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,（最
2、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-d
3、rug 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 ex
4、trapolation,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
5、,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
6、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
7、,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 Path
8、ways肠道吸收标准对照药物,Transcellular （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 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,Ch
9、ong, 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）,METHODAnimal: Male Sprague-Dawley rats (250 - 350 g), fasted overnight. Rat is anesthetized by urethane 1.5g/kg, im. before perfus
10、ion 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 inc
11、ision 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 fraction collector the perfusion segment is precleaned by passing 10 ml of blank perfusate buffer perfusion time an
12、d 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 in
13、testinal 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
14、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 ScreeningPerfusion: 4 compounds per day (4 animals) Sample siz
15、e: time 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 w
16、eeks,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 uMPreduced%= 0%,IC50 = 0.012 uMPreduced%= 0%,IC50 = 1.1 uMPreduced%= 17%,IC50 = 0.025 uMPreduced%= 15%,小结：体外和离体药物吸收实验系统体外人大肠癌细胞
17、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
18、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,H
19、eartbeat 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Live
20、r 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 s
21、lices 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
22、）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
23、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 7.4Hepatic S-9/Microsomes:0.5 mg protien/mL Species:Human/Monkey/Dog/Rat/Mouse Substrate concentration:10 mMNADPH:2.4
24、 mMUDPGA:1.5 mMIncubation:60 min at 37oCStopping procedure:chilled acetonitrile, 3 x volume,In Vitro Metabolism Assay 体外肝微粒体实验,1234,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
25、: （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,Enhanced Throughput ScreeningIncubation: 4 compounds per 24-well plate15 compounds + 1 positive control per day Sample size: T
26、ime 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 LCs24 hrsTotal manpower:PKDM tech x 3 Test article amount:0.1 mg / test articleScreening rate:one chemotype with 60 co
27、mpounds / 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？ metabo
28、lic 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 (
29、BCH-6440),Preparation of metabolite by bulk incubation,M,M,P,P,10 mg microsomal protein2 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 the
30、rapeutic 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,Inhibit
31、ors 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 Phenotyping 代谢途径鉴
32、定,Inhibitors for CYP IsoformConc (mM) Inhibition (% of NC)Tranylcypromine (CYP2A6) 5040.2Sulfaphenazole (CYP2C9) 2514.24-methylpyrazole (CYP2E1) 25067.6Ketoconazole (CYP3A4) 575.2,Metabolism Phenotyping 代谢途径鉴定,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,I
33、ND,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 Kimechanism - mechanistic（机械性） competitive （竞争性） test
34、 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: glucuronidation,IC50 (M):0.675Goodness of Fit:0.980795% Confidence Intervals:5.638.2
35、8,IC50 (M):20.4Goodness of Fit:0.973095% 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 procedureDay 0: Isolate fresh hepatocytes, viability 70%Plating hepatocytes to 24-well plate, 0.7 x 106 viable
36、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 with test articlesDay 4:same as Day 3Day 5:washing out the dosing solution and adding substrates for CYP450 isoforms as below:1A2 -
37、 ethocyresorufin O-deethylation2A6 - coumarin 7-hydroxylation2C9 - tolbutamide 4-hydroxylation2C19 - S-mephenytoin 4-hydroxylation2D6 - dextromethorphan O-demethylation2E1 - chlorzoxazone 6-hydroxylation3A4 - 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
38、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 bioavaila
39、bility 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 An
40、imal: 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
41、,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 Monk
42、ey,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): 22Or
43、al 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 hour,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 动物和人的代谢特征 (长期毒性实验的动物选择) metabolism profiling in animals and humans
44、,Evaluation of In Vitro Metabolism Profiles Cryopreserved Hepatocytes mouse rat dog monkey human实验药物 (MW = 455.59 in 1/2 tartrate salt)14C 放射性比活性 (23.7 Ci/mg),Methods,Incubation proceduresViability (Trypan blue exclusion) 70%Final cell density: 2 x 106 viable cell/mLFinal concentration of test artic
45、le: 20 MIncubation time: 4 hoursBioanalytical measurementHPLC/Fractionating/Scintillation: Metabolic profilesLC/MS/MS: Mass identification/structure elucidation,ResultsTreated vs Negative Control,D,P,Viable Human Hepatocytes 速冻人肝细胞,Heated Human Hepatocytes 热灭活人肝细胞,D,P,M2,M3,M4,Metabolism ProfilesHuman vs Dog,D,P,Viable Human Hepatocytes 速冻人肝细胞,Viable Dog Hepatocytes 速冻狗肝细胞,D,P,M2,M3,D,P,M2,M3,M4,Metabolism Profiles Human vs Monkey,Viable Human Hepatocytes速冻人肝细胞,
