药代动力学在新药研发中的作用课件.ppt

1、药物代谢及其动力学在新药研发中的应用药物代谢及其动力学在新药研发中的应用胡卓汉胡卓汉 博士博士瑞德肝脏疾病研究瑞德肝脏疾病研究(上海上海)有限公司有限公司复旦大学药学院复旦大学药学院2004年年12月月30日日中国中国.北京北京Efficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&D药物代谢动力学的任务药物代谢动力学的任务0.010.111010010000.511.522.533.54.（最大无毒性浓度）（最大无毒性浓度）(最小有效浓度）最小有效浓度）(最小药效时

2、间）最小药效时间）血浆浓度血浆浓度时间时间药效药效毒理毒理药代药代最佳最佳血浆浓度血浆浓度Efficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&DEfficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&DEfficacy HitsOptimized Lead Go or no go decisionCompound for Developm

3、ent(CD)NEW DRUGINDNDAR&DEfficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&D二五原则二五原则 5 毫克毫克 5 天天排出太快排出太快/药效时间太短药效时间太短口服吸收差口服吸收差/血浆浓度太低血浆浓度太低分布分布排泻排泻代谢问题代谢问题吸收问题吸收问题蛋白质相互作用蛋白质相互作用分布体积分布体积肾脏排泄肾脏排泄肝脏代谢肝脏代谢溶解度溶解度肠道吸收肠道吸收膜通透性膜通透性肠道消化肠道消化早期研发阶段早期研发阶段后期研发阶段后期研发阶段in

4、vitro体外体外metabolismin situ离体离体permeabilityin vivo体内体内bioavailabilityPlasma concentrations of BCH-3840 and its metabolite(BCH-6440)in mice dosed 50 mg/kg orallyPoor oral bioavailability06012018002004006008001000 Parent MetaboliteTmax(min)6.146.53Cmax(g/ml)0.0830.799AUC(g/ml/min)4.82321.810Bioavailabi

5、lity%2.52311.434R20.92920.9964BCH3840MetaboliteTime(min)Concentration ng/ml计算机计算机脂溶度脂溶度脂层转移脂层转移细胞层转移细胞层转移十二指肠灌流十二指肠灌流14absorption/distribution model 脂层转移模型脂层转移模型水相水相Aqueous phase水相水相Aqueous phase有机相有机相Organic phasepH=6.5pH=7.4Permeability Evaluation Permeability Evaluation in vitroin vitro15in vitro

6、 absorption/distribution modelIn Vitro/In Vivo Correlation PooledData from Four Biostudies020406080100020406080100formulation finding studyBE study 1BE study 2Y=4.2+1.00 X,R2=.987specification study%Distributed%AbsorpedCaco-2 Transport Pathways人大肠癌细胞模型人大肠癌细胞模型Transport Pathways药物吸收机制药物吸收机制被动被动细胞间细胞间

7、主动主动P糖蛋白糖蛋白Probes for Transport Pathways肠道吸收标准对照药物肠道吸收标准对照药物Transcellular（被动吸收）（被动吸收）Propranolol,TestosteroneParacellular（细胞间渗透）（细胞间渗透）Mannitol,InulinCarrier mediated（主动吸收）（主动吸收）GlucoseP-Glycoprotein mediated（P糖蛋白调节）糖蛋白调节）底物底物 Vinblastine抑制物抑制物 VerapamilGlucose（蔗糖）（蔗糖）vs Inulin（木香素）（木香素）主动吸收主动吸收 vsv

8、s 细胞间渗透细胞间渗透050100150200250020406080100Time(min)FluxPropranolol vs Mannitol被动吸收被动吸收 vsvs 细胞间渗透细胞间渗透由由P P蛋白所调节的药物吸收蛋白所调节的药物吸收使用使用P P糖蛋白抑制剂糖蛋白抑制剂 VerapamilVerapamilOral Absorption3-dayDrugsin HumansCaco-2(%)Kpcaffeine100227ibuprofen100201desipramine95261acetaminophen95218propranolol90265hydrazine90155

9、Ketoconazole76120terbutaline7356atenolol5030acetbutalol4029nadolol3522losartan3342mannitol1640inulin512Chong,Dando&Morrison;Pharm.Res.1997False Positive假阳性假阳性 低低False Negative假阴性假阴性 高高Caco-2 Transport Pathways 人大肠癌细胞吸收模型人大肠癌细胞吸收模型in situ rat intestinal perfusion(single pass)离体大鼠十二指肠灌流模型（单循环）离体大鼠十二指肠

