药代动力学在新药研发中的作用课件

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.北京药物代谢及其动力学在新药研发中的应用胡卓汉博士1EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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安全Safety/Toxicology药物代谢动力学DrugMetabolism/PharmcokineticsEfficacyHitsOptimizedLeadCom2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）血浆浓度时间药物代谢动力学的任务（最大无毒性浓度）(最小有效浓度）(最小3药效毒理药代最佳血浆浓度药效毒理药代最佳4EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom5EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

extrapolationEfficacyHitsOptimizedLeadCom6EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom7EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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)EfficacyHitsOptimizedLeadCom8二五原则5毫克5天二五原则9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)临床前实验药物代谢动力学的生物模型10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11PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-312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13absorption/distributionmodel脂层转移模型水相Aqueousphase水相Aqueousphase有机相OrganicphasepH=6.5pH=7.4PermeabilityEvaluation–invitro14absorption/distributionmodelinvitroabsorption/distributionmodel15invitroabsorption/distributiCaco-2TransportPathways

人大肠癌细胞模型Caco-2TransportPathways

人大肠癌16TransportPathways

药物吸收机制被动细胞间主动P糖蛋白TransportPathways

药物吸收机制被动细胞间17ProbesforTransportPathways

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 VerapamilProbesforTransportPathways

18Glucose（蔗糖）vsInulin（木香素）

主动吸收vs细胞间渗透Glucose（蔗糖）vsInulin（木香素）

19PropranololvsMannitol

被动吸收vs细胞间渗透PropranololvsMannitol

被动吸收20由P－蛋白所调节的药物吸收

－使用P－糖蛋白抑制剂Verapamil由P－蛋白所调节的药物吸收

－使用P－糖蛋白抑制剂Vera21Chong,Dando&Morrison;Pharm.Res.1997Chong,Dando&Morrison;Pharm22FalsePositive假阳性＝低FalseNegative假阴性＝高Caco-2TransportPathways人大肠癌细胞吸收模型FalsePositiveFalseNegativeCa23insituratintestinalperfusion(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/mLinsituratintestinalperfusi24PerfusionProcedures: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)PerfusionProcedures:insitur25Insituratintestinalpermeability(singlepass)Predictionwithin90%interval=19/31(61.3%)In-housevalidation假阳性假阴性Insituratintestinalpermeab26PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-327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28InSituRatIntestinalPermeability:Good阳性对照阴性对照受试药物InSituRatIntestinalPermeab29EnhancedThroughputScreeningPerfusion: 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/2weeksEnhancedThroughputScreening30pKa=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pKa=10 pKa=8.4 p31SAR: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%SAR:permeabilityvs.efficacy32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天小结：体外和离体药物吸收实验系统33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34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35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. --药代研究员死还是不死,这是个问题.36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，药物代谢实验成本动物体内模型-----------人体内(临床试验)37HeartbeatandBodyweight（心率和体重）小鼠大鼠兔猴狗人38HeartbeatandBodyweight（心率和体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39LiverweightandHepaticFlowAntipyrineclearance(l/min)ratmouserabbitmonkeydoghumanClearance40Antipyrineclearance(l/min)raInVitroModelsoftheLiver

