藥代動力學(xué)在新藥研發(fā)中的作用

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)復(fù)旦大學(xué)藥學(xué)院2004年12月30日中國.北京編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(fā)現(xiàn)藥物研發(fā)的三大任務(wù)藥效Efficacy/Pharmacodynamics平安Safety/Toxicology藥物代謝動力學(xué)DrugMetabolism/Pharmcokinetics編輯ppt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〕(最小藥效時間〕血漿濃度時間編輯ppt藥效毒理藥代最正確血漿濃度編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(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

編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(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屢次給藥和體內(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extrapolation編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(fā)現(xiàn)臨床階段長期毒性實驗的動物選擇

metabolismprofilinginanimalsandhumans

編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(fā)現(xiàn)臨床實驗準那么GoodClinicalPractice(GCP)非臨床實驗準那么GoodLaboratoryPractice(GLP)編輯ppt二五原那么5毫克5天編輯ppt臨床前實驗藥物代謝動力學(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)編輯ppt排出太快/藥效時間太短口服吸收差/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編輯pptPlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailability編輯ppt藥物吸收模型計算機脂溶度脂層轉(zhuǎn)移細胞層轉(zhuǎn)移十二指腸灌流編輯pptabsorption/distributionmodel脂層轉(zhuǎn)移模型水相Aqueousphase水相Aqueousphase有機相OrganicphasepH=6.5pH=7.4PermeabilityEvaluation–invitro14編輯pptinvitroabsorption/distributionmodel15編輯pptCaco-2TransportPathways

人大腸癌細胞模型編輯pptTransportPathways

藥物吸收機制被動細胞間主動P糖蛋白編輯pptProbesforTransportPathways

腸道吸收標準對照藥物Transcellular〔被動吸收〕 Propranolol,TestosteroneParacellular〔細胞間滲透〕 Mannitol,InulinCarriermediated〔主動吸收〕 GlucoseP-Glycoproteinmediated〔P－糖蛋白調(diào)節(jié)〕 底物 Vinblastine

抑制物 Verapamil編輯pptGlucose〔蔗糖〕vsInulin〔木香素〕

主動吸收vs細胞間滲透編輯pptPropranololvsMannitol

被動吸收vs細胞間滲透編輯ppt由P－蛋白所調(diào)節(jié)的藥物吸收

－使用P－糖蛋白抑制劑Verapamil編輯pptChong,Dando&Morrison;Pharm.Res.1997編輯pptFalsePositive假陽性＝低FalseNegative假陰性＝高Caco-2TransportPathways人大腸癌細胞吸收模型編輯pptinsituratintestinalperfusion(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/mL編輯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)編輯pptInsituratintestinalpermeability(singlepass)Predictionwithin90%interval=19/31(61.3%)In-housevalidation假陽性假陰性編輯pptPlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailability編輯ppt排出太快/藥效時間太短口服吸收差/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編輯pptInSituRatIntestinalPermeability:Good陽性對照陰性對照受試藥物編輯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編輯pptpKa=10 pKa=8.4 pKa=6.5Preduced%=0% Preduced%=7% Preduced%=12%SAR:pKavs.permeability實例：結(jié)構(gòu)優(yōu)化和吸收率分析編輯pptSAR:permeabilityvs.efficacy實例：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%編輯ppt小結(jié)：體外和離體藥物吸收實驗系統(tǒng)體外人大腸癌細胞模型(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)模型二五原那么:5毫克/5天編輯ppt血漿濃度時間化學(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編輯ppt排出太快/藥效時間太短口服吸收差/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編輯ppt死還是不死,這是個問題.Tobeornottobe,thisisaproblem. --哈默雷特體內(nèi)試驗還是體外試驗,這是個問題.Invitroorinvivo,thisisaproblem. --藥代研究員編輯ppt動物體內(nèi)模型-----------人體內(nèi)(臨床試驗)Invivoanimalsvs.invivohumans人體外模型---------------人體內(nèi)(臨床試驗)Invitrohumansvs.invivohumans選擇的指南與人相似：疾病模型，藥效，毒性，藥物代謝實驗本錢編輯pptHeartbeatandBodyweight〔心率和體重〕小鼠大鼠兔猴狗人38編輯pptLiverweightandHepaticFlowvsBodyweight〔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39編輯pptAntipyrineclearance(l/min)ratmouserabbitmonkeydoghumanClearance40編輯pptInVitroModelsoftheLiver

體外肝模型Hepatocytes肝細胞Liverslices肝切片Livermicrosomes肝微粒體LiverS-9Fraction肝S-9組分編輯pptUSFDAGuidanceforIndustry

美國藥物和食品管理局關(guān)于藥物代謝實驗的指南“Themostcompletepictureforhepaticmetabolismcanbeobtainedwithliversystems,inwhichthecofactorsareself-sufficientandthenaturalorientationforlinkedenzymesispreserved.Isolatedhepatocytesandprecision-cutsliceshavethesedesirablefeatures.〞GuidanceforIndustry,DrugMetabolism/DrugInteractionStudiesintheDrugDevelopmentProcess:StudiesInVitroCDER,CBER,U.S.FDA,1997譯文：肝系統(tǒng)〔別離的肝細胞和精確的肝切片〕能為藥物代謝實驗提供最完全的信息，因為這個系統(tǒng)含有足夠的天然水平的酶系。編輯pptHOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE-3-GlucuronideEE2-3-SulfateConjugatesEE2EE2Hepatocytes〔肝細胞〕Microsomes〔微粒體〕Hepatocytes〔肝細胞〕MetabolismofEythinylEstradiol(EE2)

