药代动力学在新药研发中的作用

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)复旦大学药学院2004年12月30日中国.北京编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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平安Safety/Toxicology药物代谢动力学DrugMetabolism/Pharmcokinetics编辑ppt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〕血浆浓度时间编辑ppt药效毒理药代最正确血浆浓度编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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屡次给药和体内积蓄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编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D临床实验临床前实验研究和发现临床实验准那么GoodClinicalPractice(GCP)非临床实验准那么GoodLaboratoryPractice(GLP)编辑ppt二五原那么5毫克5天编辑ppt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)编辑ppt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SituationAnalysisinvitro体外metabolisminsitu离体permeabilityinvivo体内bioavailability编辑pptPlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailability编辑ppt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编辑pptabsorption/distributionmodel脂层转移模型水相Aqueousphase水相Aqueousphase有机相OrganicphasepH=6.5pH=7.4PermeabilityEvaluation–invitro14编辑pptinvitroabsorption/distributionmodel15编辑pptCaco-2TransportPathways

人大肠癌细胞模型编辑pptTransportPathways

药物吸收机制被动细胞间主动P糖蛋白编辑pptProbesforTransportPathways

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编辑pptGlucose〔蔗糖〕vsInulin〔木香素〕

主动吸收vs细胞间渗透编辑pptPropranololvsMannitol

被动吸收vs细胞间渗透编辑ppt由P－蛋白所调节的药物吸收

－使用P－糖蛋白抑制剂Verapamil编辑pptChong,Dando&Morrison;Pharm.Res.1997编辑pptFalsePositive假阳性＝低FalseNegative假阴性＝高Caco-2TransportPathways人大肠癌细胞吸收模型编辑pptinsituratintestinalperfusion(singlepass)离体大鼠十二指肠灌流模型〔单循环〕METHODAnimal: MaleSprague-Dawleyrats(250-350g),fasted overnight. Ratisanesthetizedbyurethane1.5g/kg,im. beforeperfusionstarts.Perfusate:Phosphatebuffer,pH=6.5 10mMglucose Phenolred(negativecontrol) Acetaminophen(positivecontrol)

Finalconcentrationsoftestarticle =0.05-0.30mg/mL编辑pptPerfusionProcedures:ratisputonaheatingpadtomaintainbodytemperaturejejunumisexposedviaamiddlelineincisionsutures:1stismadeat5cmdistaltotheligamentofTreitz 2ndismadeatabout20cmdistalto1stonetheinletofcannula-asyringeinfusionpumptheoutletofcannula-afractioncollectortheperfusionsegmentisprecleanedbypassing10mlofblankperfusatebufferperfusiontimeandrate=0.1ml/minfor120minoutletperfusionsamplesarecollectedevery10minplasmasamplesarecollectedat30,60,90and120minafterperfusionCalculations:Permeability(Peff,cm/min)=(Q/2RLp)x(1-C’out/C’in)

C’out/C’in=(Cout/Cin)x[phenolred]in/[phenolred]outinsituratintestinalperfusion(singlepass)编辑pptInsituratintestinalpermeability(singlepass)Predictionwithin90%interval=19/31(61.3%)In-housevalidation假阳性假阴性编辑pptPlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailability编辑ppt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SituationAnalysisinvitro体外metabolisminsitu离体permeabilityinvivo体内bioavailability编辑pptInSituRatIntestinalPermeability:Good阳性对照阴性对照受试药物编辑pptEnhancedThroughputScreeningPerfusion: 4compoundsperday(4animals)

Samplesize: timepoints 7 duplicate x2 control/drug x3 sample/perfusion 42 Totalsamples/day 168

Bioanalysis: noextraction nostandardcurve(peakarea) machinetime/2LCs 24hrsTotalmanpower: animaltechx1 PKDMtechx2 Testarticleamount: 1mg/testarticleScreeningrate: onechemotypeswith30compounds/2weeks编辑pptpKa=10 pKa=8.4 pKa=6.5Preduced%=0% Preduced%=7% Preduced%=12%SAR:pKavs.permeability实例：结构优化和吸收率分析编辑pptSAR: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%编辑ppt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那么:5毫克/5天编辑ppt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编辑ppt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SituationAnalysisinvitro体外metabolisminsitu离体permeabilityinvivo体内bioavailability编辑ppt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. --药代研究员编辑ppt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，药物代谢实验本钱编辑pptHeartbeatandBodyweight〔心率和体重〕小鼠大鼠兔猴狗人38编辑pptLiverweightandHepaticFlowvsBodyweight〔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39编辑pptAntipyrineclearance(l/min)ratmouserabbitmonkeydoghumanClearance40编辑pptInVitroModelsoftheLiver

体外肝模型Hepatocytes肝细胞Liverslices肝切片Livermicrosomes肝微粒体LiverS-9Fraction肝S-9组分编辑pptUSFDAGuidanceforIndustry

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〔别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。编辑pptHOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE-3-GlucuronideEE2-3-SulfateConjugatesEE2EE2Hepatocytes〔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)编辑pptPlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailability编辑ppt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SituationAnalysisinvitro体外metabolisminsitu离体permeabilityinvivo体内bioavailability编辑pptReactionvolume: 1.0ml,DPBSpH7.4HepaticS-9/Microsomes: 0.5mgprotien/mLSpecies: Human/Monkey/Dog/Rat/MouseSubstrateconcentration: 10mMNADPH: 2.4mMUDPGA: 1.5mMIncubation: 60minat37oCStoppingprocedure: chilledacetonitrile,3xvolume

