.,藥物代謝及其動(dòng)力學(xué)在新藥研發(fā)中的應(yīng)用,胡卓漢博士瑞德肝臟疾病研究(上海)有限公司復(fù)旦大學(xué)藥學(xué)院,2004年12月30日中國.北京,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),藥物研發(fā)的三大任務(wù)藥效Efficacy/Pharmacodynamics安全Safety/Toxicology藥物代謝動(dòng)力學(xué)DrugMetabolism/Pharmcokinetics,.,藥物代謝動(dòng)力學(xué)的任務(wù),（最大無毒性濃度）,(最小有效濃度）,(最小藥效時(shí)間）,血漿濃度,時(shí)間,.,藥效,毒理,藥代,最佳血漿濃度,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段能否被吸收？permeability是否被代謝？metabolicstability代謝產(chǎn)物？metaboliteidentification代謝途徑？pathwayidentification對其它藥物的影響？drug-druginteraction,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床前階段生物利用度bioavailability血漿濃度的線性和非線性doseescalation&proportionality多次給藥和體內(nèi)積蓄multipledoses&accumulation吸收和排泄模式massbalance體內(nèi)分布distribution從動(dòng)物代謝推算人體代謝extrapolation,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床階段長期毒性實(shí)驗(yàn)的動(dòng)物選擇metabolismprofilinginanimalsandhumans,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床實(shí)驗(yàn)準(zhǔn)則GoodClinicalPractice(GCP),非臨床實(shí)驗(yàn)準(zhǔn)則GoodLaboratoryPractice(GLP),.,二五原則5毫克5天,.,臨床前實(shí)驗(yàn)藥物代謝動(dòng)力學(xué)的生物模型體外和離體模型(invitro/insitumodels)吸收模型absorption/permeability代謝模型metabolism體外推測和體內(nèi)(invitro/invivocorrelation)動(dòng)物模型(invivoanimalmodels)動(dòng)物推測人(speciesextrapolation),.,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,SituationAnalysis,invitro體外metabolism,insitu離體permeability,invivo體內(nèi)bioavailability,.,PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorally,Poororalbioavailability,.,藥物吸收模型,計(jì)算機(jī),脂溶度,脂層轉(zhuǎn)移,細(xì)胞層轉(zhuǎn)移,十二指腸灌流,.,14,absorption/distributionmodel脂層轉(zhuǎn)移模型,水相Aqueousphase,水相Aqueousphase,有機(jī)相Organicphase,pH=6.5,pH=7.4,PermeabilityEvaluationinvitro,.,15,invitroabsorption/distributionmodel,.,Caco-2TransportPathways人大腸癌細(xì)胞模型,.,TransportPathways藥物吸收機(jī)制,被動(dòng),細(xì)胞間,主動(dòng),P糖蛋白,.,ProbesforTransportPathways腸道吸收標(biāo)準(zhǔn)對照藥物,Transcellular（被動(dòng)吸收）Propranolol,TestosteroneParacellular（細(xì)胞間滲透）Mannitol,InulinCarriermediated（主動(dòng)吸收）GlucoseP-Glycoproteinmediated（P糖蛋白調(diào)節(jié)）底物Vinblastine抑制物Verapamil,.,Glucose（蔗糖）vsInulin（木香素）主動(dòng)吸收vs細(xì)胞間滲透,.,PropranololvsMannitol被動(dòng)吸收vs細(xì)胞間滲透,.,由P蛋白所調(diào)節(jié)的藥物吸收使用P糖蛋白抑制劑Verapamil,.,Chong,DandoPharm.Res.1997,.,FalsePositive假陽性低,FalseNegative假陰性高,Caco-2TransportPathways人大腸癌細(xì)胞吸收模型,.,insituratintestinalperfusion(singlepass)離體大鼠十二指腸灌流模型（單循環(huán)）,METHODAnimal:MaleSprague-Dawleyrats(250-350g),fastedovernight.Ratisanesthetizedbyurethane1.5g/kg,im.beforeperfusionstarts.Perfusate:Phosphatebuffer,pH=6.510mMglucosePhenolred(negativecontrol)Acetaminophen(positivecontrol)Finalconcentrationsoftestarticle=0.05-0.30mg/mL,.,PerfusionProcedures:ratisputonaheatingpadtomaintainbodytemperaturejejunumisexposedviaamiddlelineincisionsutures:1stismadeat5cmdistaltotheligamentofTreitz2ndismadeatabout20cmdistalto1stonetheinletofcannula-asyringeinfusionpumptheoutletofcannula