10、灌流模型（单循环）METHODAnimal:Male Sprague-Dawley rats(250-350 g),fasted overnight.Rat is anesthetized by urethane 1.5g/kg,im.before perfusion starts.Perfusate:Phosphate buffer,pH=6.5 10 mM glucose Phenol red(negative control)Acetaminophen(positive control)Final concentrations of test article =0.05-0.30 mg/

11、mLPerfusion Procedures:rat is put on a heating pad to maintain body temperature jejunum is exposed via a middle line incision sutures:1st is made at 5 cm distal to the ligament of Treitz2nd is made at about 20 cm distal to 1st one the inlet of cannula -a syringe infusion pump the outlet of cannula-a

12、 fraction collector the perfusion segment is precleaned by passing 10 ml of blank perfusate buffer perfusion time and rate=0.1 ml/min for 120 min outlet perfusion samples are collected every 10 min plasma samples are collected at 30,60,90 and 120 min after perfusion Calculations:Permeability(Peff,cm

13、/min)=(Q/2RLp)x(1-Cout/Cin)Cout/Cin=(Cout/Cin)x phenol red in/phenol red outin situ rat intestinal perfusion(single pass)In situ rat intestinal permeability(single pass)0.0000.0010.0020.0030.0040.005020406080100Permeability(cm/min)Human Oral Bioavailability(%)Prediction within 90%interval=19/31(61.3

14、%)In-house validation假阳性假阳性假阴性假阴性Plasma concentrations of BCH-3840 and its metabolite(BCH-6440)in mice dosed 50 mg/kg orallyPoor oral bioavailability06012018002004006008001000 Parent MetaboliteTmax(min)6.146.53Cmax(g/ml)0.0830.799AUC(g/ml/min)4.82321.810Bioavailability%2.52311.434R20.92920.9964BCH38

15、40MetaboliteTime(min)Concentration ng/ml排出太快排出太快/药效时间太短药效时间太短口服吸收差口服吸收差/血浆浓度太低血浆浓度太低分布分布排泻排泻代谢问题代谢问题吸收问题吸收问题蛋白质相互作用蛋白质相互作用分布体积分布体积肾脏排泄肾脏排泄肝脏代谢肝脏代谢溶解度溶解度肠道吸收肠道吸收膜通透性膜通透性肠道消化肠道消化早期研发阶段早期研发阶段后期研发阶段后期研发阶段in vitro体外体外metabolismin situ离体离体permeabilityin vivo体内体内bioavailabilityIn Situ Rat Intestinal Permea

16、bility:Good5060708090 100 110 120 130 140 1500255075100Phenol RedAcetaminophenBCH-3840Time(min)Decreased Concentration(%)阳性对照阳性对照阴性对照阴性对照受试药物受试药物Enhanced Throughput ScreeningPerfusion:4 compounds per day(4 animals)Sample size:time points 7duplicate x 2control/drug x 3sample/perfusion 42Total samples

17、/day168 Bioanalysis:no extractionno standard curve(peak area)machine time/2 LCs24 hrsTotal manpower:animal tech x 1PKDM tech x 2 Test article amount:1 mg/test articleScreening rate:one chemotypes with 30 compounds/2 weeks NONNOSOOONONHFFFOONONNOSOOOOONHFFFOONONNOSOOOOONHFFFOOpKa=10 pKa=8.4 pKa=6.5 P

18、reduced%=0%Preduced%=7%Preduced%=12%NONNOOONSNNNHFFFOONONNOSOOONSNNNHHChiralFFFOONONNOOONSNNFFFOONONNOSOOONSNN+FFFOOPreduced%=Preduced%=Preduced%=Preduced%=0.010.111010010000.511.522.533.54.血浆浓度血浆浓度时间时间化学药物化学药物化学药物化学药物+中药中药5060708090100 110 120 130 140 1500255075100Ph e n o l Re dA ce tam in o p h e

19、 nBC H-3840Tim e(m in)Decreased Concentration(%)中药的药物代谢动力学的任务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本身的药物代谢动力学问题 对其它药物吸收的作用对其它药物吸收的作用排出太快排出太快/药效时间太短药效时间太短口服吸收差口服吸收差/血浆浓度太低血浆浓度太低分布分布排泻排泻代谢问题代谢问题吸收问题吸收问题蛋白质相互作用蛋白质相互作用分布体积分布体积肾脏排泄肾脏排泄肝脏代谢肝脏代谢溶解度溶解度肠道吸收肠道吸收膜通透性膜通透性肠道消化肠道消化早期研发阶段早期研发阶段后期研发阶段后期研发阶段in vitro体外体外metaboli

