药代动力学在新药研发中的作用.ppt

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,（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？ drug-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,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),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 bioavailability,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,Probes 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 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/mL,Perfusion 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 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 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/min) = (Q/2RLp) x (1- Cout / Cin ) Cout / Cin = (Cout / Cin) x phenol red in / 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 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,In Situ Rat Intestinal Permeability: Good,阳性对照,阴性对照,受试药物,Enhanced Throughput Screening Perfusion: 4 compounds per day (4 animals) Sample size: time points 7 duplicate x 2 control/drug x 3 sample/perfusion 42 Total samples/day 168 Bioanalysis: no extraction no standard curve (peak area) machine time/2 LCs 24 hrs Total manpower: animal tech x 1 PKDM tech x 2 Test article amount: 1 mg / test article Screening 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 = 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 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 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 for 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 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 protien/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 = 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 Incubation: 4 compounds per 24-well plate 15 compounds + 1 positive control per day Sample size: Time zero duplicate (16 x 2) VC duplicate (16 x 2) NC duplicate (16 x 2) Treated duplicate (16 x 2) Total samples/day 128 Bioanalysis: no extraction no standard curve (peak area) machine time/2 LCs 24 hrs Total manpower: 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,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,Liquid Chromatography / Mass Spectrum of BCH-3840 and its metabolite (BCH-6440),Hydroxylation 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 profiles 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 stability 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 Isoform Conc (mM) Furafulline (CYP1A2) 10 Tranylcypromine (CYP2A6) 50 Sulfaphenazole (CYP2C9) 25 Omeprazole (CYP2C19) 20 Quinidine (CYP2D6) 2 4-methylpyrazole (CYP2E1) 250 Ketoconazole (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 Isoform Conc (mM) Inhibition (% of NC) Tranylcypromine (CYP2A6) 50 40.2 Sulfaphenazole (CYP2C9) 25 14.2 4-methylpyrazole (CYP2E1) 250 67.6 Ketoconazole (CYP3A4) 5 75.2,Metabolism 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 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, 2C19, 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 （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 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-demethylation 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 bioavailability,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 extrapolation,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: 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 Tim