醫(yī)學(xué)藥代動力學(xué)在新藥研發(fā)中的作用專題培訓(xùn)課件

1、藥代動力學(xué)在新藥研發(fā)中的作用藥代動力學(xué)在新藥研發(fā)中的作用Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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(wù) 藥效 Efficacy/ Pharmacodynamics 安全 Safety / Toxicology 藥物代謝動力學(xué) Drug Metabolism/PharmcokineticsEfficacy HitsOptimized LeadCom藥物代謝動力學(xué)的任務(wù)（最大無毒性濃度）(最小有效濃度）(最小藥效

2、時間）血漿濃度時間藥物代謝動力學(xué)的任務(wù)（最大無毒性濃度）(最小有效濃度）(最小藥效毒理藥代最佳血漿濃度藥效毒理藥代最佳Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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-dr

3、ug interaction Efficacy HitsOptimized LeadComEfficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 distrib

4、ution 從動物代謝推算人體代謝 extrapolation Efficacy HitsOptimized LeadComEfficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 HitsOptimized LeadComEfficacy HitsOptimized Lead Go or no go d

5、ecisionCompound for Development (CD)NEW DRUGINDNDAR&D臨床實驗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)Efficacy HitsOptimized LeadCom二五原則 5 毫克 5 天二五原則臨床前實驗藥物代謝動力學(xué)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 /

6、in vivo correlation)動物模型 (in vivo animal models)動物推測人 (species extrapolation)臨床前實驗藥物代謝動力學(xué)的生物模型排出太快/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metabolismin situ離體permeabilityin vivo體內(nèi)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Plasma conc

7、entrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orallyPoor oral bioavailabilityPlasma concentrations of BCH-3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absorption/distribution model 脂層轉(zhuǎn)移模型水相Aqueous phase水相Aqueous phase有機相Organic phasepH=6.5pH=7.4Permeability

8、Evaluation in vitro14absorption/distribution model in vitro absorption/distribution model15in vitro absorption/distributiCaco-2 Transport Pathways人大腸癌細胞模型Caco-2 Transport Pathways人大腸癌Transport Pathways藥物吸收機制被動細胞間主動P糖蛋白Transport Pathways藥物吸收機制被動細胞間Probes for Transport Pathways腸道吸收標準對照藥物Transcellular

9、（被動吸收）Propranolol, TestosteroneParacellular （細胞間滲透）Mannitol, InulinCarrier mediated （主動吸收） GlucoseP-Glycoprotein mediated （P糖蛋白調(diào)節(jié)）底物 Vinblastine抑制物 VerapamilProbes for Transport PathwaysGlucose （蔗糖） vs Inulin （木香素）主動吸收 vs 細胞間滲透Glucose （蔗糖） vs Inulin （木香素）Propranolol vs Mannitol被動吸收 vs 細胞間滲透Propranol

10、ol vs Mannitol被動吸收由P蛋白所調(diào)節(jié)的藥物吸收使用P糖蛋白抑制劑 Verapamil由P蛋白所調(diào)節(jié)的藥物吸收使用P糖蛋白抑制劑 VeraChong, Dando & Morrison; Pharm. Res. 1997Chong, Dando & Morrison; PharmFalse 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False PositiveFalse NegativeCain situ rat intestinal perfusion (single pass) 離體大

11、鼠十二指腸灌流模型（單循環(huán)）METHODAnimal: 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 a

12、rticle = 0.05-0.30 mg/mLin situ rat intestinal perfusiPerfusion 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 Treitz2nd is made at about 20 cm distal to 1st one the inlet of ca

13、nnula - 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

14、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 outin situ rat intestinal perfusion (single pass)Perfusion Procedures:in situ rIn situ rat intestinal permeability (single pass)Prediction within

15、 90% interval = 19/31 (61.3%)In-house validation假陽性假陰性In situ rat intestinal permeabPlasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orallyPoor oral bioavailabilityPlasma concentrations of BCH-3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

16、化早期研發(fā)階段后期研發(fā)階段Situation Analysis in vitro體外metabolismin situ離體permeabilityin vivo體內(nèi)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In Situ Rat Intestinal Permeability: Good陽性對照陰性對照受試藥物In Situ Rat Intestinal PermeabEnhanced Throughput ScreeningPerfusion: 4 compounds per day (4 animals) Sample size: time

17、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 weeks Enh

18、anced Throughput ScreeningpKa = 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pKa = 10 pKa = 8.4 pSAR: permeability vs. efficacy實例：結(jié)構(gòu)優(yōu)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 uMPred

