藥代動力學(xué)在新藥研發(fā)中的作用課件

藥物代謝及其動力學(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.北京藥物代謝及其動力學(xué)在新藥研發(fā)中的應(yīng)用胡卓漢博士1EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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藥物代謝動力學(xué)DrugMetabolism/PharmcokineticsEfficacyHitsOptimizedLeadCom2藥物代謝動力學(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)間藥物代謝動力學(xué)的任務(wù)（最大無毒性濃度）(最小有效濃度）(最小3藥效毒理藥代最佳血漿濃度藥效毒理藥代最佳4EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

EfficacyHitsOptimizedLeadCom5EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

從動物代謝推算人體代謝

extrapolationEfficacyHitsOptimizedLeadCom6EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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)的動物選擇

metabolismprofilinginanimalsandhumans

EfficacyHitsOptimizedLeadCom7EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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)EfficacyHitsOptimizedLeadCom8二五原則5毫克5天二五原則9臨床前實(shí)驗(yàn)藥物代謝動力學(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)動物模型(invivoanimalmodels)動物推測人(speciesextrapolation)臨床前實(shí)驗(yàn)藥物代謝動力學(xué)的生物模型10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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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11PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-312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13absorption/distributionmodel脂層轉(zhuǎn)移模型水相Aqueousphase水相Aqueousphase有機(jī)相OrganicphasepH=6.5pH=7.4PermeabilityEvaluation–invitro14absorption/distributionmodelinvitroabsorption/distributionmodel15invitroabsorption/distributiCaco-2TransportPathways

人大腸癌細(xì)胞模型Caco-2TransportPathways

人大腸癌16TransportPathways

藥物吸收機(jī)制被動細(xì)胞間主動P糖蛋白TransportPathways

藥物吸收機(jī)制被動細(xì)胞間17ProbesforTransportPathways

腸道吸收標(biāo)準(zhǔn)對照藥物Transcellular（被動吸收） Propranolol,TestosteroneParacellular（細(xì)胞間滲透） Mannitol,InulinCarriermediated（主動吸收） GlucoseP-Glycoproteinmediated（P－糖蛋白調(diào)節(jié)） 底物 Vinblastine

抑制物 VerapamilProbesforTransportPathways

18Glucose（蔗糖）vsInulin（木香素）

主動吸收vs細(xì)胞間滲透Glucose（蔗糖）vsInulin（木香素）

19PropranololvsMannitol

被動吸收vs細(xì)胞間滲透PropranololvsMannitol

被動吸收20由P－蛋白所調(diào)節(jié)的藥物吸收

－使用P－糖蛋白抑制劑Verapamil由P－蛋白所調(diào)節(jié)的藥物吸收

－使用P－糖蛋白抑制劑Vera21Chong,Dando&Morrison;Pharm.Res.1997Chong,Dando&Morrison;Pharm22FalsePositive假陽性＝低FalseNegative假陰性＝高Caco-2TransportPathways人大腸癌細(xì)胞吸收模型FalsePositiveFalseNegativeCa23insituratintestinalperfusion(singlepass)離體大鼠十二指腸灌流模型（單循環(huán)）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假陽性假陰性Insituratintestinalpermeab26PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-327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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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28InSituRatIntestinalPermeability:Good陽性對照陰性對照受試藥物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實(shí)例：結(jié)構(gòu)優(yōu)化和吸收率分析pKa=10 pKa=8.4 p31SAR: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%SAR:permeabilityvs.efficacy32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)動物藥物代謝動力學(xué)模型二五原則:5毫克/5天小結(jié)：體外和離體藥物吸收實(shí)驗(yàn)系統(tǒng)33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力學(xué)的任務(wù)本身的藥物代謝動力學(xué)問題對其它藥物吸收的作用血漿濃度時(shí)間化學(xué)藥物化學(xué)藥物+中藥中藥的藥物代謝動力學(xué)的任務(wù)34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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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35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. --藥代研究員死還是不死,這是個(gè)問題.36動物體內(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)成本動物體內(nèi)模型-----------人體內(nèi)(臨床試驗(yàn))37HeartbeatandBodyweight（心率和體重）小鼠大鼠兔猴狗人38HeartbeatandBodyweight（心率和體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39LiverweightandHepaticFlowAntipyrineclearance(l/min)ratmouserabbitmonkeydoghumanClearance40Antipyrineclearance(l/min)raInVitroModelsoftheLiver

