复旦大学药物代谢及其动力学在新药研发中的应用.ppt

药物代谢及其动力学在新药研发中的应用,胡卓汉博士瑞德肝脏疾病研究(上海)有限公司复旦大学药学院,2004年12月30日中国.北京,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,药物研发的三大任务药效Efficacy/Pharmacodynamics安全Safety/Toxicology药物代谢动力学DrugMetabolism/Pharmcokinetics,药物代谢动力学的任务,（最大无毒性浓度）,(最小有效浓度）,(最小药效时间）,血浆浓度,时间,药效,毒理,药代,最佳血浆浓度,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,研究和发现阶段能否被吸收？permeability是否被代谢？metabolicstability代谢产物？metaboliteidentification代谢途径？pathwayidentification对其它药物的影响？drug-druginteraction,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,临床前阶段生物利用度bioavailability血浆浓度的线性和非线性doseescalation&proportionality多次给药和体内积蓄multipledoses&accumulation吸收和排泄模式massbalance体内分布distribution从动物代谢推算人体代谢extrapolation,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,临床阶段长期毒性实验的动物选择metabolismprofilinginanimalsandhumans,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,临床实验准则GoodClinicalPractice(GCP),非临床实验准则GoodLaboratoryPractice(GLP),二五原则5毫克5天,临床前实验药物代谢动力学的生物模型体外和离体模型(invitro/insitumodels)吸收模型absorption/permeability代谢模型metabolism体外推测和体内(invitro/invivocorrelation)动物模型(invivoanimalmodels)动物推测人(speciesextrapolation),排出太快/药效时间太短,口服吸收差/血浆浓度太低,分布,排泻,代谢问题,吸收问题,蛋白质相互作用,分布体积,肾脏排泄,肝脏代谢,溶解度,肠道吸收膜通透性,肠道消化,早期研发阶段,后期研发阶段,SituationAnalysis,invitro体外metabolism,insitu离体permeability,invivo体内bioavailability,PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorally,Poororalbioavailability,药物吸收模型,计算机,脂溶度,脂层转移,细胞层转移,十二指肠灌流,14,absorption/distributionmodel脂层转移模型,水相Aqueousphase,水相Aqueousphase,有机相Organicphase,pH=6.5,pH=7.4,PermeabilityEvaluationinvitro,15,invitroabsorption/distributionmodel,Caco-2TransportPathways人大肠癌细胞模型,TransportPathways药物吸收机制,被动,细胞间,主动,P糖蛋白,ProbesforTransportPathways肠道吸收标准对照药物,Transcellular（被动吸收）Propranolol,TestosteroneParacellular（细胞间渗透）Mannitol,InulinCarriermediated（主动吸收）GlucoseP-Glycoproteinmediated（P糖蛋白调节）底物Vinblastine抑制物Verapamil,Glucose（蔗糖）vsInulin（木香素）主动吸收vs细胞间渗透,PropranololvsMannitol被动吸收vs细胞间渗透,由P蛋白所调节的药物吸收使用P糖蛋白抑制剂Verapamil,Chong,DandoPharm.Res.1997,FalsePositive假阳性低,FalseNegative假阴性高,Caco-2TransportPathways人大肠癌细胞吸收模型,insituratintestinalperfusion(singlepass)离体大鼠十二指肠灌流模型（单循环）,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,假阳性,假阴性,PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorally,Poororalbioavailability,排出太快/药效时间太短,口服吸收差/血浆浓度太低,分布,排泻,代谢问题,吸收问题,蛋白质相互作用,分布体积,肾脏排泄,肝脏代谢,溶解度,肠道吸收膜通透性,肠道消化,早期研发阶段,后期研发阶段,SituationAnalysis,invitro体外metabolism,insitu离体permeability,invivo体内bioavailability,InSituRatIntestinalPermeability:Good,阳性对照,阴性对照,受试药物,