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METABOLISMOFEZLOPITANT,ANONPEPTIDICSUBSTANCEPRECEPTORANTAGONIST,INLIVERMICROSOMES:ENZYMEKINETICS,CYTOCHROMEP450ISOFORMIDENTITY,ANDINVITRO-INVIVOCORRELATIONR.SCOTTOBACHDrugMetabolismDepartment,PfizerCentralResearch,Groton,Connecticut(ReceivedMarch29,2000;acceptedMay22,2000)Thispaperisavailableonlineathttp://www.dmd.orgABSTRACT:Theenzymekineticsofthemetabolismofezlopitantinlivermicro-somesfromvariousspecieshavebeendetermined.Therankorderofthespecieswithregardtotheinvitrointrinsicclearanceofezlopitantwasmonkey..guineapig>rat..dog>human.CJ-12,764,abenzylalcoholanalog,wasobservedasamajormetabolite,andadehydrogenatedmetabolite(CJ-12,458)wasequallyimportantinhumanlivermicrosomes.Scale-upofthelivermicrosomalintrinsicclearancedataandcorrectingforbothserumproteinbindingandnonspecificmicrosomalbindingyieldedpre-dictedhepaticclearancevaluesthatshowedagoodcorrelationwithinvivosystemicbloodclearancevalues.Includingmicroso-malbindingwasnecessarytoachieveagreementbetweenhepaticclearancevaluespredictedfrominvitrodataandsystemicclear-ancevaluesmeasuredinvivo.CytochromeP450(CYP)3A4,3A5,and2D6demonstratedtheabilitytometabolizeezlopitanttoCJ-12,458andCJ-12,764.However,inlivermicrosomes,theCYP3AisoformsappeartoplayasubstantiallymoreimportantroleinthemetabolismofezlopitantthanCYP2D6,asassessedthroughtheuseofCYP-specificinhibitors,correlationtoisoform-specificmarkersubstrateactivities,andappropriatescale-upofenzymekineticdatageneratedinmicrosomescontainingindividualheter-ologouslyexpressedrecombinantCYPisoforms.TheapparentpredominanceofCYP3AoverCYP2D6isconsistentwithobserva-tionsofthepharmacokineticsofezlopitantinhumansinvivo.Ezlopitant,(2S,3S,4S)-2-diphenylmethyl-3-[(5-isopropyl-2-methoxybenzylamino]-1-azabicyclo[2.2.2]octane(Fig.1),representsanovel,potent,nonpeptidicantagonistoftheSubstancePreceptorsimilarinstructuretothewellcharacterizedanalogCP-96,345(Snideretal.,1991).ItisbelievedthatanantagonistofthehumanSubstancePreceptorwillbeoftherapeuticutilityindisordersinwhichSub-stancePisbelievedtoplayarole,suchasinflammatorydiseases,depression,pain,andemesis(Krameretal.,1998;Heskethetal.,1999).Thepharmacokineticsofezlopitantandtwopharmacologicallyactivemetaboliteshavebeenmeasuredinseveralpreclinicalspeciesofpharmacologicalandtoxicologicalrelevance(Reed-Hagenetal.,2000).Inrat,guineapig,dog,andmonkey,ezlopitantischaracterizedbyahighsystemicclearanceandloworalbioavailability,presumablyduetohighfirst-passhepaticextraction.However,initialdatasuggestthattheclearanceofthiscompoundinhumansissubstantiallylowerthaninpreclinicalspecies(unpublishedobservations).Thus,despitethepoorpharmacokineticcharacteristicsinanimals,ezlopitanthaspharmacokineticsinhumansamenabletocommercialutility(i.e.,goodoralexposure,half-lifesuitableforonce-per-dayadministra-tion).Ezlopitantisconvertedprimarilytotwopharmacologicallyactivemetabolites,bothofwhichhavebeenobservedinthesystemiccircu-lationofpreclinicalspecies(Reed-Hagenetal.,2000)aswellasinhumans(unpublishedobservations).CJ-12,458,analkenemetabolite,andCJ-12,764,abenzylalcoholmetabolite(Fig.1),arecompoundswithinvitroSubstancePreceptorantagonistactivitiesinthesamerangeastheparentcompound.CJ-12,764isthemajormetaboliteinpreclinicalspecies,withlessCJ-12,458observed,althoughthetwometabolitesareobservedinnearlyequalabundanceinhumancircu-lation.Thesemetabolitescanpotentiallycontributetothepharmaco-logicalactivityobservedafteradministrationoftheparentcompound.Hepaticmicrosomescontainmanydrug-metabolizingenzymes,themostnotablebeingtheheme-thiolate-containingcytochromeP450s(CYP)1(OrtizdeMontellano,1996).Thislargefamilyofenzymescatalyzeawidevarietyofreactionsofxenobioticmetabolismaswellasmetabolismofendogenouscompoundssuchaslipidsandsteroids.BecauseCJ-12,458andCJ-12,764representmetabolitesofezlopitantthatarisefromoxidativeprocesses,themetabolismofezlopitanttothesetwocompoundswasinvestigatedinhepaticmicrosomesinvitro.Theuseofenzymekineticdataofdrugmetabolismreactionsmeasuredinhepaticmicrosomesinattemptingtoquantitativelyde-scribeinvivopharmacokineticshasbeenanareaofexplorationforapproximately20years,withthefirstdemonstrationofthepotentialutilityofthisapproachdoneusingratlivermicrosomes(Raneetal.,1977;reviewedinHouston,1994;Iwatsuboetal.,1997).Theuseofsuchdatacanbeapowerfultoolinthedrugdiscoveryanddevelop-mentprocessesbecausehumanandhuman-derivedmaterialscanbeusedtopredicttheclearanceandhepaticextractionofdrugsinhumansbeforeundertakingthelargecostsassociatedwithadminis-Sendreprintrequeststo:Dr.R.ScottObach,DrugMetabolismDepartment,CentralResearch,Pfizer,Inc.,Groton,CT,06340.E-mail:ronald_s_obach@groton.pfizer.com1Abbreviationsusedare:CYP,cytochrome450;TAO,triacetyloleandomycin;TEA,triethylamine;KMapp,Michaelisconstant;CL9int,intrinsicclearance;fu,un-boundfraction;MS,massspectrometry.0090-9556/00/2809-1069–1076$03.00/0DRUGMETABOLISMANDDISPOSITIONVol.28,No.9Copyright©2000byTheAmericanSocietyforPharmacologyandExperimentalTherapeutics66/843646DMD28:1069–1076,2000PrintedinU.S.A.1069atASPETJournalsonOctober9,2016dmd.aspetjournals.orgDownloadedfromteringnewchemicalentitiesintheclinic.Anearlydemonstrationofacorrelationbetweeninvitroenzymekineticdataandpharmacoki-neticdata,asappliedtoahumansystem,wasdescribedforfelodipine(Baarnhielmetal.,1986).Inadditiontousinghumanhepaticmicrosomesinthequantitativepredictionofinvivohumanpharmacokineticdata,thisinvitroap-proachcanbeusedinexaminingtherolesofindividualdrug-metab-olizingenzymes,mostimportantlyCYPenzymes,intheoverallmetabolismofagivencompoundaswellasspecificbiotransforma-tionreactions.MeasurementofthecontributionofindividualCYPisoformsinvolvedinthemetabolismofdrugscanhaveimportantimplicationsforinterindividualvariabilitybecausesomeCYPiso-formsareexpressedinwidelyvariableamountsduetoenvironmentalorgeneticfactors(reviewedinWrightonandStevens,1992).Further-more,suchknowledgecanprovideabetterunderstandingofthepotentialforpharmacokineticdrug-druginteractions.Therefore,ex-perimentalresultsarealsoincludedthataddresstherolesofhumanCYPisoformsinthemetabolismofezlopitanttothemajormetabo-lites,CJ-12,458andCJ-12,764.Theimpactofthefindingsastheyrelatetoexpectedpharmacokineticbehaviorinhumansandthepo-tentialimpactofCYP2D6metabolizerphenotypearediscussed.Thus,theprimaryobjectivesoftheseexperimentsareto:1)attempttomakeacross-speciescomparisonoftheenzymekineticsoflivermicrosomalmetabolismofezlopitantandtocomparetheenzymekineticsofformationoftheactivemetabolitesCJ-12,458andCJ-12,764inthedevelopmentofaninvitro-invivocorrelation,and2)attempttodeterminethecontributionofvariousisoformsofhumanCYPtoboththeoverallmetabolismofezlopitantandthespecificbiotransformationpathwaysgivingrisetoCJ-12,458andCJ-12,764.ExperimentalProceduresMaterials.EzlopitantdihydrochlorideandCJ-12,764dibenzenesulfonatewereobtainedfromtheProcessResearchandDevelopmentDepartment,PfizerCentralResearch,Groton,CT.CJ-12,458wasobtainedfromtheMedicinalChemistryDepartment,Pfizer,Nagoya,Japan.