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InVitroModelsoftheLiver

体41USFDAGuidanceforIndustry

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。USFDAGuidanceforIndustry

美国42HOHOHOHOHOHOOGLUCHOOSOOHO2-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)HOHOHOHOHOHOOGLUCHOOSOOHO2-Hyd43PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-344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45Reactionvolume: 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Reactionvolume: 1.0ml,DPBS461234ABCDEFEnhancedThroughputScreening（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1ABCDEFEnhance47EnhancedThroughputScreeningIncubation: 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/1weekEnhancedThroughputScreening48HPLCprofilesofBCH-3840anditsmetabolite(BCH-6440)BCH-3840metabolite?InvitrometabolicstabilitybyrathepaticS9HPLCprofilesofBCH-3840and49EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom50LiquidChromatography/MassSpectrumofBCH-3840anditsmetabolite(BCH-6440)HydroxylationorOxidationMH+=310MH+=294MassIdentificationLiquidChromatography/MassS51HPLCprofilesofBCH-3840anditsmetabolite(BCH-6440)PreparationofmetabolitebybulkincubationMMPP10mgmicrosomalprotein2mgBCH-3840FractioncollectionofmetabolitefractionationconcentrationHPLCprofilesofBCH-3840and52NuclearMagneticResonanceprofilesofBCH-3840anditsmetabolite(BCH-6440)C5-HBCH-3840MetaboliteStructureElucidationNuclearMagneticResonancepro53InvitrotherapeuticindexofBCH-6440Invitrotherapeuticindexof54EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom55InhibitorsforCYPIsoform 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InhibitorsforCYPIsoform Con56InhibitorsforCYPIsoform 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InhibitorsforCYPIsoform Con57EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom58Drug-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 Drug-DrugInteractions（对其它药物代59IC50(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=1uMIC50(M): 0.675 IC50(M): 60Drug-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-hydroxylationDrug-druginteraction:Inducti61Drug-druginteraction:Induction诱导作用Drug-druginteraction:Inducti62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63EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

extrapolationEfficacyHitsOptimizedLeadCom64119%236%310%Proportionality血浆浓度的非线性提示：

代谢或排泄的非线性饱和119%236%310%Proportionality血浆6590%72%Proportionality:AUC（大鼠试验）93%63%提示：药物吸收的非线性饱和90%72%Proportionality:AUC（大鼠66TOXICOKINETICS

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)TOXICOKINETICS毒物代谢动力学试验6790%72%Proportionality:AUC（大鼠试验）93%63%提示：药物吸收的非线性饱和90%72%Proportionality:AUC（大鼠6801002003004005006000102030405060FemaleRatsOralDose(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010020030040050060001020304050690.920.771.041.191.021.07AccumulationRatio药物积累率（大鼠）MaleratsFemalerats0.920.771.041.191.021.07Accumu70Proportionality:AUC（猕猴）MaleMonkeyFemaleMonkey49%34%60%38%提示：药物吸收的非线性饱和Proportionality:AUC（猕猴）Male7138%31%55%32%Proportionality:Cmax（猕猴）MaleMonkeyFemaleMonkey提示：药物吸收的非线性饱和38%31%55%32%Proportionality:C72MaleMonkeyFemaleMonkey0.791.111.120.730.761.14AccumulationRatio药物积累率（猕猴）MaleMonkeyFemaleMonkey0.791.73PhaseITrial(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): 0.5,1,1.5,2,3,4,6,8,10,12, 16,20,and24hour

PhaseITrial(Singledoseesc74EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D临床实验临床前实验研究和发现临床阶段动物和人的代谢特征(长期毒性实验的动物选择)

metabolismprofilinginanimalsandhumans

EfficacyHitsOptimizedLeadCom75EvaluationofInVitroMetabolismProfiles

CryopreservedHepatocytesmouseratdogmonkeyhuman

实验药物(MW=455.59in1/2tartratesalt) [14C]放射性比活性(23.7µCi/mg)药代动力学在新药研发中的作用课件76MethodsIncubationprocedures Viability(Trypanblueexclusion)>70% Finalcelldensity:2x106viablecell/mL Finalconcentrationoftestarticle:20µM Incubationtime:4hoursBioanalyticalmeasurement HPLC/Fractionating/Scintillation:Metabolicprofiles LC/MS/MS:Massidentification/structureelucidationMethodsIncubationprocedures77Results TreatedvsNegativeControlDPViableHumanHepatocytes速冻人肝细胞HeatedHumanHepatocytes热灭活人肝细胞DPM2M3M4Results TreatedvsNegativeCo78MetabolismProfiles HumanvsDogDPViableHumanHepatocytes速冻人肝细胞ViableDogHepatocytes

速冻狗肝细胞DPM2M3DPM2M3M4MetabolismProfiles HumanvsD79MetabolismProfilesHumanvsMonkeyViableHumanHepatocytes速冻人肝细胞ViableMonkeyHepatocytes速冻猴肝细胞DPM2M3M4M1DPM2M3M4MetabolismProfilesHumanvs80MetabolismProfilesHumanvsMouseViableHumanHepatocytes速冻人肝细胞ViableMouseHepatocytes速冻小鼠肝细胞DPM2M3M4DPM2M3M4MetabolismProfilesHumanvs81MetabolismProfilesHumanvsRatViableHumanHepatocytes速冻人肝细胞ViableRatHepatocytes速冻大鼠肝细胞DPM2M3M4DPM2M3M4MetabolismProfilesHumanvs82药代动力学在新药研发中的作用课件83致谢