-afractioncollectortheperfusionsegmentisprecleanedbypassing10mlofblankperfusatebufferperfusiontimeandrate=0.1ml/minfor120minoutletperfusionsamplesarecollectedevery10minplasmasamplesarecollectedat30,60,90and120minafterperfusionCalculations:Permeability(Peff,cm/min)=(Q/2RLp)x(1-Cout/Cin)Cout/Cin=(Cout/Cin)xphenolredin/phenolredout,insituratintestinalperfusion(singlepass),.,Insituratintestinalpermeability(singlepass),Predictionwithin90%interval=19/31(61.3%),In-housevalidation,假陽性,假陰性,.,PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorally,Poororalbioavailability,.,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,SituationAnalysis,invitro體外metabolism,insitu離體permeability,invivo體內(nèi)bioavailability,.,InSituRatIntestinalPermeability:Good,陽性對照,陰性對照,受試藥物,.,EnhancedThroughputScreeningPerfusion:4compoundsperday(4animals)Samplesize:timepoints7duplicatex2control/drugx3sample/perfusion42Totalsamples/day168Bioanalysis:noextractionnostandardcurve(peakarea)machinetime/2LCs24hrsTotalmanpower:animaltechx1PKDMtechx2Testarticleamount:1mg/testarticleScreeningrate:onechemotypeswith30compounds/2weeks,.,pKa=10pKa=8.4pKa=6.5Preduced%=0%Preduced%=7%Preduced%=12%,SAR:pKavs.permeability實(shí)例：結(jié)構(gòu)優(yōu)化和吸收率分析,.,SAR:permeabilityvs.efficacy實(shí)例：結(jié)構(gòu)優(yōu)化和吸收率和活性的分析,IC50=2uMPreduced%=0%,IC50=0.012uMPreduced%=0%,IC50=1.1uMPreduced%=17%,IC50=0.025uMPreduced%=15%,.,小結(jié)：體外和離體藥物吸收實(shí)驗(yàn)系統(tǒng)體外人大腸癌細(xì)胞模型(invitroCaco-2monolayer)離體大鼠十二指腸灌流模型(insituratintestineperfusion)體內(nèi)動(dòng)物藥物代謝動(dòng)力學(xué)模型二五原則:5毫克/5天,.,血漿濃度,時(shí)間,化學(xué)藥物,化學(xué)藥物+中藥,中藥的藥物代謝動(dòng)力學(xué)的任務(wù)本身的藥物代謝動(dòng)力學(xué)問題對其它藥物吸收的作用,.,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,SituationAnalysis,invitro體外metabolism,insitu離體permeability,invivo體內(nèi)bioavailability,.,死還是不死,這是個(gè)問題.Tobeornottobe,thisisaproblem.-哈默雷特體內(nèi)試驗(yàn)還是體外試驗(yàn),這是個(gè)問題.Invitroorinvivo,thisisaproblem.-藥代研究員,.,動(dòng)物體內(nèi)模型-人體內(nèi)(臨床試驗(yàn))Invivoanimalsvs.invivohumans人體外模型-人體內(nèi)(臨床試驗(yàn))Invitrohumansvs.invivohumans選擇的指南與人相似：疾病模型，藥效，毒性，藥物代謝實(shí)驗(yàn)成本,.,38,HeartbeatandBodyweight（心率和體重）,小鼠,大鼠,兔,猴,狗,人,.,39,LiverweightandHepaticFlowvsBodyweight（體重，肝重和肝血流量）,人,狗,猴,兔,大鼠,小鼠,人,狗,猴,兔,大鼠,小鼠,.,40,Antipyrineclearance(l/min),rat,mouse,rabbit,monkey,dog,human,Clearance,.,InVitroModelsoftheLiver體外肝模型,Hepatocytes肝細(xì)胞Liverslices肝切片Livermicrosomes肝微粒體LiverS-9Fraction肝S-9組分,.,USFDAGuidanceforIndustry美國藥物和食品管理局關(guān)于藥物代謝實(shí)驗(yàn)的指南,“Themostcompletepictureforhepaticmetabolismcanbeobtainedwithliversystems,inwhichthecofactorsareself-sufficientandthenaturalorientationforlinkedenzymesispreserved.Isolatedhepatocytesandprecision-cutsliceshavethesedesirablefeatures.”,GuidanceforIndustry,DrugMetabolism/DrugInteractionStudiesintheDrugDevelopmentProcess:StudiesInVitroCDER,CBER,U.S.FDA,1997,譯文：肝系統(tǒng)（分離的肝細(xì)胞和精確的肝切片）能為藥物代謝實(shí)驗(yàn)提供最完全的信息，因?yàn)檫@個(gè)系統(tǒng)含有足夠的天然水平的酶系。