Interaction of dexloxiglumide, a cholecystokinin type-1 receptor antagonist, with human cytochromes P450

Received 21 July 2003Revised 30 October 2003

Accepted 28 January 2004Copyright # 2004 John Wiley & Sons, Ltd.

BIOPHARMACEUTICS & DRUG DISPOSITIONBiopharm. Drug Dispos. 25: 163–176 (2004)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdd.397

Interaction of Dexloxiglumide, a Cholecystokinin Type-1Receptor Antagonist, with Human Cytochromes P450

Michael Halla,*, Stefano Persianid, Yen-Ling Cheunga, Anne Matthewsa, Z. Richard Cybulskib,Jeremy D. Holdingc, Ram Kapile, Massimo D’Amatod, Francesco Makovecd and Lucio C. Rovatid

aDepartment of In Vitro Metabolism, Huntingdon Life Sciences Ltd, Huntingdon, UKbDepartment of Mass Spectrometry, Huntingdon Life Sciences Ltd, Huntingdon, UKcDepartment of Pharmacokinetics, Huntingdon Life Sciences Ltd, Huntingdon, UKdRotta Research Laboratorium S.p.A., Monza, ItalyeForest Laboratories Inc., Harborside Financial Center, Plaza Three, Suite 602, Jersey City, NJ 07311, USA

ABSTRACT: Dexloxiglumide (DEX) is a cholecystokinin type-1 receptor antagonist underdevelopment for the treatment of constipation-predominant irritable bowel syndrome. Studies ofthe potential interaction of DEX with human cytochromes P450 (CYPs) were conducted in vitro.DEX (300 mm), both with and without a 15-min pre-incubation, was incubated with pooled humanliver microsomes and substrates selective for each of eight CYPs. This resulted in > 30% inhibitionof tolbutamide 4-methyl-hydroxylase (CYP2C9/10) and lauric acid 11-hydroxylase (CYP2E1)activities. Mean Ki (SD) for CYP2C9/10 and CYP2E1 were 69.0 (24.3) and 426 (60) mm, respectively.Incubations of [14C]DEX with pooled human liver microsomes produced one major phase Imetabolic fraction, with Vmax ¼ 131 pmol/min/mg protein and Km ¼ 23:7mm. Further incubationswith (i) liver microsomes from 16 individual donors (correlation analysis), (ii) SupersomesTM and(iii) selective chemical inhibitors, implicated CYP3A4/5, CYP2B6 and CYP2C9 in the formation ofthis component. Thus, DEX interacts with CYP2C9 both as inhibitor (Ki ¼ 69:0mm) and as substratein vitro. However, based on the maximum concentration (27 mm) after repeated oral doses of 200 mgt.i.d. and the unbound fraction (0.03) of DEX in human plasma, no clinically relevant metabolicinteractions with other CYP substrates are predicted. Copyright # 2004 John Wiley & Sons, Ltd.

Key words: dexloxiglumide; cytochromes P450; CYP2C9; drug interactions

Introduction

Medicines are often concurrently administered topatients, thereby leading to the possibility that

some degree of drug–drug interaction will occur.This might be at the level of drug absorption,plasma and/or tissue protein binding, carrier-mediated transport across membranes (includinghepatic or renal uptake and biliary or urinaryexcretion), or metabolism [1–3]. The consequenceof such interactions may be to cause changes inthe pharmacokinetic and therefore, safety andefficacy, profile of a drug. Although only a smallproportion of drug–drug interactions proveto be clinically significant, the potential existsfor serious, even fatal adverse reactions toarise, particularly with drugs having a narrowtherapeutic index [2,3]. The majority ofdrugs presently prescribed are metabolized by

*Correspondence to: Department of In Vitro Metabolism, Hun-tingdon Life Sciences Ltd, Woolley Road, Alconbury, Huntingdon,Cambs., PE28 4HS, UK. E-mail: [email protected]

Parts of this work were presented in poster form at the 6thInternational Meeting of the International Society for the Study ofXenobiotics, Munich, Germany, 7–11 October 2001 (see Hallet al. Drug Metab Rev 2001; 33 (Suppl 1): 91; Persiani et al. DrugMetab Rev 2001; 33 (Suppl 1): 101) and at the 16th Annual Meetingof the Japanese Society for the Study of Xenobiotics, Kobe, Japan,17–19 October 2001 (see Hall et al. Xenobiotic Metab Dispos 2001; 16(Suppl): S 253).

cytochrome P450-dependent monooxygenase(CYP), a superfamily of enzymes that are foundin most organs of the body, but especially in theliver [4]. Consequently the inhibition of CYP-catalysed metabolism of one drug resulting fromthe co-administration of a second therapeuticagent serves as the basis for a large number ofclinically important drug–drug interactions[2,3,5]. Due to the routine availability of hu-man-derived material, together with an increasedknowledge of the enzymes themselves, it is nowcommon practice during the development of adrug candidate to use in vitro techniques toinvestigate the potential for drug interactions totake place. The results of such in vitro studies canthen be used to predict the clinical significance ofany interactions seen and to guide in theplanning of in vivo interaction studies [1–3,5,6].