[3H]EzlopitantwaspreparedbycatalyticreductivedehalogenationofadibromoanalogbyTokaiResearchLaboratories,Ibaraki,Japan.Thespecificactivityofthismaterialwas20.0Ci/mmol(radiochemicalpurity.99%),andthepositionsoftritiumincorpo-rationwerethemetapositionsofthetwophenylringsofthediphenylmethylmoiety.[3H]CJ-12,764wasbiosynthesizedasdescribedbyReed-Hagenetal.(2000).HepaticmicrosomeswerepreparedfromSprague-Dawleyrat(CharlesRiverLabs,Wilmington,MA),Hartleyguineapig(CharlesRiverLabs),beagledog(MarshallFarms,NorthRose,NY),cynomolgusmonkey(BRF/CharlesRiverLabs,Houston,TX),andhumanlivers(IIAM,Exton,PA)usingstandardprocedures.ProteinconcentrationsweredeterminedusingtheBCAassaymethod(PierceChemicalCo.,Rockland,IL),totalCYPwasmeasuredbythemethodofOmuraandSato(1964),andmicrosomeswerecharacterizedusingCYP-specificmarkeractivitiesbytheDrugMetabolismDepartment,PfizerCentralResearch,Groton,CT.Microsomesusedinthefollowingexperimentsrepresentcombinationsofpreparationsfromthefollowingnum-bersofindividualanimals:4(rats),4(guineapigs),4(dogs),and3(monkeys).Pooledhumanlivermicrosomesrepresentanequalmixturefrom10individualdonors.Sf9cellmicrosomescontainingrecombinantheterologouslyexpressedCYPisoformsandreductasewereobtainedfromtheMolecularSciencesDepartment,PfizerCentralResearch,andmicrosomesfromhumanb-lympho-blastoidcellswereobtainedfromGentestCorp.(Woburn,MA).IncubationConditions.Ezlopitant,mixedwith[3H]ezlopitant(1.2mCi)asaradiotracer,wasincubatedinthepresenceoflivermicrosomesatvariousproteinconcentrations(0.05–4.0mg/ml),NADP(1.3mM),glucose6-phos-phate(3.3mM),MgCl2(3.3mM),andglucose6-phosphatedehydrogenase(5U)inatotalvolumeof0.2mlof25mMpotassiumphosphate,pH7.5.Inpreliminaryexperiments,timecoursesofproductformationweremeasuredtoestablishconditionsofreactionlinearity.Thefollowingmicrosomalproteinconcentrationswereusedinsubstratesaturationexperiments:rat,0.5mg/ml;FIG.1.StructuresofezlopitantandmetabolitesCJ-12,458andCJ-12,764.Theasterisksdenotethesitesoftritiumincorporation.FIG.2.HPLCradiochromatogramsofmicrosomalincubationsof[3H]ezlopitant.Individualchromatogramsarefromratlivermicrosomes(panelA;0.5mg/ml;0.33mMCYP),guineapigliver(panelB;0.2mg/ml;0.14mMCYP),dogliver(panelC;1.0mg/ml;0.42mMCYP),monkeyliver(panelD;0.05mg/ml,0.05mMCYP),humanliver(panelE;4.0mg/ml;1.0mMCYP),heterologouslyexpressedCYP2D6(panelF;0.05mg/ml;0.6nMCYP),andheterologouslyexpressedCYP3A4(panelG;0.6mg/ml;0.028mMCYP).FIG.3.HPLCradiochomatogramofhumanlivermicrosomalincubationof[3H]CJ-12,764.1070OBACHatASPETJournalsonOctober9,2016dmd.aspetjournals.orgDownloadedfromguineapig,0.2mg/ml;dog,1.0mg/ml;monkey,0.05mg/ml;andhuman,4.0mg/ml.Incubationswerecommencedbyadditionofthecofactors,incubatedfor10minat37°Cinashakingwaterbathopentotheatmosphere,followedbyterminationofthereactionsbyadditionoftwovolumesofCH3CN.Theprecipitatedmaterialswereremovedbycentrifugation,andthesupernatantwasanalyzedbyradiometricHPLCasdescribedbelow.InhibitionStudies.Inhibitionofezlopitantmetabolismbytriacetylolean-domycin(TAO)(aCYP3Ainactivator)wasassessedbycomparisonoftheactivityofanindividuallotofhumanlivermicrosomesversusthesamelotthathadbeenpreincubated30minwithTAO(50mM)andanNADPHgeneratingsystem,followedbywashingthemicrosomesofexcessinhibitor.Quinidineinhibition(CYP2D6)wasassessedbyexaminingthemetabolismofezlopitantbypooledhumanlivermicrosomesinincubationscontaining0,1.0,3.0,10,30,and100mMquinidine.Ketoconazoleinhibition(CYP3Ainhibitor)wasassessedusinginhibitorconcentrationsrangingfrom0.05to10mMandanezlopitantconcentrationof10mM.Sulfaphenazoleinhibition(CYP2C9)wasassessedbyexaminingthemetabolismofezlopitantbyanindividuallotofhumanlivermicrosomescontainingahighCYP2C9-tolbutamidehydroxylaseactivityinincubationscontaining0,1.0,3.0,10,30,and100mMsulfa-phenazole.Incubations(totalvolume50.2ml)wereconductedatanezlopi-tantconcentrationof10mM(containing1.2mCi[3H]ezlopitant)andcofactorsatconcentrationsasdescribedabove.Reactionswereincubatedandterminatedasabove,andanalyzedbyHPLCasdescribedbelow.CorrelationStudies.Ezlopitant(mixedwith1.2mCi[3H]ezlopitantasaradiotracer)atsubstrateconcentrationsof0.3,10,and100mM(hence,specificactivitiesof20,0.6,and0.06mCi/nmol)wasincubatedwithcofactorsasdescribedaboveandhumanlivermicrosomalprotein(2.5–4.6mg/ml)from11individualdonorsinduplicatedeterminations.Reactionswereincubatedandterminatedasabove,andanalyzedbyHPLCasdescribedbelow.MetabolismofEzlopitantbyHeterologouslyExpressedCYPIsoforms.Ezlopitant,atasubstrateconcentrationof50mM,wasincubatedinthepresenceofmicrosomesfromcellscontainingheterologouslyexpressedCYPisoformscoexpressedwithCYP/NADPHreductase.CYP2C9,CYP2C19,CYP2D6,CYP3A4,andCYP3A5wereexpressedinabaculovirussystem,andCYP1A1,CYP1A2,CYP2A6,andCYP2E1wereexpressedinhumanb-lym-phoblastoidcells(Gentest).Microsomeswereusedatproteinconcentrationsrangingfrom0.3to6.2mg/ml,commensuratewiththerelativeactivitiesreportedformarkersubstrateactivitiesforeachofthepreparations.Reactionswereincubatedandterminatedasabove,andanalyzedbyHPLCasdescribedbelow.SubstratesaturationexperimentsusingCYP2D6,CYP3A4,andCYP3A5wereconductedatsubstrateconcentrationsrangingfrom0.1to50mMusingunlabeledezlopitant,cofactor,andmicrosomalproteinconcentra-tionsasabove.AnalysisoftheCJ-12,458andCJ-12,764productswasaccom-plishedusingHPLC-massspectrometry(MS)asdescribedbelow.Analysisof[3H]EzlopitantIncubationMixturesbyRadiometricHPLC.IncubationmixtureswereanalyzedonanHPLCsystemconsistingofanLDCAnalytical(RivieraBeach,FL)4100ConstaMetricgradientpump,3200Spec-troMonitorvariablewavelengthultravioletdetector,membranedegasser,Per-kin-Elmer(Norwalk,CT)ISS200injector,andInusSystems(Tampa,FL)b-RAMradioactivitydetectorwitha500-mlmixingcell.ThesupernatantsFIG.4.Substratesaturationplotofezlopitantmetabolismbyhumanlivermicrosomes.Datarepresentindividuallyplottedtriplicatedeterminations.f,CJ-12,458for-mation;F,CJ-12,764formation.TABLE1Enzymekineticparametersofezlopitantmetabolismbyrat,guineapig,dog,monkey,andhumanlivermicrosomesReactionvelocitiesweremeasuredasdescribedinExperimentalProcedures.KineticparametersweredeterminedbyfittingreactionvelocityversussubstrateconcentrationdatatotheMichaelis-Mentenequation.Inthecaseofdog,thedatawasfittoasumequationoftwoMichaelis-Mententerms.SpeciesReactionKMappVmaxCL9intmMpmol/min/mgmicrosomalproteinml/min/mgmicrosomalproteinRatEzlopitantmetabolism2.86930.248CJ-12,458formation9.41540.016CJ-12,764formation2.54800.192RRT50.900formation4.1730.018GuineapigEzlopitantmetabolism2.59720.389CJ-12,458formation7.12790.039CJ-12,764formation1.95370.283RRT50.616formation9313830.015DogEzlopitantmetabolisma0.350.016552500.005CJ-12,458formation19.5290.0015CJ-12,764formationa0.350.017521530.003MonkeyEzlopitantmetabolism2.358332.54CJ-12,458formation10.425230.243CJ-12,764formation2.337871.65RRT50.616formation14.11000.00716451030.031HumanEzlopitantmetabolism987350.0077CJ-12,458formation803240.0041CJ-12,764formation892570.0029RRT50.616formation2701800.0007aTwoKMvaluesweredeterminedfordogezlopitantmetabolismandCJ-12,764formation.1071METABOLISMOFEZLOPITANTINVITROatASPETJournalsonOctober9,2016dmd.aspetjournals.orgDownloadedfromfromterminatedincubationmixtureswereinjected(120ml)ontoaWatersNovapakC-18column(3.63150mm)pre-equilibratedin20mMCH3COOH/CH3CN(87.5:12.5)containing0.1%triethylamine(TEA)ataflowrateof1.0ml/min.Initialconditionsweremaintainedfor5min,followedbyalineargradientto20mMCH3COOH/CH3CN(20:80)with0.1%TEAat30min.Thefinalconditionswereheldfor10min,afterwhichthecolumnwasre-equilibratedfor12min.Theeluentwasmonitoredforradioactivityusingb-Blendscintillationfluidat4.0ml/min.Ezlopitant,CJ-12,458,andCJ-12,764elutedat35.8,34.5,and25.5min,respectively.AnalysisofCJ-12,764andCJ-12,458byHPLC-MS.Insomecases,determinationofconcentrationsofCJ-12,458andCJ-12,764wasaccom-plishedbyliquidextractionfollowedbyHPLC-MSanalysis.Incubationmix-tureswereterminatedwithvortexmixingwith3mlofmethyltertiarybutyletherand100ngofinternalstandardCJ-11,957,in0.1mlofwaterwasadded.(CJ-11,957isidenticalwithezlopitant,withtheexceptionthattheisopropylgroupinthelatterisreplacedwithanethylgroup.)Themixtureswerevortexmixedfor1min,followedbyseparationofthelayersbyspinningat3000rpmatambienttemperatureinaJouanmodelCT422swingingbuckettabletopcentrifuge.Thesampleswereplacedinadry-iceacetonebathtoeffectfreezingoftheaqueouslayer,andtheorganiclayerwasdecantedintoafreshsilylatedglasstesttube.ThesolventwasremovedunderN2at30°CinaZymarkTurboVap,andtheresiduewasreconstitutedin0.1mlofHPLCmobilephase.TheHPLC-MSsystemconsistedofaHewlett-Packard1100HPLCsystemcoupledtoaPESciexAPI100singlequadrupolemassspectrometercontain-inganatmosphericpressurechemicalionizationinterface.ThecolumnwasaWatersSymmetryC18(3.93150mm;5-mmparticlesizepacking),andtheinitialmobilephaseconsistedof45.5%CH3CNin20mMaceticacid,adjustedtopH4withNH4OHataflowrateof0.8ml/min.Samples(75ml)wereinjected,andtheinitialmobilephasecompositionwasmaintainedfor2min,afterwhichalineargradientwasapplied,resultingin95%CH3CNat6min.Theentireflowwasintroducedintotheatmosphericpressurechemicalionizationsourceoperatedinthepositiveionmode.Theorificevoltagewas45V,andthenebulizertemperaturewassetat500°C.Foreachanalyte,proton-atedmolecularionswerefollowed(m/z471.2forCJ-12,764;m/z453.2forCJ-12,458;m/z455.2forCJ-11,974;andm/z441.0forCJ-11,957internalstandard)withadwelltimeforeachionof150ms.Theretentiontimeswere1.8minforCJ-12,764,5.2minforCJ-12,458,5.6minforCJ-11,974,and5.0minforCJ-11,957internalstandard.Quantitationwasaccomplishedbyex-trapolationfromastandardcurvewithlineardynamicrangefrom0.1to100ng/ml(1/xweighting).MetaboliteProfileof[3H]CJ-12,764inLiverMicrosomes.CJ-12,764(0.3mM)withbiosynthesized[3H]CJ-12,764asaradiotracer(1.2mCi;finalspecificactivity520mCi/nmol)wasincubatedwithlivermicrosomesfromrat,guineapig,dog,monkey,andhumanataproteinconcentrationof4.0mg/mlandcofactorsunderconditionsasdescribedabove.Aliquots(0.2ml)wereremovedattimepointsof5and30minandprocessedasdescribedabove.ResultsComparisonofLiverMicrosomalMetaboliteProfilesAcrossSpecies.HPLCradiochromatogramsoflivermicrosomalincubationsof[3H]ezlopitantareshowninFig.2,A–E.BothCJ-12,764andCJ-12,458wereformedinlivermicrosomesfromallfivespecies.CJ-12,764wasthepredominantmetaboliteinrat,guineapig,dog,andmonkey,whereasCJ-12,764andCJ-12,458wereformedtosimilarextentsinhumanlivermicrosomes.Inadditiontothesetwometab-olites,twoadditionalmetabolitesofunknownstructurewereob-served:oneinrat(retentiontime532min;referredtoasRRT50.900),andtheotheringuineapig,monkey,andhuman(retentiontime522min;referredtoasRRT50.616).ExaminationofthemetabolicprofileofCJ-12,764inlivermicrosomeswasdonewiththeprimaryintentionofdeterminingwhetherthedehydrogenatedmetab-olite,CJ-12,458,aroseviaasimpledehydrationofthebenzylicalcoholundertheincubationconditionsusedintheexaminationofthemetabolicprofileoftheparentcompoundezlopitant.InnocasewasCJ-12,458observedinanyoftheincubationsofCJ-12,764(Fig.3).SeveralothermetabolitesofCJ-12,764ofunknownstructurewereobserved.SubstrateSaturationExperiments.Anexampleofasubstratesaturationplotforthemetabolismofezlopitantinhumanlivermi-crosomesispresentedinFig.4.Apparentenzymekineticconstantsfortheoverallmetabolismofezlopitant,formationofCJ-12,764,CJ-12,458,andotherselectedmajormetabolitesarepresentedinTable1forfivespecies.Intrinsicclearancewasdeterminedbydivid-ingVmaxbyKMapp.Therankorderofthespecieswithrespecttooverallezlopitantintrinsicclearanceismonkey.guineapig.rat.dog.human.ApparentMichaelisconstants(KMapp)forallreactionsFIG.5.InhibitionofezlopitantmetabolisminhumanlivermicrosomesbyCYPisoformspecificinhibitors.Eachpointrepresentstheaverageofduplicatedeterminations.(f),CJ-12,458formation;(Œ),CJ-12,764formation.Averagecontrolactivitiesinthesulfa-phenazoleinhibitionexperimentswere15and16pmol/min/mgforCJ-12,458andCJ-12,764formation,respectively.Averagecontrolactivitiesintheketoconazoleandquinidineexperimentswere25and29pmol/min/mgforCJ-12,458andCJ-12,764formation,respectively.1072OBACHatASPETJournalsonOctober9,2016dmd.aspetjournals.orgDownloadedfrominratandguineapig(exceptone)werebelow10mM.Inbothofthesespecies,conversionofezlopitanttoCJ-12,764occurredtoagreaterextentthanconversiontoCJ-12,458,byvirtueofbothalowerKMappvalueandhigherVmaxvalue.Theintrinsicclearance(CL9int)ofthemetabolismofezlopitanttoCJ-12,764accountedforapproximatelythree-fourthsoftheCL9intofezlopitant,whereastheconversiontoCJ-12,458wasonlyabout10%ofthetotal.Indogmicrosomes,thedatasuggestedthepresenceoftwoenzymesinvolvedinthemetabolismofezlopitant,onelowKMapp-lowVmaxenzymeandonehighKMapp-highVmaxenzyme.However,limiteddatawereobtainedatsubstrateconcentrationsnearthelowKMappvalue(duetoassaysensitivitylimitations);thusthepresenceofthelowKmappactivitymustbeinterpretedwithcaution.Indog,CJ-12,764accountedforthemajorityofezlopitantmetabolism;bothoftheobservedenzymeactivitiesgeneratedthismetabolite.CJ-12,458wasformedtoamuchlesserextent,andalowKMappactivitycouldnotbeobservedundertheseassayconditions.EzlopitanthadahighCL9intvalueinmonkeylivermicrosomes,andalargeportionofthisvalueconsistedofconversiontoCJ-12,764(65%).TheportionoftotalezlopitantintrinsicclearanceconsistingofconversiontoCJ-12,458wascomparablewiththatobservedinrat,guin