POSTER SESSION ABSTRACTSSingulus Pulpitum: Microfluidics Coupled With Mass Spectrometry For...

CPSA Shanghai 2016April 20 - 22, 2014

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Thursday, April 21 9:00 am - 5:00 pm

Friday, April 22 9:00 am - 5:00 pm

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POSTER SESSION ABSTRACTS

Inspiration and Education

POSTERABSTRACT

Where Technology and Solutions Meet Where East Meets West

#102DeterminationofLimaprost,ananalogueofPGE1inhumanplasmabyQTRAP® 6500+andSelexION®+technology

Gangyi.Liu1,ChaoZhang2,WenhaiJin2

1.XuhuiCenterHospital,Shanghai,China;2.SCIEXAsiaPacificApplicationSupportCenter,China

Introduction:Limaprost,ananalogueofprostaglandinE1analogue,isapromisingdrugthathasstrongvasodilatory andantiplateletactivityforthetreatmentofvariousischemicsymptoms,suchasulcers,pain,andcold sensations associatedwith thromboangitis obliterans (TAO) andsubjective symptoms associatedwith acquiredlumberspinalcanalstenosis(LCS).Requirementofultra-lowlimitofquantitation(sub- pg/mLlevel)and separation fromendogenous interferences inhumanplasmabecomesabigobstacleforitspharmacokineticresearch.Bynow2D-LC/MS/MSsystem [1-2]istheprincipalsolutiontoreduce orseparate the endogenous interferences. However, it suffers from complexity of 2D-systemoptimizationandmethoddevelopment,especiallythepretty longanalyticaltime(>50min).Wepresent hereamoresimplified1D-LC/MS/MSassaybasedonaSCIEXQTRAP®6500+LC-MS/MSsystem equippedwithaSelexION®+differentialmobilityseparationtechnologydeviceaimedatbetter usabilityandhigherefficiencyincomparisonofcurrent2D-LC/MS/MS.

References[1]KomabaJ,MasudaY,HashimotoY,NagoS,TakamotoM,ShibakawaK,NakadeS,MiyataY. UltrasensitivedeterminationofLimaprost,aprostaglandinE1analogue, inhumanplasmausingon line two-dimensional reversed-phase liquidchromatography– tandemmass spectrometry. J Chromatogr B. 2007;852:590–7.[2]ParkYS,ParkJH,KimSH,LeeMH,LeeYS,YangSC,KangJS.Pharmacokineticcharacteristics ofavasodilatoryandantiplateletagent,Limaprostalfadex, inthehealthyKoreanvolunteers.ClinAppl Thromb Hemost. 2010;16:326–33.

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#103

AGenericKit-BasedApproachforQuantifyingMonoclonalAntibodyDrugsThroughDirectDigestionofDiscoveryStudySamplesMaryLame,HuaYang,SherriNaughton,andErinChambersWatersCorporation,Milford,MA,USAINTRODUCTIONOverthepast5–10years,therehasbeenasignificantshifttowardsagreater%ofbiologicsinpharmaceuticalpipelines.However,theindustryfindsitselfinthemiddleofpatentexpiryformanyofthecriticalmonoclonalantibodyandotherprotein-baseddrugs,withpatentexpirationdatesrangingfrom2012–2020ThishasresultedinafocusonproteinquantificationinBioanalyticallabs,innovatorpharmaandCRO’saswellasbiomarkerresearchlabs.Whileimmunoassay(IA)methodsaresensitiveandsimpletoexecute,poorreagentreproducibility,lackofstandardization,cross-reactivity,limitedlineardynamicrange,andothershort-comingshaveledthedrivetoconverttoLC-MS.TheseLC-MSworkflowshowever,encompassamultitudeofsub-segments,eachhavingmanysteps.Thosethatarecommontomostworkflowsmayincludeaffinitypurification,denaturation,reduction,alkylation,digestion,andSPEclean-up(eachrequiringoptimization).Suchtraditionalproteinquantificationprotocolsoftenrequireasmuchasadayandhalfforcompletion.Furthermore,themarginandpossibilityoferrorissignificantwithineachindividualstep.Thereisastrongneedforsimpler,morestandardizedworkflowswhichenablescientiststocompletesamplepreparationandstartananalyticalrunbymid-day.Atthesametime,ideallyusinggeneric,kittedmethods,assaysensitivitymustbehighenoughtoaccuratelyandpreciselyquantifylowenoughlevelsofthetargetproteintomakecriticaldecisionsindiscovery.Thetypicalworkflowcomplexity(seeposter),oftenleadstoerrorsandpoorreproducibilityorsensitivity.Inthisapplicationnote,wehaveusedtheProteinWorks™eXpressDirectDigestKittosimplifyandstreamlinetheworkflowprocessusingthesameuniversalprotocolandreagentsforallmonoclonalantibodydrugstested.Infliximab,bevacizumab,trastuzumab,andadalimumab(Figures2–5)inplasmaweredirectlydigested,andpeptidesextractedusingSPEinunder4hourstotaltime.Thisenableddatatobegintobeacquiredthesameday,withseveral96-wellplatesbeingrunbythefollowingmorning.

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#104CharacterizationandcollisioncrosssectiondeterminationofobesityrelatedlipidswithinmousemodelsusingtravellingwaveIMS-QtofmassspectrometryGertjanKramer1,NicholasDekker1,LeeAGethings2,JohnPShockcor3,VictoriaLee4,AndersFeldthus2,RobertJBeynon4,JamesILangridge2,JohannesPCVissers2,JohannesMFGAerts11DepartementofMedicalBiochemistry,AcademicMedicalCentre,UniversityofAmsterdam,Netherlands,2WatersCorporation,Wilmslow,UnitedKingdom,3WatersCorporation,Milford,MA,USA,4InstituteofIntegrativeBiology,UniversityofLiverpool,UnitedKingdomIntroductionObesityisarisk-factorassociatedwithmetabolicsyndrome,causingexcessbodyfattobeaccumulatedtotheextentthatitadverselyaffectshealthandlifeexpectancy.Thiscanleadtofurtherhealthimplicationssuchastype2diabetes,heartandliverdiseaseandpotentiallinkstovariousformsofcancer.Ithasbeendemonstratedthatglucosylceramidesplayacrucialpartinmetabolicsyndrome.Themanipulationofthefunctionofglucosylceramideswithsmallmoleculedrugcompoundshasshownthatsymptomscanbenegated.Apreviousmulti-omicstudyshoweddifferent-tiationbetweensubjectstreatedwithglucosylceramidesynthaseinhibitors.Lipidanalyseshavebeenconductedusingalabel-freeLC-DIA-IM-MSapproach,providingqualitativeandquantitativeinformationfromasingleexperiment.Thisworkisanextension,providingadditionalcharacterizationofthelivercelllipidcomplementusingion-mobilityandassociatedcollisioncrosssectiondatabases,obtainedwithanovelgeometrytravellingwaveIMS-QTofMSplatform,forobesemousemodelswhichhaveundergonetreatmenttopreventorrevertobesity.Thecurateddatasetsweretheninterrogatedusingpathwayanalysistools,indicatingthatphysiologicalprocessessuchashepaticsystemdevelopment,inflammatoryresponseandcarbohydratemetabolismareinfluencedfollowingMZ-21treatment.

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#105SingulusPulpitum:MicrofluidicsCoupledWithMassSpectrometryForMulti-OmicsAndTargetedAssaysInTranslationalResearchPaulD.Rainville1,GuiseppeAstarita1,JamesP.Murphy1,IanD.Wilson2,andJamesI.Langridge11WatersCorporation34MapleStreetMilford,MA01757USA2ImperialCollege,DivisionofSurgeryandCancer,SouthKensington,London,SW72AZ,UnitedKingdomAbstract

Translationmedicineisaninterdisciplinarysciencethataimsatcombiningtheinformationtakenfrombenchtobedside.Inthisprocessmoleculesareisolatedandidentifiedindiscoveryandthenutilizedintheclinicalsettingasbiomarkersofhealthanddiseasetobetterdeveloptherapies.Ithasbecomerecentlyapparentthatproteomics,metabolomics,lipidomics,andglycomicsdatacombinedarenecessarytoaddressthechallengeoftranslationalresearchwhichplacesstrainonavailablesampleandinstrumentutilization[1-4].Duetothecomplexityofderivingmeaningfulinformationfromthesestudies,thedevelopmentofnewanalyticaltechnologiesiscritical[5-6].HerewepresenttheutilizationofamicrofluidicLCcoupledwithmassspectrometryforbothdiscoveryandtargetedstudiesintranslationalresearch.AndTherobustness,reproducibilityandabilitytoanalyzemultiplepreparationsofbiofluidsformulti-omicsexperimentsindicatesthattheuseofmicrofluidicLC/MSmayplayafuturekeyroleinthedevelopmentoftranslationalandpersonalizedmedicine.