20、smin situ离体离体permeabilityin vivo体内体内bioavailability38Heartbeat and Bodyweight（心率和体重）心率和体重）小鼠小鼠大鼠大鼠兔兔猴猴狗狗人人39）人人狗狗猴猴兔兔大鼠大鼠小鼠小鼠人人狗狗猴猴兔兔大大鼠鼠小鼠小鼠40Antipyrine clearance(l/min)ratmouserabbitmonkeydoghumanClearanceIn Vitro Models of the Liver体外肝模型体外肝模型 Hepatocytes 肝细胞肝细胞 Liver slices 肝切片肝切片 Liver microsome

21、s 肝肝微粒体微粒体 Liver S-9 Fraction 肝肝S-9组分组分 USFDA Guidance for Industry美国药物和食品管理局关于药物代谢实验的指南美国药物和食品管理局关于药物代谢实验的指南“The most complete picture for hepatic metabolism can be obtained with liver systems,in which the cofactors are self-sufficient and the natural orientation for linked enzymes is preserved.Iso

22、lated hepatocytes and precision-cut slices have these desirable features.”Guidance for Industry,Drug Metabolism/Drug InteractionStudies in the Drug Development Process:Studies In VitroCDER,CBER,U.S.FDA,1997HOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE -3-GlucuronideEE2-3-SulfateConjugatesEE2EE2Hepatocyt

23、es （肝细胞）（肝细胞）Microsomes（微粒体）（微粒体）Hepatocytes（肝细胞）（肝细胞）Metabolism of Eythinyl Estradiol(EE2)肝微粒体和肝细胞的代谢功能差异肝微粒体和肝细胞的代谢功能差异Plasma concentrations of BCH-3840 and its metabolite(BCH-6440)in mice dosed 50 mg/kg orallyPoor oral bioavailability06012018002004006008001000 Parent MetaboliteTmax(min)6.146.53Cm

24、ax(g/ml)0.0830.799AUC(g/ml/min)4.82321.810Bioavailability%2.52311.434R20.92920.9964BCH3840MetaboliteTime(min)Concentration ng/ml排出太快排出太快/药效时间太短药效时间太短口服吸收差口服吸收差/血浆浓度太低血浆浓度太低分布分布排泻排泻代谢问题代谢问题吸收问题吸收问题蛋白质相互作用蛋白质相互作用分布体积分布体积肾脏排泄肾脏排泄肝脏代谢肝脏代谢溶解度溶解度肠道吸收肠道吸收膜通透性膜通透性肠道消化肠道消化早期研发阶段早期研发阶段后期研发阶段后期研发阶段in vitro体外体外

25、metabolismin situ离体离体permeabilityin vivo体内体内bioavailabilityReaction volume:1.0 ml,DPBS pH 7.4Hepatic S-9/Microsomes:0.5 mg protien/mL Species:Human/Monkey/Dog/Rat/Mouse Substrate concentration:10 MNADPH:2.4 mMUDPGA:1.5 mMIncubation:60 min at 37oCStopping procedure:chilled acetonitrile,3 x volumeIn V

26、itro Metabolism Assay 体外肝微粒体实验体外肝微粒体实验1234 A B C D E FEnhanced Throughput Screening（增速筛选增速筛选）A-B:（空白对照）：（空白对照）：test article+buffer=vehicle control (VC)C-D:（阴性对照）：阴性对照）：test article+microsomes=negative control(NC)E-F:（实验样品）：（实验样品）：test article+microsomes+cofactors=treatedDosing solution=time zero(T=0

27、)4 compounds including positive reference*/plate*7 ethoxycoumarinEnhanced Throughput ScreeningIncubation:4 compounds per 24-well plate15 compounds+1 positive control per day Sample size:Time zeroduplicate(16 x 2)VCduplicate(16 x 2)NCduplicate(16 x 2)Treatedduplicate(16 x 2)Total samples/day 128Bioan

28、alysis:no extractionno standard curve(peak area)machine time/2 LCs24 hrsTotal manpower:PKDM tech x 3 Test article amount:0.1 mg/test articleScreening rate:one chemotype with 60 compounds/1 week HPLC profiles of BCH-3840 and its metabolite(BCH-6440)BCH-3840metabolite?In vitro metabolic stability by r

29、at hepatic S9Efficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&DLiquid Chromatography/Mass Spectrum of BCH-3840 and its metabolite(BCH-6440)NNNHOOHydroxylation or OxidationMH+=310MH+=294Mass IdentificationHPLC profiles of BCH-3840 and its metabolite(BCH-6440