,.,2-Hydroxy-EE2,Conjugates,EE2,EE2,Hepatocytes（肝細(xì)胞）,Microsomes（微粒體）Hepatocytes（肝細(xì)胞）,MetabolismofEythinylEstradiol(EE2)肝微粒體和肝細(xì)胞的代謝功能差異,Li,Hartman,Lu,CollinsandStrong,BrJClinPharmacol48,733-742(1999),.,PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorally,Poororalbioavailability,.,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,SituationAnalysis,invitro體外metabolism,insitu離體permeability,invivo體內(nèi)bioavailability,.,Reactionvolume:1.0ml,DPBSpH7.4HepaticS-9/Microsomes:0.5mgprotien/mLSpecies:Human/Monkey/Dog/Rat/MouseSubstrateconcentration:10mMNADPH:2.4mMUDPGA:1.5mMIncubation:60minat37oCStoppingprocedure:chilledacetonitrile,3xvolume,InVitroMetabolismAssay體外肝微粒體實(shí)驗(yàn),.,1234,ABCDEF,EnhancedThroughputScreening（增速篩選）,A-B:（空白對照）：testarticle+buffer=vehiclecontrol(VC)C-D:（陰性對照）：testarticle+microsomes=negativecontrol(NC)E-F:（實(shí)驗(yàn)樣品）：testarticle+microsomes+cofactors=treatedDosingsolution=timezero(T=0)4compoundsincludingpositivereference*/plate*7ethoxycoumarin,陰性對照,空白對照,測試樣本,.,EnhancedThroughputScreeningIncubation:4compoundsper24-wellplate15compounds+1positivecontrolperdaySamplesize:Timezeroduplicate(16x2)VCduplicate(16x2)NCduplicate(16x2)Treatedduplicate(16x2)Totalsamples/day128Bioanalysis:noextractionnostandardcurve(peakarea)machinetime/2LCs24hrsTotalmanpower:PKDMtechx3Testarticleamount:0.1mg/testarticleScreeningrate:onechemotypewith60compounds/1week,.,HPLCprofilesofBCH-3840anditsmetabolite(BCH-6440),BCH-3840,metabolite?,InvitrometabolicstabilitybyrathepaticS9,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段能否被吸收？permeability是否被代謝？metabolicstability代謝產(chǎn)物？metaboliteidentification代謝途徑？pathwayidentification對其它藥物的影響？drug-druginteraction,.,LiquidChromatography/MassSpectrumofBCH-3840anditsmetabolite(BCH-6440),HydroxylationorOxidation,MH+=310,MH+=294,MassIdentification,.,HPLCprofilesofBCH-3840anditsmetabolite(BCH-6440),Preparationofmetabolitebybulkincubation,M,M,P,P,10mgmicrosomalprotein2mgBCH-3840,Fractioncollectionofmetabolite,fractionation,concentration,.,NuclearMagneticResonanceprofilesofBCH-3840anditsmetabolite(BCH-6440),C5-H,BCH-3840,Metabolite,StructureElucidation,.,InvitrotherapeuticindexofBCH-6440,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段能否被吸收？permeability是否被代謝？metabolicstability代謝產(chǎn)物？metaboliteidentification代謝途徑？pathwayidentification對其它藥物的影響？drug-druginteraction,.,InhibitorsforCYPIsoformConc(mM)Furafulline(CYP1A2)10Tranylcypromine(CYP2A6)50Sulfaphenazole(CYP2C9)25Omeprazole(CYP2C19)20Quinidine(CYP2D6)24-methylpyrazole(CYP2E1)250Ketoconazole(CYP3A4)5,ChemicalInhibition（化學(xué)抑制）,Pureenzyme（純酶）CorrelationAnalysis（相關(guān)分析）,MetabolismPhenotyping代謝途徑鑒定,.,InhibitorsforCYPIsoformConc(mM)Inhibition(%ofNC)Tranylcypromine(CYP2A6)5040.2Sulfaphenazole(CYP2C9)2514.24-methylpyrazole(CYP2E1)25067.6Ketoconazole(CYP3A4)575.2,MetabolismPhenotyping代謝途徑鑒定,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段能否被吸收？