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实例：结构优化和吸收率分析,SAR:permeabilityvs.efficacy实例：结构优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%,小结：体外和离体药物吸收实验系统体外人大肠癌细胞模型(invitroCaco-2monolayer)离体大鼠十二指肠灌流模型(insituratintestineperfusion)体内动物药物代谢动力学模型二五原则:5毫克/5天,血浆浓度,时间,化学药物,化学药物+中药,中药的药物代谢动力学的任务本身的药物代谢动力学问题对其它药物吸收的作用,排出太快/药效时间太短,口服吸收差/血浆浓度太低,分布,排泻,代谢问题,吸收问题,蛋白质相互作用,分布体积,肾脏排泄,肝脏代谢,溶解度,肠道吸收膜通透性,肠道消化,早期研发阶段,后期研发阶段,SituationAnalysis,invitro体外metabolism,insitu离体permeability,invivo体内bioavailability,死还是不死,这是个问题.Tobeornottobe,thisisaproblem.-哈默雷特体内试验还是体外试验,这是个问题.Invitroorinvivo,thisisaproblem.-药代研究员,动物体内模型-人体内(临床试验)Invivoanimalsvs.invivohumans人体外模型-人体内(临床试验)Invitrohumansvs.invivohumans选择的指南与人相似：疾病模型，药效，毒性，药物代谢实验成本,38,HeartbeatandBodyweight（心率和体重）,小鼠,大鼠,兔,猴,狗,人,39,LiverweightandHepaticFlowvsBodyweight（体重，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体外肝模型,Hepatocytes肝细胞Liverslices肝切片Livermicrosomes肝微粒体LiverS-9Fraction肝S-9组分,USFDAGuidanceforIndustry美国药物和食品管理局关于药物代谢实验的指南,“Themostcompletepictureforhepaticmetabolismcanbeobtainedwithliversystems,inwhichthecofactorsareself-sufficientandthenaturalorientationforlinkedenzymesispreserved.Isolatedhepatocytesandprecision-cutsliceshavethesedesirablefeatures.”,GuidanceforIndustry,DrugMetabolism/DrugInteractionStudiesintheDrugDevelopmentProcess:StudiesInVitroCDER,CBER,U.S.FDA,1997,译文：肝系统（分离的肝细胞和精确的肝切片）能为药物代谢实验提供最完全的信息，因为这个系统含有足够的天然水平的酶系。,2-Hydroxy-EE2,Conjugates,EE2,EE2,Hepatocytes（肝细胞）,Microsomes（微粒体）Hepatocytes（肝细胞）,MetabolismofEythinylEstradiol(EE2)肝微粒体和肝细胞的代谢功能差异,Li,Hartman,Lu,CollinsandStrong,BrJClinPharmacol48,733-742(1999),PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorally,Poororalbioavailability,排出太快/药效时间太短,口服吸收差/血浆浓度太低,分布,排泻,代谢问题,吸收问题,蛋白质相互作用,分布体积,肾脏排泄,肝脏代谢,溶解度,肠道吸收膜通透性,肠道消化,早期研发阶段,后期研发阶段,SituationAnalysis,invitro体外metabolism,insitu离体permeability,invivo体内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体外肝微粒体实验,1234,ABCDEF,EnhancedThroughputScreening（增速筛选）,A-B:（空白对照）：testarticle+buffer=vehiclecontrol(VC)C-D:（阴性对照）：testarticle+microsomes=negativecontrol(NC)E-F:（实验样品）：testarticle+microsomes+cofactors=treatedDosingsolution=timezero(T=0)4compoundsincludingpositivereference*/plate*7ethoxycoumarin,阴性对照,空白对照,测试样本,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,临床实验,临床前实验,研究和发现,研究和发现阶段能否被吸收？permeability是否被代谢？metabolicstability代谢产物？metaboliteidentification代谢途径？pathwayidentification对其它药物的影响？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,临床实验,临床前实验,研究和发现,研究和发现阶段能否被吸收？