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#106Tissue-engineeredlongtermhepatocyteco-culturemodelanddataindependentmassspectrometryacquisitiontoidentifymultigenerationmetabolites1YvonneSchaus,1JackMcGeehan,1VinceZuwaraski,2ShawXia2SumaRamagiri1Hepregen,Boston,MA,USA,2Sciex,Framingham,MA,USAForResearchUseOnly,NotforDiagnosticUseIntroductionThedeterminationofmetabolicstability(Clint)andaccuratepredictionofmajorhumanmetabolicprofilesiscriticaltooptimizingdrugcandidateselection.Traditionalmodelssuchassubcellularfractionsandsuspendedhepatocytesareshort-livedandareoftenlimitedintheirabilitytoresolvemulti-generationmetabolitesordrugclearance,particularlyforslowlymetabolizedcompounds.Furthermore,manycurrentLC/MSsystemsusedformetaboliteidentificationlackthesensitivitytogeneratequantitativebioanalyticalmeasurementswithinthetherapeuticdosingrange.Thus,identificationofmetabolitesandevaluationofdrugclearancearetraditionallycollectedfromseparatesamples.Inthisstudy,wesimultaneouslyidentifiedandquantifiedmetabolitesalongwithdrugclearanceusing1µMconcentrationinalong-termhepatocyteco-culturemodelusingQ-ToFanddataindependentacquisitionstrategyMethodsandResultsTolbutamidewaspurchasedcommerciallyand24-wellhumanhepatocyteco-cultureplatesweremanufacturedatHepregenCorproation.Compound(@1µM&10µM)wasincubatedat0,4,48,and168hoursinhumanhepatocyteco-culture.Afterincubation,400µLacetonitrilesolutionwasaddeddirectlytothewellandlysedcellsandmedia(totalcellfraction)werecollectedandtransferred(withscraping/disruptionofthecellsamples)toacollectionreservoir.Directinjectionsofsampleat10µlwasusedforLC/MSfor1µMtolbutamide.MobilephaseAwascomposedofwater(0.1%formicacid),andmobilephaseBwascomposedofacetonitrile(0.1%formicacid).ThemetaboliteprofilingmethodwasdevelopedonaTripleTOF®6600systemcoupledwithaShimadzuNexeraUHPLC.Humanhepatocyteco-culturesincubationsoftolbutamidewerepreparedat1µM&10µMconcentrationsoverthetimecourseof7days.Thebioanalysisofsampleswasconductedonahighresolutionaccuratemassspectrometertosimultaneouslya)toidentifyandconfirmallpossiblephaseI,IImetabolitesinsingleanalysisb)resolveastrongcorrelationbetweenthedisappearanceofparentcompoundandtheappearanceofmetabolites.DatawereacquiredwithhighresolutionTOF-MSsurveyscanandSWATHMS/MS,andprocessedusingseveralalgorithmssuchasprincipalcomponentvariablegrouping(PCVG),multiplemassdefectfiltering(MMDF),isotopepatternfiltering(IPF),commonproductionandneutrallossndgenericbackgroundsubtractiontoidentifyandelucidatechemicalstructuresoftheobservedmetabolites.Thedisappearance(%)oftolbutamideoccurredat87%by2daysincultureand50%by7daysinculture.Hydroxytolbutamide(m/z299.0702)andcarboxytolbutamide(m/z299.0809)werethepredominantmetabolitesoftolbutamide(m/z269.0965)upon168hincubationinhumanhepatocyteco-culture.Comparisonsofmetaboliteprofilespostincubationofeither1µM&10µMtolbutamiderevealedtheneedlookingupreferencestocomparemetabolitesfoundinourstudytoclinicaltrialdataThepreliminaryresultsfromthisworkshowcasetheabilitytosimultaneouslyresolvequantitativeandqualitativedatafordrugmetabolismoflowturnovercompoundsinahumanhepatocyteco-culturemodelusingUHPLC-Q-TOF-MSwithdataindependentacquisitiontodrawinvitro-invivocorrelation.ForResearchUseOnly,NotforDiagnosticUse

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#107QuantitativedeterminationofendogenousbiomarkerlysophosphatidicacidsinmouseplasmabyLC-MS/MS

MingfeiZeng,MarcHCao;LillyChinaResearchandDevelopmentCenter,Shanghai,China

Lysophosphatidicacid(LPA)isanimportantclassofendogenousbiomarkerforseveralpotentialdrugtargetsbecauseofitsvarioussignalingexpressionthroughGprotein-coupledreceptors.LPAlevelinhumanbiofluidshasbeenshowntocorrelatewithvariousdiseases,suchasosteoarthritis,cancerandmyeloma.Theproductionoflysophosphatidicacid(LPA)iscatalyzedbyAutotaxintoremovecholinegroupfromlysophosphatidylcholines(LPC),oneofwell-characterizedLPAbiosynthesispathwayinvivo.Herewedevelopedahighlyrobustandsensitiveliquidchromatography–tandemmassspectrometry(LC-MS/MS)methodtosimultaneouslymeasuretheconcentrationofendogenousLysophosphatidicacid(16:0,18:0,18:1,20:4LPA).LPA(17:0),undetectableinbiologicalmatrices,wasusedasinternalstandardinquantification.Proteinprecipitationbymethanolextractedtargetanalyteswithhighrecovery.ChromatographicseparationwasperformedonashortWatersXbridgeC8columnattheflowrateof0.4ml/min.Mobilephasesconsistedofwatercontaining0.1mMammoniumacetatewith0.05%ammoniumandMeOHcontaining0.02%ethylenediamine(DEA)and0.05%ammonium.Theadditionofionpairreagentethylenediamine(DEA)helpstogenerateshaperpeaksandsuperiorresolution.TheslopeofLPAcalibrationcurvesincharcoal-strippedplasma(CSP)parallelsthatinuntreatedplasmathereforecalibratorswerepreparedinCSPovertherangeof2-250ng/mL.Matrixeffect,recovery,selectivity,accuracy,precisionandstabilitywereevaluatedinauthenticmatrix.Eachinjectiontakesabout3.5mintoallowtheelutionofabundantendogenousLPCthatcaninterferewithaccuratequantificationofLPA.Inaddition,severalfactors,whichcouldpotentiallyinfluencetheaccuracyandprecisionofquantification,wereevaluatedduringmethoddevelopment,suchasadsorption,ion-sourcefragment,pHofextractioncondition,LPC&LPAconversion.AbundantendogenousLPC,LPIandotherslysophosphatidicspeciescoulddegradetorespectiveLPAduetoin-sourcefragmentoccurredontheweakbondconnectingcholine/inosineheadgroupwithLPAbackbond.Thiswouldsubsequentlyintroducesignificantbias/interferenceduringLC-MS/MSanalysiswhenchromatographicbaselineseparationwasnotachieved.TheartificialformationofLPAdemonstratedthatacidicpHandconcomitantendogenousenzymeinplasmacouldaccelerateLPCconvertingtoLPA.Hencesamplehandlingconditionshouldbecarefullycontrolled.ThevalidatedLC-MS/MSmethodwassuccessfullyappliedtoquantifyLPAcontentsinmouseplasmainapilotstudy.

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#108QuantificationofAcetaminophenProteinAdductsasaBiomarkerinHumanPlasmabyusingLC-MS/MS*HuiHong,GaochaoYue,HuafangJiang,WenzhongLiang,XinpingFang,XinZhangWuXiAppTecCo.,DepartmentofBioanalyticalServices,288FuteZhongRd,WaigaoqiaoFreeTradeZone,Shanghai,P.R.China,200131Abstract:Acetaminophen(APAP)isapopularOTCdrugcommonlyuseasanalgesicandantipyreticagent.APAPisconsideredsafeatthetherapeuticdosesbutoverdosesofitsacetaminophen-containingproductscanleadtohepatotoxicityandprogressedtoacuteliverfailure.Acetaminophen-inducedhepatotoxicityhasbeenattributedtocovalentbindingofreactivemetaboliteN-acetyl-p-benzoquinoneiminetocysteinegroupsonproteinsasanacetaminophen-cysteineconjugate(APAP-Cys).EarlystudiesofAPAPproteinadductsreliedonradiometricandimmunochemicalmethodsfortheanalysisofAPAP-Cys.Later,HPLC-ECDmethodwasdeveloped.Thematrixwerefirstdialyzedtoremovenon-protein-boundAPAP-CysandthedialyzedsamplesweresubjectedtodigestionwithproteaseandthenpreparedforHPLC-ECDanalysis.Recently,LC-MS/MSmethodsusingdialysisorgelfiltrationwerereportedtoquantifyAPAP-Cysinhumanserum.However,themethodsusedrelativelylargevolumeofserumsampleandobtaineda10nMLLOQ.Inpresentstudy,amoresensitiveandrapidLC-MS/MSmethodonAPI6500weredevelopedandappliedinaclinicalstudytomonitorAPAPproteinadductasbiomarker.NovelAspect:ItisthefirstreportedsensitiveandspecificLC-MS/MSmethodtoachieve1ng/mLLLOQusing0.1mLhumanplasmaforthedeterminationofAPAP-Cys.PreliminaryResults:Duringmethoddevelopment,weutilizeddialysisdevicesin96-wellplateformattosignificantlyreducethecostandachievehighersamplepreparationthroughput.Inaddition,positivecontrolsampleusingmicrosomalincubationwasusedtoevaluatedigestionefficiency.Figure1.RepresentativeLC-MS/MSchromatogramofAPAP-Cys(positivecontrolsample)inhumanlivermicrosome(SeePoster)

