医学药代动力学在新药研发中的作用专题培训课件

1、药代动力学在新药研发中的作用药代动力学在新药研发中的作用Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 Drug Metabolism/PharmcokineticsEfficacy HitsOptimized LeadCom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

2、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）(最小药效毒理药代最佳血浆浓度药效毒理药代最佳Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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？ metabolic stability 代谢产物？ metabolite identification 代谢途径？ pathway identification 对其它药物的影响？ drug-dr

3、ug interaction Efficacy HitsOptimized LeadComEfficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 dose escalation & proportionality 多次给药和体内积蓄 multiple doses & accumulation 吸收和排泄模式 mass balance 体内分布 distrib

4、ution 从动物代谢推算人体代谢 extrapolation Efficacy HitsOptimized LeadComEfficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 metabolism profiling in animals and humans Efficacy HitsOptimized LeadComEfficacy HitsOptimized Lead Go or no go d

5、ecisionCompound for Development (CD)NEW DRUGINDNDAR&D临床实验临床前实验研究和发现临床实验准则Good Clinical Practice (GCP)非临床实验准则Good Laboratory Practice (GLP)Efficacy HitsOptimized LeadCom二五原则 5 毫克 5 天二五原则临床前实验药物代谢动力学的生物模型体外和离体模型 (in vitro / in situ models)吸收模型 absorption/permeability代谢模型 metabolism体外推测和体内 (in vitro /

6、in vivo correlation)动物模型 (in vivo animal models)动物推测人 (species extrapolation)临床前实验药物代谢动力学的生物模型排出太快/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Situation Analysis in vitro体外metabolismin situ离体permeabilityin vivo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Plasma conc

7、entrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orallyPoor oral bioavailabilityPlasma concentrations of BCH-3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absorption/distribution model 脂层转移模型水相Aqueous phase水相Aqueous phase有机相Organic phasepH=6.5pH=7.4Permeability

8、Evaluation in vitro14absorption/distribution model in vitro absorption/distribution model15in vitro absorption/distributiCaco-2 Transport Pathways人大肠癌细胞模型Caco-2 Transport Pathways人大肠癌Transport Pathways药物吸收机制被动细胞间主动P糖蛋白Transport Pathways药物吸收机制被动细胞间Probes for Transport Pathways肠道吸收标准对照药物Transcellular

9、（被动吸收）Propranolol, TestosteroneParacellular （细胞间渗透）Mannitol, InulinCarrier mediated （主动吸收） GlucoseP-Glycoprotein mediated （P糖蛋白调节）底物 Vinblastine抑制物 VerapamilProbes for Transport PathwaysGlucose （蔗糖） vs Inulin （木香素）主动吸收 vs 细胞间渗透Glucose （蔗糖） vs Inulin （木香素）Propranolol vs Mannitol被动吸收 vs 细胞间渗透Propranol

10、ol vs Mannitol被动吸收由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 VeraChong, Dando & Morrison; Pharm. Res. 1997Chong, Dando & Morrison; PharmFalse Positive假阳性 低False Negative假阴性 高Caco-2 Transport Pathways 人大肠癌细胞吸收模型False PositiveFalse NegativeCain situ rat intestinal perfusion (single pass) 离体大

11、鼠十二指肠灌流模型（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 a

12、rticle = 0.05-0.30 mg/mLin situ rat intestinal perfusiPerfusion 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 ca

13、nnula - a syringe infusion pump the outlet of cannula - a 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

14、and 120 min after perfusion Calculations: Permeability (Peff, cm/min) = (Q/2RLp) x (1- Cout / Cin ) Cout / Cin = (Cout / Cin) x phenol red in / phenol red outin situ rat intestinal perfusion (single pass)Perfusion Procedures:in situ rIn situ rat intestinal permeability (single pass)Prediction within

15、 90% interval = 19/31 (61.3%)In-house validation假阳性假阴性In situ rat intestinal permeabPlasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orallyPoor oral bioavailabilityPlasma concentrations of BCH-3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

16、化早期研发阶段后期研发阶段Situation Analysis in vitro体外metabolismin situ离体permeabilityin vivo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In Situ Rat Intestinal Permeability: Good阳性对照阴性对照受试药物In Situ Rat Intestinal PermeabEnhanced Throughput ScreeningPerfusion: 4 compounds per day (4 animals) Sample size: time

17、points 7duplicate x 2control/drug x 3sample/perfusion 42Total samples/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 Enh

18、anced Throughput ScreeningpKa = 10 pKa = 8.4 pKa = 6.5 Preduced%= 0% Preduced%= 7% Preduced%= 12%SAR: pKa vs. permeability实例：结构优化和吸收率分析pKa = 10 pKa = 8.4 pSAR: permeability vs. efficacy实例：结构优化和吸收率和活性的分析IC50 = 2 uMPreduced%= 0%IC50 = 0.012 uMPreduced%= 0%IC50 = 1.1 uMPreduced%= 17%IC50 = 0.025 uMPred

19、uced%= 15%SAR: permeability vs. efficacy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 (in vitro Caco-2 monolayer)离体大鼠十二指肠灌流模型 (in situ rat intestine perfusion)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

20、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Situation Analysis in vitro体外metabolismin situ离体permeabilityin vivo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死还是不死,这是个问题.To be or not to be, this is a problem.- 哈默雷特体内试验还是体外试验, 这是个问题.In vitro or in vivo, this is a problem.-药代研究员死还是不死,这是个问题.动物体内模型 -

21、人体内(临床试验)In vivo animals vs. in vivo humans人体外模型 -人体内(临床试验)In vitro humans vs. in vivo humans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)Heartbeat and Bodyweight （心率和体重）小鼠大鼠兔猴狗人38Heartbeat and Bodyweight （心率和体Liver weight and Hepatic Flow vs Bodyweight （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39Liver weight a

22、nd Hepatic Flow Antipyrine clearance (l/min)ratmouserabbitmonkeydoghumanClearance40Antipyrine clearance (l/min)raIn Vitro Models of the Liver体外肝模型Hepatocytes 肝细胞Liver slices 肝切片Liver microsomes 肝微粒体Liver S-9 Fraction 肝S-9组分 In Vitro Models of the Liver体USFDA Guidance for Industry美国药物和食品管理局关于药物代谢实验的指

23、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. Isolated hepatocytes and precision-cut slices have these desirable features.”Guidance for Industry, Drug Metab

24、olism/Drug InteractionStudies in the Drug Development Process: Studies In VitroCDER, 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。USFDA Guidance for Industry美国HOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE -3-GlucuronideEE2 -3-SulfateConjugatesEE2EE2Hepatocytes （肝细胞）Micros

25、omes（微粒体）Hepatocytes（肝细胞）Metabolism of Eythinyl Estradiol (EE2) 肝微粒体和肝细胞的代谢功能差异Li, Hartman, Lu, Collins and Strong, Br J Clin Pharmacol 48, 733-742(1999)HOHOHOHOHOHOOGLUCHOOSOOHO2-HydPlasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orallyPoor oral bioavailabilit

26、yPlasma concentrations of BCH-3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Situation Analysis in vitro体外metabolismin situ离体permeabilityin vivo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Reaction volume:1.0 ml, DPBS pH 7.4Hepatic S-9/Microsomes:0.5 mg protien/m

27、L Species:Human/Monkey/Dog/Rat/Mouse Substrate concentration:10 mMNADPH:2.4 mMUDPGA:1.5 mMIncubation:60 min at 37oCStopping procedure:chilled acetonitrile, 3 x volumeIn Vitro Metabolism Assay 体外肝微粒体实验Reaction volume:1.0 ml, DPBS1234 A B C D E FEnhanced Throughput Screening （增速筛选）A-B: （空白对照）：test art

28、icle + buffer = vehicle control (VC)C-D:（阴性对照）：test article + microsomes = negative control (NC)E-F: （实验样品）：test article + microsomes + cofactors = treatedDosing solution = time zero (T = 0)4 compounds including positive reference* / plate* 7 ethoxycoumarin阴性对照空白对照测试样本1 A B C D E FEnhanceEnhanced Th

29、roughput 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 128Bioanalysis: no extractionno standard curve (peak area)machine time/2 LCs

30、24 hrsTotal manpower:PKDM tech x 3 Test article amount:0.1 mg / test articleScreening rate:one chemotype with 60 compounds / 1 week Enhanced Throughput ScreeningHPLC profiles of BCH-3840 and its metabolite (BCH-6440)BCH-3840metabolite?In vitro metabolic stability by rat hepatic S9HPLC profiles of BC

31、H-3840 and Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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？ metabolic stability 代谢产物？ metabolite identification 代谢途径？ pathway identification 对其它药物的影响？ drug-drug interaction Efficacy HitsOptimized LeadComLiq

32、uid Chromatography / Mass Spectrum of BCH-3840 and its metabolite (BCH-6440)Hydroxylation or OxidationMH+ = 310MH+ = 294Mass IdentificationLiquid Chromatography / Mass SHPLC profiles of BCH-3840 and its metabolite (BCH-6440)Preparation of metabolite by bulk incubationMMPP10 mg microsomal protein2 mg

33、 BCH-3840Fraction collection of metabolitefractionation concentration HPLC profiles of BCH-3840 and Nuclear Magnetic Resonance profiles of BCH-3840 and its metabolite (BCH-6440)C5-HBCH-3840MetaboliteStructure Elucidation Nuclear Magnetic Resonance proIn vitro therapeutic index of BCH-6440In vitro th

34、erapeutic index of Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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？ metabolic stability 代谢产物？ metabolite identification 代谢途径？ pathway identification 对其它药物的影响？ drug-drug interaction Efficacy HitsOptimized Le

35、adComInhibitors for CYP IsoformConc (mM)Furafulline (CYP1A2) 10Tranylcypromine (CYP2A6) 50Sulfaphenazole (CYP2C9) 25Omeprazole (CYP2C19) 20Quinidine (CYP2D6) 24-methylpyrazole (CYP2E1) 250Ketoconazole (CYP3A4) 5 Chemical Inhibition （化学抑制） Pure enzyme （纯酶） Correlation Analysis （相关分析）Metabolism Phenot

36、yping 代谢途径鉴定Inhibitors for CYP IsoformConInhibitors for CYP IsoformConc (mM) Inhibition (% of NC)Tranylcypromine (CYP2A6) 5040.2Sulfaphenazole (CYP2C9) 2514.24-methylpyrazole (CYP2E1) 25067.6Ketoconazole (CYP3A4) 575.2Metabolism Phenotyping 代谢途径鉴定Inhibitors for CYP IsoformConEfficacy HitsOptimized L

37、ead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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？ metabolic stability 代谢产物？ metabolite identification 代谢途径？ pathway identification 对其它药物的影响？ drug-drug interaction Efficacy HitsOptimized LeadComDrug-Drug Interactions （对其它药物代谢的影响）Inh

38、ibition （抑制）potential - IC50 and Kimechanism - mechanistic（机械性） competitive （竞争性） test system: liver microsomes （肝微粒体）cryopreserved hepatocytes （冷冻肝细胞）Induction（诱导）test system: fresh isolated hepatocytes （肝细胞）Target EnzymesCytochrome P450s: 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4Phase II conjugation

39、: glucuronidationDrug-Drug Interactions （对其它药物代IC50 (M):0.675Goodness of Fit:0.980795% Confidence Intervals:5.638.28IC50 (M):20.4Goodness of Fit:0.973095% Confidence Intervals:16.9-26.3CYP3A4 CYP3A4Drug-drug interaction: inhibition 抑制作用体外药效浓度 = 1 uMIC50 (M):0.675IC50 (M):Drug-drug interaction: Induc

40、tion （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 cells/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

41、 with test articlesDay 4:same as Day 3Day 5:washing out the dosing solution and adding substrates for CYP450 isoforms as below:1A2 - ethocyresorufin O-deethylation2A6 - coumarin 7-hydroxylation2C9 - tolbutamide 4-hydroxylation2C19 - S-mephenytoin 4-hydroxylation2D6 - dextromethorphan O-demethylation

42、2E1 - chlorzoxazone 6-hydroxylation3A4 - testosterone 6b - hydroxylationDrug-drug interaction: InductiDrug-drug interaction: Induction 诱导作用Drug-drug interaction: Inducti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Situation Analysis in vitro体外metabolismin situ离体pe

43、rmeabilityin vivo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Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 dose escalation & proportionality 多次给药和体内积蓄 multiple doses & accumulation 吸收和排泄模式 mass ba

44、lance 体内分布 distribution 从动物代谢推算人体代谢 extrapolation Efficacy HitsOptimized LeadCom119%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90%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TOXICOKINETICS 毒物代谢动力学试验 Animal: Sprague-Dawley rats (m

45、ale & female) Cynomolgus monkey (male & female) Single dose escalation （线性动力学） (50, 250, 500 mg/kg) Multiple dose escalation （药物体内积累） (50, 250, 500 mg/kg, daily for 14 days)TOXICOKINETICS 毒物代谢动力学试验90%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0100200300400500600

46、0102030405060Female RatsOral Dose (mg/kg)010020030040050060001020304050Male RatsOral Dose (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 0100200300400500600010203040500.920.771.041.191.021.07Accumulation Ratio 药物积累率 （大鼠）Male ratsFemale rats0.920.771.041.191.021.07AccumuProp

47、ortionality: AUC （猕猴）Male MonkeyFemale Monkey49%34%60%38%提示 ：药物吸收的非线性饱和 Proportionality: AUC （猕猴）Male 38%31%55%32%Proportionality: Cmax （猕猴）Male MonkeyFemale Monkey提示 ：药物吸收的非线性饱和 38%31%55%32%Proportionality: CMale MonkeyFemale Monkey0.791.111.120.730.761.14Accumulation Ratio 药物积累率 （猕猴）Male MonkeyFemale Monkey0.791.Phase I Trial (Single dose escalation)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