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Enzyme Inhibition
抑制of cytochrome P450 (CYP) and UGT enzymes is a major cause of clinically relevant drug-drug interactions(see DDIs)。抑制潜在的测试条的sessed by determining its effect on the metabolism of selective probe substrates for human CYP enzymes in pooled human hepatic microsome-based incubations. Inhibitory enzyme kinetics can be further characterized and the resultant data used to predict whether a clinically significant DDI may occur following administration of the drug.
法规considerations for enzyme inhibition studies
These studies are recommended by both FDA and EMA drug-drug interactions (DDI) guidelines to generate data on both reversible (direct) and irreversible (time-dependent) cytochrome P450 inhibition before going to first-in-human trials. Inhibition data is used in determining the requirement and scope of clinical DDI studies.
Method
- 测试系统：汇集人肝微粒体（HLM）
- CYP Enzymes Evaluated: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,CYP2E1 and CYP3A4/5
- UGT enzymes evaluated in direct inhibition assays upon request: UGT1A1, UGT1A3, UGT1A4, UGT11A6, UGT1A9, UGT2B7, UGT2B15
- Test Article Concentrations: 8 concentrations in triplicate
- 所有样品制剂和孵育都是使用Hamilton MicroLab Star自动化液体处理系统进行的
CYP抑制在直接和依赖性测定中测量。使用CYP选择性底物，以达到最大反应速度的一半的底物浓度（km) for each CYP enzyme (see below). Known inhibitors are used as positive controls for both direct and metabolism-dependent inhibition assays. All incubations are terminated by addition of chilled acetonitrile, containing a stable-labeled internal metabolite standard specific to the CYP substrate.
直接抑制
在不存在和存在的情况下进行测定以确定抑制潜力并在可能的情况下定义IC50。
通过确定抑制常数，可以进一步表征直接抑制（ki）和使用5个浓度的探针基板观察抑制的类型。
Time-Dependent (Irreversible) Inhibition
在稀释到探针基板测定混合物中，使用8次浓度试验制品的测定在不存在和存在下孵育30分钟。如果观察到值得注意的时间依赖性抑制，则可以确定额外的动力学参数，包括灭活常数（kinact）和抑制常数（kI). These parameters, determined experimentally by varying the pre-incubation time and inhibitor concentration, can help define the potential for drug-drug interactions.
CYP抑制测定条件
阳性控制 |
||||
|---|---|---|---|---|
Cytochrome P450 |
基质 |
分析物 |
Direct inhibition |
Time-dependent Inhibition |
CYP1A2 |
phenacetin. |
对乙酰氨基酚 |
Fluvoxamine |
Furafylline. |
CYP2B6 |
Bupropion |
羟基布丙酮 |
orphenadrine. |
thootep. |
CYP2C8 |
am |
脱乙酰氨基 |
Montelukast |
Gemfibrozil 1-O-β-glucuronide |
CYP2C9 |
双氯芬太奇 |
4'-Hydroxydiclofenac |
Sulfaphenazole |
Tienilic acid. |
CYP2C19 |
Mephenytoin |
4'-羟基飞待 |
nootkatone. |
Esomeprazole |
CYP2D6 |
Dextromethan. |
Dextronan. |
奎尼丁 |
帕罗西汀 |
CYP3A4/5 |
Testosterone |
6β-Hydroxytestosterone |
Ketoconazole |
Erythromycin |
CYP3A4/5 |
Midazolam |
1'-羟基咪唑胺 |
Ketoconazole |
Troleandomycin. |
可交付成果
这些测定将提供IC50values for direct or irreversible inhibition of CYP enzymes. If significant direct inhibition is observed, the inhibition constant (Ki）可以确定。If significant time-dependent inhibition is observed, the mechanism-based inactivation parameters (KI和K.inact）可以确定。使用这些参数，药物测量学在评估需要临床试验时可以允许额外的指导。