30、)Preparation of metabolite by bulk incubationMMPP10 mg microsomal protein2 mg BCH-3840Fraction collection of metaboliteNuclear Magnetic Resonance profiles of BCH-3840 and its metabolite(BCH-6440)NNNHOONNOHNHOOC5-HBCH-3840MetaboliteStructure Elucidation-3-2-1012340255075100BCH-3840 IC50=1.21 g/ml;TI=

31、15.97Toxicity:TC50=19.17 g/mlBCH-6440 IC50=9.99 g/ml;TI=1.91Concentration g/mlEfficacy/Cytotoxicity%NNOHNHOOIn vitro therapeutic index of BCH-6440Efficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&DInhibitors for CYP IsoformConc(M)Furafulline(CYP1A2)10Tranylc

32、ypromine(CYP2A6)50Sulfaphenazole(CYP2C9)25Omeprazole(CYP2C19)20Quinidine(CYP2D6)24-methylpyrazole(CYP2E1)250Ketoconazole(CYP3A4)5Metabolism Phenotyping 代谢途径鉴定代谢途径鉴定Inhibitors for CYP IsoformConc(M)Inhibition(%of NC)Tranylcypromine(CYP2A6)5040.2Sulfaphenazole(CYP2C9)2514.24-methylpyrazole(CYP2E1)2506

33、7.6Ketoconazole(CYP3A4)575.2NC1A22A62C92C192D62E13A401020304050Chemical Inhibition of CYP Isoform activityFormation of Metabolite(peak area)Metabolism Phenotyping 代谢途径鉴定代谢途径鉴定Efficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&DDrug-Drug Interactions（对其它药物代谢的影

34、响）对其它药物代谢的影响）Inhibition（抑制）抑制）potential-IC50 and Kimechanism-mechanistic（机械性）（机械性）competitive（竞争性）（竞争性）test system:liver microsomes（肝微粒体）（肝微粒体）cryopreserved hepatocytes（冷冻肝细胞）（冷冻肝细胞）Induction（诱导）（诱导）test system:fresh isolated hepatocytes（肝细胞）（肝细胞）Target EnzymesCytochrome P450s:1A2,2A6,2C8,2C9,2C19,2

35、D6,2E1,3A4Phase II conjugation:glucuronidationIC50(M):0.675Goodness of Fit:0.980795%Confidence Intervals:5.638.28-5-4-3-2-10123450255075100125RWJ-67657 log MFormation of Bilirubin Monoglucuronide(Percent of NC)IC50(M):20.4Goodness of Fit:0.973095%Confidence Intervals:16.9-26.3Drug-drug interaction:i

36、nhibition 抑制作用抑制作用体外药效浓度体外药效浓度=1 uM-7-6-5-4-3-2-1012345670255075100RWJ-351958 log MFormation of Bilirubin Monoglucuronide(Percent of NC)Drug-drug interaction:Induction（肝细胞诱导模型）肝细胞诱导模型）5 days procedureDay 0:Isolate fresh hepatocytes,viability 70%Plating hepatocytes to 24-well plate,0.7 x 106 viable c

37、ells/wellPlating media replaced with sandwich after 7-hour attachment Day 1:incubation for establishing basal levels of CYP450 isoforms.Day 2:same as Day 1Day 3:dosing with test articlesDay 4:same as Day 3Day 5:washing out the dosing solution and adding substrates for CYP450 isoforms as below:1A2-et

38、hocyresorufin O-deethylation2A6-coumarin 7-hydroxylation2C9-tolbutamide 4-hydroxylation2C19-S-mephenytoin 4-hydroxylation2D6-dextromethorphan O-demethylation2E1-chlorzoxazone 6-hydroxylation3A4-testosterone 6b-b-hydroxylationCYP1A2VCPC2 20 200 2000 012345348260 MPC=omeprazole(50 M)Resorufin formatio

39、n(pmol/million cells/min)CYP3A4VCPC2 20 200 2000 0255075348260 MPC=rifampin(25 M)6b b-hydroxytestosterone Formation(pmol/million cells/min)Drug-drug interaction:Induction 诱导作用诱导作用排出太快排出太快/药效时间太短药效时间太短口服吸收差口服吸收差/血浆浓度太低血浆浓度太低分布分布排泻排泻代谢问题代谢问题吸收问题吸收问题蛋白质相互作用蛋白质相互作用分布体积分布体积肾脏排泄肾脏排泄肝脏代谢肝脏代谢溶解度溶解度肠道吸收肠道吸收膜