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#109

Hepatocyte-BasedinVitroModelforEvaluationofDrug-InducedCholestasisandHepatotoxicity

Na Long, Hong Yu, Suxing Zhang, Feipeng Zhang, Xueqin Lv, Peizhen Ye and Kezhi ZhangCarysBioHoldingsLimited,Foshan,GuangdongProvince,PRCPurpose: to explore the toxicity and intracellular of bile acids and the linkbetweendrug-inducedcholestasisandhepatictransportofbileacids(BA)andbilirubin.Methods:First,toxicityandintracellularaccumulationofvariousbileacidsincludingDCA,CDCA,UDCAandGCDCAweretested inbothday-1andday-3sandwichculturedrathepatocytes (SCRH) model. And then, with SCRH model and biomarkers detectionincludingALT,AST,ALP,LDHandureageneration,fifteencompoundsincludingcompoundsknownassociatingwithliverinjury(suchasCyclosporinA,Labetolol,Diclofenac, Sorafenib, Chlorpromazine, Verapamil, Benzbromarone, Acetominophen) weretested in the following assay: (i) drug-induced hepatotoxicity with bile acids at 10-foldconcentrationofratserumlevelor40-foldconcentrationofhumanserumlevel;(ii) Inhibitionof test compound on Ntcp-mediated hepatic uptake of d5-taurocholate; (iii) Inhibitionoftest compoundon Bsep-mediated biliary effluxofd5-taurocholate; (iv) drug-inducedhepatotoxicitywith100μMbilirubininpresenceorabsenceofbileacidsat 10-foldconcentration of rat serum level. Briefly, on day-0, seeded fresh prepared rat hepatocytesand formed 2-layer configuration with ice cold BD Matrigel™ after 4hr incubation.Thehepatotoxicitystudywasperformedonday-1andday-3,whencellswere treatedwithtestcompoundsaloneorco-incubatedwithbileacidsmixtureorbilirubinfor 24hours.After24h-treatmentpipettedthesupernatantmediatodeterminetheleakage of AST, ALT, LDH andALP. And then urea generation was determined to calculate drug-induced cholestasis index(DICI). The inhibition studies of test compounds on hepaticuptakeandbiliaryeffluxofd5-taurocholatewereperformedonday-1andday-4, respectively.Onday-4formedwithbilecanaliculus,theaccumulationofd5-taurocholate inhepatocyteandbilecanaliculiwasquantifiedundertheincubationwithregularHBSS, whiletheaccumulationonlyinhepatocytewasquantifiedinthepresenceof5mMEGTAto chelate Ca2+/Mg2+ and disrupt bile canaliculi tight junction. The concentration ofd5-taurocholateinbothstudieswasdeterminedbyLC/MS/MS.Result:Theresultsdemonstratedthatincreasingintracellularaccumulationofbileacids wouldcause cholestasis and hepatotoxicity. And the inhibition effect of compounds on hepatictransportofbileacidscouldaggravatethehepatotoxicity,suchasdrug-inducedcholestasis.Andsandwichculturedrathepatocyteswasausefultooltoevaluate drug-inducedhepatotoxicityinvitro.Conclusion:Thesandwichculturedrathepatocytesareanexcellentmodeltocharacterize druginducedhepatotoxicityespeciallytransporter-basedcholestasis

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#110OptimizationofdetectionsensitivityforderivatizedandunderivatizedneurotransmittersondifferenttriplequadrupolemassspectrometerplatformJinlianLu,WeiqunCao,WeiminHu,WenzhongLiang,YiTao,XinZhangWuXiAppTecCo.,Non-GLPBioanalyticalService,90DelinRd,WaigaoqiaoFreeTradeZone,Shanghai,P.R.China,200131Abstract:Norepinephrine(NE),serotonin(5-HT)anddopamine(DA)playimportantrolesasneurotransmittersandareclassicalbiomarkersfordiseasessuchashypertensionandneuroblastoma.Thereforehigh-sensitivitymeasurementofcatecholaminesbyLCMSisverymuchwarranted.OneofmanyapproachesforimprovingthesensitivityofcatecholaminedetectionbyLCMSisderivatizationofcatecholaminestoattachahydrophobicmoietysothattheyretainbetteronreverse-phasecolumns.Whilethisapproachiseffectivefromachromatographicpointofview,theextentofthiseffectontheoverallsensitivityofdetectiondependsonionizationefficiencyandiontransmissioninmassspectrometry,whichvaryamongstinstrumenttypeduetodifferentarchitecture.Wecomparedhereinthesensitivity,backgroundnoiselevelandrepeatabilityoftheneurotransmittersmeasurementbetweenShimadzuandABSciexinstruments.Standardsolutionsofnorepinephrine(NE),serotonin(5-HT)anddopamine(DA)werepreparedat0.002-1,0.002-1and0.02-10ng/mL,respectively.Internalstandardsolutionswerepreparedbymixing2ng/mLd6-NE,2ng/mLd4-5-HTand10ng/mLd4-DAinethanol.Asaqualitycontrolsample,artificialCSFcomposedofNaCl,KCl,CaCl2,MgCl2andsodiumphosphatebufferwerespikedwithstandardsolutions.Priortomeasurement,10μLofstandardsolutionsormodelsamplesweremixedwithequalvolumeofinternalstandard,andthenderivatizedbyadding50μLofderivatizationreagent.ThereactionproductwasdirectlysubjectedtoLCMSanalysis.Alternatively,thesamplesweredirectlysubjectedtoanalysiswithoutderivatizationandquantifiedbyexternalcalibration.NovelAspect:Thesensitivityofneurotransmitterswasdirectlycomparedusingdifferenthigh-endmassspectrometersunderidenticalLCcondition.PreliminaryResults:WithABSciexTripleQuad5500,thelowerlimitofquantification(LLOQ)ofderivatizedNE,5-HTandDAweredeterminedtobe0.005,0.005and0.05ng/mL,respectively.ThepeakarearatioreproducedwellwithqualitycontrolsampleatLLOQ,showingthatsamplepreparationandderivatizationresultedinnosignificantlossofanalyte.However,LOD(withoutderivatization)waslimitedto0.2ng/mLNE,0.02ng/mL5-HTand0.1ng/mLDAinartificialCSF.Ontheotherhand,theLLOQofintactcatecholaminesonShimadzuLCMS-8060wasdeterminedtobe0.005ng/mLforNE,5-HT,and0.01ng/mLforDA,withoutderivatization.TheLLOQofderivatizedNE,5-HTandDAweredeterminedtobe0.01,0.002and0.02ng/mL,respectively.Wearecurrentlystandardizingthesampleandchromatographicconditionsfordirectcomparisonbetweenthetwoinstruments.

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#111ImprovedpredictabilityofMDCKII-MDR1-BCRPassaybylowersubstrateconcentrationto0.1µM:potentiallyovercomingsaturationoftransportactivityYumeiYan-AstrazenecaAbstractP-glycoprotein(P-gp)andbreastcancerresistanceprotein(BCRP),bothATP-dependenteffluxtransporters,areexpressedattheapicalmembraneofblood-brainbarrier(BBB)whichplayanimportantroleinlimitingentryofdrugstoCNS.Double-transducedMDCKIIcellswithhumanP-gpandBCRPhasbeenwidelyemployedasanin-vitrotooltostudyeffluxpotentialofdrugsinCNSdrugdiscovery.InoneofAstraZenecaCNSprojects,weobservedthatin-vitroP-gp&BCRPcellsfailedtoefficientlyscreenoutlowbrainpenetrablecompoundsinratCNSmodelatsubstrateconcentrationof1µM,whichisthemostcommonlyadaptedconcentration.Forthisassay,oneofthereasonsisthattransportactivityofP-gpandBCRPmaybesaturatedat1µMtestcompoundconcentrationleadingtoincapableofidentifyeffluxpotential1.Herewepresentedtransportexperimentbyloweringthetestcompoundconcentrationto0.1µMtoavoidthepossiblesaturationofeffluxprotein.ThemodifiedassaywasvalidatedbyusingCNSdrugs.Themodelwasthensuccessfullyappliedfortheprojectwithgoodinvitro-invivocorrelationbetweenP-gpandBCRPmediatedtransportactivityandinvivobrainpenetrationinrats.Thepresentfindingsareconsistentwithliteraturereports,indicatingthatlowerconcentrationwasmoresensitivetoidentifydrugeffluxpotential.Theresultsprovideanefficientwayinselectingbrainpenetrablecandidateforhuman.

