed with MassLynx 4.0 sp4 (Waters Ltd., Manchester).
For LC-MS/MS quantitation a triple quadrupole mass spectrometer API4000 (AB
Sciex Instrument) was interfaced with Jasco X-LC binary pumps (JASCO UK, Ltd.,
Great Dunmow, Essex, UK) and a CTC analytics autosampler HTS PAL (Presearch
Ltd, Basingstoke, Hants, UK). The instrument was operated in the positive ion mode
using a Turbo Ionspray interface. Compounds were injected onto a Phenomenex
Synergi 2.5 µm fusion 30 x 2.0 mm column and eluted using a gradient method
starting at 97% aqueous , going down to 10% over 0.6 min, back to 97% at 0.61 min
and holding at 97% aqueous for a further 0.5 min, all at a flow rate of 1 mL/min.
Aqueous solvent contained 95% HPLC grade water, 5% acetonitrile and 0.1% formic
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
11
acid; organic solvent consisted of 100% acetonitrile and 0.1% formic acid. The
different transitions selected for each of the compounds are detailed in table 1.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
12
Results
Identification of glucuronide metabolites.
All of the compounds tested underwent glucuronidation to various extents. Based
upon peak intensity, econazole, miconazole, tioconazole, bifonazole, sulconazole and
ketoconazole showed more extensive glucuronidation with glucuronide formation
being at least an order of magnitude higher than that for alprazolam, fluconazole,
voriconazole and itraconazole. For all imidazole containing compounds, based upon
fragmentation patterns, there was evidence of N-glucuronide formation on the
imidazole ring. There was however insufficient MS/MS data to unambiguously
assign the site of glucuronidation for the triazole containing compounds. Based upon
precedence with tioconazole (Macrae et al., 1990), posaconazole (Ghosal et al., 2004)
and croconazole (Takeuchi et al., 1989) it is assumed that glucuronides forming on
the imidazole or triazole rings are quaternary N-glucuronides.
Enzyme Kinetics of Azoles Glucuronidation in Human Liver Microsomes.
Time linearity varied from one azole compound to the other and incubation times are
detailed in table 1. Glucuronide formation rate was proportional to microsomal
protein concentration up to 1 mg protein/mL for all compounds studied. Itraconazole
and all imidazole antifungal compounds fitted the substrate-inhibition model
described in the methods section as glucuronide formation rate started decreasing at
high substrate concentrations. The remaining substrates showed Michaelis-Menten
behaviour. Examples of typical Michaelis-Menten kinetics and substrate-inhibition
kinetics in HLM are shown in figure 2.
Relative Vmax, KM and Ki values obtained from incubations of azole compounds in
HLM are summarised in table 2. Relative Vmax values are reported as peak
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
13
area/min/mg protein as authentic standards of the glucuronides of interest were not
available. Relative Vmax values ranged from 29 to 447 peak area/min/mg protein for
triazole compounds and from 1158 peak area/min/mg protein to 25600 peak
area/min/mg protein for imidazole compounds. KM values ranged from 14.8 µM to
144 µM with imidazoles and from 8.4 µM to 3037 µM with triazoles.
rUGT phenotyping.
All compounds were incubated with 12 individual rUGT enzymes 1A1, 1A3, 1A4,
1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17 in order to identify the
enzyme(s) responsible for the glucuronidation of azoles. Examples of typical bar
charts of the screening results are shown in figure 3. Table 3 summarizes the
involvement of individual UGT enzymes in the metabolism of azole compounds.
rUGT1A4 was the main enzyme responsible for azoles glucuronidation except
fluconazole for which rUGT2B7 was flagged as the main enzyme involved.
Enzyme Kinetics in rUGT1A4 (rUGT2B7 for fluconazole).
With the exception of fluconazole, rUGT1A4 was found to be the major catalysing
enzyme for drug glucuronidation in vitro. Hence enzyme kinetics were determined
using rUGT1A4 for all compounds but fluconazole, for which UGT2B7 was used.
