HPLC-UV法測定新型抗腫瘤候選化合物N-芐氧羰基-甘氨酰脯氨酸-阿霉素在大鼠膽汁中的濃度及其膽汁排泄研究

1/1HPLC-UV法測定新型抗腫瘤候選化合物N-芐氧羰基-甘氨酰脯氨酸-阿霉素在大鼠膽汁中的濃度及其膽汁排泄研究AsimpleHPLC-UVmethodforthedeterminationofanovelanticancercandidatecompoundZ-Gly-Pro-doxorubicininratbileanditsapplicationtobiliaryexcretionstudy#HUANGWeixin,WANGJingjing,MALi,HANHai,XUJun,CAIShaohui**(CollegeofPharmacy,JinanUniversity,Guangzhou510632)Abstract:Z-Gly-Pro-doxorubicin(Z-GP-DOX),aprodrugofdoxorubicin(DOX),hasbeenprovedinourpreviousstudytobeagoodprodrugtoachievetargeteddeliveryofDOX.Also,thepharmacokineticstudyofZ-GP-DOXinratplasmahasbeencompleted.Inthispaper,asimple,sensitiveandspecificHPLC-UVmethodforthedeterminationofZ-GP-DOXinratbilewasestablishedandvalidated.Followingliquid-liquidextraction,chromatographicseparationwasaccomplishedbythemobilephaseacetonitrile-0.1%trifluoroaceticacid(50:50,v/v)withaC18chromatographycolumnataflowrateof1mL/min,roomtemperatureanddetectionwavelengthof495nm.TheretentiontimeofZ-GP-DOXwas6.6min.Alinearcurveovertheconcentrationrangeof1-1200g/mL(r20.999)wasestablished,andtheLODandLOQforZ-GP-DOXwere0.5and1g/mL,respectively.Goodprecisionandaccuracyatconcentrationsof2,600and1000g/mLwereobtained.ThemeanextractionrecoveryofZ-GP-DOXinbilewasover82.92%atthestudiedconcentrations.Theintra-dayandinter-dayrelativestandarddeviationsweregenerallylessthan10%.ThismethodwassuccessfullyappliedtobiliaryexcretionstudyinratsafterintravenousadministrationofZ-GP-DOX.Bilesampleswerecollectedfrombileductcannulatedratsafteranintravenousbolusdoseof10mg/kgor20mg/kgZ-GP-DOX,andtheconcentrationsweremeasuredbyHPLC-UV.TheresultsshowedthattheconcentrationofZ-GP-DOXinratbilewasmuchhigherthanthatinplasma.Afterdosing,28.142.13%and20.253.59%ofthedosewereexcretedintobileinunchangedformaftera12-hcollection.ThepresentstudywillcontributetosupplementingthepreviouspharmacokineticstudyofZ-GP-DOXinratsandwillbehelpfultoimprovethedruggabilitystudyofZ-GP-DOX.Keywords:Z-GP-DOX,HPLC-UV,ratbile,excretion510152025300IntroductionDoxorubicin(DOX),oneoftheanthracyclineglycosideantibiotics,iswidelyusedinthechemotherapyofhumancancerbecauseofitsbroadspectrumofantineoplasticactivity[1-3].However,cumulativedose-relatedcardiotoxicityisamajoradversereactionofDOX,inadditiontotheacutetoxicities,suchasstomatitis,alopecia[1],myelosuppression[4],nauseaandvomiting[1,4].Therefore,retentionofitsanticanceractivitywithoutincreasingitstoxicitiesisachallengeinthefieldofcancerchemotherapy.Formanyyears,endeavorshavebeenmadetoimprovetheselectivityandreducethetoxicityusingnontoxicprodrugsthatarepreferentiallyconvertedintoactiveanticanceragentsatthetumorsitewheresomeenzymesareexpressedathighlevelsandtheactivityoftheseenzymesiselevatedcomparedwiththatinthenormaltissuesfromwhichthe40tumorsarederived[5].Fibroblastactivationprotein(FAP),whichisaserineprotease[6],isexpressedinover90%ofthestromaofmalignantepithelialtumors,whilenotinnormaltissues[7,8].FAPexhibitsadipeptidylpeptidaseIV(DPPIV)-likeexopeptidaseactivity.Moreover,FAPbutnotDPPIV,possessesendopeptidaseactivitytowardN-terminalbenzyloxycarbonyl(Z)-blockedpeptides[9].Consequently,FAPisincreasinglyconsideredasapromisingtumor45targetfordesigningtumor-targetedprodrugsbyconjugatingananticancercompoundwithaFAP-