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#112LC-MS/MSmethodforthedeterminationofbileacidfromserumsampleusingCleanertMAS-MAWWarrenChen,SuziQin,WanWang-Bonna-AgelaTechnologies,Tianjin300462,ChinaCleanertMAS-MAWwasusedtopurifytheserumsamplewhichwithamixmechanismofreversephaseandanionexchangeinteraction.Ithasproventobeanefficientandsimplewaytoextractbileacidfromserumsample.Themix-modematerialeliminatedmatrixeffectofserumsuchasproteinsandphospholipidsandobtainedsufficientrecoverywithacceptableprecision.Samplepreparation100μLofserumsampledilutedwith100μLof1%formicacidaqueoussolutionwasloadedintoCleanertMAS-MAWwhichwaspre-conditionedby2mLofacetonitrileand2mLof3%formicacidaqueoussolutionsequentially.Thecartridgewaswashedwith2mLofmethanol/water(50/50,v/v),thendiscardedtheeluate.Thetargetcompoundswereelutedwith2mLofmethanoland2mLoftriethylamine/water/methanol(2/10/88,v/v/v)sequentially.Theeluateswerecollectedtogetherandconcentratedat40℃underagentlestreamofnitrogentodryness.Theresidueswerereconstitutedwith100μLofmethanol/water(7/3,v/v)andfiltratedbyPTFEfiltrationwiththeporesizeof0.22μm,andthenanalysisbyLC-MS/MS.ResultsanddiscussionCleanertMAS-MAWisamixed-modemechanismcartridgewithreversephaseandanionexchangematerial.Reversephaseinteractionexistbetweenpolystyrene/divinylbenzenematerialsandbileacidswiththefunctionalgroupsofcarboxylicacidandhydroxyl.Anionexchangeinteractionwassuppliedbyaminogroupwhichextractedthebileacidswithsulphonatefunctionalgroup.FormicacidwasusedtoadjustpHofloadingsample,whichcouldmaintainapartofbileacidsasmolecularstate.Thestepwasimportanttogetadmiredrecoverydataofbileacidswiththefunctionalgroupsofcarboxylicacidandhydroxyl.Mostoftheproteinandphospholipidswerewashedoutby50%methanolinwaterwithoutlossofbileacids.Theeluteprocesswasdividedintotwosteps.Reversephaseinteractionwasdisruptedby100%methanolinthefirststepwhichobtainedbileacidswiththefunctionalgroupsofcarboxylicacidandhydroxyl.Inthesecondstep,mixtureoftriethylamineandhighpercentageofmethanolelutedthebileacidswithfunctionalgroupofsulphonate.TriethylamineasorganicbasewasusedinionexchangeSPEmodewhileammoniumhydroxideincompetenttorestraintorionizethetargetcompoundandabsorbentmaterial.Inthisapplication,triethylaminewasusedasionizationsuppressiontocontrolthestateofabsorbentmaterial.Theaveragerecoveriesofbileacidfromthesamplepretreatmentmethodwithtwoconcentrationlevelsat50ng/mLand500ng/mLarerangefrom82.7%to130.8%,LODdataofbileacidarerangedfrom0.33to2.39ng/mL.ConclusionsThemixed-modeSPEplatecouldbeusedforeliminatingmatrixeffectofphospholipidsandproteinspriortotheanalysisofbileacidinserumbyLC-MS/MS.Asufficientrecoveryandgreatprecisionwereobtained.Themethodcanbeappliedfortheassayofbileacidinpatient'sserumsamples.

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#113N-ethylmaleimide(NEM)asthestabilizerforthesmallmolecule-drugconjugateswithdisulfidebondLingliZhang,ShaorongLi,JinqiangZhang,ZiqiangChengDMPK,ICC,Asia&EmergingMarketsiMed,AstraZenecaSmallmolecule-drugconjugates(SMDCs)directedagainsttumormarkerssuchasFR[1~3],PSMA[4],CAIX[5],somatostatinreceptor[6]representanefficientchemicalstrategyforthedeliverychemotherapeuticagentstotumorstogenerateanti-tumoractivitywithoutcausingmuchoftheassociatedoff-targettoxicities.SMDCsarebuiltwiththreemodules:ahighaffinitytargetingligand,asuitablelinkerandacytotoxicpayload(Fig.1)[1,2].DisulfidebondisanattractivelinkerwidelyusedinSMDCsproduct,whichcanbeefficientlycleavedtoreleaseactivedrugfromligandbyantioxidants(eg.Glutathione,GSH)insideendosomeaftertheSMDCistakenupbythediseasedcells[7,8].ThechallengeforDMPKscientististopreciselyquantifySMDClevelinbiologicalsampleslikeplasmawhichcontainsGSHandotherthiolcontainingmoleculesandcandegradeSMDC.Here,wehavedescribedtheutilityofN-Ethylmaleimide(NEM)asthestabilizerofdisulfidebondSMDCsinplasma.2mMofNEMinPBScansignificantlyreducethegenerationofpayloadfromSMDC.TheperformanceofNEMonstabilizingSMDCinplasmawastestedwithdifferentconcentrationsanddifferenttemperature.NEMat10mMiscapableofblockingGSHandthusstabilizingthedisulfidebondSMDCinplasma.Themethodwasappliedsuccessfullyinthestudyofmousepharmacokinetics(PK)ofSMDC:plasmasamplesaftercollectionwereimmediatelypretreatedby10mMofNEM.Inconclusion,ourstudyprovidedawayofdeterminingpreciselydisulfidebondSMDClevelinplasmaandhelpprojectteamtounderstandin-vivoPKofSMDC.Reference:1.JasgitSachdevetal,Aphase1studyof2differentschedulesofthefolicacid-tubulysinsmall-moleculedrugconjugateEC1456inpatientswithadvancedsolidtumorsAbstractP1.082.MaurerA.H.etal.Imagingthefolatereceptoroncancercellswith99mTc-Etarfolatide:properties,clinicaluse,andfuturepotentialoffolatereceptorimaging.J.Nucl.Med.2014,55:701-704.3.LeamonC.P.etal.ReducingUndesirableHepaticClearanceofaTumor-TargetedVincaAlkaloidviaNovelSaccharopeptidicModifications.JPET.,2011336(2):336-343.4.KularatneS.A.etal.Prostate-SpecificMembraneAntigenTargetedImagingandTherapyofProstateCancerUsingaPSMAInhibitorasaHomingLigand.Mol.Pharmaceutics2009,6(3):780-789.5.KrallN.etal.Abivalentsmallmolecule-drugconjugatedirectedagainstcarbonicanhydraseIXcanelicitcompletetumourregressioninmice.Chem.Sci.2014,5；3640-3644.6.MartinR.E.etal.DiscoveryoftheFirstNonpeptidic,Small-Molecule,HighlySelectiveSomatostatinReceptorSubtype5Antagonists:AChemogenomicsApproach.J.Med.Chem.2007,50:6291-6294.7.LeamonC.P.etal.Propertiesinfluencingtherelativebindingaffinityofpteroatederivativesanddrugconjugatesthereoftothefolatereceptor.Pharm.Res.2009,26(6):1315-1323.8.YangJ.etal.Evaluationofdilsulfidereductionduringreceptor-mediatedendocytosisbyusingFRETimaging.PNAS2006,103；13872-13877.

POSTERABSTRACT


#114ComprehensiveworkflowforsequencevariantanalysisusinganewbioinformaticsplatformandaUHPLCcoupledtoatribridmassspectrometer.StephaneHouel,TerryZhang,JessicaWang,JenniferSutton,MichaelA.Blank,JonathanJosephsThermoFisherScientific,SanJose,CA.Misincorporationofanunintendedaminoacidwillgenerateasequencevariantofthetargetprotein.Duringthedevelopmentofrecombinantproteintherapeutics,sequencevariantproteinsareaconcernforthebiopharmaceuticalindustrybecausetheycanpotentiallyinduceimmuneresponse.Sequencevariantproteins,presentatverylowlevel,areclassifiedasproduct-relatedimpurities.Herewepresentaworkflowtailoredsequencevariantanalysis.Totesttheworkflow,syntheticpeptidesrepresentingsequencevariantpeptideswerespikedintotheNISTIgGtrypticdigestatdifferentlevels.TriplicateLC-MS/MSrunswereacquiredindatadependentacquisitionmodeusingatribridmassspectrometercoupledtoanUHPLCforeachcondition.TherawfileswereprocessedwithBioPharmaFinder1.0softwareandonlythenativesequenceoftheIgGwasusedasaninput.InDDAmode,spikedpeptideswereselectedandhighqualityMS/MSwereacquiredevenwhenthesequencevariantpeptideswerespikedinatlessthan0.5%ofthenativepeptide.Thebiopharmasoftwaresuccessfullyidentifiedandquantifiedspikedsequencevariantpeptidesandalsoreportedcoefficientofvariation(%CV)below10%fortriplicateruns.PreviouslyidentifiedsequencevariantpeptidesoftheNISTdigestIgGwerealsoidentifiedwiththepresentworkflow.SequencevariantanalysisisachallenginganalyticalexperimentandthepreliminarydatashowedthattheLC-MShardwareaswellasthesoftwarearewellsuitedforthistypeofassay.