All imidazole antifungal compounds fitted the substrate-inhibition model as for
HLMs. Alprazolam, fluconazole and voriconazole fitted the Michaelis-Menten
model. Examples of the Michaelis-Menten kinetics and substrate-inhibition kinetics
in rUGT are shown in figure 4.
Relative Vmax, KM and Ki values obtained from incubations in rUGT enzymes are
summarised in table 4. Relative Vmax values ranged from 170 to 436 peak
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
14
area/min/mg protein for triazole compounds. Relative Vmax values ranged from 541
peak area/min/mg protein to 31445 peak area/min/mg protein for imidazole
compounds. KM values ranged from 7 to 124 µM with imidazoles and from 261 µM
to 1667 µM with triazoles. Poor assay sensitivity for itraconazole precluded accurate
determination of a KM value, however the KM appeared to be low (<10µM), in
keeping with the value observed in human liver microsomes.
Consideration of physicochemical properties.
Lipophilicity values (clogP) and dissociation constants relative to the azole moiety
(pKa) were obtained from ACDLABS 11.0 database and are tabulated in table 5. All
imidazoles had high pKa values compared to triazoles, whilst in terms of clogP the
triazoles were generally less lipophilic than the imidazoles, with the exception of
itraconazole and posaconazole with clogP values in the same range as the imidazoles.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
15
Discussion
While studies have been carried out on the effects of antifungals acting as inhibitors
of glucuronidation (Sampol et al., 1995; Takeda et al., 2006) this study highlights
their ability to also act as substrates of UGT. Whilst for some compounds
glucuronide metabolites have previously been reported ;(Takeuchi et al., 1989;
Macrae et al., 1990; Ghosal et al., 2004), this is the first report of the enzyme kinetic
characterisation of the UGT-mediated route of metabolism for this class of
compounds.
The MS/MS fragmentation of quaternary ammonium-linked glucuronide metabolites
(N+-glucuronides) under atmospheric pressure ionisation and fast bombardment
ionisation can provide useful information about the metabolite. An essential
component of this form of analysis is the monitoring of the ion corresponding to the
protonated aglycone that is formed from cleavage of the glycosolic bond (i.e. (M-
176)+). This method was utilised successfully in the present study to demonstrate that
all of the examined compounds were susceptible to glucuronidation. The size of the
glucuronide peaks for the imidazoles were at least an order of magnitude greater than
the triazoles, suggesting that imidazoles may be more susceptible to glucuronidation
than triazole compounds.
Transitions from mass spectrometric data suggest that with the exception of
fluconazole all of the glucuronides detected were formed at nitrogen atom(s) in the
imidazole or triazole ring, hence confirming that N+-glucuronidation is a relevant
pathway in human metabolism of drugs containing a tertiary amine group. Whilst C-
glucuronidation cannot be ignored, existing data for posaconazole, croconazole and
tioconazole support N-glucuronidation for this series of compounds (Takeuchi et al.,
1989; Macrae et al., 1990; Ghosal et al., 2004) It has been reported that the various
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
16
types of N-glucuronide metabolites differ markedly in their susceptibility to chemical
hydrolysis and, in general, the N-glucuronide metabolites of primary and secondary
amines are very unstable in solution below pH 7. N+-glucuronides on the other hand
tend to be stable in acidic solutions (Hawes, 1998). In the current study, incubations
were terminated by addition of acidified acetonitrile and glucuronides could be
detected which provides further evidence that quaternary ammonium-linked
glucuronides of azole compounds were being formed.
In the current study UGT enzyme kinetics of various imidazole and triazole antifungal
agents and one benzodiazepine (fused triazole-benzodiazepine ring) were initially
investigated in human liver microsomes. Following reaction phenotyping with 12
different recombinant UGT enzymes UGT1A4 was identified as the main enzyme
involved in the glucuronidation of all compounds tested with the exception of
fluconazole for which UGT2B7 was primarily responsible for its glucuronidation.
Enzyme kinetics with recombinant UGT1A4 and 2B7 in the case of fluconazole were
subsequently investigated.