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InVitroModelsoftheLiver

體41USFDAGuidanceforIndustry

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。USFDAGuidanceforIndustry

美國42HOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE-3-GlucuronideEE2-3-SulfateConjugatesEE2EE2Hepatocytes（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)HOHOHOHOHOHOOGLUCHOOSOOHO2-Hyd43PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-344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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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45Reactionvolume: 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)Reactionvolume: 1.0ml,DPBS461234ABCDEFEnhancedThroughputScreening（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

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

EfficacyHitsOptimizedLeadCom55InhibitorsforCYPIsoform Conc(mM)Furafulline(CYP1A2) 10Tranylcypromine(CYP2A6) 50Sulfaphenazole(CYP2C9) 25Omeprazole(CYP2C19) 20Quinidine(CYP2D6) 24-methylpyrazole(CYP2E1) 250Ketoconazole(CYP3A4) 5ChemicalInhibition（化學(xué)抑制）Pureenzyme（純酶）

CorrelationAnalysis（相關(guān)分析）MetabolismPhenotyping代謝途徑鑒定InhibitorsforCYPIsoform Con56InhibitorsforCYPIsoform Conc(mM)

Inhibition(%ofNC)Tranylcypromine(CYP2A6) 50 40.2Sulfaphenazole(CYP2C9) 25 14.24-methylpyrazole(CYP2E1) 250 67.6Ketoconazole(CYP3A4) 5 75.2MetabolismPhenotyping代謝途徑鑒定InhibitorsforCYPIsoform Con57EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

EfficacyHitsOptimizedLeadCom58Drug-DrugInteractions（對其它藥物代謝的影響）Inhibition（抑制） potential-IC50andKi mechanism- 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 Drug-DrugInteractions（對其它藥物代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抑制作用體外藥效濃度=1uMIC50(M): 0.675 IC50(M): 60Drug-druginteraction:Induction（肝細(xì)胞誘導(dǎo)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誘導(dǎo)作用Drug-druginteraction:Inducti62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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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63EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

從動物代謝推算人體代謝

extrapolationEfficacyHitsOptimizedLeadCom64119%236%310%Proportionality血漿濃度的非線性提示：

代謝或排泄的非線性飽和119%236%310%Proportionality血漿6590%72%Proportionality:AUC（大鼠試驗(yàn)）93%63%提示：藥物吸收的非線性飽和90%72%Proportionality:AUC（大鼠66TOXICOKINETICS

毒物代謝動力學(xué)試驗(yàn)

Animal:Sprague-Dawleyrats(male&female)Cynomolgusmonkey(male&female)

Singledoseescalation（線性動力學(xué)）(50,250,500mg/kg)

Multipledoseescalation（藥物體內(nèi)積累）(50,250,500mg/kg,dailyfor14days)TOXICOKINETICS毒物代謝動力學(xué)試驗(yàn)6790%72%Proportionality:AUC（大鼠試驗(yàn)）93%63%提示：藥物吸收的非線性飽和90%72%Proportionality:AUC（大鼠6801002003004005006000102030405060FemaleRatsOralDose(mg/kg)010020030040050060001020304050MaleRatsOralDose(mg/kg)Cmax(mg/mL)73%47%56%49%Proportionality:Cmax（大鼠試驗(yàn)）提示：藥物吸收的非線性飽和010020030040050060001020304050690.920.771.041.191.021.07AccumulationRatio藥物積累率（大鼠）MaleratsFemalerats0.920.771.041.191.021.07Accumu70Proportionality:AUC（獼猴）MaleMonkeyFemaleMonkey49%34%60%38%提示：藥物吸收的非線性飽和Proportionality:AUC（獼猴）Male7138%31%55%32%Proportionality:Cmax（獼猴）MaleMonkeyFemaleMonkey提示：藥物吸收的非線性飽和38%31%55%32%Proportionality:C72MaleMonkeyFemaleMonkey0.791.111.120.730.761.14AccumulationRatio藥物積累率（獼猴）MaleMonkeyFemaleMonkey0.791.73PhaseITrial(Singledoseescalation)臨床一期單劑量藥代動力學(xué)試驗(yàn)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臨床實(shí)驗(yàn)臨床前實(shí)驗(yàn)研究和發(fā)現(xiàn)臨床階段動物和人的代謝特征(長期毒性實(shí)驗(yàn)的動物選擇)