POSTERABSTRACT


#115HPLCSeparationPolarCompoundswithaFocusontheRoleStationaryPhaseESIndustriesThechromatographicseparationandtheanalysisofpolarcompoundsusingHPLChasalwaysbeenachallenging.ItwouldseemthatHPLCanalysiswouldbewellsuitedforthedeterminationofthepolarcompoundshoweverroutinereversephaseHPLCanalysishasyieldedpoorqualityresultsforthesetypesofcompounds.TheanalysisofpolarcompoundsviaroutineHPLCanalysishasbeendeficientinseveralmainaspectsincludingpoorretention,unacceptablelowk’valuesandpoorpeakshapes.OneoffewHPLCtechniquescapableofanalyzingpolarcompoundshasbeentraditionalnormalphasechromatography(polarstationaryphasewithanon-polarmobilephase).Unfortunately,normalphasechromatographysuffersfromseveralmajordeficienciesincludingpoorretentiontimereproducibility,poorcolumntocolumnreproducibilityandverylowornosolubilityofthetargetanalytes.AnumberofHPLCstationaryphaseshaveintroducedtoimprovetheanalysispolarcompounds.TheseHPLCstationaryphasesincludehighaqueousstablephases,HILIC(hydrophilicinteractionchromatography)phasesandpolarembeddedphasesallofwhichutilizevariouslevelsofwaterinthemobilephase.ItisthefocusofthispresentationtocompareandcontrasttheHPLCstationaryphasesdevelopedfortheanalysisofpolarcompounds.Inaddition,wewillintroducenewstationaryphasesoptimizedfortheseparationofpolarcompounds.TheultimategoalofthisworkistopresentastrategyfortheHPLCanalysisofpolarcompoundsandtoprovidethechromatographerwiththeinformationnecessarytomatchtheirpolarcompounds/sampleswiththeappropriateHPLCtechnique.

POSTERABSTRACT


#116UniqueChemicallyModifiedCarbohydrateBasedChiralStationaryPhasestoImproveChiralSeparationsESIndustriesThechromatographicseparationofchiralcompoundsisanimportanttoolinthesearchfornewpharmaceuticalentities.BothHPLCandSFCseparationsofchiralchemicalsareimportanttoolsforanalyticaldeterminationandpreparativeisolationofenantiomericmixtures.Existingchiralstationaryphasescanseparateamanychiralmixtures.Manyofthesechiralstationaryphasesarebasedonchemicallymodifiedcarbohydrates.However,evenwiththeexistingchemicallymodifiedcarbohydratesstationaryphasestherearestillmanyenantiomericmixturesthataredifficulttoseparatelimitingtheabilitytocharacterizeandpurifychemicalmixturescontainingchiralcompounds.Inthisstudywearechemicallymodifyingcarbohydrates,suchascelluloseandamylose,withfunctionalgroupsthathavenotbeenroutinelyemployed.Chemicalmodificationsofthecarbohydratesincludehalogenated,aromaticandhetero-aromaticfunctionalgroups.Wewillpresentinformationonthechiralseparationcharacteristicsandoverallseparationcapabilitiesforthesechemicallymodifiedcarbohydratebasedchiralstationaryphases.

POSTERABSTRACT


#117

TheDevelopmentofUniqueHPLCandSFCStationaryPhasesthatUtilizeAdvancedParticleTechnologies

ESIndustries

BothReversed-phaseHPLCandSFCiswidelyusedforseparationofmanychemicalcompounds.AmajorityoftheseseparationsarebasedonODStypecolumns.However,retentionandseparationofvariouscompoundshaveproventobeachallenge.Manyofthesetypesofcompoundsareunretained,poorlyretainedorunseparatedonmostconventionalODSreversed-phasecolumns,evenwhentheseODScolumnarepackedwithhighlyefficienctsub2particles.Fortunately,todealwiththesetypesofanalyteswecanemployalternativemodesofchromatographythatuseuniquestationaryphasescontainingpolargroups,organicbases,fluorinatedgroupsandothernon-hydrocarbonfunctionalgroups.ThesecolumnscantobeusedinthetraditionalreversephasemodeaswellasbothSFCand“hydrophilicinteractionchromatography”orHILIC.SFCusessupercriticalCO2alongwithanorganicmodifiersuchasmethanol.HILICchromatographyusesmobilephasescontainingbetween5-20%waterfortheretentionofpolarcompounds.Theseuniquestationaryphasesarebondedtosupportmaterialsthatutilizeadvancedparticletechnologies.Itwillbedemonstratedthatthecombinationofuniquestationaryphasesbondedtoadvancedparticletechnologieswillimprovedseparationsandaddflexibilitytooperatingconditions.

POSTERABSTRACT


#118AnintegratedChip-basedLC-MSSystemforHighPerformanceMicrospray.HelenaSvobodova,AmandaBerg,PeterWang,GaryValaskovicNewObjective,Inc.2ConstitutionWay,WoburnMA01801Packed-tipcolumnshavesuccessfullyenablednanoflowLC-MSapplicationsbydeliveringoptimalchromatographicperformanceatnanoflowrates.Incorporatingpacked-tip-columnsintoachip-likedeviceenablesease-of-usewhilepreservingtheperformanceofthisuniquecolumnformat.Couplingthischip-basedcolumnsolutiontopneumaticallyenabledsourcehardwareexpandsthefunctionalapplicationwindow,deliveringawiderrangeofoperatingflowrates(100nl/min.to10µl/min.)inasingledevice.Usingpneumaticallyenabledsourcehardwareandchip-basedcolumns,theeffectofpneumaticallyassistedESIwasevaluatedfor150µmIDchip-basedconsumablesat1µl/min.to5µl/min.Theeffectofheat,previouslyinvestigatedfor75µmIDcolumns,willbeexploredformicroflowLC-MSusing150µmand200µmIDcolumns.Theeffectofsheathgasonthespraystabilitywasevaluatedbycalculatingtherelativestandarddeviation(RSD)fortheextractedionchromatogramoftheangiotensinMH3+ion(349.9Da).Theoptimalconditionsfor1and2µl/min.wereachievedatasheathgassettingof0(RSD10.0and5.1respectively).TheadditionofsheathgasresultedinincreasedRSDvalues,withamaximumRSDof26.6at1µl/min.and10.4at2µl/min.,observedatasheathgassettingof10.Theadditionofsheathgaswasbeneficialatthehigherflowratesof4and5µl/min.At4µl/min.thespraywasmoststablewithasheathgassettingof20resultinginanRSDof3.4comparedwithanRSDof17.7withoutsheathgas.Thebestspraystabilityat5µl/minwasachievedwithasheathgassettingof30(RSD2.8),aneight-foldimprovementwhencomparedtoasheathgassettingof0(RSD23.1).ExtractedionchromatogramsfortheMH2+(524.2Da)andMH3+(349.9Da)angiotensinIIionsshowedchangesintheintensityratiosbetweenthesetwoionswhichwasparameterdependent.Increasingtheflowratefrom1µl/min.to5µl/min.resultedintheratioofMH3+/MH2+increasingfrom0.9to2.4at5µl/min.withagassettingof0.Increasingthegasfrom0to30resultedinadecreasedMH3+/MH2+ratiofrom1.2to0.6at3µl/min.Thespraystabilitydatawillbeusedtooptimizetheconditionsforthechromatographicrunsandthechromatographicdatapresented.

POSTERABSTRACT


#119

PharmacokineticsstudyofXGXinjectioninChinesenon-cancerpainpatients

WenXu-ClinicalPharmacologyResearchCenter,PUMCHXGXinjectionisanopioidreceptoranalgesic.Itisregisteredasatype1.1innovativedruginChinaFoodandDrugAdministration(CFDA).CurrentlyphaseIclinicaltrialofXGXisbeingevaluatedinPekingUnionMedicalCollegeHospital.Inthisthesisweinvestigatedthesafety,toleranceandsingle-dosepharmacokineticsofXGXinChinesenon-cancerpainpatients.ThemajorobjectiveofthispartwastodevelopandvalidateaUPLC-MS/MSmethodforthedeterminationofXGX,XGX-2andXGX-3inhumanplasma.TheUPLC-MS/MSmethodwasfullyvalidatedbasedonthebioanalyticalguidelineissuedbyCFDA.Thevalidationparametersincludedspecificity,linearity,sensitivity,accuracy,precision,stability,extractionrecoveryandmatrixeffect.ThestudyresultsshowedthattheUPLC-MS/MSmethodwasaccurate,rapidandsensitive.Withthishigh-throughputbioanalyticalmethod,theplasmaconcentrationsofXGX,XGX-2andXGX-3weresimultaneouslydetermined.ThismethodwascompletelysuitableforthehumanpharmacokineticstudyofXGXinChinesesubjects.Then,theplasmaconcentrationsofXGX,XGX-2andXGX-3weredeterminedusingtheUPLC-MS/MSdevelopedabove.

InordertofullyunderstandthepharmacokineticsofXGXinpatients,andtoidentifythevariabilityaffectingthepharmacokinetics,apopulationcovariatepharmacokineticmodelofXGXwasestablishedbymeansofnon-linearmixed-effectsmodelingusingPhoenix™NLMEsoftware.Thefollowingcovariatesweretested:creatinine(CR),directbilirubin(DBIL),albumin(ALB),Aspartatetransaminase(AST),totalbilirubin(TBIL),gender,totalbodyweight(TBW),heightandleanbodyweight(LBW).ThispopulationpharmacokineticmodelwouldbegreatlyhelpfulinthefutureclinicalevaluationofXGX.