The triazoles, alprazolam, voriconazole and fluconazole obeyed Michaelis-Menten
kinetics and showed low affinity for the enzyme responsible for their glucuronidation,
as shown by the high KM values in summary table 5. It is thought that because of the
presence of the hydroxyl group (-OH) in fluconazole’s chemical structure the O-
glucuronide is preferentially formed. Further evidence that the O-glucuronide of
fluconazole was preferentially formed over the N-glucuronide was that UGT2B7
rather than UGT1A4 was identified as the main enzyme responsible for its
metabolism. Current literature information suggests that only UGT1A3, UGT1A4
and UGT2B10 can form quaternary ammonium linked glucuronides (Miners et al.,
2004; Kaivosaari et al., 2008) hence a fluconazole glucuronide formed UGT2B7 is
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
17
unlikely to be the N-glucuronide. UGT1A4 was also found to be involved in
fluconazole metabolism but low LC-MS/MS signal precluded from running enzyme
kinetics with rUGT1A4. A second triazole, alprazolam, was found to be solely
metabolised by UGT1A4.
Itraconazole and all the imidazoles investigated did not obey Michaelis-Menten
kinetics as above a critical substrate concentration a decrease in metabolite formation
rate was observed, hence a substrate-inhibition model was fitted to the data. It is
often the case that a Michaelis-Menten model fits UGT enzyme kinetics satisfactorily,
however in recent years non Michaelis-Menten kinetics for the glucuronidation of a
number of drugs have been described, including biphasic and auto-activation kinetics
(Fisher et al., 2000; Lewis et al., 2007; Iwuchukwu and Nagar, 2008; Uchaipichat et
al., 2008; Hyland et al., 2009). Substrate inhibition, as observed here, is characterised
by a second molecule of substrate binding to the enzyme-substrate complex thereby
forming a ternary complex substrate-enzyme-substrate. Ki represents the dissociation
constant of the ternary complex, i.e. it is an (inverse) estimation of the substrate’s
affinity to a second inhibitory binding site on the enzyme. High Ki values will
indicate a low affinity for the enzyme-substrate complex (Pufall and Graves, 2002).
Substrate inhibition has been reported in the literature for a number of UGT enzymes
including UGT1A4 (Uchaipichat et al., 2006a; Iwuchukwu and Nagar, 2008).
Itraconazole, the benzodiazepine midazolam (Klieber et al., 2008; Hyland et al., 2009)
and the imidazole antifungals currently investigated all showed higher affinity for
UGT enzymes than alprazolam, voriconazole or fluconazole in HLM (see summary
table 5). They were all metabolised by UGT1A4 primarily, even though the
involvement of other UGT enzymes (UGT1A3 and UGT2B7 mainly) was identified
with all imidazoles, except ketoconazole. Posaconazole, an antifungal agent
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
18
structurally related to itraconazole, is reported to have similar KM values to
itraconazole: KM values of 27.9 µM in human liver microsomes and 15.9 µM in
UGT1A4 (Ghosal et al., 2004). In the current study the KM values determined in
rUGT were within 2-3 fold of values observed in HLM (with the exception of
tioconazole). Similar trends were observed in terms of alprazolam, voriconazole and
fluconazole showing lower affinity for UGT enzymes compared to the remaining
compounds in the series.
Lipophilicity values, dissociation constants relative to the azole moiety (pKa) and
enzyme kinetic values are tabulated in summary table 5 for each of the azole agent
studied and for midazolam (Hyland et al., 2009) and posaconazole (Ghosal et al.,
2004). In both HLM and rUGT a similar trend was observed in terms of KM values,
with all the imidazoles having higher affinity for UGT metabolism, along with
itraconazole and posaconazole. In terms of SAR, it has previously been reported that
the steric and electronic character immediately local to the nucleophilic atom is an
important predictor for glucuronidation (Sorich et al., 2006) and for many UGTs the
attachment of an aromatic ring to the nucleophile and the type of nucleophile
increases the likelihood for glucuronidation. From an evaluation of physicochemical
properties it was observed that all the imidazole nucleophiles were more basic than
the triazoles (pKA 6.03-6.88 for imidazoles compared to 2.44-2.94 for triazoles).
However for this group of compounds pKa alone could not account for the differences
observed in affinity for UGT1A4, with two of the triazoles (itraconazole and
posaconazole) having KM’s of a similar order of magnitude to the imidazole
compounds. For UGT1A4 a 2D-QSAR modelling technique has highlighted the
importance of hydrophobicity in substrate-enzyme binding (Smith et al., 2003). For
these azole compounds the impact of lipophilicity is also clear, with the compounds
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
19
exhibiting higher substrate affinity (all imidazoles, posaconazole and itraconazole)
being more lipophilic than the remaining low affinity triazoles (clog P values between
3.8 and 4.97 for imidazoles, 4.99 for itraconazole, 4.67 for posaconazole vs 0.45-1.92
for triazoles). In addition a study by Vashishtha (Vashishtha et al., 2002) has shown
that quaternary ammonium-linked glucuronidation of 1-substituted imidazoles by
liver microsomes was also influenced by lipophilicity. They reported that the greater
the lipophilicity of the imidazole derivative the higher the enzyme affinity. A similar
relationship has been observed here.
It is important to also consider the role microsomal binding will play on Km for this
group of compounds. In particular, for the more lipophilic molecules with clogP >4
the free Km will be significantly lower than total Km. Two algorithms for the
prediction of fraction unbound in the incubation have been proposed (Austin et al.,
2002; Hallifax and Houston, 2006) and both are based upon the lipophilicity of
compounds. All the UGT1A4 substrates here have a clogP > 1 and will likely have a
lower free Km. If correction for microsomal binding were incorporated it is expected
that the relationship discussed above would become more distinct, with the most
lipophilic compounds (clogP >3.8) having an even lower free Km than less lipophilic
triazoles (clogP 1.21, 1.92). The situation is slightly different for the UGT2B7
substrate fluconazole, since addition of albumen to incubations indicates that for
UGT2B7 a lower true Km is observed as a result of sequestering inhibitory fatty acids
released from the incubation (Rowland et al., 2007).
In summary, it has been demonstrated that all of the imidazole and triazole
compounds investigated are substrates for UGT enzymes, particularly UGT1A4. The
imidazole containing compounds were shown to have a greater affinity for UGT1A4
than triazole containing compounds with the exception of itraconazole and
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
20
posaconazole. The higher affinity of itraconazole and posaconazole for UGT
metabolism compared to other triazoles could be explained by physicochemical
properties. Whilst data already exists in the literature indicating that some of these
compounds can inhibit UGT2B7, with imidazoles being more potent inhibitors than
triazoles (Sampol et al., 1995; Takeda et al., 2006), it is highly likely that they will
also be inhibitors of UGT1A4. Based upon the KM values for UGT1A4, it is also
likely that imidazoles will exhibit greater inhibitory potency than triazole agents, with
itraconazole and posaconazole as the exceptions.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
21
Acknowledgements
This study was sponsored by Pfizer and all authors were Pfizer employees at the time
the work was performed, with the exception of Jacqueline Maximilien, who was a
student in the Department of Chemistry, Loughborough University, Loughborough,
UK, and under contract to Pfizer.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
22
References Austin RP, Barton P, Cockroft SL, Wenlock MC and Riley RJ (2002) The influence
of nonspecific microsomal binding on apparent intrinsic clearance, and its prediction from physicochemical properties. Drug Metab Dispos 30:1497-1503.
Breyer-Pfaff U, Fischer D and Winne D (1997) Biphasic kinetics of quaternary ammonium glucuronide formation from amitriptyline and diphenhydramine in human liver microsomes. Drug Metab Dispos 25:340-345.
Fischer D and Breyer-Pfaff U (1997) Variability of diphenhydramine N-glucuronidation in healthy subjects. Eur J Drug Metab Pharmacokinet 22:151-154.
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.
