Transport Pathways 药物吸收机制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 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:	5.63–8.28 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 诱导作用被动细胞间主动P糖蛋白Chong, Dando & Morrison; 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- C’out / C’in ) C’out / C’in = (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Heartbeat and Bodyweight （心率和体重）小鼠大鼠兔猴狗人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Antipyrine clearance (l/min) rat mouse rabbit monkey dog human Clearance “The most complete picture for hepatic metabolism can be obtained with liver systems,in which the