波立维生物等效性1

PharmacogeneticSelectionofVolunteersIncreasesStringencyofBioequivalenceStudies;TheCaseofClopidogrelJ.Garcés-Eisele,2A.Ruiz-Argüelles,1,2LarisaEstrada-Marín,1VirginiaReyes-Núñez,R.Vázquez-Pérez,1OlgaGuzmán-García,1R.Coutiño-Medina,2LeticiaAcosta-Sandria,1andBeatrizCedillo-Carvallo1,2,*Authorinformation►Articlenotes►CopyrightandLicenseinformation►ThisarticlehasbeencitedbyotherarticlesinPMC.AbstractClopidogrelisaprodrugthatrequiresoxidationtoitsintermediatemetabolite,2-oxo-clopidogrel,andthentothethiolderivative,2-oxo-clopidogrel[1].Thisactivethiolmetaboliteinhibitsadenosinediphosphate(ADP)-inducedplateletaggregationbyblockingtheplateletP2Y12receptorresultinginimportantreductioninADP-mediatedplateletaggregation[2].Clopidogrelisstandardofcareinmostpatientsundergoingpercutaneouscoronaryinterventionandthoseexperiencingacutecoronarysyndromes.However,ithasbeensuggestedthatresponsetoclopidogrelvarieswidelywithnonresponseratesrangingfrom4%to30%at24h[3,4].Suggestedmechanismsforthisvariabilityhaveincludedunder-dosing,druginteractionsandintrinsicinterindividualdifferencesresultingfromgeneticpolymorphismsinthepathwaysofclopidogrelpharmacokineticsandpharmacodynamics[5,6].CytochromeP450-2C19(CYP2C19)isoneoftheprincipalenzymesinvolvedinthebioactivationoftheprodrug[7,8].Acommonloss-of-functionallele,CYP2C19*2(c.681GA;rs4244285),isassociatedwithincreasedriskforseriousadversecardiovasculareventsinbothheterozygousandhomozygouspatientswithacutecoronarysyndromeswhoarereceivingclopidogrel,particularlyamongthoseundergoingpercutaneouscoronaryintervention[9,10].GuidelinesforCYPC19genotype–directedantiplatelettherapyhavebeenpublished[11,12,13,14].UponexpirationofthepharmaceuticalpatentofclopidogrelinMay2012(DailyFinancePosted02/27/11),genericcompanieshavestartedtomanufactureinterchangeableformulationsofthisdrugandhence,tosubjectthemtobioequivalencestudies.Herein,weprovedthatselectionofvolunteershomozygousfortheCYP2C19*1haplotype,increasedthestringencyofbioequivalencestatisticsandresultedinbioinequivalenceofagenericcompoundthatotherwiseprovedequivalentwhentestedinanopenpopulation.MATERIALSANDMETHODSVolunteers:Atotalof36Mexican-Mestizovolunteersofbothgenderswereincludedinthestudy.AllwereaccruedthroughopeninvitationandprovedtofulfillthecriteriaestablishedbyMexicanFederalbylawsandregulations.Briefly,volunteers(20male)hadamedianageof26years(range18-54),theirweight,heightandbodymassindexwere65.2±8.45kg,166.1±6.39cmand23.6±2.18,respectively(mean±standarddeviation);carefulclinicalexaminationruledoutpresentorpastrelevantdiseases,vitalsignswerewithinnormalaswerelaboratoryresults(completebloodcellcount,basicclinicalchemistry,hepaticenzymes,urineanalysis,drugsofabuseandpregnancytest),electrocardiogramandchestX-rays.AllvolunteerswereawareoftherisksandsignedaClinicalInvestigationAgreementtoparticipateinthestudy.Theresearchprotocolandinformedconsent(LCPB-11-009Dated2011/07/15DI-F012Rev.0/2011-05-11andLCPB-11-009Dated2011/07/15UC-F003Rev.0/2009-01-09)wereapprovedonJuly25th2011bytheEthicsCommitteeofCentrodeHematologíayMedicinaInterna,LaboratoriosClínicosdePueblayLaboratoriosClínicosdePuebladeBioequivalencia,andfurtherendorsedbytheFederalCommissionfortheProtectionAgainstSanitaryRisks(COFEPRISApprovalNumberCAS/OR/01/CMN/113300410B0192-3259/2011)onAugust9th2011.Clinicalstudydesign:Asingle-doseadministration,cross-overstudywasused,withawash-outperiodofsevendays(≈21timesT½).Afterrandomizationofsequences,eithera75mgtabletofgenericclopidogrelorasimilartabletofPlavix®wereadministeredtothefastingvolunteersinthefirstperiod,andtheoppositesequenceduringthesecondsession.Atotalof13bloodsamplesalonga36hperiodweredrawnintoheparin-containingtubesfromeachsubjectineachexperimentalsessiontomeasureclopidogrelplasmalevels.AnadditionalEDTApretreatmentsamplewasobtainedfromeachvolunteerintoanEDTAK2-containingtubeforgenotyping.Measurementofclopidogrelplasmalevels:Ultra-performanceliquidchromatography(UPLC)coupledtotandemmassspectrometry(MS/MS)wasusedtodevelopamethodtomeasureclopidogrelplasmaconcentrationsinthevolunteers’samples[15].Solidphaseextraction(SPE)wasperformedatacidicpHemployingOasisMCX™(Waters)96-wellplates.AnAcquity®UPLCBEHC18,1.7μm,2.1×50mmcolumn(Waters®,PartNo186002350)wasusedwithisocraticacetonitrile:formicacid0.1%(75:25v/v).Thetotalruntimewas3.5minandtheretentiontimeswere1.897±0.02and1.450±0.03minforclopidogrelandticlopidine(internalstandard,IS),respectively.ChromatographyandtandemspectrometrywereperformedinaWatersQuattroPremierXE™tandemquadrupolemassspectrometerwithAcquityUPLC™systemandanalyzedinthemultiplereactionmonitoringmodeusingtherespective[M+H]+ions,m/z321.9154.75and263.95153.77forclopidogrelandIS,respectively.ThemethodwasvalidatedinaccordancewiththerecommendationsofboththeUnitedStatesFoodandDrugAdministrationandtheMexicanComisiónFederalparalaProteccióncontraRiesgosSanitarios[16,17].Theoverallmeanrecovery,usingSPEextraction,wasfoundtobe82.70,82.06and80.0%,forlow,mediumandhighconcentrations,respectively.Calibrationcurveswerelinearintheconcentrationrang