Franklin RB (1998) The N-glucuronidation of xenobiotics. An aspet-supported symposium held at the 1996 faseb meeting in washington, dc. Drug Metab Dispos 26:829.
Ghosal A, Hapangama N, Yuan Y, Achanfuo-Yeboah J, Iannucci R, Chowdhury S, Alton K, Patrick JE and Zbaida S (2004) Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of posaconazole (Noxafil). Drug Metab Dispos 32:267-271.
Hallifax D and Houston JB (2006) Binding of drugs to hepatic microsomes: comment and assessment of current prediction methodology with recommendation for improvement. Drug Metab Dispos 34:724-726; author reply 727.
Hawes EM (1998) N+-glucuronidation, a common pathway in human metabolism of drugs with a tertiary amine group. Drug Metab Dispos 26:830-837.
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.
Hyland R, Osborne T, Payne A, Kempshall S, Logan YR, Ezzeddine K and Jones B (2009) In vitro and in vivo glucuronidation of midazolam in humans. Br J Clin Pharmacol 67:445-454.
Iwuchukwu OF and Nagar S (2008) Resveratrol (trans-resveratrol, 3,5,4'-trihydroxy-trans-stilbene) glucuronidation exhibits atypical enzyme kinetics in various protein sources. Drug Metab Dispos 36:322-330.
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 glucuronosyltransferase (UGT) 1A4 and UGT2B10 account for the complex kinetics of human liver microsomes. Drug Metab Dispos 36:1529-1537.
King CD, Rios GR, Green MD and Tephly TR (2000) UDP-glucuronosyltransferases. Curr Drug Metab 1:143-161.
Klieber S, Hugla S, Ngo R, Arabeyre-Fabre C, Meunier V, Sadoun F, Fedeli O, Rival M, Bourrie M, Guillou F, Maurel P and Fabre G (2008) Contribution of the N-glucuronidation pathway to the overall in vitro metabolic clearance of midazolam in humans. Drug Metab Dispos 36:851-862.
Lehman JP, Fenselau C and Depaulo JR (1983) Quaternary ammonium-linked glucuronides of amitriptyline, imipramine, and chlorpromazine. Drug Metab Dispos 11:221-225.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
23
Lewis BC, Mackenzie PI, Elliot DJ, Burchell B, Bhasker CR and Miners JO (2007) Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation. Biochem Pharmacol 73:1463-1473.
Macrae PV, Kirrs M, Pullen FS and Tarbit MH (1990) Characterization of a quaternary, N-glucuronide metabolite of the imidazole antifungal, tioconazole. Drug Metab Dispos 18:1100-1102.
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.
Pufall MA and Graves BJ (2002) Autoinhibitory domains: modular effectors of cellular regulation. Annu Rev Cell Dev Biol 18:421-462.
Rowland A, Gaganis P, Elliot DJ, Mackenzie PI, Knights KM and Miners JO (2007) Binding of inhibitory fatty acids is responsible for the enhancement of UDP-glucuronosyltransferase 2B7 activity by albumin: implications for in vitro-in vivo extrapolation. J Pharmacol Exp Ther 321:137-147.
Sampol E, Lacarelle B, Rajaonarison JF, Catalin J and Durand A (1995) Comparative effects of antifungal agents on zidovudine glucuronidation by human liver microsomes. Br J Clin Pharmacol 40:83-86.
Sawamura R, Sato H, Kawakami J and Iga T (2000) Inhibitory effect of azole antifungal agents on the glucuronidation of lorazepam using rabbit liver microsomes in vitro. Biol Pharm Bull 23:669-671.
Smith PA, Sorich MJ, McKinnon RA and Miners JO (2003) Pharmacophore and quantitative structure-activity relationship modeling: complementary approaches for the rationalization and prediction of UDP-glucuronosyltransferase 1A4 substrate selectivity. J Med Chem 46:1617-1626.
Sorich MJ, McKinnon RA, Miners JO and Smith PA (2006) The importance of local chemical structure for chemical metabolism by human uridine 5'-diphosphate-glucuronosyltransferase. J Chem Inf Model 46:2692-2697.