19、uced%= 15%SAR: permeability vs. efficacy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(xué)模型二五原則: 5毫克/5天小結(jié)：體外和離體藥物吸收實驗系統(tǒng)血漿濃度時間化學(xué)藥物化學(xué)藥物+中藥中藥的藥物代謝動力學(xué)的任務(wù) 本身的藥物代謝動力學(xué)問題 對其它藥物吸收的作用血漿濃度時間化學(xué)藥物化學(xué)藥物+中藥中藥的藥物代謝動力學(xué)的任務(wù)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

20、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metabolismin situ離體permeabilityin vivo體內(nèi)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.-藥代研究員死還是不死,這是個問題.動物體內(nèi)模型 -

21、人體內(nèi)(臨床試驗)In vivo animals vs. in vivo humans人體外模型 -人體內(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，藥物代謝實驗成本動物體內(nèi)模型 - 人體內(nèi)(臨床試驗)Heartbeat and Bodyweight （心率和體重）小鼠大鼠兔猴狗人38Heartbeat and Bodyweight （心率和體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39Liver weight a

22、nd Hepatic Flow Antipyrine clearance (l/min)ratmouserabbitmonkeydoghumanClearance40Antipyrine clearance (l/min)raIn Vitro Models 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 In Vitro Models of the Liver體USFDA Guidance for Industry美國藥物和食品管理局關(guān)于藥物代謝實驗的指

23、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 Metab

24、olism/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息，因為這個系統(tǒng)含有足夠的天然水平的酶系。USFDA Guidance for Industry美國HOHOHOHOHOHOOGLUCHOOSOOHO2-Hydroxy-EE22EE -3-GlucuronideEE2 -3-SulfateConjugatesEE2EE2Hepatocytes （肝細胞）Micros

25、omes（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)HOHOHOHOHOHOOGLUCHOOSOOHO2-HydPlasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orallyPoor oral bioavailabilit

26、yPlasma concentrations of BCH-3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metabolismin situ離體permeabilityin vivo體內(nèi)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Reaction volume:1.0 ml, DPBS pH 7.4Hepatic S-9/Microsomes:0.5 mg protien/m

27、L Species:Human/Monkey/Dog/Rat/Mouse Substrate concentration:10 mMNADPH:2.4 mMUDPGA:1.5 mMIncubation:60 min at 37oCStopping procedure:chilled acetonitrile, 3 x volumeIn Vitro Metabolism Assay 體外肝微粒體實驗Reaction volume:1.0 ml, DPBS1234 A B C D E FEnhanced Throughput Screening （增速篩選）A-B: （空白對照）：test art

28、icle + 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 = treatedDosing 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1 A B C D E FEnhanceEnhanced Th

29、roughput ScreeningIncubation: 4 compounds per 24-well plate15 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 128Bioanalysis: no extractionno standard curve (peak area)machine time/2 LCs

30、24 hrsTotal manpower:PKDM tech x 3 Test article amount:0.1 mg / test articleScreening rate:one chemotype with 60 compounds / 1 week Enhanced Throughput ScreeningHPLC profiles of BCH-3840 and its metabolite (BCH-6440)BCH-3840metabolite?In vitro metabolic stability by rat hepatic S9HPLC profiles of BC

31、H-3840 and Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 Efficacy HitsOptimized LeadComLiq

32、uid Chromatography / Mass Spectrum of BCH-3840 and its metabolite (BCH-6440)Hydroxylation or OxidationMH+ = 310MH+ = 294Mass IdentificationLiquid Chromatography / Mass SHPLC profiles of BCH-3840 and its metabolite (BCH-6440)Preparation of metabolite by bulk incubationMMPP10 mg microsomal protein2 mg

33、 BCH-3840Fraction collection of metabolitefractionation concentration HPLC profiles of BCH-3840 and Nuclear Magnetic Resonance profiles of BCH-3840 and its metabolite (BCH-6440)C5-HBCH-3840MetaboliteStructure Elucidation Nuclear Magnetic Resonance proIn vitro therapeutic index of BCH-6440In vitro th

34、erapeutic index of Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 Efficacy HitsOptimized Le

35、adComInhibitors for CYP IsoformConc (mM)Furafulline (CYP1A2) 10Tranylcypromine (CYP2A6) 50Sulfaphenazole (CYP2C9) 25Omeprazole (CYP2C19) 20Quinidine (CYP2D6) 24-methylpyrazole (CYP2E1) 250Ketoconazole (CYP3A4) 5 Chemical Inhibition （化學(xué)抑制） Pure enzyme （純酶） Correlation Analysis （相關(guān)分析）Metabolism Phenot