肝微粒體和肝細胞的代謝功能差異Li,Hartman,Lu,CollinsandStrong,BrJClinPharmacol48,733-742(1999)編輯pptPlasmaconcentrationsofBCH-3840anditsmetabolite(BCH-6440)inmicedosed50mg/kgorallyPoororalbioavailability編輯ppt排出太快/藥效時間太短口服吸收差/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編輯pptReactionvolume: 1.0ml,DPBSpH7.4HepaticS-9/Microsomes: 0.5mgprotien/mLSpecies: Human/Monkey/Dog/Rat/MouseSubstrateconcentration: 10mMNADPH: 2.4mMUDPGA: 1.5mMIncubation: 60minat37oCStoppingprocedure: chilledacetonitrile,3xvolume

InVitroMetabolismAssay

體外肝微粒體實驗編輯ppt1234ABCDEFEnhancedThroughputScreening〔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:〔實驗樣品〕：testarticle+microsomes+cofactors=treatedDosingsolution=timezero(T=0)4compoundsincludingpositivereference*/plate*7ethoxycoumarin陰性對照空白對照測試樣本編輯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編輯pptHPLCprofilesofBCH-3840anditsmetabolite(BCH-6440)BCH-3840metabolite?InvitrometabolicstabilitybyrathepaticS9編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(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

編輯pptLiquidChromatography/MassSpectrumofBCH-3840anditsmetabolite(BCH-6440)HydroxylationorOxidationMH+=310MH+=294MassIdentification編輯pptHPLCprofilesofBCH-3840anditsmetabolite(BCH-6440)PreparationofmetabolitebybulkincubationMMPP10mgmicrosomalprotein2mgBCH-3840Fractioncollectionofmetabolitefractionationconcentration編輯pptNuclearMagneticResonanceprofilesofBCH-3840anditsmetabolite(BCH-6440)C5-HBCH-3840MetaboliteStructureElucidation編輯pptInvitrotherapeuticindexofBCH-6440編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(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

編輯pptInhibitorsforCYPIsoform 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代謝途徑鑒定編輯pptInhibitorsforCYPIsoform Conc(mM)

Inhibition(%ofNC)Tranylcypromine(CYP2A6) 50 40.2Sulfaphenazole(CYP2C9) 25 14.24-methylpyrazole(CYP2E1) 250 67.6Ketoconazole(CYP3A4) 5 75.2MetabolismPhenotyping代謝途徑鑒定編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(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

編輯pptDrug-DrugInteractions〔對其它藥物代謝的影響〕Inhibition〔抑制〕 potential-IC50andKi mechanism- mechanistic〔機械性〕 competitive〔競爭性〕testsystem: livermicrosomes〔肝微粒體〕 cryopreservedhepatocytes〔冷凍肝細胞〕Induction〔誘導(dǎo)〕testsystem: freshisolatedhepatocytes〔肝細胞〕TargetEnzymesCytochromeP450s: 1A2,2A6,2C8,2C9,2C19,2D6,2E1,3A4PhaseIIconjugation:glucuronidation 編輯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抑制作用體外藥效濃度=1uM編輯pptDrug-druginteraction:Induction〔肝細胞誘導(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-hydroxylation編輯pptDrug-druginteraction:Induction誘導(dǎo)作用編輯ppt排出太快/藥效時間太短口服吸收差/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編輯pptEfficacyHitsOptimizedLeadGoornogodecisionCompoundforDevelopment(CD)NEWDRUGINDNDAR&D臨床實驗臨床前實驗研究和發(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屢次給藥和體內(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extrapolation編輯ppt119%236%310%Proportionality血漿濃度的非線性提示：代謝或排泄的非線性飽和編輯ppt90%72%Proportionality:AUC〔大鼠試驗〕93%63%提示：藥物吸收的非線性飽和編輯pptTOXICOKINETICS毒物代謝動力學(xué)試驗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)編輯ppt90%72%Proportionality:AUC〔大鼠試驗〕93%63%提示：藥物吸收的非線性飽和編輯ppt01002003004005006000102030405060FemaleRatsOralDose(mg/kg)010020030040050060001020304050MaleRatsOralDose(mg/kg)Cmax(mg/mL)73%47%56%49%Proportionality:Cmax〔大鼠試驗〕提示：藥物吸收的非線性飽和編輯ppt0.920.771.041.191.021.07AccumulationRatio藥物積累率〔大鼠〕MaleratsFemalerats編輯pptProportionality:AUC〔獼猴〕MaleMonkeyFemaleMonkey49%34%60%38%提示：藥物吸收的非線性飽和編輯ppt38%31%55%32%Proportionality:Cmax〔獼猴〕MaleMonkeyFemaleMonkey提示：藥物吸收的非線性飽和編輯pptMaleMonkeyFemaleMonkey0.791.111.120.730.761.14AccumulationRatio藥物積累率〔獼猴〕編輯pptPhaseITrial(Singledoseescalation)臨床一期單劑量藥代動力學(xué)試驗HealthyMaleSubject(n):22OralDoses(4): 100,200,400,and800mg

Timepoints(13)