permeability是否被代謝？metabolicstability代謝產(chǎn)物？metaboliteidentification代謝途徑？pathwayidentification對其它藥物的影響？drug-druginteraction,.,Drug-DrugInteractions（對其它藥物代謝的影響）Inhibition（抑制）potential-IC50andKimechanism-mechanistic（機(jī)械性）competitive（競爭性）testsystem:livermicrosomes（肝微粒體）cryopreservedhepatocytes（冷凍肝細(xì)胞）Induction（誘導(dǎo)）testsystem:freshisolatedhepatocytes（肝細(xì)胞）TargetEnzymesCytochromeP450s:1A2,2A6,2C8,2C9,2C19,2D6,2E1,3A4PhaseIIconjugation:glucuronidation,.,IC50(M):0.675GoodnessofFit:0.980795%ConfidenceIntervals:5.638.28,IC50(M):20.4GoodnessofFit:0.973095%ConfidenceIntervals:16.9-26.3,CYP3A4,CYP3A4,Drug-druginteraction:inhibition抑制作用,體外藥效濃度=1uM,.,Drug-druginteraction:Induction（肝細(xì)胞誘導(dǎo)模型）,5daysprocedureDay0:Isolatefreshhepatocytes,viability70%Platinghepatocytesto24-wellplate,0.7x106viablecells/wellPlatingmediareplacedwithsandwichafter7-hourattachmentDay1:incubationforestablishingbasallevelsofCYP450isoforms.Day2:sameasDay1Day3:dosingwithtestarticlesDay4:sameasDay3Day5:washingoutthedosingsolutionandaddingsubstratesforCYP450isoformsasbelow:1A2-ethocyresorufinO-deethylation2A6-coumarin7-hydroxylation2C9-tolbutamide4-hydroxylation2C19-S-mephenytoin4-hydroxylation2D6-dextromethorphanO-demethylation2E1-chlorzoxazone6-hydroxylation3A4-testosterone6b-hydroxylation,.,Drug-druginteraction:Induction誘導(dǎo)作用,.,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,SituationAnalysis,invitro體外metabolism,insitu離體permeability,invivo體內(nèi)bioavailability,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床前階段生物利用度bioavailability血漿濃度的線性和非線性doseescalation&proportionality多次給藥和體內(nèi)積蓄multipledoses&accumulation吸收和排泄模式massbalance體內(nèi)分布distribution從動(dòng)物代謝推算人體代謝extrapolation,.,119%,236%,310%,Proportionality血漿濃度的非線性,提示：代謝或排泄的非線性飽和,.,90%,72%,Proportionality:AUC（大鼠試驗(yàn)）,93%,63%,提示：藥物吸收的非線性飽和,.,TOXICOKINETICS毒物代謝動(dòng)力學(xué)試驗(yàn)Animal:Sprague-Dawleyrats(male&female)Cynomolgusmonkey(male&female)Singledoseescalation（線性動(dòng)力學(xué)）(50,250,500mg/kg)Multipledoseescalation（藥物體內(nèi)積累）(50,250,500mg/kg,dailyfor14days),.,90%,72%,Proportionality:AUC（大鼠試驗(yàn)）,93%,63%,提示：藥物吸收的非線性飽和,.,0,100,200,300,400,500,600,0,10,20,30,40,50,60,FemaleRats,OralDose(mg/kg),0,100,200,300,400,500,600,0,10,20,30,40,50,MaleRats,OralDose(mg/kg),Cmax,(,m,g/mL),73%,47%,56%,49%,Proportionality:Cmax（大鼠試驗(yàn)）,提示：藥物吸收的非線性飽和,.,0.92,0.77,1.04,1.19,1.02,1.07,AccumulationRatio藥物積累率（大鼠）,Malerats,Femalerats,.,Proportionality:AUC（獼猴）,MaleMonkey,FemaleMonkey,49%,34%,60%,38%,提示：藥物吸收的非線性飽和,.,38%,31%,55%,32%,Proportionality:Cmax（獼猴）,MaleMonkey,FemaleMonkey,提示：藥物吸收的非線性飽和,.,MaleMonkey,FemaleMonkey,0.79,1.11,1.12,0.73,0.76,1.14,AccumulationRatio藥物積累率（獼猴）,.,PhaseITrial(Singledoseescalation)臨床一期單劑量藥代動(dòng)力學(xué)試驗(yàn)HealthyMaleSubject(n):22OralDoses(4):100,200,400,and800mgTimepoints(13):0.5,1,1.5,2,3,4,6,8,10,12,16,20,and24hour,.,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA