药代动力学在新药研发中的作用.pptx

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2021/10/1,#,药品代谢及其动力学在新药研发中应用,胡卓汉 博士,瑞德肝脏疾病研究(上海)有限企业,复旦大学药学院,12月30日,中国.北京,药代动力学在新药研发中的作用,1/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,药品研发三大任务,药效,Efficacy/Pharmacodynamics,安全,Safety/Toxicology,药品代谢动力学,Drug Metabolism/Pharmcokinetics,药代动力学在新药研发中的作用,2/84,药品代谢动力学任务,（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,药代动力学在新药研发中的作用,3/84,药效,毒理,药代,最正确,血浆浓度,药代动力学在新药研发中的作用,4/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,研究和发觉阶段,能否被吸收？,permeability,是否被代谢？,metabolic stability,代谢产物？,metabolite identification,代谢路径？,pathway identification,对其它药品影响？,drug-drug interaction,药代动力学在新药研发中的作用,5/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,临床前阶段,生物利用度,bioavailability,血浆浓度线性和非线性,dose escalation&proportionality,屡次给药和体内积蓄,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,药代动力学在新药研发中的作用,6/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,临床阶段,长久毒性试验动物选择,metabolism profiling in animals and humans,药代动力学在新药研发中的作用,7/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,临床试验准则,Good Clinical Practice(GCP),非临床试验准则,Good Laboratory Practice(GLP),药代动力学在新药研发中的作用,8/84,二五标准,5 毫克,5 天,药代动力学在新药研发中的作用,9/84,临床前试验药品代谢动力学生物模型,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),药代动力学在新药研发中的作用,10/84,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,药代动力学在新药研发中的作用,11/84,Plasma concentrations of BCH-3840 and its metabolite(BCH-6440),in mice dosed 50 mg/kg orally,Poor oral bioavailability,药代动力学在新药研发中的作用,12/84,药品吸收模型,计算机,脂溶度,脂层转移,细胞层转移,十二指肠灌流,药代动力学在新药研发中的作用,13/84,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,14,药代动力学在新药研发中的作用,14/84,in vitro absorption/distribution model,15,药代动力学在新药研发中的作用,15/84,Caco-2 Transport Pathways,人大肠癌细胞模型,药代动力学在新药研发中的作用,16/84,Transport Pathways,药品吸收机制,被动,细胞间,主动,P糖蛋白,药代动力学在新药研发中的作用,17/84,Probes for Transport Pathways,肠道吸收标准对照药品,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糖蛋白调整）,底物 Vinblastine抑制物 Verapamil,药代动力学在新药研发中的作用,18/84,Glucose（蔗糖）vs Inulin（木香素）,主动吸收 vs 细胞间渗透,药代动力学在新药研发中的作用,19/84,Propranolol vs Mannitol,被动吸收 vs 细胞间渗透,药代动力学在新药研发中的作用,20/84,由P蛋白所调整药品吸收使用P糖蛋白抑制剂 Verapamil,药代动力学在新药研发中的作用,21/84,Chong,Dando Pharm.Res.1997,药代动力学在新药研发中的作用,22/84,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,药代动力学在新药研发中的作用,23/84,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,药代动力学在新药研发中的作用,24/84,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/2RL,p,)x(1-Cout/Cin),Cout/Cin,=(Cout/Cin)x phenol red in/phenol red out,in situ rat intestinal perfusion(single pass),药代动力学在新药研发中的作用,25/84,In situ rat intestinal permeability(single pass),Prediction within 90%interval=19/31(61.3%),In-house validation,假阳性,假阴性,药代动力学在新药研发中的作用,26/84,Plasma concentrations of BCH-3840 and its metabolite(BCH-6440),in mice dosed 50 mg/kg orally,Poor oral bioavailability,药代动力学在新药研发中的作用,27/84,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,药代动力学在新药研发中的作用,28/84,In Situ Rat Intestinal Permeability:,Good,阳性对照,阴性对照,受试药品,药代动力学在新药研发中的作用,29/84,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/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 rate,:one chemotypes with 30 compounds/2 weeks,药代动力学在新药研发中的作用,30/84,pKa=10 pKa=8.4 pKa=6.5,P,reduced,%=0%P,reduced,%=7%P,reduced,%=12%,SAR:pKa vs.permeability,实例：结构优化和吸收率分析,药代动力学在新药研发中的作用,31/84,SAR:permeability vs.efficacy,实例：结构优化和吸收率和活性分析,IC,50,=2 uM,P,reduced,%=,0%,IC,50,=0.012 uM,P,reduced,%=,0%,IC,50,=1.1 uM,P,reduced,%=,17%,IC,50,=0.025 uM,P,reduced,%=,15%,药代动力学在新药研发中的作用,32/84,小结：体外和离体药品吸收试验系统,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),体内动物药品代谢动力学模型,二五标准:5毫克/5天,药代动力学在新药研发中的作用,33/84,血浆浓度,时间,化学药品,化学药品+中药,中药药品代谢动力学任务,本身药品代谢动力学问题,对其它药品吸收作用,药代动力学在新药研发中的作用,34/84,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,药代动力学在新药研发中的作用,35/84,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,药代动力学在新药研发中的作用,36/84,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,选择指南,与人相同：疾病模型，药效，毒性，药品代谢,试验成本,药代动力学在新药研发中的作用,37/84,Heartbeat and Bodyweight（,心率和体重）,小鼠,大鼠,兔,猴,狗,人,38,药代动力学在新药研发中的作用,38/84,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,39,药代动力学在新药研发中的作用,39/84,Antipyrine clearance(l/min),rat,mouse,rabbit,monkey,dog,human,Clearance,40,药代动力学在新药研发中的作用,40/84,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,药代动力学在新药研发中的作用,41/84,USFDA Guidance for Industry,美国药品和食品管理局关于药品代谢试验指南,“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（分离肝细胞和准确肝切片）能为药品代谢,试验提供最完全信息，因为这个系统含有足够天然,水平酶系。,药代动力学在新药研发中的作用,42/84,H,O,H,O,H,O,H,O,H,O,H,O,O,G,L,U,C,H,O,O,S,O,O,H,O,2-Hydroxy,-EE,2,2,EE,-3-,Glucuronide,EE,2,-3-Sulfate,Conjugates,EE,2,EE,2,Hepatocytes,（肝细胞）,Microsomes,（微粒体）,Hepatocytes,（肝细胞）,Metabolism of Eythinyl Estradiol(EE,2,),肝微粒体和肝细胞代谢功效差异,Li,Hartman,Lu,Collins and Strong,Br J Clin Pharmacol 48,733-742(1999),药代动力学在新药研发中的作用,43/84,Plasma concentrations of BCH-3840 and its metabolite(BCH-6440),in mice dosed 50 mg/kg orally,Poor oral bioavailability,药代动力学在新药研发中的作用,44/84,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,药代动力学在新药研发中的作用,45/84,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,m,M,NADPH:2.4 mM,UDPGA:1.5 mM,Incubation:60 min at 37,o,C,Stopping procedure:chilled acetonitrile,3 x volume,In Vitro Metabolism Assay,体外肝微粒体试验,药代动力学在新药研发中的作用,46/84,1,2,3,4,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:,（试验样品）：,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,药代动力学在新药研发中的作用,47/84,Enhanced Throughput Screening,Incubation,: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 manpower:,PKDM tech x 3,Test article amount,:0.1 mg/test article,Screening rate,:one chemotype with 60 compounds/1 week,药代动力学在新药研发中的作用,48/84,HPLC profiles of BCH-3840 and its metabolite(BCH-6440),BCH-3840,metabolite?,In vitro metabolic stability by rat hepatic S9,药代动力学在新药研发中的作用,49/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,研究和发觉阶段,能否被吸收？,permeability,是否被代谢？,metabolic stability,代谢产物？,metabolite identification,代谢路径？,pathway identification,对其它药品影响？,drug-drug interaction,药代动力学在新药研发中的作用,50/84,Liquid Chromatography/Mass Spectrum of BCH-3840 and its,metabolite(BCH-6440),Hydroxylation or Oxidation,MH,+,=310,MH,+,=294,Mass Identification,药代动力学在新药研发中的作用,51/84,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,药代动力学在新药研发中的作用,52/84,Nuclear Magnetic Resonance profiles of BCH-3840 and its metabolite,(BCH-6440),C5-H,BCH-3840,Metabolite,Structure Elucidation,药代动力学在新药研发中的作用,53/84,In vitro,therapeutic index of BCH-6440,药代动力学在新药研发中的作用,54/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,研究和发觉阶段,能否被吸收？,permeability,是否被代谢？,metabolic stability,代谢产物？