Dexloxiglumide (DEX; Figure 1), (R)-4-(3,4-dichlorobenzamido)-5-[N-(3-methoxypropyl)-N-pentylamino]-5-oxopentanoic acid, is a selectiveand potent cholecystokinin (CCK) type-1 (CCK1)receptor antagonist. The peptide hormone CCKinteracts with receptors located both in the brainand in the gastrointestinal tract and acts a majorregulator of gall bladder contraction and pan-creatic enzyme secretion and is itself secreted inresponse to food intake [7,8]. It also regulatesmotor and sensory functions in the gut [9]. Dueto its antagonist activity toward CCK1 receptors[10,11], DEX entered clinical development for the

treatment of constipation-predominant irritablebowel syndrome (IBS) [12–16]. In contrast to thepharmacological effects of DEX, the ðSÞ- enantio-mer is devoid of any pharmacological activity[17].

After oral administration of single and re-peated doses of 200 mg t.i.d. (the therapeuticposology), the pharmacokinetics of DEX weredose- and time-independent in healthy malevolunteers [18]. The clearance of DEX in vivofrom humans is mainly through metabolism.Approximately 70% of the ½14C�DEX that wasadministered to healthy male volunteers, both byoral and intravenous routes was excreted in thefaeces, while another approximately 25% of thedose was found in the urine [19]. A proportion ofthe radiolabelled components of both excreta wasidentified as metabolites of DEX.

IBS patients are usually treated with severaldrugs, including anti-inflammatories, antacids,myorelaxants, prokinetics, laxatives, immuno-suppressants and antibiotics. Since several ofthese drugs are metabolized by CYPs, the presentinvestigation was conducted to determine (A) thepotential of DEX to inhibit human CYPs, and (B)the CYP enzymes involved in the human hepaticphase I metabolism of DEX in vitro. From theresults obtained, a prediction was made of anypossible clinically significant inhibitory meta-bolic interactions that might occur following theadministration of DEX with other therapeuticproducts.

Method

Chemicals

½14C�DEX (specific activity 32.83 mCi/mmol,radiochemical purity > 93%) was synthesized atHuntingdon Life Sciences Ltd. Non-radiolabelledDEX and all non-radiolabelled reference stan-dards were synthesized by Rotta ResearchLaboratorium S.p.A. All other chemicals werehigh grade materials purchased from commercialsources.

Microsomes

Pooled human liver microsomes (HLM)were prepared by mixing microsomes from six

DEX

*denotes position of the14 C radiolabel

NH

N

CH3

OH

CO2H

Cl

Cl

O

O

CR 3529

O

NH

N

CH3

OCH3

CO2H

ClCl

O

*

Figure 1. Chemical structures of DEX and of CR 3529

Copyright # 2004 John Wiley & Sons, Ltd. Biopharm. Drug Dispos. 25: 163–176 (2004)

M. HALL ET AL.164

individual donors (three male and three female,from various ethnic backgrounds) that wereobtained from Tissue Transformation Technolo-gies (Edison, NJ). Microsomes from 16 individualdonors used for the correlation analysis werefrom XenoTech LLC (Kansas City, KS), suppliedas their Reaction Phenotyping Kit. CYP2B6,CYP2C9, CYP3A4 and insect cell controlSupersomesTM were purchased from GentestCorporation (Woburn, MA).

The protein and total CYP concentrations ofpooled HLM were determined by establishedmethods [20,21], while those of HLM fromindividual donors and of SupersomesTM weretaken from the data sheets supplied with theseproducts.

CYP assays

7-Ethoxyresorufin O-deethylase (CYP1A1/2) wasassayed [22] using 0.25 mg microsomal protein ina total volume of 2 ml 100 mm phosphate buffer,pH 7.4. The reaction was started by the additionof 7-ethoxyresorufin (2.05 mm final concentration)in DMSO and the production of resorufinfollowed for up to 3 min at 308C with anHitachi F-4500 fluorescence spectrophotometer(Nissei Sangyo Co. Ltd, Wokingham, Berkshire,UK). Coumarin 7-hydroxylase (CYP2A6) wasassayed [23] using 0.25 mg microsomal proteinin a total volume of 2.5 ml 50 mm phosphatebuffer, pH 7.4. The reaction was started by theaddition of coumarin (100 mm final concentration)in methanol and the production of 7-hydroxy-coumarin followed for up to 4 min at 378Cwith an Hitachi F-4500 fluorescence spectropho-tometer. S-Mephenytoin-N-demethylase (CYP2B6)was assayed [24] using either 0.25 mg microso-mal protein or 25 pmoles CYP2B6 SupersomesTM

in a total volume of 0.25 ml 100 mm phosphatebuffer, pH 7.4. The reaction was started by theaddition of ½14C�S-mephenytoin (100 mm finalconcentration; specific activity 56 mCi/mmol,radiochemical purity 599%) in acetonitrile. Aftereither 60 min (HLM) or 90 min (SupersomesTM)incubation at 378C, the reaction was terminatedby addition of perchloric acid. Separation ofS-mephenytoin from its metabolite, R-ð-Þnirvanol,was achieved by linear gradient reversed-phase HPLC. Tolbutamide 4-methyl-hydroxylase