POSTERABSTRACT


#120ComplementaryinvitrotoolstoinvestigaterenaldrugtransportLaszloSzilagyi,SOLVOBiotechnology

Renalexcretionisanimportantpathwayfortheeliminationofendogenousand

xenobioticsubstances.Awiderangeofeffluxanduptaketransportersareexpressedintherenalepithelialcellstoregulatetheexcretionandthereabsorptionofvariouskindsoforganicanions,cations,peptidesandnucleosides.

Theprimaryproximaltubulecell(PTC)monolayerassay,establishedbyDrColinBrown(NewcastleUniversity,UK)isauniqueinvitroapproachforinvestigatingrenaldrughandling,identifyingdrug-transporterinteractions,potentialnephrotoxicityandtransportermediateddrug-druginteractions.Unlikeotherprimaryrenalcellmodels,thismonolayerassaymaintainsthefullcomplementandexpressionlevelofendogenousrenaltransporters,resultinginamorephysiologicallyrelevant,andthereforepredictive,modelofdrughandlingintheclinicalsetting.

WhilethePTCmodelservesasaholisticapproachtostudyrenalhandlingofdrugmoleculesandendogenoussubstrates,doubletransfectedmonolayerassayscouldcompletetheresultsgainedinPTCmodelwithfunctionalcharacteristics,suchassubstratespecificityandtransportmechanismsinvolvedintherenalelimination.Certaindoubletransfectedmonolayerassayshavebeendevelopedtoinvestigatetheunderlyingmechanismsofrenaleliminationofbothorganicanion(MDCKII-OAT1/BCRPandMDCKII-OAT3/BCRP)andcation(MDCKII-OCT2/MATE1andMDCKII-OCT2/MATE2-K)compounds.Transportofselecteddrugsandphysiologicallyrelevantendogenoussubstratessuchasmetformin,methotrexate,PAH,urate,TEAandE3ShasbeeninvestigatedwithbothPTCandappropriatedoubletransfectantmonolayers.Theresultshighlightthecomplementarynatureofthetwomodels.

POSTERABSTRACT


#121DeterminationoftheliposomeandnonliposomevincristineinhumanplasmabyUltraPerformanceLiquidChromatography-tandemmassspectrometry YeTianBeijingUnionMedicalCollegeHospitalClinicalPharmacologyCenterBackground:Liposomalencapsulationofvincristinesulfateisdesignedtoincreasesafetyandtolerabilitybydecreasingneurotoxicityandgastrointestinaltoxicity.Liposomescanofferthepotentialofselectivedeliverytothesiteofaction,thusincreasingtheexposureprofiletodiseasecellsortissues.EMAandFDAhavepointedoutthatshoulddevelopedaneffectivebiologicalanalyticalmethodsofseparatingtheliposomeandnon-liposomedrug.Here,wedevelopedanultra-performanceliquidchromatography-tandemmassspectrometry(UPLC-MS/MS)methodtoquantifythefreevincristine(F-VCR)andtotalvincristine(T-VCR)inhumanplasmaafterintravenousadministrationofvincristinesulfateliposomeinjection(VSLI).Method:TheanalyticalcolumnwasHSST31.8µm（2.1×50mm）,andthemobilephasewascomposedofmethanol-watercontaining0.2%formicacid.Theflowratewas0.4mL/min.Detectionwasperformedwithmultiplereactionsmonitoring(MRM)usingpositiveelectrosprayionization(ESI).Result:Itwasindicatedthatthecalibrationcurvewaslinearovertheconcentrationrangeof0.2-50ng/mLforF-VCRand0.5-400ng/mLforT-VCR.Inter-andintra-dayprecisionwerelessthan15%andaccuracywaswithin85-115%aboutF-VCRandT-VCR.Conclusion:Thismethodprovedtoberapid,sensitiveandspecific,andsuitableforthepharmacokineticstudyofVSLIinhumans.

POSTERABSTRACT


#122HighQualityBioanalyticalServicesforTK/PK/BEStudiesBoLi*,XirongLi,XiaojuanWang,YuxuanLiu,WanxinWu,DongjunLi,PinYe,TianyiZhangDepartmentofBiologicsServices,FrontageLaboratories(Shanghai)Co.,Ltd.*Correspondingauthor,Email:[email protected](Shanghai)Co.,Ltd.wasestablishedin2006,theDepartmentofBiologicsServiceshasbeendedicatedtosupplyhighqualitybioanalyticalservicesonTK/PK/BEStudiesandonbiomarker/immunogenicityevaluationsforpreclinicalandclinicalstudies.About50projectswereconductedincludingmethodtransfer,methodvalidation,andsampleanalysisbyusingimmunoassaysandbiochemicalassaysonplatformsofSpectraMaxM2andMSDSectorImager6000.Thedrugsoftheprojectscoveredfromproteins(includingantibodies,polypeptidesorhormones),polysaccharides,tosmallmolecules(fortestsonPD/biomarkers).Wehavehighcapabilityonbioanalyticalservices:

1. MethodDevelopmentNotonlyusingmethodssuppliedbysponsorswhenworkingwell,ormodifyingmethodstomakethemworkbetter,butalsodeveloping/establishingmethodswhennoqualifiedmethodssuppliedbysponsorsRecentlywedevelopedmethodsforanti-drug-antibodydetectionsuccessfullywithgoodsensitivityandspecificitybyusingMSDplatform.

2. MethodTransferandValidationAfterwegetmethodsandreagents,methodstransferwasnormallydonewithin1~2weeks,andmethodvalidationdonewithin2~3weeksaccordingtoapprovedvalidationplan.

3. SampleAnalysisNormallyonescientistruns4~6plates,about100~ 200samplesononeassayday.Goodperformanceisalwaysrequired.ForexampleonaTKstudybyusingMSDplatform,over3000sampleswereanalyzedinmorethan150runs,lessthan0.5%ofrunsfailed,lessthan0.8%ofsampleswerereassayedduetoCV>20%,andmorethan97%ofISRsamplesmetrequirements.

DepartmentofBiologicsServicesatFrontageLaboratories(Shanghai)Co.,Ltd.willcontinuouslysupplyhighqualityservicesusingGLPprinciplesasaqualitystandard.

POSTERABSTRACT


#123

Developmentoftheinvitrofluorescence-basedhumanOATP1B1andOATP1B3ModelforDDIevaluation

Haiwei Xiong, Hui Dai, Hongxia Zhang, Ting Zhang, Jianling Wang, Genfu Chen

Department of DMPK Service, Lab Testing Division, WuXi AppTec Co. Ltd., 90 Delin Rd, Waigaoqiao Free Trade Zone, Shanghai, 200131, P.R. China

Theobjectiveofthecurrentstudywastoestablishthefluorescence-basedassaysfortheassessmentofdruginteractionwiththehumantransportersOATP1B1andOATP1B3stablyexpressedinHEK-293cellsusing8-fluorescein-cAMP(8-FcA)asasubstrate.TheOATP1B1orOATP1B3-mediatedtransportof8-FcAwastimedependentandsaturable(Kmwas3.49±0.734μMforOATP1B1,and1.70±0.190μMforOATP1B3,respectively)undertheconditionstested.EachofpositiveinhibitorsforOATPs,includingCyclosporineA,Bromosulfophthalein,RitonavirandRifampicin,demonstratedaconcentration-dependentinhibitionof8-FcAtransportbyOATP1B1andOATP1B3.Theinvitrofluorescence-basedassaysdescribedhereusing8-FcAasthesubstrateareconvenient,rapidandhaveutilityinscreeningdrugcandidatesforpotentialdrug–druginteractionswithOATP1B1andOATP1B3.