specificZ-blockedpeptide.Inthisway,thesynthesizedprodrugcouldreleaseitsparentdrugatthetumorsitesexpressingFAP,whilequitestableinthenormaltissues.Thus,theanticanceractivitycouldberetainedwhilethetoxicactivitieswouldbeavoided.AFAP-targetingprodrugofDOX,Z-Gly-Pro-Doxorubicin(Z-GP-DOX,Fig.1)wasFoundations:NationalNaturalScienceFoundationofChina(NO.30973565),NationalImportantTechnologyProjectBriefauthorintroduction:黃偉鑫，(1987-)，男，研究生，藥動學(xué)Correspondanceauthor:蔡紹暉，(1956-)，女，教授，免疫與腫瘤藥理學(xué).csh5689@35-1-synthesizedbyconjugatingtheaminogroupofDOXwiththehydroxylgroupofaFAP-specificdipeptideZ-Gly-Pro(Z-GP)[10].WepreviouslydemonstratedthatuponthehydrolysisofFAPaswellastheincubationwithtumorhomogenateof4T1tumor(FAP-positivetumor),DOXcouldbeeffectuallyreleasedbyZ-GP-DOX,whichwerehighlystableinmouseplasmaandavarietyoftissuehomogenatesincludingheart,liver,andsoon.Additionally,Z-GP-DOXproducedsimilarantitumorefficacyin4T1tumor-bearingmicetofreeDOXwithoutobviouscardiotoxiceffect.Moreover,accumulationofZ-GP-DOXreducedsignificantlyinheartcomparedtofreeDOX[10].ThesefindingssuggestedthatZ-GP-DOXcouldbeapromisinganticancerprodrugtoachievetargeteddeliveryofantitumoragents.Also,asensitiveandstableHPLCmethodfordeterminingtheconcentrationofZ-GP-DOXinratplasmahasbeenestablishedandvalidated.Afteri.v.doseof20mg/kg,theconcentrationofZ-GP-DOXinratplasmareachedtothepeakvalueof19.911.30mg/kgat3minandthendecreasedrapidlyduringthenext5h[11].ThismethodhasbeensuccessfullyappliedtothepharmacokineticstudyofZ-GP-DOXinratplasma[12].Thepresentstudyisperformedonthebasisoftheachievementsabove.Fig.1ChemicalstructureofZ-GP-DOX.50556065DOXismainlyexcretedintobile[13,14],thereforetheinvestigationonZ-GP-DOXbiliaryexcretionisofgreatimportance.Also,biliaryexcretionstudyisoneofthecriticalcomponentsofpreclinicalpharmacokineticstudiesindrugdevelopment.Tofacilitatesuchstudy,asuitableanalyticalmethodforquantificationofZ-GP-DOXinbileisnecessary.Therefore,inthispaper,a70highlysensitiveandselectiveHPLC-UVassayforZ-GP-DOXdeterminationinratbilewasestablishedandvalidatedaccordingtothecriteriabasedonFDAguidelines[15].Furthermore,asimpleliquid-liquidextractionmethodwasusedinthesamplepreparationbeforequantification.Finally,thisanalyticalmethodwasappliedtothebiliaryexcretionstudyofZ-GP-DOX.1Experimental1.1Chemicalsandreagents75Z-GP-DOX(purity99%)wasobtainedfromCollegeofPharmacyofSunYat-senUniversity(Guangzhou,China).MethanolandacetonitrileofHPLCgradewerepurchasedfromMerck(Darmstad,Germany).AllotherchemicalsofanalyticalgradewerefromTianjinDamaoChemicalReagentFactory(Tianjin,China).1.2Animals80MaleSprague-Dawleyrats,weighing220-280g,werepurchasedfromtheMedicalLaboratoryAnimalCenterofGuangdongProvince(Guangzhou,China).Animalswerehousedundercontrolledenvironmentalconditionswithfreeaccesstotapwaterandastandarddiet.AnimalexperimentswereapprovedbytheAnimalEthicsCommitteeoftheChineseAcademyofMedical85Sciences.