(CYP2C9/10) was assayed essentially as de-scribed [25]. For the evaluation of the inhibitionpotential of DEX, 0.5 mg microsomal protein in atotal volume of 0.25 ml 50 mm Tris-HCl buffer,pH 7.4 was used. For a positive controlassay, either 0.25 mg microsomal protein or25 pmoles CYP2C9 SupersomesTM in a totalvolume of 0.25 ml 100 mm phosphate buffer,pH 7.4 was used. In both cases, the reactionwas started by the addition of ½14C�tolbutamide(100 mm final concentration; specific activity61 mCi/mmol, radiochemical purity 599%) inacetonitrile. After 20 min (inhibition potential) or60 min (positive control assay) incubation at378C, the reaction was terminated by additionof perchloric acid. Following extraction intodiethyl ether, separation of tolbutamide from itsmetabolite, 1-butyl-3-(4-hydroxymethylphenyl)-sulphonylurea, was achieved by isocratic re-versed-phase HPLC. S-Mephenytoin 40-hydroxylase (CYP2C19) was assayed [24] using0.5 mg microsomal protein in a total volume of0.25 ml 100 mm phosphate buffer, pH 7.4. Thereaction was started by the addition of½14C�S-mephenytoin (200 mm final concentration)in acetonitrile. After 60 min incubation at 378C,the reaction was terminated by addition ofperchloric acid. Separation of S-mephenytoinfrom its metabolite, 40-hydroxymephenytoin,was achieved by gradient reversed-phase HPLC.Debrisoquine 4-hydroxylase (CYP2D6) was as-sayed [26] using 0.5 mg microsomal protein in atotal volume of 0.1 ml 50 mm Tris-HCl buffer, pH7.69. The reaction was started by the addition of[14C]debrisoquine sulphate (500 mm final concen-tration; specific activity 53 mCi/mmol, radio-chemical purity 599%) in buffer. After 90 minincubation at 378C, the reaction was terminatedby addition of perchloric acid. Separation ofdebrisoquine from its metabolite, 4-hydroxydeb-risoquine, was achieved by linear gradientreversed-phase HPLC. Lauric acid 11- and 12-hydroxylases (CYP2E1 and CYP4A9/11, respec-tively) were assayed [27] using 0.5 mg micro-somal protein in a total volume of 1.0 ml 500 mm

Tris-HCl buffer, pH 7.69. The reaction was startedby the addition of ½14C�lauric acid (100 mm finalconcentration; specific activity 55 mCi/mmol,radiochemical purity 599%) in buffer. After20 min incubation at 378C, the reaction was


DEXLOXIGLUMIDE INTERACTION WITH HUMAN CYP 165

terminated by addition of hydrochloric acid.Separation of lauric acid from its metabolites,11- and 12-hydroxylauric acids was achieved bylinear gradient reversed-phase HPLC. Testoster-one 6b-hydroxylase (CYP3A4/5) was assayedbased on the methods described [28,29]. For theevaluation of the inhibition potential of DEX,0.5 mg microsomal protein in a total volume of1.0 ml 80 mm Tris-HCl buffer, pH 7.32, containing240 mm KCl, 1.6 mm EDTA and 16 mm MgCl2 wasused. For a positive control assay, either 0.5 mgmicrosomal protein or 20 pmoles CYP3A4SupersomesTM in a total volume of 1.0 ml100 mm phosphate buffer, pH 7.4 was used.The reaction was started by the addition of½14C�testosterone (final concentration of either175 mm or 50 mm; specific activity 56 mCi/mmol,radiochemical purity 599%) in either methanolor acetonitrile for inhibition potential andpositive control assays, respectively. After10 min (inhibition potential) or 60 min (positivecontrol assay) incubation at 378C, the reactionwas terminated by addition of either ethylacetate or acetonitrile. Separation of testosteronefrom its metabolite, 6b-hydroxytestosterone, wasachieved by concave gradient reversed-phaseHPLC [30]. For all assays involving radiolabelledsubstrates, eluted radioactive components werequantified using a b-RAM radioisotope systemwith Laura software (LabLogic, Sheffield, UK).The linear response of the radiodetector waschecked by constructing a standard curve fromthe results obtained from analysis of a minimumof five different concentrations of each radiola-belled substrate. These ranged from approxi-mately the lower limit of detection to themaximum amount of radioactivity that wasanalysed. For each substrate, the lower limit ofquantification was equivalent to the lowestconcentration of compound analysed that wason the linear portion of the standard curve.

Potential inhibition of human CYPs by DEX

Preliminary incubation conditions. DEX (final con-centration: 300 mm) was incubated with pooledHLM and either NADPH generator or NADPH,depending on the assay, for both 0 and 15 min.This was followed by the addition of one of arange of CYP substrates that are generally

considered to be selective for CYP1A1=2, 2A6,2C9=10, 2C19, 2D6, 2E1, 3A4=5 and 4A9=11[31,32]. The effects of DEX on these activitieswere compared with those elicited by a range ofCYP-selective chemical inhibitors (furafylline(30 mm; CYP1A1=2), 8-methoxypsoralen (1 mm;CYP2A6), sulphaphenazole (20 mm; CYP2C9/10),tranylcypromine (100 mm; CYP2C19), quinidine(5 mm; CYP2D6), diethyldithiocarbamate (DEDC,300 mm; CYP2E1) and troleandomycin (TAO,100 mm; CYP3A4=5); the effects of DEDC onCYP4A9=11 were evaluated incidental to its useas a selective inhibitor of CYP2E1 in the sameassay).

Ki determinations for inhibition of CYP2C9=10 andCYP2E1 by DEX. DEX was incubated at finalconcentrations of 10, 30, 100, 300 and either600 mm (CYP2C9/10 assay) or 1000 mm (CYP2E1assay) with pooled HLM and NADPH, withoutany extended pre-incubation period. Final sub-strate concentrations of tolbutamide were 68, 203,606 and 1812 mm and of lauric acid were 33, 100,300 and 1000 mm.