40、通透性膜通透性肠道消化肠道消化早期研发阶段早期研发阶段后期研发阶段后期研发阶段in vitro体外体外metabolismin situ离体离体permeabilityin vivo体内体内bioavailabilityEfficacy HitsOptimized Lead Go or no go decisionCompound for Development(CD)NEW DRUGINDNDAR&D01020304050600500100015002000250030003500LegendIV Dose(mg/kg)AUCinf(g*min/mL)Proportionality 血浆浓度

41、的非线性血浆浓度的非线性01002003004005006000255075100125Female RatsOral Dose(mg/kg)AUCinf(g*hr/mL)Proportionality:AUC（大鼠试验）（大鼠试验）01002003004005006000255075100125Male RatsOral Dose(mg/kg)AUCinf(g*hr/mL)TOXICOKINETICS 毒物代谢动力学试验毒物代谢动力学试验 AnimalAnimal:Sprague-Dawley rats(male&female)Cynomolgus monkey(male&female)Si

42、ngle dose escalation Single dose escalation（线性动力学）（线性动力学）(50,250,500 mg/kg)Multiple dose escalation Multiple dose escalation（药物体内积累）药物体内积累）(50,250,500 mg/kg,daily for 14 days)01002003004005006000255075100125Female RatsOral Dose(mg/kg)AUCinf(g*hr/mL)Proportionality:AUC（大鼠试验）（大鼠试验）01002003004005006000

43、255075100125Male RatsOral Dose(mg/kg)AUCinf(g*hr/mL)01002003004005006000102030405060Female RatsOral Dose(mg/kg)010020030040050060001020304050Male RatsOral Dose(mg/kg)Cmax(g/mL)Proportionality:Cmax（大鼠试验）（大鼠试验）50250500020406080100day 1day 14Dose(mg/kg)AUCinf(g*hr/mL)50250500020406080100Dose(mg/kg)AUCi

44、nf(g*hr/mL)0.920.771.041.191.021.07Accumulation Ratio 药物积累率药物积累率（大鼠）（大鼠）Male ratsFemale rats0100200300400500600050100150200250FemaleOral Dose(mg/kg)AUCinf(g*hr/mL)0100200300400500600050100150200250MaleOral Dose(mg/kg)AUCinf(g*hr/mL)Proportionality:AUC（猕猴）（猕猴）Male MonkeyFemale Monkey01002003004005006

45、00020406080100120Oral Dose(mg/kg)AUCinf(g*hr/mL)0100200300400500600020406080100120Male RatsOral Dose(mg/kg)Cmax(g/mL)Proportionality:Cmax（猕猴）（猕猴）Male MonkeyFemale MonkeyMale MonkeyFemale Monkey502505000255075100125Dose(mg/kg)AUCinf(g*hr/mL)502505000255075100125Dose(mg/kg)AUCinf(g*hr/mL)day 1day 140.

46、791.111.120.730.761.14Accumulation Ratio 药物积累率药物积累率（猕猴）（猕猴）Phase I Trial(Single dose escalation)临床一期单剂量药代动力学试验临床一期单剂量药代动力学试验Healthy Male Subject(n):22Oral Doses(4):100,200,400,and 800 mgTime points(13):0.5,1,1.5,2,3,4,6,8,10,12,16,20,and 24 hourEfficacy HitsOptimized Lead Go or no go decisionCompoun

47、d for Development(CD)NEW DRUGINDNDAR&DMethodsIncubation proceduresViability(Trypan blue exclusion)70%Final cell density:2 x 106 viable cell/mLFinal concentration of test article:20 MIncubation time:4 hoursBioanalytical measurementHPLC/Fractionating/Scintillation:Metabolic profilesLC/MS/MS:Mass ident

48、ification/structure elucidation ResultsTreated vs Negative Control0102030405002004006008001000120012007200Minute0102030405002004006008001000120012007200Minute14C-Radioactivity(dpm)Metabolism ProfilesHuman vs Dog0102030405002004006008001000120012007200Minute0102030405002004006008001000120012007200Min

49、ute14C-Radioactivity(dpm)Metabolism Profiles Human vs Monkey0102030405002004006008001000120012007200Minute0102030405002004006008001000120012007200Minute14C-Radioactivity(dpm)Metabolism Profiles Human vs Mouse0102030405002004006008001000120012007200Minute0102030405002004006008001000120012007200Minute

50、14C-Radioactivity(dpm)Metabolism Profiles Human vs Rat0102030405002004006008001000120012007200Minute0102030405002004006008001000120012007200Minute14C-Radioactivity(dpm)Samples14CConc.ParentD1M1M2M3M4Total*MCdpm11648172NA25151512108%of total96.21.42NA0.210.120.1298.2Mousedpm14451423NA45511333.015656%