藥物代謝及其動力學在新藥研發(fā)中的應用

1、藥物代謝及其動力學在新藥研發(fā)中的應用,胡卓漢 博士 瑞德肝臟疾病研究(上海)有限公司 復旦大學藥學院,2004年12月30日 中國.北京,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),藥物研發(fā)的三大任務 藥效 Efficacy/ Pharmacodynamics 安全 Safety / Toxicology 藥物代謝動力學 Drug Metabolism/Pharmcokinetics,藥物代謝動力學的任務

2、,（最大無毒性濃度）,(最小有效濃度）,(最小藥效時間）,血漿濃度,時間,藥效,毒理,藥代,最佳 血漿濃度,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對其它藥物的影響？ dr

3、ug-drug interaction,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),臨床前階段 生物利用度 bioavailability 血漿濃度的線性和非線性 dose escalation & proportionality 多次給藥和體內(nèi)積蓄 multiple doses & accumulation 吸收和排泄模式 mass balance 體內(nèi)分布 distribution 從動物代謝推算人體

4、代謝 extrapolation,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),臨床階段 長期毒性實驗的動物選擇 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,臨床實驗

5、,臨床前實驗,研究和發(fā)現(xiàn),臨床實驗準則 Good Clinical Practice (GCP),非臨床實驗準則 Good Laboratory Practice (GLP),二五原則 5 毫克 5 天,臨床前實驗藥物代謝動力學的生物模型 體外和離體模型 (in vitro / in situ models) 吸收模型 absorption/permeability 代謝模型 metabolism 體外推測和體內(nèi) (in vitro / in vivo correlation) 動物模型 (in vivo animal models) 動物推測人 (species extrapolation)

6、,排出太快/藥效時間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orally,Poor oral bioavaila

7、bility,藥物吸收模型,計算機,脂溶度,脂層轉(zhuǎn)移,細胞層轉(zhuǎn)移,十二指腸灌流,14,absorption/distribution model 脂層轉(zhuǎn)移模型,水相 Aqueous phase,水相 Aqueous phase,有機相 Organic phase,pH=6.5,pH=7.4,Permeability Evaluation in vitro,15,in vitro absorption/distribution model,Caco-2 Transport Pathways 人大腸癌細胞模型,Transport Pathways 藥物吸收機制,被動,細胞間,主動,P糖蛋白,Pro

8、bes for Transport Pathways 腸道吸收標準對照藥物,Transcellular （被動吸收） Propranolol, Testosterone Paracellular （細胞間滲透） Mannitol, Inulin Carrier mediated （主動吸收） Glucose P-Glycoprotein mediated （P糖蛋白調(diào)節(jié)） 底物 Vinblastine 抑制物 Verapamil,Glucose （蔗糖） vs Inulin （木香素） 主動吸收 vs 細胞間滲透,Propranolol vs Mannitol 被動吸收 vs 細胞間滲透,由P

9、蛋白所調(diào)節(jié)的藥物吸收 使用P糖蛋白抑制劑 Verapamil,Chong, Dando Pharm. Res. 1997,False Positive 假陽性 低,False Negative 假陰性 高,Caco-2 Transport Pathways 人大腸癌細胞吸收模型,in situ rat intestinal perfusion (single pass) 離體大鼠十二指腸灌流模型（單循環(huán)）,METHOD Animal: Male Sprague-Dawley rats (250 - 350 g), fasted overnight. Rat is anesthetized by

10、 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 jej

11、unum 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 bl

12、ank 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

13、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,假陽性,假陰性,Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orally,Poor oral

14、bioavailability,排出太快/藥效時間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,In Situ Rat Intestinal Permeability: Good,陽性對照,陰性對照,受試藥物,Enhanced Throughput Screening Perfusion:

15、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 articl

16、e 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 實例：結(jié)構(gòu)優(yōu)化和吸收率分析,SAR: permeability vs. efficacy 實例：結(jié)構(gòu)優(yōu)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、%= 17%,IC50 = 0.025 uM Preduced%= 15%,小結(jié)：體外和離體藥物吸收實驗系統(tǒng) 體外人大腸癌細胞模型 (in vitro Caco-2 monolayer) 離體大鼠十二指腸灌流模型 (in situ rat intestine perfusion) 體內(nèi)動物藥物代謝動力學模型 二五原則: 5毫克/5天,血漿濃度,時間,化學藥物,化學藥物+中藥,中藥的藥物代謝動力學的任務 本身的藥物代謝動力學問題 對其它藥物吸收的作用,排出太快/藥效時間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜

18、通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,死還是不死,這是個問題. To be or not to be, this is a problem. - 哈默雷特 體內(nèi)試驗還是體外試驗, 這是個問題. In vitro or in vivo, this is a problem. -藥代研究員,動物體內(nèi)模型 - 人體內(nèi)(臨床試驗) In vivo animals vs. in vivo humans 人體外模型 -人

19、體內(nèi)(臨床試驗) In vitro humans vs. in vivo humans 選擇的指南 與人相似：疾病模型，藥效，毒性，藥物代謝 實驗成本,38,Heartbeat and Bodyweight （心率和體重）,小鼠,大鼠,兔,猴,狗,人,39,Liver weight and Hepatic Flow vs Bodyweight （體重，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 M