Takeda S, Kitajima Y, Ishii Y, Nishimura Y, Mackenzie PI, Oguri K and Yamada H (2006) Inhibition of UDP-glucuronosyltransferase 2b7-catalyzed morphine glucuronidation by ketoconazole: dual mechanisms involving a novel noncompetitive mode. Drug Metab Dispos 34:1277-1282.
Takeuchi M, Nakano M, Mizojiri K, Iwatani K, Nakagawa Y, Kikuchi J and Terui Y (1989) Quaternary ammonium glucuronide of croconazole in rabbits. Xenobiotica 19:1327-1336.
Tephly TR and Burchell B (1990) UDP-glucuronosyltransferases: a family of detoxifying enzymes. Trends Pharmacol Sci 11:276-279.
Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581-616.
Uchaipichat V, Galetin A, Houston JB, Mackenzie PI, Williams JA and Miners JO (2008) Kinetic modeling of the interactions between 4-methylumbelliferone, 1-naphthol, and zidovudine glucuronidation by udp-glucuronosyltransferase 2B7 (UGT2B7) provides evidence for multiple substrate binding and effector sites. Mol Pharmacol 74:1152-1162.
Uchaipichat V, Mackenzie PI, Elliot DJ and Miners JO (2006a) Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone)
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
24
"probes" for human udp-glucuronosyltransferases. Drug Metab Dispos 34:449-456.
Uchaipichat V, Winner LK, Mackenzie PI, Elliot DJ, Williams JA and Miners JO (2006b) Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation. Br J Clin Pharmacol 61:427-439.
Vashishtha SC, Hawes EM, McCann DJ, Ghosheh O and Hogg L (2002) Quaternary ammonium-linked glucuronidation of 1-substituted imidazoles by liver microsomes: interspecies differences and structure-metabolism relationships. Drug Metab Dispos 30:1070-1076.
Vashishtha SC, Hawes EM, McKay G and McCann DJ (2001) Quaternary ammonium-linked glucuronidation of 1-substituted imidazoles: studies of human UDP-glucuronosyltransferases involved and substrate specificities. Drug Metab Dispos 29:1290-1295.
Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR and Ball SE (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32:1201-1208.
Yong WP, Ramirez J, Innocenti F and Ratain MJ (2005) Effects of ketoconazole on glucuronidation by UDP-glucuronosyltransferase enzymes. Clin Cancer Res 11:6699-6704.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
25
Figure Legends Figure 1. Structures of the various azole compounds studied.
Figure 2. Examples of enzyme kinetic plots obtained for A) Michaelis-Menten kinetics B)
substrate inhibition kinetics in human liver microsomes (each point represents mean and
standard deviation of 3 determinations). Eadie Hofstee plots as insets
Figure 3. Examples of reaction phenotyping plots obtained for, A) alprazolam (triazole), B)
sulconazole (imidazole). Data are replicates of 4 determinations ± standard deviation
Figure 4. Examples of enzyme kinetic plots obtained for A) Michaelis-Menten kinetics B)
kinetics , in recombinant UGT1A4 (each point represents mean and standard deviation of 3
determinations). Eadie Hofstee plots as insets
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
26
Compound
Optimised incubation
conditions Transitions
Collision
energy Incubation
time (min)
Protein conc
(mg/mL) Parent Q1-Q3
Glucuronide
Q1-Q3
Alprazolam 120 1 309 - 281 485 – 309 35 eV
Bifonazole 45 1 311 – 115 487 – 243 15 eV
Econazole 15 0.8 381 - 125 557 – 381 40 eV
Fluconazole 60 1 307 - 220 483 – 307 20 eV
Itraconazole 15 0.8 705 - 89 781 - 705 40 eV
Ketoconazole 15 0.8 531 - 489 707 - 531 55 eV
Miconazole 15 0.8 415 - 158 591 - 415 40 eV
Sulconazole 45 1 397 - 89 573 - 125 49 eV
Tioconazole 15 0.8 386 - 130 563 - 386 40 eV
Voriconazole 120 1 350 – 127 526 - 127 60eV
Table 1. Incubation times and protein concentrations optimised for each compound; parent
and glucuronide transitions and corresponding collision energies.