metabolismprofilinginanimalsandhumans

EfficacyHitsOptimizedLeadCom75EvaluationofInVitroMetabolismProfiles

CryopreservedHepatocytesmouseratdogmonkeyhuman

實(shí)驗(yàn)藥物(MW=455.59in1/2tartratesalt) [14C]放射性比活性(23.7μCi/mg)藥代動力學(xué)在新藥研發(fā)中的作用課件76MethodsIncubationprocedures Viability(Trypanblueexclusion)>70% Finalcelldensity:2x106viablecell/mL Finalconcentrationoftestarticle:20μM Incubationtime:4hoursBioanalyticalmeasurement HPLC/Fractionating/Scintillation:Metabolicprofiles LC/MS/MS:Massidentification/structureelucidationMethodsIncubationprocedures77Results TreatedvsNegativeControlDPViableHumanHepatocytes速凍人肝細(xì)胞HeatedHumanHepatocytes熱滅活人肝細(xì)胞DPM2M3M4Results TreatedvsNegativeCo78MetabolismProfiles HumanvsDogDPViableHumanHepatocytes速凍人肝細(xì)胞ViableDogHepatocytes

速凍狗肝細(xì)胞DPM2M3DPM2M3M4MetabolismProfiles HumanvsD79MetabolismProfilesHumanvsMonkeyViableHumanHepatocytes速凍人肝細(xì)胞ViableMonkeyHepatocytes速凍猴肝細(xì)胞DPM2M3M4M1DPM2M3M4MetabolismProfilesHumanvs80MetabolismProfilesHumanvsMouseViableHumanHepatocytes速凍人肝細(xì)胞ViableMouseHepatocytes速凍小鼠肝細(xì)胞DPM2M3M4DPM2M3M4MetabolismProfilesHumanvs81MetabolismProfilesHumanvsRatViableHumanHepatocytes速凍人肝細(xì)胞ViableRatHepatocytes速凍大鼠肝細(xì)胞DPM2M3M4DPM2M3M4MetabolismProfilesHumanvs82藥代動力學(xué)在新藥研發(fā)中的作用課件83致謝

Acknowledgements

軍事醫(yī)學(xué)科學(xué)院輸血研究所/藥理毒理研究所杉山雄一博士/小澤正吾博士國家外專局引進(jìn)國外智力項(xiàng)目2004-A-034致謝軍事醫(yī)學(xué)科學(xué)院輸血研究所/藥理毒理研究所84藥物代謝及其動力學(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.北京藥物代謝及其動力學(xué)在新藥研發(fā)中的應(yīng)用胡卓漢博士85EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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藥物代謝動力學(xué)DrugMetabolism/PharmcokineticsEfficacyHitsOptimizedLeadCom86藥物代謝動力學(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)間藥物代謝動力學(xué)的任務(wù)（最大無毒性濃度）(最小有效濃度）(最小87藥效毒理藥代最佳血漿濃度藥效毒理藥代最佳88EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

EfficacyHitsOptimizedLeadCom89EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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