36、yping 代謝途徑鑒定Inhibitors for CYP IsoformConInhibitors for CYP IsoformConc (mM) Inhibition (% of NC)Tranylcypromine (CYP2A6) 5040.2Sulfaphenazole (CYP2C9) 2514.24-methylpyrazole (CYP2E1) 25067.6Ketoconazole (CYP3A4) 575.2Metabolism Phenotyping 代謝途徑鑒定Inhibitors for CYP IsoformConEfficacy HitsOptimized L

37、ead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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 Efficacy HitsOptimized LeadComDrug-Drug Interactions （對其它藥物代謝的影響）Inh

38、ibition （抑制）potential - IC50 and Kimechanism - 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(dǎo)）test system: fresh isolated hepatocytes （肝細胞）Target EnzymesCytochrome P450s: 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4Phase II conjugation

39、: glucuronidationDrug-Drug Interactions （對其它藥物代IC50 (M):0.675Goodness of Fit:0.980795% Confidence Intervals:5.638.28IC50 (M):20.4Goodness of Fit:0.973095% Confidence Intervals:16.9-26.3CYP3A4 CYP3A4Drug-drug interaction: inhibition 抑制作用體外藥效濃度 = 1 uMIC50 (M):0.675IC50 (M):Drug-drug interaction: Induc

40、tion （肝細胞誘導(dǎo)模型）5 days procedureDay 0: Isolate fresh hepatocytes, viability 70%Plating hepatocytes to 24-well plate, 0.7 x 106 viable 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

41、 with test articlesDay 4:same as Day 3Day 5:washing out the dosing solution and adding substrates for CYP450 isoforms as below:1A2 - ethocyresorufin O-deethylation2A6 - coumarin 7-hydroxylation2C9 - tolbutamide 4-hydroxylation2C19 - S-mephenytoin 4-hydroxylation2D6 - dextromethorphan O-demethylation

42、2E1 - chlorzoxazone 6-hydroxylation3A4 - testosterone 6b - hydroxylationDrug-drug interaction: InductiDrug-drug interaction: Induction 誘導(dǎo)作用Drug-drug interaction: Inducti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metabolismin situ離體pe

43、rmeabilityin vivo體內(nèi)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Efficacy HitsOptimized Lead Go or no go decisionCompound for Development (CD)NEW DRUGINDNDAR&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

44、lance 體內(nèi)分布 distribution 從動物代謝推算人體代謝 extrapolation Efficacy HitsOptimized LeadCom119%236%310%Proportionality 血漿濃度的非線性提示： 代謝或排泄的非線性飽和119%236%310%Proportionality 血漿90%72%Proportionality: AUC （大鼠試驗）93%63%提示 ：藥物吸收的非線性飽和 90%72%Proportionality: AUC （大鼠TOXICOKINETICS 毒物代謝動力學(xué)試驗 Animal: Sprague-Dawley rats (m

45、ale & female) Cynomolgus monkey (male & female) Single dose escalation （線性動力學(xué)） (50, 250, 500 mg/kg) Multiple dose escalation （藥物體內(nèi)積累） (50, 250, 500 mg/kg, daily for 14 days)TOXICOKINETICS 毒物代謝動力學(xué)試驗90%72%Proportionality: AUC （大鼠試驗）93%63%提示 ：藥物吸收的非線性飽和 90%72%Proportionality: AUC （大鼠0100200300400500600

46、0102030405060Female RatsOral Dose (mg/kg)010020030040050060001020304050Male RatsOral Dose (mg/kg)Cmax(mg/mL)73%47%56%49%Proportionality: Cmax （大鼠試驗）提示 ：藥物吸收的非線性飽和 0100200300400500600010203040500.920.771.041.191.021.07Accumulation Ratio 藥物積累率 （大鼠）Male ratsFemale rats0.920.771.041.191.021.07AccumuProp

47、ortionality: AUC （獼猴）Male MonkeyFemale Monkey49%34%60%38%提示 ：藥物吸收的非線性飽和 Proportionality: AUC （獼猴）Male 38%31%55%32%Proportionality: Cmax （獼猴）Male MonkeyFemale Monkey提示 ：藥物吸收的非線性飽和 38%31%55%32%Proportionality: CMale MonkeyFemale Monkey0.791.111.120.730.761.14Accumulation Ratio 藥物積累率 （獼猴）Male MonkeyFemale Monkey0.791.Phase I Trial (Single dose escalation)臨床一期單劑量藥代動力學(xué)試驗Healthy Male Subject (n): 22Oral Doses (4): 100, 200, 400, and 800 mgTime points(13):0.5, 1, 1.5, 2, 3, 4, 6, 8