InVitroMetabolismAssay

体外肝微粒体实验编辑ppt1234ABCDEFEnhancedThroughputScreening〔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编辑pptEnhancedThroughputScreeningIncubation: 4compoundsper24-wellplate 15compounds+1positivecontrolperday

Samplesize: Timezero duplicate(16x2) VC duplicate(16x2) NC duplicate(16x2) Treated duplicate(16x2) Totalsamples/day 128

Bioanalysis: noextraction nostandardcurve(peakarea) machinetime/2LCs 24hrsTotalmanpower: PKDMtechx3 Testarticleamount: 0.1mg/testarticleScreeningrate: onechemotypewith60compounds/1week编辑pptHPLCprofilesofBCH-3840anditsmetabolite(BCH-6440)BCH-3840metabolite?InvitrometabolicstabilitybyrathepaticS9编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

编辑pptLiquidChromatography/MassSpectrumofBCH-3840anditsmetabolite(BCH-6440)HydroxylationorOxidationMH+=310MH+=294MassIdentification编辑pptHPLCprofilesofBCH-3840anditsmetabolite(BCH-6440)PreparationofmetabolitebybulkincubationMMPP10mgmicrosomalprotein2mgBCH-3840Fractioncollectionofmetabolitefractionationconcentration编辑pptNuclearMagneticResonanceprofilesofBCH-3840anditsmetabolite(BCH-6440)C5-HBCH-3840MetaboliteStructureElucidation编辑pptInvitrotherapeuticindexofBCH-6440编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

编辑pptInhibitorsforCYPIsoform Conc(mM)Furafulline(CYP1A2) 10Tranylcypromine(CYP2A6) 50Sulfaphenazole(CYP2C9) 25Omeprazole(CYP2C19) 20Quinidine(CYP2D6) 24-methylpyrazole(CYP2E1) 250Ketoconazole(CYP3A4) 5ChemicalInhibition〔化学抑制〕Pureenzyme〔纯酶〕CorrelationAnalysis〔相关分析〕MetabolismPhenotyping代谢途径鉴定编辑pptInhibitorsforCYPIsoform Conc(mM)

Inhibition(%ofNC)Tranylcypromine(CYP2A6) 50 40.2Sulfaphenazole(CYP2C9) 25 14.24-methylpyrazole(CYP2E1) 250 67.6Ketoconazole(CYP3A4) 5 75.2MetabolismPhenotyping代谢途径鉴定编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

编辑pptDrug-DrugInteractions〔对其它药物代谢的影响〕Inhibition〔抑制〕 potential-IC50andKi mechanism- 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 编辑pptIC50(

M): 0.675 GoodnessofFit: 0.9807 95%ConfidenceIntervals: 5.63–8.28IC50(

M): 20.4 GoodnessofFit: 0.9730 95%ConfidenceIntervals: 16.9-26.3 CYP3A4

CYP3A4Drug-druginteraction:inhibition抑制作用体外药效浓度=1uM编辑pptDrug-druginteraction:Induction〔肝细胞诱导模型〕5daysprocedureDay0: Isolatefreshhepatocytes,viability>70% Platinghepatocytesto24-wellplate,0.7x106viablecells/well Platingmediareplacedwithsandwichafter7-hourattachmentDay1: incubationforestablishingbasallevelsofCYP450isoforms.Day2: sameasDay1Day3: dosingwithtestarticlesDay4: sameasDay3Day5: washingoutthedosingsolutionandaddingsubstratesfor CYP450isoformsasbelow: 1A2- ethocyresorufinO-deethylation 2A6- coumarin7-hydroxylation 2C9- tolbutamide4-hydroxylation 2C19- S-mephenytoin4-hydroxylation 2D6- dextromethorphanO-demethylation 2E1- chlorzoxazone6-hydroxylation 3A4- testosterone6b-hydroxylation编辑pptDrug-druginteraction:Induction诱导作用编辑ppt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SituationAnalysisinvitro体外metabolisminsitu离体permeabilityinvivo体内bioavailability编辑pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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屡次给药和体内积蓄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编辑ppt119%236%310%Proportionality血浆浓度的非线性提示：代谢或排泄的非线性饱和编辑ppt90%72%Proportionality:AUC〔大鼠试验〕93%63%提示：药物吸收的非线性饱和编辑pptTOXICOKINETICS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)编辑ppt90%72%Proportionality:AUC〔大鼠试验〕93%63%提示：药物吸收的非线性饱和编辑ppt01002003004005006000102030405060FemaleRatsOralDose(mg/kg)010020030040050060001020304050MaleRatsOralDose(mg/kg)Cmax(mg/mL)73%47%56%49%Proportionality:Cmax〔大鼠试验〕提示：药物吸收的非线性饱和编辑ppt0.920.771.041.191.021.07AccumulationRatio药物积累率〔大鼠〕MaleratsFemalerats编辑pptProportionality:AUC〔猕猴〕MaleMonkeyFemaleMonkey49%34%60%38%提示：药物吸收的非线性饱和编辑ppt38%31%55%32%Proportionality:Cmax〔猕猴〕MaleMonkeyFemaleMonkey提示：药物吸收的非线性饱和编辑pptMaleMonkeyFemaleMonkey0.791.111.120.730.761.14AccumulationRatio药物积累率〔猕猴〕编辑pptPhaseITrial(Singledoseescalation)临床一期单剂量药代动力学试验HealthyMaleSubject(n):22OralDoses(4): 100,200,400,and800mg

Timepoints(13)