POSTERABSTRACT


YoungScientistExcellenceSubmission#124Relationshipsbetweenilaprazole/esomeprazoletreatmentandplasmaasymmetricdimethylargininelevelinhealthysubjectsandpatientswithduodenalulcerXuemeiLiu,HongyunWang,PeiHu,JiJiang*ClinicalPharmacologyResearchCenter,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalScience,Beijing,ChinaAbstractBackground:Protonpumpinhibitors(PPIs)havedemonstratedasuperiorefficacyprofileinthemanagementofacid-relateddisorders.Recently,muchmoreconcernshavebeenraisedaboutthepotentialforPPIstoincreasethecardiovasculardiseaserisk,whichmaybeillustratedbytheelevationofplasmaasymmetricdimethylarginine(ADMA),anendogenouscompetitiveinhibitorofendothelialnitricoxidesynthase(eNOS).However,thereislittleclinicaldataevaluatingtherelationshipbetweenplasmaADMAandPPIsexposure,andwhethertheuseofPPIsdirectlycausescardiovascularharm.Method:AtotaloftwoprospectiveclinicaltrialswerecarriedouttoinvestigatethepharmacokineticsofilaprazoleandesomeprazoleinChinesehealthyindividuals(ILA-I)andpatientswithduodenalulcer(ILA-IIA).ForILA-Istudy,16individualswith18-45yearsofageingeneralgoodhealthwererandomizedinto4treatmentsequences,whichwereseparatedby3periodsof1-weekwashoutintervalstoevaluateboth3ascendingdoses(5mg,10mg,20mg)ofilaprazolewithinjectionformulation(iv)and10mgofilaprazolewithenteric-coatedtablet(ETC)formulation.ForILA-I,20duodenalulceractivevolunteers,confirmedbygastroscopywithinanulcerdiameter≤15mm,mild,non-consolidatedulcerbleeding,wereincludedtoreceiveeither20mgilaprazole(iv)or40mgesomeprazole(iv).Inbothtwostudies,theplasmasamplescollectedforPKstudieswouldalsobeutilizedtoassessthefluctuationofADMAinplasma,rangingfrombaselineto24hafteradministration.Asimple,sensitiveandrapidhydrophilicinteractionchromatographicmethodcoupledtotandemmassspectrometrywasdevelopedandvalidatedforthedeterminationofplasmaADMA.ThepotentialcorrelationbetweentheADMAandconcentrationofPPIs(eitherilaprazoleoresomeprazole)wereevaluatedusingspearmancorrelationcoefficient.Results:TheprimitiveADMAplasmalevelswere0.44±0.05μmol/Lforhealthyvolunteers(n=15)and0.53±0.09μmol/Lforpatientswithduodenalulcer(n=20),andMann-WhitneyU-testshowstatisticallysignificantdifferencebetweentwostudies(p<0.05).However,therelationshipbetweenilaprazoleandesomeprazoleplasmalevelsandchange(%)inADMAfrombaselineshowmarginalcorrelation(r=0.036forhealthyvolunteersand0.085forpatients)withnostatisticalsignificance(p=0.599and0.903).Conclusion:Inthosetwostudiesconductedamonghealthysubjectsandpatientswithduodenalulcer,theADMAbaselineweremuchmorehigherinpatients,andPPIusedidnotsignificantlyinfluenceplasmaADMAlevel.Larger,long-termandblindedtrialsareneededtomechanisticallyexplainthecorrelationbetweenPPIuseandadverseclinicaloutcomes,whichhasrecentlybeenreportedinretrospectivecohortstudies.

POSTERABSTRACT


YoungScientistExcellenceSubmission#125

ASemiPBPKmodelconstructingthetransportermediatedclearanceandP450inductioninlivercompartment,todemonstratethedrugdruginteractionwhenco-administrationofRifampin,LopinavirandRitonavirinHIV-InfectedAdultsXunTao-ChempartnerLopinavir-RitonavirCocktailhasbeenwidelyappliedinthetherapyforHIV-infectedpatients.BothLopinavirandRitonavirhavebeenprovenassubstrateofCYP3A4withextensivemetabolismrateinhumanlivermicrosomes.However,neitherLopinavirnorRitonavirhasthegoodpassivepermeabilityintohepatocyte,Ontheotherhand,LopinavirandRitonavirarereportedasthesubstrateofOATP.Hence,theinvivoclearancesofLopinavirandRitonavirlikelyarelimitedbythestepofhepaticuptakerate.Theco-administrationofLopinavirandRitonavirutilizedthecompetitionofOATPoccupancytoslowdownthehepaticuptakerate,andthenachievetheAUCboost.Ontheotherhand,RifampincoadministrationdramaticallyreducesplasmalopinavirandRitonavirconcentrations,probablybecauseofitspotentCYP3A4induction.Arecentclinicaltrialdemonstratesthatdoublingthedoseofalopinavir-ritonavircapsuleformulationovercametheinteractionofRifampin.Basedonthatclinicaldata,aswellasotherpublishedin-vitrodata,asemi-PBPKmodelisestablished,whichconstructingthetransportermediatedclearance,andP450inductionprogressinlivercompartment,todemonstratetheaforementioneddrugdruginteraction.Themodelwillbehelpfulfortheprotocoloptimizationinfutureclinicaltrial.

POSTERABSTRACT



Modeling and Simulation of pharmacokinetics for Trastuzumabemtansinefrompre-clinicalspeciestohumanXianPan-ChempartnerThe researchanddevelopmentof therapeuticbiologicalproductshavebeen surgingover lastthreedecades.Humandoseandpharmacokinetics(PK)predictionbasedonthePKdataofpre-clinical species showsgreat importance inbridgingpre-clinical and clinical research, however,prediction of human PK profile can be very challenging, especially for the biologics showednonlinear PK causedby target-mediateddrugdisposition (TMDD). Trastuzumabemtansine (T-DM1)isapprovedforpatientswithhumanepidermalgrowthfactorreceptor2(HER2)-positivemetastaticbreastcancer.T-DM1bindstonon-humanprimateHER2butnottherodenthomolog.Therefore, target-dependent and non-target-dependent drug disposition can be evaluated inmonkeys and rats, respectively. In this study, total antibodyplasma concentration in rats andmonkeys was measured by generic enzyme-linked immunosorbent assay. Two-compartmentmodelwasappliedtoobtainT-DM1specificparametersinrats.Thentwo-compartmentmodelwithTMDDapproximationswasdevelopedtopredictT-DM1PKprofileinNHPsupondifferentdoseregimensusingBerkeleyMadonna9.0software.ThepredictedPKinNHPsofTDM1wasingood agreementwith the observed nonlinear PK. Using human physiological parameters andestimatedPKparametersviaallometricmethod,themodelsuccessfullypredictedthehumanPKprofileofT-DM1.Takentogether,themodelappliedinthepresentstudywasprovedtobeaneffectiveapproachinhumanPKpredictionforT-DM1,whichinturn,couldbehelpfulinthePKpredictionforthebiologicswithTMDD-causednonlinearelimination.

POSTERABSTRACT



Determinationofmajorclearancepathwayofilaprazoleinhumanbyprofilingandcharacterizinginvitroandinvivometabolitesofnon-radiolabeledilaprazoleJiePu

1,2

,FengWang1

,XiaomeiZhang3

,FeiMa1

,AliciaDu1

,MingsheZhu4

,WeiTang1

1

DMPKDept.ChemPartner,Shanghai,China;2

SchoolofPharmacy,EastChinaUniversityofScienceandTechnology,

Shanghai,China;3

BeijingInstituteofToxicologyandPharmacology,Beijing,China;4

Bristol-MyersSquibb,NJ,USA.AbstractIlaprazole,anewprotonpumpinhibitor(PPI),isdevelopedinChinaandapprovedbyCFDArecentlyforthetreatmentofpepticulcers.IlaprazolehassignificantadvantagesovertraditionalPPIssuchasomeprazole,includingfavorablelongerhalf-lifeandnoassociationwithpolymorphicCYP2C19.Metabolismstudyinhumanlivermicrosomes(HLM)showedthatilaprazolemainlyundergoesCYP3A4-mediatedsulfuroxidationtoformthesulfonemetabolite.However,thereisnocorrelationbetweentheCYP3Aphenotypeandilaprazoleexposureinhuman.Furthermore,co-administrationofilaprazolewithclarithromycin,apotentCYP3A4inhibitor,andclarithromycindoesnotincreasetheexposureofilaprazoleinhuman.Althoughilaprazolesulfideanditsfurthermetabolitearefoundinhumanurine,radiolabeledhumanADMEstudyhasnotbeenconducted.Thus,metabolismanddispositionofilaprazoleinhuman,especiallythemajorclearancepathwayandassociatedenzymeortransporter,remainsunclear.Theprimaryobjectiveofthisstudywastodeterminethemajorclearancepathwayofilaprazoleinhumans.Threesetsofexperimentswerecarriedouttoaccomplishtheobjective:(1)identification,characterizationandquantitativeestimationofilaprazolemetabolitesinHLM,(2)determinationofmetabolicratesandprofilesofilapraozolesulfideandilaprazolesulfoneinHLM,and(3)detectionandidentificationofilaprazole-relatedcomponentsinhumanurineandfeces.Invitroand/orinvivometabolitesoftestcompounds(ilaprazole,ilaprazolesulfide,andilaprazolesulfone)weredetectedandcharacterizedusinghighresolutionmassspectrometryandquantitativelyanalyzedusingLC/UVorLC/MSwithchemicalstandards.ResultsshowedthatilaprazolesulfideanditsdemethylatedproductweremajormetabolitesinHLM,whilepreviouslyreportedilaprazolesulfoneandmonohydroxylatedmetaboliteswereminormetabolites.ThereductionofilaprazoletosulfideinHLMdidnotrequireNADPHandoccurredafterHLMenzymesweredeactivated.AmajorityoftheseilaprazolemetaboliteswerealsoobservedinHLMincubationwithilaprazolesulfide.MetabolicstabilitytestingshowedthatT1/2valueswere34minforilaparazole,144minforilaprazolesulfonemetaboliteand8minforilaprazolesulfideinHLM.Alargenumberofilaprazolemetaboliteswerefoundinhumanurinebutnoparentdrugafterasingleoraldose(10mg),mostofwhichwereassociatedwithilaprazolesulfide.Basedontheseobservations,weproposethatnon-enzymeticreductionofilaprazoletothesulfidemetabolitefollowedbyfastoxidativemetabolismisthemajorclearancepathwayofilaprazoleinhuman.Ifmetaboliteprofilingdatainhumanfecessupporttheproposal,ilaprazolewouldhavenodrug-druginteractionswhenco-administeredwithaninhibitorofametabolizingenzymeortransporter.Theinformationwouldgreatlyhelpthesafeuseofilaprazoleinpatients.

