Measurement of Michaelis constants for cytochrome P450-mediated biotransformation reactions using a substrate depletion approach
Measurement of Michaelis constants for cytochrome P450-mediated biotransformation reactions using a substrate depletion approach
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DOI：
10.1124/dmd.30.7.831
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发表时间：
2002-07-01
影响因子：
3.9
通讯作者：
Reed-Hagen, AE
中科院分区：
文献类型：
--
作者：
Obach, RS;Reed-Hagen, AE
细胞色素P450介导的药物生物转化反应的米氏常数（K-M）可以是理解药物在体内表现出饱和代谢和非线性剂量-暴露关系的潜力的重要参数。使用重组异源表达的人酶测量几种药物生物转化反应的K-M值。这些测定是使用在多种底物浓度下监测底物损失的方法（“体外t（1/2）”方法）进行的，目的是将通过该方法测定的K-M值与使用在几种底物浓度下测量产物形成速率的常规方法测定的K-M值进行比较。检查的反应为CYP 2C 9催化的双氯芬酸4 '-羟基化、CYP 2D 6催化的美沙芬O-脱甲基化和硫利达嗪S-氧化、CYP 2C 19催化的丙咪嗪N-脱甲基化、CYP 3A 4催化的咪达唑仑1'-羟基化和CYP 1A 2催化的他克林1-羟基化。K-M值跨越80倍范围，从0.12 μ M（CYP 2D 6催化的硫利达嗪S-氧化）到9.8 μ M（CYP 2C 19催化的丙咪嗪N-去甲基化）。平均而言，K-M值确定的底物消耗的方法是在1.54倍的测量产品形成所确定的。因此，可以确定药物代谢反应的K-M值，而不需要代谢物结构的知识或需要用于构建定量生物分析的标准曲线的代谢物的真实标准。确定K-M值的体外t（1/2）方法应有助于早期药物发现工作，以识别具有低K-M值的化合物，从而更有可能表现出超比例的剂量-暴露关系。
The Michaelis constant (K-M) for cytochrome P450-mediated drug biotransformation reactions can be an important parameter in understanding the potential for a drug to exhibit saturable metabolism in vivo and nonlinear dose-exposure relationships. K-M values were measured for several drug biotransformation reactions using recombinant heterologously expressed human enzymes. These determinations were made using an approach of monitoring substrate loss ("in vitro t(1/2)" method) at multiple substrate concentrations, with the objective of comparing K-M values determined by this approach with K-M values determined using the conventional approach of measuring product formation rates at several substrate concentrations. The reactions examined were CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation and thioridazine S-oxidation, CYP2C19-catalyzed imipramine N-demethylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, and CYP1A2-catalyzed tacrine 1-hydroxylation. K-M values spanned an 80-fold range from 0.12 muM (CYP2D6-catalyzed thioridazine S-oxidation) to 9.8 muM (CYP2C19-catalyzed imipramine N-demethylation). On average, K-M values determined by the substrate depletion approach were within 1.54-fold of those determined by measuring product formation. Thus, K-M values can be determined for drug metabolism reactions without requiring knowledge of metabolite structures or requiring authentic standards of metabolites for use in construction of standard curves for quantitative bioanalysis. The in vitro t(1/2) approach of determining K-M values should be useful in early drug discovery efforts to identify those compounds with low K-M values and, hence, a greater probability of exhibiting supraproportional dose-exposure relationships.