Acknowledgements

军事医学科学院输血研究所/药理毒理研究所杉山雄一博士/小澤正吾博士国家外专局引进国外智力项目2004-A-034致谢军事医学科学院输血研究所/药理毒理研究所84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.北京药物代谢及其动力学在新药研发中的应用胡卓汉博士85EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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安全Safety/Toxicology药物代谢动力学DrugMetabolism/PharmcokineticsEfficacyHitsOptimizedLeadCom86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）血浆浓度时间药物代谢动力学的任务（最大无毒性浓度）(最小有效浓度）(最小87药效毒理药代最佳血浆浓度药效毒理药代最佳88EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom89EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

extrapolationEfficacyHitsOptimizedLeadCom90EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(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

EfficacyHitsOptimizedLeadCom91EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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)EfficacyHitsOptimizedLeadCom92二五原则5毫克5天二五原则93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)临床前实验药物代谢动力学的生物模型94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95PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-396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97absorption/distributionmodel脂层转移模型水相Aqueousphase水相Aqueousphase有机相OrganicphasepH=6.5pH=7.4PermeabilityEvaluation–invitro98absorption/distributionmodelinvitroabsorption/distributionmodel99invitroabsorption/distributiCaco-2TransportPathways

人大肠癌细胞模型Caco-2TransportPathways

人大肠癌100TransportPathways

药物吸收机制被动细胞间主动P糖蛋白TransportPathways

药物吸收机制被动细胞间101ProbesforTransportPathways

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 VerapamilProbesforTransportPathways

102Glucose（蔗糖）vsInulin（木香素）

主动吸收vs细胞间渗透Glucose（蔗糖）vsInulin（木香素）

103PropranololvsMannitol

被动吸收vs细胞间渗透PropranololvsMannitol

被动吸收104由P－蛋白所调节的药物吸收

－使用P－糖蛋白抑制剂Verapamil由P－蛋白所调节的药物吸收

－使用P－糖蛋白抑制剂Vera105Chong,Dando&Morrison;Pharm.Res.1997Chong,Dando&Morrison;Pharm106FalsePositive假阳性＝低FalseNegative假阴性＝高Caco-2TransportPathways人大肠癌细胞吸收模型FalsePositiveFalseNegativeCa107insituratintestinalperfusion(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/mLinsituratintestinalperfusi108PerfusionProcedures: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)PerfusionProcedures:insitur109Insituratintestinalpermeability(singlepass)Predictionwithin90%interval=19/31(61.3%)In-housevalidation假阳性假阴性Insituratintestinalpermeab110PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-3111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112InSituRatIntestinalPermeability:Good阳性对照阴性对照受试药物InSituRatIntestinalPermeab113EnhancedThroughputScreeningPerfusion: 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/2weeksEnhancedThroughputScreening114pKa=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pKa=10 pKa=8.4 p115SAR: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%SAR:permeabilityvs.efficacy116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天小结：体外和离体药物吸收实验系统117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118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119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. --药代研究员死还是不死,这是个问题.120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，药物代谢实验成本动物体内模型-----------人体内(临床试验)121HeartbeatandBodyweight（心率和体重）小鼠大鼠兔猴狗人122HeartbeatandBodyweight（心率和体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123LiverweightandHepaticFlowAntipyrineclearance(l/min)ratmouserabbitmonkeydoghumanClearance124Antipyrineclearance(l/min)raInVitroModelsoftheLiver

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InVitroModelsoftheLiver

体125USFDAGuidanceforIndustry

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。USFDAGuidanceforIndustry

美国126HOHOHOHOHOHOOGLUCHOOSOOHO2-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)HOHOHOHOHOHOOGLUCHOOSOOHO2-Hyd127PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-3128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129Reactionvolume: 1.0ml,DPBSpH7.4HepaticS-9/Microsomes: 0.5mgprotien/mLSpecies: Human/Monkey/Dog/Rat/MouseSubs