POSTERABSTRACT



PredictionofInVivoHepaticUptakeofRosuvastatininChinesePeopleBasedonTransporterProteinQuantificationandInVitroHepatocyteUptakeModelTaoChen1,2,NaLong1,HongYu1,SuxingZhang1,FeipengZhang1,XueqinLv1,PeizhenYe1,LaiyouWang2,KezhiZhang1*1CarysBioHoldingsLimited,Foshan,GuangdongProvince,PRC2GuangdongPharmaceuticalUniversity,GuangdongProvince,PRCPurpose:Thisresearchistoexplorethecorrelationbetweentransporterexpressionanditsbiologicalfunctions,andpredicttheinvivotransportermediatedhepaticuptakeofRosuvastatinbasedontransporterproteinquantificationandInVitroHepatocyteUptakeModel.Method:DrugtransportersincludingOATP1B1,OATP1B3,OATP2B1andNTCPplayanimportantroleinRosuvastatinhepaticclearance.However,noeffectivemethodcouldbeusedtoevaluatetheuptakeactivitiesinvivo.So,thisresearchaimedtoestablishamodelbasedonPBPKmodelthroughdeterminationoftransporterproteinexpressionanduptakeactivityinvitrotopredicttransporter-mediateduptakeinvivo.Firstly,theliquidchromatography-tandemmassspectrometry(LC-MS/MS)methodwasdevelopedandvalidatedforsimultaneousdeterminationofOATP1B1,OATP1B3,OATP2B1andNTCPexpressionlevelin23donorsofChinesehumancryopreservedhepatocytesandHEK293cellsstablytransfectedwithhumanOATP1B1/OATP1B3/OATP2B1/NTCPgene,withsynthesizingsurrogatepeptidesofOATP1B1,OATP1B3,OATP2B1andNTCPascalibratedstandard,andcorrespondingstableisotopelabeledsurrogatepeptidesasinternalstandardandoptimizingthechromatographandmassspectrometryconditions.

Second,KmandVmaxvaluesofRosuvastatinweredeterminedwithstabletransfectedcelllinesincludinghOATP1B1-293,hOATP1B3-293,/hOATP2B1-293andhNTCP-293,andappliedinpredictionofuptakeactivityinvivocombinedwiththeresultofproteinquantificationbasedonMichaelis-Mentenequation.

Besides,withthemodelofsandwichculturedhumanhepatocytes,hepaticuptakeactivitiesofRosuvastatinwerealsodeterminedindifferentbatchesofhumanhepatocytes.Andtheresultwasusedtoanalyzethecorrelationbetweentransporterproteinexpressionanduptakeactivityinvitroandthedifferencesofpredictingactivityinvivoanddetermineddatainvitro.Result:TheLC-MS/MSmethodwasestablishedandvalidatedforsimultaneousquantificationofOATP1B1,OATP1B3,OATP2B1andNTCP.Boththeintra-dayandinter-dayprecisionandaccuracyofanalyteswerewithintheacceptablecriterionofmacromoleculesbioanalysis.Thestandardcurvesweredemonstratedlinearintherangeof0.2-50nMforOATP1B1,OATP1B3andOATP2B1and0.4-100nMforNTCP,respectively(r＞0.995).Astheresult,theproteinexpressionlevelsofOATP1B1,OATP1B3,OATP2B1andNTCPin23donorsofChinesehumancryopreservedhepatocyteswere4.74±1.26,1.39±0.82,3.02±0.71and3.69±1.15fmol/μg,respectively.TheresultsshowedthatthesefourtransportershaddifferentexpressionlevelonhepatocytesmembraneinwhichOATP1B1wasthehighest.Besides,transporterproteinexpressionhadindividualdifferences.Bothcouldbeusefulfordrugresearchandclinicalindividualizedmedication.WiththeKmandVmaxresultinstabletransfectedcellmodel,Rosuvastatinwasthesubstrateofallfourtransporterwithdifferentaffinity(Km:OATP1B3<NTCP<OATP1B1<OATP2B1).Combinedwithtransporterquantification,WiththeKmandVmaxresultinstabletransfectedcellmodel,Rosuvastatinwasthesubstrateofallfourtransporterswithdifferentaffinity(Km:OATP1B3<NTCP<OATP1B1<OATP2B1).Combinedwithtransporterquantification,NTCPshowedamajorcontributioninthehepaticuptakeofrosuvastainwith68%ofoverallactiveuptake,andpredictingactivitywas56.9±37.0pmol/min/mgbasedonMichaelis-Mentenequation.Besides,theresultofhepaticuptakeclearancewithsandwichculturedhumanhepatocytesshowedthat74.94%±15.04%ofhepaticuptakeofRosuvastatinwasmediatedbytransporters,andthedeterminedactiveuptakeactivitywas21.0±19.7pmol/min/mg.Asthecomparisonofthevaluesofpredictingclearanceanddeterminedhepaticactiveclearance,linearregressioncorrelationcoefficientwas0.66forRosuvastain.Andtheresultswithspearmananalysisshowedpositivecorrelationwithrswas0.3604.Conclusion:proteinquantificationofhepatictransporterwasveryimportantforpredictionandevaluationofdrughepaticclearanceinvivo.AndapreliminaryPBPKmodelwasestablishedwithtransporterquantificationandhepaticuptakemodelinvitrowithwellcorrelationbetweenpredictinganddetermineddata.

POSTERABSTRACT


YoungScientistExcellenceSubmission#129 ASystemsPharmacologyModelforPredictingAnticoagulantEffectsofFXaInhibitorsinHealthySubjects:AssessmentofPharmacokineticandBindingKineticProperties*XuanZhou1,DymphyR.H.Huntjens2andRonAHJ.Gilissen3.1ClinicalPharmacology,Novartis,China;2Model-BasedDrugDevelopment,Janssen,Belgium;3DepartmentofPharmacokinetics,MetabolismandDynamics,DiscoverySciences,Janssen,Belgium. Forthelastdecade,theconceptofdrug-targetbindingkineticshasbeeninfocusinthedrugdiscovery,becausethereismountingevidencethattheoftenignoredkineticaspectsoftheinteractionbetweenasmallmoleculedruganditsproteintargetarehighlyrelevantforinvivoefficacyandclinicalsuccess.TheexplorationofpharmacokineticsandbindingkineticsofdrugsfocusingonthedurationofeffecthasbeendescribedusingtraditionalPK/PDmodels.However,thecomplexbiologicalprocessanddetaileddrugactionareusuallyunclear,whichmakesitdifficulttopredictclinicaloutcomeatthedrugdiscoverystage.Inthisstudy,weselectedthetargetofFactorXa(FXa)asanexample,whichemergedasapromisingtargetforeffectiveanticoagulation.Alarge-scalesystemspharmacologymodelwasdevelopedbasedonthepublisheddata.Ittakesintoaccountthepathwaysofthecoagulationnetwork,andcapturesdrug-specificfeature:PKandbindingkinetics.Astheresults,themodelpredictstheclottingtimeandanti-FXaeffectsandcouldserveasapredictivetoolfortheanticoagulantpotentialofanewcompound.

POSTERABSTRACT


YoungScientistExcellenceSubmission#130Applicationofamulti-compartmentmodeltopredictthedistributionofasteroidglycosideingut,liverandplasmainrats.CuifengZhang1,2,HongcanRen3,YunfeiLin2,ZhitaoWu2,XuanNi2,LeWang2, HaitangXie*1,GuoyuPan*2.(1.YijishanhospitalofWannanMedicalCollege2.ShanghaiInstituteofMateria& Medica,UniversityofChineseAcademyofSciences3.HutchisonMediPharma)

Introduction: TX-1 is a major bioactive steroid glycoside isolated from traditional Chinesemedicine. Ithasvariouspharmacologicalactivities,suchasanti-oxidative, anti-inflammatory,andanti-plateletaggregation.PreviouslystudyshowedTX-1was mainlymetabolizedintoTX-2andTX-3bythedeglycosylationfromintestinal microbiota.Thisstudyaimedtoestablishamulti-compartmentmodeltosimulatethe PKprofileofTX-1ingut, liverandplasmainrats,whichmayfacilitatethe understandingofthemetabolickineticsofTX-1anditsmetabolites.Methods: TX-1metabolism in gut was determined by get SD rat colon contents incubatedin vitro. After single oral administration in SD rats respectively, the plasma and liverconcentration of TX-1, TX-2 andTX-3werequantified byHPLC-MS/MS. Their individual PKparameters were calculated. Amulti-compartment model was constructed forTX-1and itsmetabolites TX-2 and TX-3 using Phoenix WinNonlin (6.1). Data from literature and ourpartner’sgroup(ProfessorChenggangHuang)were usedtovalidatetheaccuracyofthismodel.ThemodelwasemployedtopredictTX-1, TX-2andTX-3liverconcentrationinrats.Results: An integrated multi-compartment model was established successfully. Our modelworked well in the dose range from 30-90mg/kg.When the dose increased to 180mg/kg,the predicted plasma concentrations were below the observed concentrationswhichmaycamefrommetabolicsaturation.Conclusions:An integratedmulti-compartment model was successfully constructed, whichwasimportanttounderstandthedispositionofTX-1,TX-2andTX-3inplasma, liverandgut.

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