Investigation into UDP-glucuronosyltransferase enzyme ......2010/03/19 · Enzyme kinetic...

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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

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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


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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.


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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,


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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


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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).


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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.


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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.


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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.


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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


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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.


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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


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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


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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.


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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


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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


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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


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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


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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


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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.


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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.


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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


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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.


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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)


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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


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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)


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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).


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