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
27
Compound * Relative Vmax (peak area of
glucuronide/mg protein/min)
KM (μM) Ki (μM)
Alprazolam 447 158 N/A
Fluconazole 365 3037 N/A
Itraconazole 29.3 8.4 421 (n = 2)
Voriconazole 427 550 N/A
Tioconazole 25613 14.8 170
Ketoconazole 1158 (n = 2) 23.3 (n = 2) 326 (n = 2)
Econazole 3743 29.5 286
Miconazole 3345 33.4 517
Bifonazole 17640 83.9 71.7
Sulconazole 3025 144 371
Table 2. Enzyme kinetic constants for glucuronide formation of various antifungals in HLM.
All values represent the geometric mean of three independent experiments (unless otherwise
indicated). (N/A = not applicable)
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
28
Compound UGT enzymes involved in increasing order of peak area
Alprazolam UGT1A4
Fluconazole UGT2B7>UGT1A4
Itraconazole UGT1A4
Voriconazole UGT1A4>>UGT2B17=UGT1A9=UGT1A6>UGT1A7=UGT1A3
Tioconazole UGT1A4>UGT2B7>UGT1A3
Ketoconazole UGT1A4
Econazole UGT1A4>UGT1A3>UGT2B7>UGT1A6
Miconazole UGT1A4>UGT1A3=UGT2B7>UGT2B4>>UGT1A1
Bifonazole UGT1A4>UGT1A3>UGT2B7>UGT1A9
Sulconazole UGT1A4>UGT1A3>UGT2B7>UGT1A1
Table 3. Recombinant UGT reaction phenotyping summary results
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
29
Compound * Relative Vmax (peak area of
glucuronide/mg protein/min)
KM (μM) Ki (μM)
Alprazolam 169 261 N/A
Fluconazole 158 1667 N/A
Itraconazole No data No data No data
Voriconazole 436 1130 N/A
Tioconazole 31445 124 245
Ketoconazole 541 (n = 2) 7 (n = 2) 941 (n = 2)
Econazole 6532 115 192
Miconazole 7513 58.7 475
Bifonazole 13268 28 47
Sulconazole 4902 35 290
Table 4. Enzyme kinetic constants for glucuronide formation of various antifungals in
recombinant enzyme UGT1A4 except for fluconazole for which UGT2B7 was the main
enzyme involved in its glucuronidation. All values represent the geometric mean of three
independent experiments (unless otherwise indicated). (N/A = not applicable)
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
DMD #30676
30
Compound KM (µM) in HLM Ki (µM) in HLM KM (μM) in rUGT Ki (μM) in rUGT pKA clogP
Triazoles
Alprazolam 158 N/A 261 N/A 2.37 1.92
Fluconazole 2691 N/A UGT2B7 substrate 2.44 0.45
Itraconazole 8.4 421 No data 2.94 4.99
Posaconazole 27.9* N/A 15.9* N/A 2.75 4.67
Voriconazole 550 N/A 1130 N/A 2.72 1.21
Imidazoles
Midazolam 46** 58** 64** 79** 6.03 3.8
Tioconazole 14.8 170 124 245 6.66 4.28
Ketoconazole 23.3 326 7 (n = 2) 941 (n = 2) 6.88 4.04
Econazole 30 286 115 192 6.68 4.48
Miconazole 33 517 58.7 475 6.64 4.97
Bifonazole 84 72 28 47 6.55 4.69
Sulconazole 144 371 35 290 6.55 4.36
* Ghosal et al 2004 ** Hyland et al 2009 Table 5. Summary table of KM and Ki values in HLM and rUGT1A4 and physicochemical properties (pKA and clogP).
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.109.030676
at ASPE
T Journals on A
ugust 28, 2021dm
d.aspetjournals.orgD
ownloaded from