20、odels of the Liver 體外肝模型,Hepatocytes 肝細胞 Liver slices 肝切片 Liver microsomes 肝微粒體 Liver S-9 Fraction 肝S-9組分,USFDA Guidance for Industry 美國藥物和食品管理局關于藥物代謝實驗的指南,“The most complete picture for hepatic metabolism can be obtained with liver systems,in which the cofactors are self-sufficient and the natural

21、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,譯文： 肝系統(tǒng)（分離的肝細胞和精確的肝切片）能為藥物代謝 實驗提供最完全的信

22、息，因為這個系統(tǒng)含有足夠的天然 水平的酶系。,2-Hydroxy-EE2,Conjugates,EE2,EE2,Hepatocytes （肝細胞）,Microsomes（微粒體） Hepatocytes（肝細胞）,Metabolism of Eythinyl Estradiol (EE2) 肝微粒體和肝細胞的代謝功能差異,Li, Hartman, Lu, Collins and Strong, Br J Clin Pharmacol 48, 733-742(1999),Plasma concentrations of BCH-3840 and its metabolite (BCH-6440)

23、 in mice dosed 50 mg/kg orally,Poor oral bioavailability,排出太快/藥效時間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,Reaction volume: 1.0 ml, DPBS pH 7.4 Hepatic S-9/Microsom

24、es: 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 體外肝微粒體實驗,1 2 3 4,A B C D E F,Enhanced Throughput Screening （增速篩選）,A-B: （空白對照）

25、：test article + buffer = vehicle control (VC) C-D:（陰性對照）：test article + microsomes = negative control (NC) E-F: （實驗樣品）：test article + microsomes + cofactors = treated Dosing solution = time zero (T = 0) 4 compounds including positive reference* / plate * 7 ethoxycoumarin,陰性對照,空白對照,測試樣本,Enhanced Thro

26、ughput 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

27、 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 Lea

28、d Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對其它藥物的影響？ drug-drug interaction,Liquid Chromatography / Mass Spectrum of BCH-3840 and its met

29、abolite (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,Nucl

30、ear 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,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeab

31、ility 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對其它藥物的影響？ 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-methylpyraz

32、ole (CYP2E1) 250 Ketoconazole (CYP3A4) 5,Chemical Inhibition （化學抑制）,Pure enzyme （純酶） Correlation Analysis （相關分析）,Metabolism Phenotyping 代謝途徑鑒定,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

33、 Ketoconazole (CYP3A4) 5 75.2,Metabolism Phenotyping 代謝途徑鑒定,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對其它藥物的

34、影響？ drug-drug interaction,Drug-Drug Interactions （對其它藥物代謝的影響） Inhibition （抑制） potential - IC50 and Ki mechanism - mechanistic（機械性） competitive （競爭性） test system: liver microsomes （肝微粒體） cryopreserved hepatocytes （冷凍肝細胞） Induction（誘導） test system: fresh isolated hepatocytes （肝細胞） Target Enzymes Cytoc

35、hrome 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,體外藥效濃度

36、 = 1 uM,Drug-drug interaction: Induction （肝細胞誘導模型）,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

37、 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

38、4-hydroxylation 2D6 - dextromethorphan O-demethylation 2E1 - chlorzoxazone 6-hydroxylation 3A4 - testosterone 6b - hydroxylation,Drug-drug interaction: Induction 誘導作用,排出太快/藥效時間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabol

39、ism,in situ離體 permeability,in vivo體內(nèi) bioavailability,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實驗,臨床前實驗,研究和發(fā)現(xiàn),臨床前階段 生物利用度 bioavailability 血漿濃度的線性和非線性 dose escalation & proportionality 多次給藥和體內(nèi)積蓄 multiple doses & accumulation 吸收和排泄模式 mass ba

40、lance 體內(nèi)分布 distribution 從動物代謝推算人體代謝 extrapolation,119%,236%,310%,Proportionality 血漿濃度的非線性,提示： 代謝或排泄的非線性飽和,90%,72%,Proportionality: AUC （大鼠試驗）,93%,63%,提示 ：藥物吸收的非線性飽和,TOXICOKINETICS 毒物代謝動力學試驗 Animal: Sprague-Dawley rats (male & female) Cynomolgus monkey (male & female) Single dose escalation （線性動力學） (

41、50, 250, 500 mg/kg) Multiple dose escalation （藥物體內(nèi)積累） (50, 250, 500 mg/kg, daily for 14 days),90%,72%,Proportionality: AUC （大鼠試驗）,93%,63%,提示 ：藥物吸收的非線性飽和,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),

42、Cmax,(,m,g/mL),73%,47%,56%,49%,Proportionality: Cmax （大鼠試驗）,提示 ：藥物吸收的非線性飽和,0.92,0.77,1.04,1.19,1.02,1.07,Accumulation Ratio 藥物積累率 （大鼠）,Male rats,Female rats,Proportionality: AUC （獼猴）,Male Monkey,Female Monkey,49%,34%,60%,38%,提示 ：藥物吸收的非線性飽和,38%,31%,55%,32%,Proportionality: Cmax （獼猴）,Male Monkey,Female Monkey,提示 ：藥物吸收的非線性飽和,Male Monkey,Female Monkey,0.79,1.11,1.12,0.73,0.76,1.14,Accumulation Ratio 藥物積累率 （獼猴）,Phase I Trial (Single dose escalation) 臨床一期單劑量藥代動力學試驗 Healthy Male Subject (n): 22 Oral Doses (4): 100, 200, 400, and 800 mg Tim