Microsomal metabolism of ½14C�DEX

Incubation conditions. Incubation mixtures con-tained NADPH (2 mm) and either human livermicrosomal protein (0.5 mg) or SupersomesTM

(100 pmoles CYP) in 100 mm phosphate buffer,pH 7.4, to a total volume of 1 ml. Reactions wereinitiated by the addition of ½14C�DEX (finalconcentration: 25 mm, unless otherwise stated),in acetonitrile. The total proportion of organicsolvent was restricted to 1% (by volume) in allreaction mixtures. All incubations were per-formed at 378C in vessels open to the atmo-sphere. Reactions were terminated after 60 min(unless otherwise stated) by addition of chilledacetonitrile (0.5 ml). Following centrifugation,supernatants from samples containing final½14C�DEX concentrations of 520 mm were directlyanalysed by HPLC. Supernatants from samplescontaining final ½14C�DEX concentrations of520 mm were dried under N2 and reconstitutedin 200 ml Na=K phosphate buffer (0.1m, pH 7.4) :100 ml acetonitrile prior to analysis.


M. HALL ET AL.166

Metabolite profiling and quantification. Analysis ofincubation sample supernatants was by reversed-phase HPLC, using a Waters Alliance 2690separation module (Millipore Waters, HemelHempstead, Hertfordshire, UK). Samples wereapplied to an Econosphere C18 column (5 mm,250� 4.6 mm i.d.; supplied by Hichrom, Reading,Berkshire, UK), fitted with a C18 pre-column(Hichrom), and were eluted using an ammoniumformate (100 mm, pH 6.6):acetonitrile:water lineargradient, at a flow rate of 1 ml/min and ambienttemperature. The gradient was 95:5:0 (by vo-lume) to 5:95:0 (v/v) over 25 min, holding at thisfor 3 min, changing to 0:95:5 (by volume) over2 min, holding at this for a further 5 min,changing to 0:5:95 (by volume) over 5 min andthen back to 95:5:0 (by volume) over 5 min, beforeinjection of the next sample. The eluate wasmonitored for UV absorbance at a wavelength of244 nm, using a Waters 486 tunable absorbancedetector and for radioactivity using a b-Ramradioisotope system. Eluted radioactive compo-nents were quantified using Laura software. Theamount of radioactivity in a selected componentwas expressed as a percentage of total chromato-gram radioactivity, which was then related to theamount of ½14C�DEX present in the originalincubation solution. The lower limit of quantifi-cation of ½14C�DEX was 22.3 pmoles. The amountof radioactivity eluting with parent DEX in amicrosomal incubate was subtracted from thevalue of a control incubate (without microsomes),which was considered to be 100%, whilefor metabolite components the amount ofradioactivity present in components havingthe same elution time in control incubationswas subtracted from the microsomal incubatevalue.

LC-MS analysis of selected samples

Non-radiolabelled CR 3529 standard and super-natant from the incubation of ½14C�DEX (100 mm)with pooled HLM for 60 min were analysed byon-line LC-MS. A Finnigan TSQ 7000 LC-MS/MSsystem (Finnigan MF, San Jose, CA) coupled toon-line UV (Shimadzu SPD-10A; Dysons Instru-ments Ltd, Tyne & Wear, UK) and radioactivity(b-RAM) detectors were used. The HPLC columnwas a Phenomenex Luna C5 (150� 4.6 mm i.d.),

and the gradient mobile phase was 0:05% (v/v)ammonium acetate in water and acetonitrile, at aflow rate of 1 ml/min and ambient temperature.The gradient was 80:20 (v/v) for 1 min, changingto 5:95 (v/v) over the next 9 min and then back to80:20 (v/v) over 1 min and holding at this for afurther 3 min before injection of the next sample.The eluate from the HPLC was passed throughthe UV detector and then a stream splitter suchthat 90% of the eluate flowed through theradioactivity detector and the remainder wasintroduced into the source of the mass spectro-meter for electrospray ionization operating innegative ion mode. The spray voltage andcapillary temperature of the ionization sourcewere set at 4.5 kV and 2508C, respectively.Nitrogen was used as the sheath gas at a pressureof 66 p.s.i. and also as the auxiliary gas at ca10 units. Mass spectrometric data wereacquired over a designated scan range at a rateof 1 s/scan.

Possible DEX-related components were locatedfrom the chromatograms of both the UV and theradioactivity detectors so that their mass spectracould be obtained by averaging several scansacross the analogous region of the mass chroma-togram after background subtraction. Deproto-nated molecular ions of interest ð½M-H��Þ wereselected for MS/MS analysis in product ion scanmode where the precursor ion was fragmentedby collision induced dissociation (argon, ca2 mTorr) in the Q2 region at collision energy(25 eV) empirically optimized so as to providemaximum structural information. A mass spec-trum of the fragments produced was obtained byscanning Q3 across the appropriate mass range ata rate of 1 s/scan. Several scans were averaged soas to produce the product ion mass spectrum ofthe putative metabolites.

Data processing and statistical analysis

For the determination of Ki, the rates of CYPactivity (at varying substrate concentrations) inthe presence of differing concentrations of DEXwere analysed by simultaneous non-linear re-gression analysis, using the software WinNonlinPro version 3 (Pharsight Corporation, MountainView, CA). The non-linear expression for compe-titive enzyme inhibition (below) was used to



model each data set.

v ¼Vmax: ½S�

Km 1 þ½I�Ki

� �þ ½S�

where v is the rate of metabolite formation, Vmax

is the maximal rate of metabolite formation, Km isthe Michaelis constant, Ki is the inhibitor con-stant, [I] is the inhibitor concentration and ½S� isthe substrate concentration. No weighting wasapplied to the data.

