Caco-2 Transport Pathways 人大肠癌细胞吸收模型
False Positive 假阳性 ＝ 低
False Negative 假阴性 ＝ 高
in situ rat intestinal perfusion (single pass) 离体大鼠十二指肠灌流模型（单循环）
METHOD
NDA 临床实验
非临床实验准则
Good Laboratory Practice (GLP)
Compound for Development (CD)
IND 临床前实验
Efficacy Hits
R&D
Optimized Lead
Go or no go decision
研究和发现
二五原则
• 5 毫克 •5天
• 对其它药物的影响？ drug-drug interaction
NEW DRUG
NDA 临床实验
Compound for Development (CD)
IND 临床前实验
Efficacy Hits
R&D
Optimized Lead
Go or no go decision
研究和发现
临床前阶段
• 生物利用度
8/23/99 30
13
21
28 10-5 11-1 11-8 12-6 12-13 12-20 10
17
24
Test Day
由P－蛋白所调节的药物吸收 －使用P－糖蛋白抑制剂 Verapamil
Papp (nm/sec)
120
Vinblastine
100
Vinblastine/verapa
80
mil
60
40
20
0
day1 1999-8- 1999-9- 1999-9- 1999-9- 1999-
8/23/99 30
13
21
28 10-5
199911-1
199911-8
199912-6
199912-13
1999- 2000-1- 2000-1- 2000-1-
12-20 10
17
24
Test Day
NEW DRUG
NDA 临床实验
Compound for Development (CD)
IND 临床前实验
Efficacy Hits
R&D
Optimized Lead
Go or no go decision
研究和发现
临床实验准则
Good Clinical Practice (GCP)
NEW DRUG
(最小药效时间）
0 .5
1
1 .5
时间2.
2 .5
(最小有效浓度）
3
3 .5
4
药效
最佳 血浆浓度
毒理
药代
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
Final concentrations of test article = 0.05-0.30 mg/mL
in situ rat intestinal perfusion (single pass)
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
In-house validation
Human Oral Bioavailability (%)
100
假阴性
80
60
40
20 0
0.000
假阳性
0.001 0.002 0.003 0.004
Permeability (cm/min)
药物吸收模型
计算机 脂溶度 脂层转移 细胞层转移 十二指肠灌流
Permeability Evaluation – in vitro
absorption/distribution model 脂层转移模型
有机相 Organic phase
水相 Aqueous phase
pH=6.5
水相 Aqueous phase
Drugs
caffeine ibuprofen desipramine acetaminophen propranolol hydrazine Ketoconazole terbutaline atenolol acetbutalol nadolol losartan mannitol inulin
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
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
Oral Absorption in Humans (%)
100 100 95 95 90 90 76 73 50 40 35 33 16
5
3-day Caco-2
Kp
227 201 261 218 265 155 120 56 30 29 22 42 40 12
Chong, Dando & Morrison; Pharm. Res. 1997
NEW DRUG
NDA 临床实验
Compound for Development (CD)
IND 临床前实验
Efficacy Hits
R&D
Optimized Lead
Go or no go decision
研究和发现
药物代谢动力学的任务
血浆浓度
100 10 1 0 .1
0 .0 1 0
（最大无毒性浓度）
pH=7.4
14
in vitro absorption/distribution model
In Vitro/ In Vivo Correlation Pooled Data from Four Biostudies
% Absorped
100
formulation finding study
BE study 1
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 permeability (single pass)
被动
细胞间 主动
P糖蛋白
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）
80 100
Propranolol vs Mannitol 被动吸收 vs 细胞间渗透
Papp (nm/sec)
450
Mannitol
400
Propranolol
350
300
250
200
150
100
50
0
day1 1999-8- 1999-9- 1999-9- 1999-9- 1999- 1999- 1999- 1999- 1999- 1999- 2000-1- 2000-1- 2000-1-
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)