從動物代謝推算人體代謝

extrapolationEfficacyHitsOptimizedLeadCom90EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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)的動物選擇

metabolismprofilinginanimalsandhumans

EfficacyHitsOptimizedLeadCom91EfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&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)EfficacyHitsOptimizedLeadCom92二五原則5毫克5天二五原則93臨床前實(shí)驗(yàn)藥物代謝動力學(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)動物模型(invivoanimalmodels)動物推測人(speciesextrapolation)臨床前實(shí)驗(yàn)藥物代謝動力學(xué)的生物模型94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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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95PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-396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97absorption/distributionmodel脂層轉(zhuǎn)移模型水相Aqueousphase水相Aqueousphase有機(jī)相OrganicphasepH=6.5pH=7.4PermeabilityEvaluation–invitro98absorption/distributionmodelinvitroabsorption/distributionmodel99invitroabsorption/distributiCaco-2TransportPathways

人大腸癌細(xì)胞模型Caco-2TransportPathways

人大腸癌100TransportPathways

藥物吸收機(jī)制被動細(xì)胞間主動P糖蛋白TransportPathways

藥物吸收機(jī)制被動細(xì)胞間101ProbesforTransportPathways

腸道吸收標(biāo)準(zhǔn)對照藥物Transcellular（被動吸收） Propranolol,TestosteroneParacellular（細(xì)胞間滲透） Mannitol,InulinCarriermediated（主動吸收） GlucoseP-Glycoproteinmediated（P－糖蛋白調(diào)節(jié)） 底物 Vinblastine

抑制物 VerapamilProbesforTransportPathways

102Glucose（蔗糖）vsInulin（木香素）

主動吸收vs細(xì)胞間滲透Glucose（蔗糖）vsInulin（木香素）

103PropranololvsMannitol

被動吸收vs細(xì)胞間滲透PropranololvsMannitol

被動吸收104由P－蛋白所調(diào)節(jié)的藥物吸收

－使用P－糖蛋白抑制劑Verapamil由P－蛋白所調(diào)節(jié)的藥物吸收

－使用P－糖蛋白抑制劑Vera105Chong,Dando&Morrison;Pharm.Res.1997Chong,Dando&Morrison;Pharm106FalsePositive假陽性＝低FalseNegative假陰性＝高Caco-2TransportPathways人大腸癌細(xì)胞吸收模型FalsePositiveFalseNegativeCa107insituratintestinalperfusion(singlepass)離體大鼠十二指腸灌流模型（單循環(huán)）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假陽性假陰性Insituratintestinalpermeab110PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-3111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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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112InSituRatIntestinalPermeability:Good陽性對照陰性對照受試藥物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實(shí)例：結(jié)構(gòu)優(yōu)化和吸收率分析pKa=10 pKa=8.4 p115SAR: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%SAR:permeabilityvs.efficacy116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)動物藥物代謝動力學(xué)模型二五原則:5毫克/5天小結(jié)：體外和離體藥物吸收實(shí)驗(yàn)系統(tǒng)117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力學(xué)的任務(wù)本身的藥物代謝動力學(xué)問題對其它藥物吸收的作用血漿濃度時(shí)間化學(xué)藥物化學(xué)藥物+中藥中藥的藥物代謝動力學(xué)的任務(wù)118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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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119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. --藥代研究員死還是不死,這是個(gè)問題.120動物體內(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)成本動物體內(nèi)模型-----------人體內(nèi)(臨床試驗(yàn))121HeartbeatandBodyweight（心率和體重）小鼠大鼠兔猴狗人122HeartbeatandBodyweight（心率和體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123LiverweightandHepaticFlowAntipyrineclearance(l/min)ratmouserabbitmonkeydoghumanClearance124Antipyrineclearance(l/min)raInVitroModelsoftheLiver

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InVitroModelsoftheLiver

體125USFDAGuidanceforIndustry

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。USFDAGuidanceforIndustry

美國126HOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE-3-GlucuronideEE2-3-SulfateConjugatesEE2EE2Hepatocytes（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)HOHOHOHOHOHOOGLUCHOOSOOHO2-Hyd127PlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailabilityPlasmaconcentrationsofBCH-3128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SituationAnalysisinvitro體外metabolisminsitu離體permeabilityinvivo體內(nèi)bioavailability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129Reactionvolume: 1.0ml,DPBSpH7.4HepaticS-9/Microsomes: 0.5mgprotien/mLSpecies: Human/Monkey/Dog/Rat/MouseSubs