复旦大学药物代谢及其动力学在新药研发中的应用.ppt

1、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,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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/Pharmcokinetics,药物代谢动力学的任务

2、,（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,药效,毒理,药代,最佳 血浆浓度,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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？ dr

3、ug-drug interaction,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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 distribution 从动物代谢推算人体

4、代谢 extrapolation,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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 Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,临床实验

5、,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),二五原则 5 毫克 5 天,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 / in vivo correlation) 动物模型 (in vivo animal models) 动物推测人 (species extrapolation)

6、,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 metabolism,in situ离体 permeability,in vivo体内 bioavailability,Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orally,Poor oral bioavaila

7、bility,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,14,absorption/distribution model 脂层转移模型,水相 Aqueous phase,水相 Aqueous phase,有机相 Organic phase,pH=6.5,pH=7.4,Permeability Evaluation in vitro,15,in vitro absorption/distribution model,Caco-2 Transport Pathways人大肠癌细胞模型,Transport Pathways药物吸收机制,被动,细胞间,主动,P糖蛋白,Probe

8、s for Transport Pathways肠道吸收标准对照药物,Transcellular （被动吸收） Propranolol, Testosterone Paracellular （细胞间渗透） Mannitol, Inulin Carrier mediated （主动吸收） Glucose P-Glycoprotein mediated （P糖蛋白调节） 底物 Vinblastine抑制物 Verapamil,Glucose （蔗糖） vs Inulin （木香素）主动吸收 vs 细胞间渗透,Propranolol vs Mannitol被动吸收 vs 细胞间渗透,由P蛋白所调节的

9、药物吸收使用P糖蛋白抑制剂 Verapamil,Chong, Dando Pharm. Res. 1997,False 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,in situ rat intestinal perfusion (single pass) 离体大鼠十二指肠灌流模型（单循环）,METHOD Animal: Male Sprague-Dawley rats (250 - 350 g), fasted overnight. Rat is anesthetized by uretha

10、ne 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/mL,Perfusion Procedures: rat is put on a heating pad to maintain body temperature jejunum is

11、 exposed via a middle line incision sutures: 1st is made at 5 cm distal to the ligament of Treitz 2nd is made at about 20 cm distal to 1st one the inlet of cannula - a syringe infusion pump the outlet of cannula - a fraction collector the perfusion segment is precleaned by passing 10 ml of blank per

12、fusate 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/min) = (Q/2RLp) x (1- Cout / Cin ) Cout / Cin = (Cout / Cin) x phenol red in

13、/ phenol red out,in situ rat intestinal perfusion (single pass),In situ rat intestinal permeability (single pass),Prediction within 90% interval = 19/31 (61.3%),In-house validation,假阳性,假阴性,Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orally,Poor oral bioavai

14、lability,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 metabolism,in situ离体 permeability,in vivo体内 bioavailability,In Situ Rat Intestinal Permeability: Good,阳性对照,阴性对照,受试药物,Enhanced Throughput Screening Perfusion: 4 compo

15、unds per day (4 animals) Sample size: time points 7 duplicate x 2 control/drug x 3 sample/perfusion 42 Total samples/day168 Bioanalysis: no extraction no standard curve (peak area) machine time/2 LCs24 hrs Total manpower:animal tech x 1 PKDM tech x 2 Test article amount:1 mg / test article Screening

16、 rate:one chemotypes with 30 compounds / 2 weeks,pKa = 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,SAR: permeability vs. efficacy 实例：结构优化和吸收率和活性的分析,IC50 = 2 uM Preduced%= 0%,IC50 = 0.012 uM Preduced%= 0%,IC50 = 1.1 uM Preduced%= 17%,IC50

17、= 0.025 uM Preduced%= 15%,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,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,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

18、发阶段,后期研发阶段,Situation Analysis,in vitro体外 metabolism,in situ离体 permeability,in vivo体内 bioavailability,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,动物体内模型 - 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

19、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,38,Heartbeat and Bodyweight （心率和体重）,小鼠,大鼠,兔,猴,狗,人,39,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,40,Antipyrine clearance (l/min),rat,mouse,rabbit,monkey,dog,human,Clearance,In Vitro Models of the

20、 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,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,“The most complete picture for hepatic metabolism can be obtained with liver systems,in which the cofactors are self-sufficient and the natural orientation fo

21、r linked enzymes is preserved. Isolated 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, 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

22、然 水平的酶系。,2-Hydroxy-EE2,Conjugates,EE2,EE2,Hepatocytes （肝细胞）,Microsomes（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),Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed

23、 50 mg/kg orally,Poor oral 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 metabolism,in situ离体 permeability,in vivo体内 bioavailability,Reaction volume:1.0 ml, DPBS pH 7.4 Hepatic S-9/Microsomes:0.5 mg proti

24、en/mL Species:Human/Monkey/Dog/Rat/Mouse Substrate concentration:10 mM NADPH:2.4 mM UDPGA:1.5 mM Incubation:60 min at 37oC Stopping procedure:chilled acetonitrile, 3 x volume,In Vitro Metabolism Assay 体外肝微粒体实验,1 2 3 4,A B C D E F,Enhanced Throughput Screening （增速筛选）,A-B: （空白对照）：test article + buffer

25、 = 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 = treated Dosing 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,Enhanced Throughput Screening Incub

26、ation: 4 compounds per 24-well plate 15 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 128 Bioanalysis: no extraction no standard curve (peak area) machine time/2 LCs24 hrs Total ma

27、npower:PKDM tech x 3 Test article amount:0.1 mg / test article Screening rate:one chemotype with 60 compounds / 1 week,HPLC profiles of BCH-3840 and its metabolite (BCH-6440),BCH-3840,metabolite?,In vitro metabolic stability by rat hepatic S9,Efficacy Hits,Optimized Lead Go or no go decision,Compoun

28、d for Development (CD),NEW DRUG,IND,NDA,R&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,Liquid Chromatography / Mass Spectrum of BCH-3840 and its metabolite (BCH-6440),Hydroxylati

29、on or Oxidation,MH+ = 310,MH+ = 294,Mass Identification,HPLC profiles of BCH-3840 and its metabolite (BCH-6440),Preparation of metabolite by bulk incubation,M,M,P,P,10 mg microsomal protein 2 mg BCH-3840,Fraction collection of metabolite,fractionation,concentration,Nuclear Magnetic Resonance profile

30、s of BCH-3840 and its metabolite (BCH-6440),C5-H,BCH-3840,Metabolite,Structure Elucidation,In vitro therapeutic index of BCH-6440,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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 stabili

31、ty 代谢产物？ metabolite identification 代谢途径？ pathway identification 对其它药物的影响？ drug-drug interaction,Inhibitors for CYP IsoformConc (mM) Furafulline (CYP1A2) 10 Tranylcypromine (CYP2A6) 50 Sulfaphenazole (CYP2C9) 25 Omeprazole (CYP2C19) 20 Quinidine (CYP2D6) 2 4-methylpyrazole (CYP2E1) 250 Ketoconazole (

32、CYP3A4) 5,Chemical Inhibition （化学抑制）,Pure enzyme （纯酶） Correlation Analysis （相关分析）,Metabolism Phenotyping 代谢途径鉴定,Inhibitors for CYP IsoformConc (mM) Inhibition (% of NC) Tranylcypromine (CYP2A6) 5040.2 Sulfaphenazole (CYP2C9) 2514.2 4-methylpyrazole (CYP2E1) 25067.6 Ketoconazole (CYP3A4) 575.2,Metabo

33、lism Phenotyping 代谢途径鉴定,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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,Drug-Drug

34、Interactions （对其它药物代谢的影响） Inhibition （抑制） potential - IC50 and Ki mechanism - 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 Enzymes Cytochrome P450s: 1A2, 2A6, 2C8, 2C9, 2C1

35、9, 2D6, 2E1, 3A4 Phase II conjugation: glucuronidation,IC50 (M):0.675 Goodness of Fit:0.9807 95% Confidence Intervals:5.638.28,IC50 (M):20.4 Goodness of Fit:0.9730 95% Confidence Intervals:16.9-26.3,CYP3A4,CYP3A4,Drug-drug interaction: inhibition 抑制作用,体外药效浓度 = 1 uM,Drug-drug interaction: Induction （

36、肝细胞诱导模型）,5 days procedure Day 0: Isolate fresh hepatocytes, viability 70% Plating hepatocytes to 24-well plate, 0.7 x 106 viable cells/well Plating media replaced with sandwich after 7-hour attachment Day 1:incubation for establishing basal levels of CYP450 isoforms. Day 2:same as Day 1 Day 3:dosing

37、 with test articles Day 4:same as Day 3 Day 5:washing out the dosing solution and adding substrates for CYP450 isoforms as below: 1A2 - ethocyresorufin O-deethylation 2A6 - coumarin 7-hydroxylation 2C9 - tolbutamide 4-hydroxylation 2C19 - S-mephenytoin 4-hydroxylation 2D6 - dextromethorphan O-demeth

38、ylation 2E1 - chlorzoxazone 6-hydroxylation 3A4 - testosterone 6b - hydroxylation,Drug-drug interaction: Induction 诱导作用,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 metabolism,in situ离体 permeability,in vivo体内 bioavailab

39、ility,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&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 distribution 从动物代谢推算人体代谢 extrapolati

40、on,119%,236%,310%,Proportionality 血浆浓度的非线性,提示： 代谢或排泄的非线性饱和,90%,72%,Proportionality: AUC （大鼠试验）,93%,63%,提示 ：药物吸收的非线性饱和,TOXICOKINETICS 毒物代谢动力学试验 Animal: Sprague-Dawley rats (male & female) Cynomolgus monkey (male & female) Single dose escalation （线性动力学） (50, 250, 500 mg/kg) Multiple dose escalation （药

41、物体内积累） (50, 250, 500 mg/kg, daily for 14 days),90%,72%,Proportionality: AUC （大鼠试验）,93%,63%,提示 ：药物吸收的非线性饱和,0,100,200,300,400,500,600,0,10,20,30,40,50,60,Female Rats,Oral Dose (mg/kg),0,100,200,300,400,500,600,0,10,20,30,40,50,Male Rats,Oral Dose (mg/kg),Cmax,(,m,g/mL),73%,47%,56%,49%,Proportionality:

42、 Cmax （大鼠试验）,提示 ：药物吸收的非线性饱和,0.92,0.77,1.04,1.19,1.02,1.07,Accumulation Ratio 药物积累率 （大鼠）,Male rats,Female rats,Proportionality: AUC （猕猴）,Male Monkey,Female Monkey,49%,34%,60%,38%,提示 ：药物吸收的非线性饱和,38%,31%,55%,32%,Proportionality: Cmax （猕猴）,Male Monkey,Female Monkey,提示 ：药物吸收的非线性饱和,Male Monkey,Female Monkey,0.79,1.11,1.12,0.73,0.76,1.14,Accumulation Ratio 药物积累率 （猕猴）,Phase I Trial (Single dose escalation) 临床一期单剂量药代动力学试验 Healthy Male Subject (n): 22 Oral Doses (4): 100, 200, 400, and 800 mg Time