-2-1.3Preparationofstocksolutions,workingsolutionsandqualitycontrolsamplesPrimarystocksolutionofZ-GP-DOXwaspreparedinmethanolataconcentrationof10mg/mL.WorkingsolutionsofZ-GP-DOXwereobtainedbyserialdilutionofthestocksolutionwith9095methanol.Thestocksolutionandtheworkingsolutionswerestoredat4C.TheworkingsolutionswereaddedtoblankSDratbiletopreparecalibrationstandardsamplesovertherangeof1to1200g/mL.Freshlypreparedcalibrationstandardswereusedinonecalibrationcurvewhichwasconstructedoneachanalysisbatch.Thequalitycontrolsamples(QCs)werepreparedwithblankbileinthesamemethodatlow,mediumandhighconcentrationsof2,600and1000g/mL.Liquidchromatographyinstrumentsandconditions1.4AnalysisofthesampleswasperformedonaWatersHPLCsystem(WatersCompany,USA)equippedwithaWaters515pump,717plusautosampler,2487UVdetectorandEmpowersoftware.Thedetectionwavelengthforquantificationwas495nm.100105Theanalyticalcolumnwasareversed-phasecolumn(UltimateXB-C18,5m,2504.6mmi.d.,WelchMaterials,Inc.,USA)withaC18guardcolumn(10m,8mm4.6mmi.d.).Thecolumntemperaturewasroomtemperature.Theanalytewaselutedisocraticallywithamobilephaseconsistingofacetonitrile-0.1%trifluoroacetic(50:50,v/v)ataflowrateof1mL/min.Thetotalanalyticalruntimeforeachsamplewas8min.Themobilephasewasfilteredundervacuumthrougha0.2mmembranefilter(JintengExperimentEquipmentCo.,Ltd.,Tianjin,China)anddegassedfor15minbyultrasonicationbeforebeingused.Samplepreparation1.5Theblankbile,calibrationstandards,QCsamplesandbilesampleswerepreparedusingliquid-liquidextraction,where2mLextractingsolution(acetonitrile-dichloromethane=1:4,v/v)110wasaddedinto100Lbilesamplewhichwasthenvortexedfor90s.Subsequently,centrifugation(9000gfor15min)wasperformedtoseparatetheaqueousandorganiclayer.Theorganiclayerwastransferredtoa4mLeppendorftubeandevaporatedtodrynessat60Cunderastreamofnitrogen.Thedriedresiduewasredissolvedin200Lmethanolbyvortexing,followedbycentrifugation(7000gfor5min).10LofthesupernatantwasinjectedintotheHPLCsystem.1.6Methodvalidation115TheanalysisofbiologicalsampleswascarriedoutandtheestablishedmethodwasvalidatedaccordingtotheguidelinesofUSFDA[15].1.6.1SpecificityThespecificityofthemethodwasevaluatedbyanalyzingblankbilesamplesfromfiverats.120Eachblanksamplewaspreparedusingtheliquid-liquidextractionprocedureandtestedforendogenousinterferencewiththeHPLCconditionsdescribedabove.1.6.2LinearityandsensitivityThelinearityoftheHPLCmethodforthedeterminationofZ-GP-DOXwasevaluatedbyacalibrationcurveintheconcentrationrangeof1to1200g/mLwitha1/x2weightingfactor.The125calibrationcurvewasconstructedbyplottingthepeakareaofZ-GP-DOXversusitsconcentration.Thelimitofdetection(LOD)wasdefinedasthelowestconcentrationofZ-GP-DOXinspikedbileresultinginasignal-to-noiseratioof3:1;forthelimitofquantification(LOQ),asignal-to-noise-3-ratioof10:1wasused.1.6.3Precisionandaccuracy130135TheprecisionandaccuracyoftheHPLCmethodwereassessedbyassayingfivereplicateQCsamplesatthreedifferentconcentrations(2,600and1000g/mL),whichwerepreparedinthesamemethodasthecalibrationsamples.Thepercentageofobservedvaluetotruevalueandthepercentagerelativestandarddeviations(RSD,%)werecalculatedtoshowtheaccuracyandtheprecision,respectively.Toevaluatetheintra-andinter-dayprecision,fivereplicatesofeachconcentrationlevelwereanalyzedin1dayandoverthreeconsecutivedays,respectively.1.6.4ExtractionrecoveryTheextractionrecoveryshowedanabilitytoextracttheanalytefromthebiologicalsamples.TherecoveryofZ-GP-DOXfrombilewasdeterminedbyassayingfivereplicateQCsamplesatthreedifferentconcentrationlevels(2,600and1000g/mL)andwascalculatedbycomparingthe140peakareaofZ-GP-DOXinQCsamplesthathadbeenspikedwithZ-GP-DOXpriortoextractiontothatofZ-GP-DOXaddedinblankbileextract.1.6.5StabilityStabilitystudieswerecarriedoutonQCsamplesatthreedifferentconcentrationlevelsof2,600and1000g/mLtoensurethereliabilityoftheresultswithregardtohandlingandstoringofthe145bilesamplesandworkingsolutions.Short-termstabilitywasperformedbyplacingQCsamplesforaperiodof4hatroomtemperature(RT).Long-termstabilitywasevaluatedbyfreezingQCsamplesforaperiodof4weeksat-80C.FreezeandthawstabilityforthreecycleswasdeterminedbythawingatRTandthenrefreezingat-80Cfor24h.Workingsolutionsstabilitywastestedfor30daysat4C.1.7Applicationtobiliaryexcretionstudy150Animalswerefastedovernightwithfreeaccesstowaterbeforetheexperiment.Jugularveincanulationwasfirstcarriedoutinratsafterinductionofanesthesiawith2%pentobarbitalsodium(40mg/kg,i.p.).Bileductcanulationwasthenperformed.Afterthesurgery,bilesampleswerecollectedfor15minutesbeforeZ-GP-DOXadministration.Z-GP-DOXdissolvedinamixture155160165170(propyleneglycol-isotonicsodiumchloridesolution=1:4,v/v)wasthenadministeredtoratsviathejugularveincannula.Twodosagegroups,10and20mg/kg,whichwerebasedontheprevioustoxicologicalstudies[16]andthenusedinthepreviouspharmacokineticstudyinratplasma[12],wereappliedtothepresentstudy.Bilesampleswerecollectedat60-minintervalfor12hoursafterdosingandat15-minintervalduringthefirst60minandthebilevolumewasrecorded.Bilesampleswerestoredat-80CuntilHPLCanalysis.Frozenratbilesampleswereleftonthebenchtothawnaturallyandwerevortexedpriortotheiruse.Fiveanimalswereusedforeachdosagegroup.Theanimalswerekeptunderanesthesiaandbalancedsaltsolutionwasinjectedviathejugularveinusingasyringeinfusionpump(KDScientific100,Holliston,USA)tosupplementthelostbodyfluidthroughoutthewholeexperiment.ThebileZ-GP-DOXconcentrationswerequantifiedbytheestablishedHPLCmethod.TheamountofZ-GP-DOXexcretedintobileduringeachintervalwascalculatedbymultiplyingthebileconcentrationwiththevolumeofbilesample.CumulativeamountofZ-GP-DOXexcretedintobileoveracertaintimeperiodwascalculatedbyaddingalltheamountwithinthetimeinterval,andcumulativeexcretionwasexpressedintheformof%ofdosebydividingwiththetotaldoseadministrated.-4-Pharmacokineticnon-compartmentalmodelusingDrugAndStatisticsversion3.0(DAS3.0,BontzInc.,Beijing,parametersofZ-GP-DOXinbileweredeterminedbasedonChina).AStudentst-testwasperformedtocomparethepharmacokineticparametersofthetwodosagegroups.Aconfidencelevelof0.05wasconsideredasstatisticallysignificant.2ResultsMethodvalidationSpecificity1752.12.1.1Therepresentativechromatogramsofblankratbile,ratbilespikedwithZ-GP-DOX(20g/mL),andaratbilesample8hafterintravenousinjectionofZ-GP-DOXareshowninFig.2,showing180185theendogenouscomponentsandZ-GP-DOXpeakswereseparatedsufficiently.TheretentiontimeofZ-GP-DOXwasabout6.6min.Twosuspectedmetabolites(M1andM2)werealsodetectableandseparatedwithretentiontimesofabout5.4and6.0min,respectively.Fig.2HPLC-UVchromatogramsof(A)blankbile;(B)blankbilespikedwithZ-GP-DOX(20g/mL);(C)aratbilesample8hafteri.v.administrationofZ-GP-DOX(20mg/kg).Z-GP-DOX(retentiontimeof6.6min);M1(retentiontimeof5.4min)andM2(retentiontimeof6.0min):twosuspectedmetabolitesinratbile.2.1.2LinearityandsensitivityThecalibrationcurveofZ-GP-DOXinratbilewaslinearintheconcentrationrangeof1-1200g/mLwitharegressioncorrelationcoefficientof0.9999.Theregressionequationofthe190calibrationcurvewasy=4630x+276.96,whereywasthepeakareaofZ-GP-DOXinratbilesamplesdetectedinHPLC-UV,andxwasthebileconcentrationofZ-GP-DOX.TheLODandtheLOQforZ-GP-DOXwere0.5and1g/mL,respectively.TheseresultsshowedagoodlinearitybetweenthepeakareasandconcentrationsanddemonstratedthatthemethodcouldbeappliedtobilesamplesinawiderangeofZ-GP-DOXconcentrationlevels.2.1.3Precisionandaccuracy195TheprecisionandaccuracydataisshowninTable1.Theintra-dayandinter-dayprecisionforconcentrationsof2,600and1000g/mLZ-GP-DOXwerewithin8.58%,andtheaccuracywas-5-around91.76%.Theseresultswereallwithintheacceptablerange,whichdemonstratedthattheestablishedmethodwasapplicabletothequantificationofZ-GP-DOXandtheresultsdidnot200dependontheconcentrationofthesampleoronthedayoftheassay.Tab.1Thevalidationofintra-andinter-dayprecisionandaccuracyofZ-GP-DOXinQCsamples(n=5).Concentration(g/ml)Intra-dayvariabilityPrecisionAccura(RSD,%)cy(%)5.725.866.15Inter-dayvariabilityPrecision(RSD,%)(%)8.586.684.98Accuracy26001000103.69101.8891.76106.00104.1298.922.1.4ExtractionrecoveryThemeanextractionrecoveriesofZ-GP-DOXfromspikedratbileundertheliquid-liquidextractionconditionswere82.924.72,83.804.92and83.185.12%(n=5)atconcentrations205of2,600and1000g/mL,respectively.AlloftheextractionrecoveriesweresufficientforthedeterminationofZ-GP-DOX.2.1.5StabilityThestabilityofZ-GP-DOXduringsamplehandling(short-termtemperature,long-termandfreeze-thaw)isshowninTable2.Themeanrecoveriesfromnominalconcentrationsafterthree210215freezeandthawcyclesweremorethan92.54%.Z-GP-DOXwasstableinbilesampleswhenstoredat-80Cfora4-weekperiod,andmeanrecoveriesfromthenominalconcentrationswereallmorethan94.44%.AfterstoredatRTfor4h,atleast87.87%ofZ-GP-DOXwasstillpresentinratbile.However,inthepreliminaryexperiment,Z-GP-DOXwasnotstablefor12hatRTinbilesamples,andmeanrecoveriesfromthenominalconcentrationsdeclinedtolessthan50%,whichindicatedthatthebilesamplesshouldbestoredinthecoldimmediatelyaftercollection.Themeanrecoveriesofstocksolutionsafterstoredat4Cforatleast30dayswereover99.712.29(showninTable2),indicatingthatthestocksolutionswerestableinthiscondition.Tab.2StabilityofZ-GP-DOXinratbile(n=3;meanSD).Concentration(g/ml)Freeze-thaw92.543.88Recovery(%)Short-termLong-termStocksolution99.712.292600100095.843.2696.148.7190.241.4992.262.0187.871.3894.443.9097.126.6998.837.94102.662.07104.890.022.2Biliaryexcretionstudy220225230ThebiliaryexcretionofZ-GP-DOXwasmeasuredupto12hafteri.v.administration(10mg/kgor20mg/kg).TheconcentrationofZ-GP-DOXinbileversustimeatdifferentdosesviai.v.administrationisshowninFig.3(A).TheconcentrationofZ-GP-DOXinbilereachedto834.87194.77g/mLwiththedosageof10mg/kgand848.82158.86g/mLwiththedosageof20mg/kgaround15-30min.Then,theconcentrationsdecreasedrapidly.Duringthewholeresearchperiod,theconcentrationsofZ-GP-DOXinplasma,whichwerepreviouslyquantified[11],weremuchlowerthanthoseinbile.Afterani.v.doseof20mg/kg,thebile/plasmaratiorangedfrom149at0.25hto43at5h,whiletheconcentrationofZ-GP-DOXinplasmawasdetectableonlyupto5h[11].Incontrasttoplasmasamples,inwhichZ-GP-DOXwasexposedonlyasoriginalform[11],twosuspectedmetabolites(M1andM2)weredetectableinbilesamples.However,ourdeterminationofbiliaryexcretionisbasedonunchangedZ-GP-DOX.TheamountofZ-GP-DOXexcretedinbileover12hcanbeexpressedaspercentageofthetotalZ-GP-DOXdoseadministeredineachrat.ThecumulativebiliaryexcretioncurvesofZ-GP-DOX-6-afteri.v.administrationarepresentedinFig.3(B).28.142.13%and20.253.59%oftheZ-GP-DOXi.v.dosewasexcretedunchangedinthebileduringthestudyperiodwiththedosage235240245of10mg/kgand20mg/kg,respectively,comparedtoitsparentdrugDOXwith20%and28%excretedinbileover24and48h,respectivelyfollowingani.v.doseof4mg/kg[17].Thepharmacokineticparametersinratbileafterintravenousdoseof20mg/kgand10mg/kgarelistedinTable3.TheanalysisofvarianceofCmax,TmaxandCLshowednodifferencesbetweenthetwodosagegroupsafteri.v.administration(P0.05),whileT1/2,AUC0-t,VdandMRTvariedwithdose(P＜0.05or0.01).Fig.3(A)Concentrationsand(B)cumulativeexcretionofZ-GP-DOXinratbile(i.v.20mg/kgor10mg/kg).EachdatapointrepresentsmeanS.D.offivedifferentrats.Tab.3Estimatedpharmacokineticparametersinratbileafteri.v.administrationof10mg/kgand20mg/kg;eachvaluerepresentsthemeanSD(n=5).ParametersCmaxTmaxT1/2AUC0-tCLVdMRTUnitmg/Lhhmg/L*hL/h/kgL/kgh10mg/kg889.98254.090.400.142.881.05b1145.37141.06a0.0090.0010.0340.011b1.270.09a20mg/kg961.63275.990.500.187.222.18b1601.07332.31a0.0120.0030.130.04b2.740.60aCmax:peakplasmaconcentration;Tmax:timetoreachaCmax;T1/2:halflife;AUC0-t:areaundertheconcentration-timecurvetothefinalsamplingpoint;CL:clearance;Vd:volumeofdistribution;MRT:meanresidencetime.aP＜0.05,bP＜0.01.-7-.WhetherZ-GP-DOXisthesubstrateofP-glycoproteinand/orMrp2needtobefurtherelucidatedinthefuture.Inthepresentstudy,anHPLC-UVmethodwasestablishedandvalidatedforthequantification3Discussion250255260265270275280Inthispaper,,asimpleHPLC-UVmethodforthequantificationofZ-GP-DOXinratbilehasbeenestablishedandvalidated.ComparedwiththeHPLCmethodusedintheprevioustoxicologicalstudyandpharmacokineticstudy,inwhichagradientelutionfor40minandanisocraticelutionforatleast20minwasused,respectively[10-12,16],theproposedmethodinthispaperisfeasible,uncomplicatedandtime-saving.ThismethodwassuccessfullyappliedtothebiliaryexcretionstudyofZ-GP-DOXinrats.Directproteinprecipitationwithproteinprecipitatingagentssuchasmethanolandacetonitrilewasusedtopreparethebilesamplesduringthedevelopmentofthesamplepreparationprocedure.However,thismethodwasassociatedwithseverematrixeffectandproducedlowaccuracy.Moreover,thesolidphaseextraction(SPE)wasalsotested.Despiteitshighperformance,itwasexpensiveandtime-consumingforthepreparationofalargenumberofbilesamplesandmatrixeffectwasstillpersistent.Therefore,liquid-liquidextractionwaschosetocarryoutthesamplepreparation.Inourprevioustrial,severalagentswereusedasthecomparableextractingagent,suchasdichloromethane,ethylacetate,chloroform,methane,acetonitrileandthemixturesofthemofdifferentratios.Finally,mixturesofdichloromethaneandacetonitrilewitharatioof4:1(v/v)werechosenastheappropriateextractingagentbecauseofitshighrecoveryandhardlyanyexogenousinterference.Thecollectionperiodofbilewasextendedto24hininitialstudies,inwhichsomeconcentrationswerebeyondLODandonlylessthan0.5%ofthetotalamountofZ-GP-DOXwasexcretedintobileintheratsdosedateither20mg/kgor10mg/kg.Therefore,inthefollowingstudies,bilewascollectedfor12h.ThehighconcentrationofZ-GP-DOXinratbilesuggestedthatZ-GP-DOXwashighlyconcentratedinbileandactiveeffluxtransportersmightbeinvolved.Also,thefindingsshowedthatZ-GP-DOXcanbeapromisingcompoundappliedtothetreatmentofhepatobiliaryorevenintestinalandcolonictumorsbecauseofitshighconcentrationinbile.Theretentiontimeofthetwosuspectedmetabolites(M1andM2)waslessthanthatofZ-GP-DOX,suggestingthatthesuspectedmetabolitesmightbemorepolarthanZ-GP-DOX.ThepossiblemetabolicpathwayofZ-GP-DOXinratbileneedtobefurtherinvestigatedinthefuture.Biliaryexcretioniswellrecognizedasamajorpathwayfortheeliminationofamphipathic,hydrophobicandhighmolecularweightxenobiotics[18],thereforebiliaryexcretionofZ-GP-DOX,whichhasamolecularweightof831andisnotsolubleinwater,isreasonable.Adose-dependentpharmacokineticbehaviorofZ-GP-DOXwasobservedwiththedosesof10-20mg/kg.Asismentionedabove,cumulativeexcretiondecreasedfromabout28%to20%asthedoseincreased,indicatingthathepatobiliaryeffluxtransportersweresaturatedatthedoseof20mg/kg.Thesaturationcouldleadtononlinearpharmacokinetics,evidencedbythesignificantchangeofhalflife(T1/2)betweenthetwodosagegroupsandthenonproportionalincreaseofAUC0-12.ThepossibleeffluxtransportersmightbeP-glycoproteinandmultidrug285290resistance-associatedprotein2(Mrp2),becauseDOXisexcretedintotheratbileviabothofthem[19]ofZ-GP-DOXinratbile.Thismethodwassuitableforthebiliaryexcretionstudyinrats.Toourknowledge,thisisthefirstreportofanHPLC-UVmethodusedinthebiliaryexcretionstudyofZ-GP-DOXinrats.TheinformationgiveninthispaperwillbeusefultosupplementthepreviouspharmacokineticstudyofZ-GP-DOXinratsandwillbehelpfultoimprovethedruggabilitystudyofZ-GP-DOX.-8-Acknowledgements295300305310315320325330335340345ThisresearchworkwassupportedbygrantsfromNationalNaturalScienceFoundationofChina(NO.30973565),NationalImportantTechnologyProjectCreationofMajorNewDrugs(NO.2009ZX09103-040),andNationalNaturalScienceFoundationofChinaforYoungScholars(NO.81202461).References[1]BlumRH,CarterSK.Adriamycin[J].AnnInternMed,1974,80(2):249-59.[2]DanesiR,FogliS,GennariA,ConteP,DelTaccaM.Pharmacokinetic-pharmacodynamicrelationshipsoftheanthracyclineanticancerdrugs[J].ClinPharmacokinet,2002,41(6):431-44.[3]MaudensKE,StoveCP,LambertWE.Quantitativeliquidchromatographicanalysisofanthracyclinesinbiologicalfluids[J].JChromatogrB,2011,879:2471-86.[4]HoubaPH,BovenE,vanderMeulen-MuilemanIH,LeendersRG,ScheerenJW,PinedoHM,etal.Anoveldoxorubicin-glucuronideprodrugDOX-GA3fortumour-selectivechemotherapy:distributionandefficacyinexperimentalhumanovariancancer[J].BritJCancer,2001,84(4):550-7.[5]DevalapallyH,RajanKS,AkkinepallyRR,DevarakondaRK.Safety,pharmacokineticsandbiodistributionstudiesofabeta-galactosideprodrugofdoxorubicinforimprovementoftumorselectivechemotherapy[J].DrugDevIndPharm,2008,34(8):789-95.[6]ChenWT,KellyT.Seprasecomplexesincellularinvasiveness[J].CancerMetastRev,2003,22(2-3):259-69.[7]GeY,ZhanF,BarlogieB,EpsteinJ,ShaughnessyJ,Jr.,YaccobyS.Fibroblastactivationprotein(FAP)isupregulatedinmyelomatousboneandsupportsmyelomacell