,metabolite identification,代谢路径？,pathway identification,对其它药品影响？,drug-drug interaction,药代动力学在新药研发中的作用,55/84,Inhibitors for CYP IsoformConc(,m,M),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,代谢路径判定,药代动力学在新药研发中的作用,56/84,Inhibitors for CYP IsoformConc(,m,M),Inhibition(%of NC),Tranylcypromine(CYP2A6)5040.2,Sulfaphenazole(CYP2C9)2514.2,4-methylpyrazole(CYP2E1)25067.6,Ketoconazole(CYP3A4)575.2,Metabolism Phenotyping,代谢路径判定,药代动力学在新药研发中的作用,57/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,研究和发觉阶段,能否被吸收？,permeability,是否被代谢？,metabolic stability,代谢产物？,metabolite identification,代谢路径？,pathway identification,对其它药品影响？,drug-drug interaction,药代动力学在新药研发中的作用,58/84,Drug-Drug Interactions,（,对其它药品代谢影响）,Inhibition,（,抑制）,potential-IC,50,and K,i,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,药代动力学在新药研发中的作用,59/84,IC,50,(,M):0.675,Goodness of Fit:0.9807,95%Confidence Intervals:5.638.28,IC,50,(,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,药代动力学在新药研发中的作用,60/84,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 10,6,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 6,b-,hydroxylation,药代动力学在新药研发中的作用,61/84,Drug-drug interaction:Induction,诱导作用,药代动力学在新药研发中的作用,62/84,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,药代动力学在新药研发中的作用,63/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,临床前阶段,生物利用度,bioavailability,血浆浓度线性和非线性,dose escalation&proportionality,屡次给药和体内积蓄,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,药代动力学在新药研发中的作用,64/84,119%,236%,310%,Proportionality,血浆浓度非线性,提醒：,代谢或排泄非线性饱和,药代动力学在新药研发中的作用,65/84,90%,72%,Proportionality:,AUC,（大鼠试验）,93%,63%,提醒：药品吸收非线性饱和,药代动力学在新药研发中的作用,66/84,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（,药品体内积累）,(50,250,500 mg/kg,daily for 14 days),药代动力学在新药研发中的作用,67/84,90%,72%,Proportionality:,AUC,（大鼠试验）,93%,63%,提醒：药品吸收非线性饱和,药代动力学在新药研发中的作用,68/84,0,100,200,300,400,500,600,0,10,20,30,40,50,60,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:,C,max,（大鼠试验）,提醒：药品吸收非线性饱和,药代动力学在新药研发中的作用,69/84,0.92,0.77,1.04,1.19,1.02,1.07,Accumulation Ratio,药品积累率（大鼠）,Male rats,Female rats,药代动力学在新药研发中的作用,70/84,Proportionality:,AUC,（猕猴）,Male Monkey,Female Monkey,49%,34%,60%,38%,提醒：药品吸收非线性饱和,药代动力学在新药研发中的作用,71/84,38%,31%,55%,32%,Proportionality:,C,max,（猕猴）,Male Monkey,Female Monkey,提醒：药品吸收非线性饱和,药代动力学在新药研发中的作用,72/84,Male Monkey,Female Monkey,0.79,1.11,1.12,0.73,0.76,1.14,Accumulation Ratio,药品积累率（猕猴）,药代动力学在新药研发中的作用,73/84,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 points(13):0.5,1,1.5,2,3,4,6,8,10,12,16,20,and 24 hour,药代动力学在新药研发中的作用,74/84,Efficacy Hits,Optimized Lead,Go or no go decision,Compound for,Development(CD),NEW DRUG,IND,NDA,R&D,临床试验,临床前试验,研究和发觉,临床阶段,动物和人代谢特征,(长久毒性试验动物选择),metabolism profiling in animals and humans,药代动力学在新药研发中的作用,75/84,Evaluation of,In Vitro,Metabolism Profiles,Cryopreserved Hepatocytes,mouse,rat,dog,monkey,human,试验药品,(MW=455.59 in 1/2 tartrate salt),14,C,放射性比活性,(23.7 Ci/mg),药代动力学在新药研发中的作用,76/84,Methods,Incubation procedures,Viability(Trypan blue exclusion)70%,Final cell density:2 x 10,6,viable cell/mL,Final concentration of test article:20 M,Incubation time:4 hours,Bioanalytical measurement,HPLC/Fractionating/Scintillation:Metabolic profiles,LC/MS/MS:Mass identification/structure elucidation,药代动力学在新药研发