To estimate Vmax and Km; the rates of formationof the major radiolabelled metabolite fraction(termed MF) from ½14C�DEX at eight substrateconcentrations in the range 1–100 mm were ana-lysed by non-linear regression analysis, using thesoftware WinNonlin Pro version 3. The Michae-lis-Menten equation (below) was fitted to thedata

v ¼Vmax: ½S�Km þ ½S�

(see above for definitions). No weighting wasapplied to the data.

For correlation analysis, all parameters werefirst logarithmically transformed. Pearson corre-lation coefficients were then calculated betweenthe rate of formation of MF and the activities ofdifferent CYP in the 16 individual human livermicrosomes examined. Data were transferredfrom Excel spreadsheets to SAS datasets. Thestatistical analysis was performed in SAS 6.12(SAS Institute Inc., Cary, NC).

Results

Potential inhibition of cytochromes P450 by DEX

Preliminary incubations. The effects on CYP-cata-lysed reactions of the co-incubation of DEX(300 mm) with each selective substrate are sum-marized in Figure 2. These data indicate that, atthis concentration, DEX caused substantial(> 30%) inhibition of both tolbutamide 4-methyl-hydroxylase (CYP2C9=10) and lauricacid 11-hydroxylase (CYP2E1) activities. Themagnitude of these effects was similar regardlessof whether or not DEX had been pre-incubatedwith HLM in the presence of NADPH. Thus,

there was no evidence that DEX was acting as amechanism-based inhibitor of either of theseCYP.

Ki determinations for inhibition of CYP2C9/10 andCYP2E1 by DEX. Five concentrations of DEXwere each incubated with pooled HLM and fourconcentrations of both tolbutamide and lauricacid, in order to provide data to estimate the Ki

for inhibition of CYP2C9/10 and CYP2E1, re-spectively. However, the results that were ob-tained from incubations with the highestconcentration of tolbutamide (1812 mm) were notused in the calculation of Ki. Examination of theremaining data for both tolbutamide 4-methyl-hydroxylase and lauric acid 11-hydroxylase, bysimultaneous non-linear regression analysis gaveestimated values of Ki�standard deviation forCYP2C9=10 ¼ 69:0 � 24:3 mm and of CYP2E1 ¼426 � 60 mm: The kinetic plots (Figures 3 and 4)that were generated were reasonably consistentwith DEX acting as a competitive inhibitor ofboth CYP [32,33].

It can be assumed that the maximum concen-tration of unbound compound around the hepa-tic enzyme ðCi;maxÞ is approximately equal tothe unbound concentration in the blood closeto the entrance of the liver ðIin;uÞ [1]. In turn,Iin;u ¼ fuðImax þ ðka:D:Fa=QhÞÞ, where fu is thefraction of unbound drug in the blood (¼ 0:03for DEX [19]), Imax is the maximum concentrationin the systemic circulation (¼ approximately27 mm for DEX, at steady-state after repeated oraldoses of 200 mg given t.i.d. [18]), ka is theabsorption rate constant (¼ 0:1=min)*, D is thedose (¼ 434 mmoles for DEX, corresponding to anoral dose of 200 mg; MW ¼ 461), Fa is the fractionabsorbed ð¼ 1Þ*, Qh is the hepatic blood flowð¼ 1:61 l=minÞ* ð* these are theoretical maximumvalues, to avoid making false–negative predic-tions [1]). On this basis, the Ci;max forDEX ¼ 1:62 mm and the ratio of Ci;max=Ki (forCYP2C9/10)¼ 0:023.

Microsomal metabolism of ½14C�DEX

Initially, ½14C�DEX, at nominal final concentra-tions of 1, 3, 10, 30 and 100 mm, was incubatedwith pooled HLM for 5, 10, 20 and 60 min. Theincubations were stopped and the solutions


M. HALL ET AL.168

processed as described in the Method. Thesewere then analysed by reversed-phase HPLC andthe radioactive components in each eluate weredetermined by on-line radiodetection. The pro-files obtained indicated the presence of one majorradiolabelled fraction (termed MF; Rt� 17 min)in addition to parent compound (Rt� 19 min).Co-chromatography of microsomal incubateswith authentic non-radiolabelled reference stan-dards indicated the chromatographic equiva-lence of MF and the O-demethylated derivativeof DEX, CR 3529 (Figure 1). However, thepresence of additional DEX derivatives withinMF could not be precluded. Under the incubationconditions employed here, the rate of formationof MF was linear with time for up to 60 min at allinitial ½14C�DEX concentrations examined.

Kinetic parameters for the formation of MFwere determined from incubations of ½14C�DEX(nominal final concentrations of 1, 2.5, 5, 10, 20,40, 60 and 100 mm) with pooled HLM for 60 min.Non-linear regression analysis indicated that asingle enzyme model (correlation coefficient of0.967) adequately described the data, as shown inFigure 5. From these data, it was calculated thatthe formation of MF from [14C]DEX was cata-lysed with Vmax¼ 131 pmol=min=mg protein andan estimated Km¼ 23:7 mm.