permeability是否被代谢？metabolicstability代谢产物？metaboliteidentification代谢途径？pathwayidentification对其它药物的影响？drug-druginteraction,InhibitorsforCYPIsoformConc(mM)Furafulline(CYP1A2)10Tranylcypromine(CYP2A6)50Sulfaphenazole(CYP2C9)25Omeprazole(CYP2C19)20Quinidine(CYP2D6)24-methylpyrazole(CYP2E1)250Ketoconazole(CYP3A4)5,ChemicalInhibition（化学抑制）,Pureenzyme（纯酶）CorrelationAnalysis（相关分析）,MetabolismPhenotyping代谢途径鉴定,InhibitorsforCYPIsoformConc(mM)Inhibition(%ofNC)Tranylcypromine(CYP2A6)5040.2Sulfaphenazole(CYP2C9)2514.24-methylpyrazole(CYP2E1)25067.6Ketoconazole(CYP3A4)575.2,MetabolismPhenotyping代谢途径鉴定,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,研究和发现阶段能否被吸收？permeability是否被代谢？metabolicstability代谢产物？metaboliteidentification代谢途径？pathwayidentification对其它药物的影响？drug-druginteraction,Drug-DrugInteractions（对其它药物代谢的影响）Inhibition（抑制）potential-IC50andKimechanism-mechanistic（机械性）competitive（竞争性）testsystem:livermicrosomes（肝微粒体）cryopreservedhepatocytes（冷冻肝细胞）Induction（诱导）testsystem:freshisolatedhepatocytes（肝细胞）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,体外药效浓度=1uM,Drug-druginteraction:Induction（肝细胞诱导模型）,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诱导作用,排出太快/药效时间太短,口服吸收差/血浆浓度太低,分布,排泻,代谢问题,吸收问题,蛋白质相互作用,分布体积,肾脏排泄,肝脏代谢,溶解度,肠道吸收膜通透性,肠道消化,早期研发阶段,后期研发阶段,SituationAnalysis,invitro体外metabolism,insitu离体permeability,invivo体内bioavailability,EfficacyHits,OptimizedLeadGoornogodecision,CompoundforDevelopment(CD),NEWDRUG,IND,NDA,R&D,临床实验,临床前实验,研究和发现,临床前阶段生物利用度bioavailability血浆浓度的线性和非线性doseescalation&proportionality多次给药和体内积蓄multipledoses&accumulation吸收和排泄模式massbalance体内分布distribution从动物代谢推算人体代谢extrapolation,119%,236%,310%,Proportionality血浆浓度的非线性,提示：代谢或排泄的非线性饱和,90%,72%,Proportionality:AUC（大鼠试验）,93%,63%,提示：药物吸收的非线性饱和,TOXICOKINETICS毒物代谢动力学试验Animal:Sprague-Dawleyrats(male&female)Cynomolgusmonkey(male&female)Singledoseescalation（线性动力学）(50,250,500mg/kg)Multipledoseescalation（药物体内积累）(50,250,500mg/kg,dailyfor14days),90%,72%,Proportionality:AUC（大鼠试验）,93%,63%,提示：药物吸收的非线性饱和,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（大鼠试验）,提示：药物吸收的非线性饱和,0.92,0.77,1.04,1.19,1.02,1.07,AccumulationRatio药物积累率（大鼠）,Malerats,Femalerats,Proportionality:AUC（猕猴）,MaleMonkey,FemaleMonkey,49%,34%,60%,38%,提示：药物吸收的非线性饱和,38%,31%,55%,32%,Proportionality:Cmax（猕猴）,MaleMonkey,FemaleMonkey,提示：药物吸收的非线性饱和,MaleMonkey,FemaleMonkey,0.79,1.11,1.12,0.73,0.76,1.14,AccumulationRatio药物积累率（猕猴）,PhaseITrial(Singledoseescalation)临床一期单剂量药代动力学试验HealthyMaleSubject(n):22OralDoses(4):100,200,400,and800mgTimepoints(13):0.