Characterization of the CYP(s) catalysing forma-tion of MF

Correlation analysis. ½14C�DEX (25 mm) was incu-bated with hepatic microsomes prepared from 16

Figure 2. Effects of DEX and of selective chemical inhibitors on CYP-dependent activities. Substrates selective for the eight CYPsshown were incubated with pooled human liver microsomes and NADPH in the presence of either DEX (300mm), both with andwithout a 15 min pre-incubation, selective CYP inhibitor, or appropriate solvent. Each column represents the activity measured inthe presence of either DEX or the selective inhibitor expressed as a percentage of the solvent control (taken as 100%) andrepresents the mean of two separate incubations. Selective activities and chemical inhibitors used were as follows. For CYP1A1/2,7-ethoxyresorufin O-deethylase and furafylline (30mm); for CYP2A6, coumarin 7-hydroxylase and 8-methoxypsoralen (1mm); forCYP2C9/10, tolbutamide 4-methyl-hydroxylase and sulphaphenazole (20 mm); for CYP2C19, S-mephenytoin 40-hydroxylase andtranylcypromine (100mm); for CYP2D6, debrisoquine 4-hydroxylase and quinidine (5mm); for CYP2E1, lauric acid 11-hydroxylaseand DEDC (300mm); for CYP3A4/5, testosterone 6b-hydroxylase and TAO (100 mm); for CYP4A9/11, lauric acid 12-hydroxylase(the effects of DEDC on CYP4A9/11 were evaluated incidental to its use as a selective inhibitor of CYP2E1 in the same assay)



individual human donors and the radioactivecomponents present in the incubates separatedand quantified as before. The rates of formationof MF ranged by 7-fold among the 16 individualmicrosomal preparations. Comparisons of theserates with activities of individual CYPs measuredin the same microsomes (data supplied byXenoTech LLC) indicated that there were statis-tically significant correlations between MF for-mation and CYP2B6 (r ¼ 0:748, p ¼ 0:0009),CYP2C8 (r ¼ 0:609, p ¼ 0:0123), CYP2C9/10(r ¼ 0:531, p ¼ 0:0342) and CYP3A4/5 (r ¼ 0:885,p ¼ 0:0001) (Table 1). Within the bank of humanliver microsomes used here, there was statisti-cally significant (p50.01) correlation betweenmarker activities of CYP1A2 and CYP4A9/11(r ¼ 0:62) and CYP2B6 and CYP2C8 (r ¼ 0:69)(data supplied by XenoTech LLC).

Further investigations were then made of theinvolvement of CYPs 2B6, 2C9=10 and 3A4=5 inthe metabolism of DEX.

Selective chemical inhibitors. ½14C�DEX (25 mm) wasincubated with pooled HLM in the presence ofchemical inhibitors effective against CYP2B6(orphenadrine), CYP2C9=10 (sulphaphenazole)and CYP3A4=5 (TAO), each at concentrations of0, 2.5, 25 and 250 mm. Both sulphaphenazole andTAO inhibited the formation of MF in a concen-tration-dependent manner, by 32%, 54% and 72%in the case of sulphaphenazole and by 50%, 59%and 66% in the case of TAO. Both of theseinhibitors also effected inhibition of the markeractivities tolbutamide 4-methyl-hydroxylase andtestosterone 6b-hydroxylase, respectively. There

-600 -500 -400 -300 -200 -100 0 100 200 300 400 500 600

DEX concentration (µM)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

1/(R

ate

of to

lbut

amid

e m

etab

olite

form

atio

n, p

mol

/min

/mg

prot

ein)

68 µM Tolbutamide203 µM Tolbutamide606 µM Tolbutamide

Figure 3. Effect of the incubation of each of five separateconcentrations (10, 30, 100, 300 and 600mm) of DEX on the rateof formation of 1-butyl-3-(4-hydroxymethylphenyl)sulphony-lurea from tolbutamide, incubated at three separate concen-trations (68, 203 and 606mm) with pooled human livermicrosomes and NADPH, presented as the reciprocal of therate of formation of 1-butyl-3-(4-hydroxymethylphenyl)sul-phonylurea versus DEX concentration (Dixon plot)

-1000 -800 -600 -400 -200 0 200 400 600 800 1000

DEX concentration (µM)

0

1

2

3

4

5

6

7

8

1/ (

Rat

e of

11-

hydr

oxyl

aura

te fo

rmat

ion,

nm

ol/m

in/m

g pr

otei

n)

33 µM Lauric acid100 µM Lauric acid300 µM Lauric acid1000 µM Lauric acid

Figure 4. Effect of the incubation of each of five separateconcentrations (10, 30, 100, 300 and 1000mm) of DEX on therate of formation of 11-hydroxylaurate from lauric acid,incubated at four separate concentrations (33, 100, 300 and1000mm) with pooled human liver microsomes and NADPH,presented as the reciprocal of the rate of formation of 11-hydroxylaurate versus DEX concentration (Dixon plot)


M. HALL ET AL.170

was no consistent evidence for an effect oforphenadrine in these incubations.

Expressed cytochromes P450. The incubation of½14C�DEX (25 mm) with CYP2B6, CYP2C9 (both100 pmol CYP/ml) and CYP3A4 SupersomesTM

(20 pmol CYP/ml) gave rates of MF formation of0.02, 1.38 and 0.57 pmol/min/pmol CYP, respec-tively. Each of the CYP SupersomesTM metabo-lized their respective selective substrates,S-mephenytoin, tolbutamide and testosterone to

R-(-)nirvanol, 1–butyl-3-(4-hydroxymethylphe-nyl)sulphonylurea and 6b�hydroxytestosterone,respectively. Thus the SupersomesTM were cata-lytically active under the incubation conditionsused here.

Structural identification of metabolites

LC-MS analysis of the 100 mm ½14C�DEX 60 minmicrosomal incubation sample indicated thepresence of unchanged ½14C�DEX ðm=z 459Þ and

0 1 2 3 4 5

(Rate of MF formation, pmol/min/mg protein) / (DEX concentration, µM)

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te o

f M

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orm

atio

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ol/m

in/m

g p

rote

in)

Figure 5. Eadie-Hofstee plot showing the rate of formation of MF versus rate of MF formation relative to DEX concentration,following incubation of [14C]DEX, at eight concentrations, with pooled human liver microsomes and NADPH



at least one other related component (m=z 445).Both of these gave ion clusters, with m=z 459/461/463 and 445/447/449, respectively, whichare a characteristic isotope pattern indicating thepresence of two chlorine atoms within themolecule. The product ion mass spectra of thesecomponents obtained following LC-MS/MS ana-lysis of the microsomal incubation sample areshown in Figures 6 and 7, respectively. Thefragmentation observed for ½14C�DEX can berationalized as shown in Figure 6. LC-MS/MSanalysis of non-radiolabelled CR 3529 standardgave a product ion mass spectrum that wassimilar to that shown in Figure 7 and which canbe rationalized according to the structure shown.Therefore the fragmentation observed for theradiolabelled microsomal component with m=z445 is consistent with this proposed structure.

Discussion

It is generally accepted that the major hepaticisoforms of cytochrome P450 that are involved inxenobiotic biotransformations in humans areCYP1A2, 2C9/10, 2C19, 2D6 and 3A4 [34]. Wheninvestigating the disposition of a drug candidatein a human, it is therefore important to determineif the compound is capable of interactingwith these isoforms, either as substrate, induceror inhibitor, as this may affect both the safety

and efficacy of co-administered drugs, orrender prodrugs pharmacologically ineffective.Although most interactions that can occur aremanageable, usually by appropriate dosageadjustment, a few may be potentially life threa-tening [2].

In the present study, the potential inhibitoryproperties of DEX were investigated by incubat-ing the compound with human liver microsomespooled from six individual donors and substratesselective for eight CYPs. DEX was both added tothe incubation mixture immediately before theselective substrate, used to initiate the reaction,and was pre-incubated with HLM and NADPHfor about 15 min prior to addition of the selectivesubstrate. This latter method was designed toaccommodate the possibility of the test com-pound requiring metabolic activation to exert aninhibitory effect [32]. DEX was incubated at aconcentration of 300 mm, since this concentrationis well in excess of the maximum plasmaconcentration (plasma protein-bound plus un-bound) observed in human at steady-state(27 mm), after repeated oral doses of 200 mgadministered t.i.d. ([18] and unpublished re-sults). These adopted experimental conditionswere considered to be clinically relevant sinceDEX is eliminated mainly by hepatic biotrans-formation [19] and the 200 mg t.i.d. dosingschedule is that chosen for ongoing trials inwhich the efficacy of the drug is being tested inconstipation-predominant IBS patients.

Table 1. Correlation of rates of formation of MF from [14C]DEX (25 mm) in incubations with liver microsomes from 16 individualhuman donors, with activities selective for various CYPs

CYP Activitya MF formation

r p

1A1/2 7-Ethoxyresorufin O-deethylase �0.178 >0.052A6 Coumarin 7-hydroxylase 0.196 >0.052B6 S-Mephenytoin N-demethylase 0.748 0.00092C8 Paclitaxel 6a-hydroxylase 0.609 0.01232C9/10 Diclofenac 40-hydroxylase 0.531 0.03422C19 S-Mephenytoin 40-hydroxylase 0.199 >0.052D6 Dextromethorphan O-demethylase 0.207 >0.052E1 Chlorzoxazone 6-hydroxylase 0.008 >0.053A4/5 Testosterone 6b-hydroxylase 0.885 0.00014A9/11 Lauric acid 12-hydroxylase 0.155 >0.05

a Data provided by XenoTech LLC.

r, Pearson correlation coefficient based on log data; p, value for significance of correlation coefficient versus zero.


M. HALL ET AL.172

Of the CYP activities examined, only two,tolbutamide 4-methyl-hydroxylase and lauricacid 11-hydroxylase, were inhibited by > 30%following incubation with DEX (300 mm), effectswhich were considered to be significant. Conse-quently, the Ki for the inhibition of both of theseactivities by the test compound was determined.The results from the evaluation of the Ki fortolbutamide 4-methyl-hydroxylase inhibition in-dicated that DEX is likely to be a competitive

inhibitor of CYP2C9/10, with a Ki of 69.0 mm anda calculated Ci;max=Ki ratio of 0.023 (where Ci;max

is the estimated maximum concentration ofunbound compound around the hepatic en-zyme). A Ki> 50 mm; together with a Ci;max=Ki

ratio of 50.1, is generally considered to indicatethat the possibility of an interaction occurringin vivo is remote [1,35]. There was no evidencefrom incubations of [14C]DEX for non-specificbinding of DEX to human liver microsomes.Therefore, the results obtained in the present

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-CH2=CHCO2H-CH2=CHCO2H-CO2

m/z 145

m/z 188/189

m/z 441

m/z 415

m/z 300

m/z 387m/z 228m/z 256

(A)

(B)

Figure 6. Mass spectrum of component with m/z 459(unchanged parent compound) in the 100mm [14C]DEX60 min microsomal incubation sample. The sample wasintroduced into the mass spectrometer via HPLC. LC-MSanalysis was conducted with electrospray ionization operat-ing in negative ion mode. LC-MS/MS analysis was subse-quently performed in product ion scan mode. (A) Product ionmass spectrum, (B) rationalization of the observed fragmenta-tion pattern

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m/z 145

m/z 188/189

m/z 427

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m/z 373m/z 228m/z 256

(A)

(B)

Figure 7. Mass spectrum of component with m/z 445 in the100mm [14C]DEX 60 min microsomal incubation sample. Thesample was introduced into the mass spectrometer via HPLC.LC-MS analysis was conducted with electrospray ionizationoperating in negative ion mode. LC-MS/MS analysis wassubsequently performed in product ion scan mode. (A)Product ion mass spectrum, (B) rationalization of theobserved fragmentation pattern obtained for authentic stan-dard CR 3529 (m/z 445)



study indicate that DEX would not be expectedto produce any clinically relevant inhibitoryinteraction with substrates of CYP2C9 (such asnon-steroidal anti-inflammatories, phenytoin andwarfarin [36]). This presumes that DEX is notactively transported into the liver, which couldcause the maximum concentration of unboundcompound around the hepatic enzyme to begreater than the unbound concentration in theblood close to the entrance of the liver. Theresults from the Ki determination for inhibition oflauric acid 11-hydroxylase activity indicated thatDEX is also likely to be a competitive inhibitor ofCYP2E1, with a Ki of approximately 426 mm. Thisimplies that no clinically relevant inhibitoryinteractions of DEX with CYP2E1 substrates(such as isoflurane and chlorzoxazone) areexpected. In neither case was the extent ofinhibition affected by the pre-incubation regimeemployed.

In the present study, incubation conditionsleading to the linear formation of MF, the majorradiolabelled fraction formed from [14C]DEX bypooled human liver microsomes were estab-lished with respect to time and substrate con-centrations, prior to determination of theMichaelis-Menten kinetics. The Km value ob-tained (23.7 mm, when fitted to a single enzymekinetic model) is similar to the total (bound plusunbound) maximum DEX Cmax of 27 mm ob-served in human subjects after repeated dosesof 200 mg t.i.d., i.e. those intended to be em-ployed during the clinical use of DEX [18]. Sincethe data fitted a single enzyme kinetic model, itsuggests that only one enzyme plays a major rolein this reaction under the incubation conditionsused in vitro. Nonetheless, the possibility thattwo or more CYP isoforms are involved, eachhaving similar kinetic properties with regard toDEX metabolism, cannot be excluded. Based onthese findings, a concentration (25 mm) approx-imating to the calculated Km value, deemedsuitable for reaction phenotyping, was used inall subsequent experiments with [14C]DEX in thisstudy [37,38].

A combination of co-chromatography withauthentic non-radiolabelled reference standardsand LC-MS/MS analysis indicated that MF inpart at least comprised O-demethylated DEX(equivalent to CR 3529). This is consistent with

other data, where CR 3529 has been detected inplasma of healthy volunteers receiving DEX as asingle oral dose of 200 mg (S Persiani, unpub-lished results).

The CYP(s) involved in the formation of MFwere characterized by a combination of correla-tion analysis, co-incubation of DEX with selectivechemical CYP inhibitors and incubation withSupersomesTM [32,37,38]. Statistically significantcorrelations were obtained for MF formation andCYP2B6, 2C8, 2C9/10 and 3A4/5. CYP2B6,CYP2C9 and CYP3A4 were further investigated,since these were considered most important inthe context of the clinical action of DEX. CYP2C8was not included in these experiments since ithas been implicated in the metabolism of only asmall number of drugs [34]. In addition, acorrelation between CYP2B6 (S-mephenytoin N-demethylase) and CYP2C8 (paclitaxel 6a-hydro-xylase) marker activities was seen in the bank ofhuman liver microsomes used, which suggestedthat the two activities were not completelyindependent. Nonetheless, the involvement ofCYP2C8 in the metabolism of DEX cannot becompletely discounted. Of the CYP inhibitorstested, only sulphaphenazole (CYP2C9/10) andtroleandomycin (CYP3A4/5) had any significantconcentration-dependent inhibitory effect on therate of formation of MF. Orphenadrine, used asan inhibitor of CYP2B6 in this study, has beenpreviously shown to be relatively non-selective,inhibiting for example CYPs 2C19 and 2D6,in addition to 2B6 in native human livermicrosomes [39]. CYP2C9 Supersomes

TM

wereapproximately 2–3 times more efficient at catalys-ing the formation of MF from [14C]DEX thanwere CYP3A4 Supersomes

TM

, while CYP2B6Supersomes

TM

barely catalysed this reaction.Overall, these results suggest that both CYP2C9and CYP3A4 may be the predominant CYPenzymes catalysing the O-demethylation ofDEX. Both of these CYPs are variably expressed,especially within the Caucasian population[34,35,40]. It is interesting to note that thepharmacokinetics of DEX showed high inter-subject variability in healthy Caucasian malesadministered the compound for 7 days [18].

In conclusion, the data presented here indicatethat DEX can interact with human CYP2C9, bothas inhibitor and substrate. Extrapolation from the


M. HALL ET AL.174

in vitro results of this study suggests that theseinteractions are unlikely to be clinically signifi-cant, particularly given that CYP3A4/5 alsoappears to be capable of metabolizing DEX.

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Documents

Interaction of dexloxiglumide, a cholecystokinin type-1 receptor antagonist, with human cytochromes P450