Integrated Cytochrome P450 Reaction Phenotyping: Attempting to Bridge the Gap Between cDNA-expressed Cytochromes P450 and Native Human Liver Microsomes
Integrated Cytochrome P450 Reaction Phenotyping: Attempting to Bridge the Gap Between cDNA-expressed Cytochromes P450 and Native Human Liver Microsomes
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DOI：
10.1016/s0006-2952(98)00268-8
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发表时间：
1999-03
影响因子：
5.8
通讯作者：
Rodrigues Ad
中科院分区：
文献类型：
--
作者：
Rodrigues Ad
随着人肝组织、重组（cDNA表达的）细胞色素P450蛋白（rCYP）的可用性增加，以及对人β-内酰胺酶池的了解（例如天然肝微粒体中每种β-内酰胺酶形式的免疫定量水平），现在可以以综合方式进行体外”β-内酰胺酶反应表型分析”。反应表型分析允许人们使用从天然人肝微粒体和重组蛋白获得的数据的组合来鉴定哪种形式参与给定药物的代谢。下面描述如何尝试整合这些数据。分析中共包括10种药物，由12种反应（6种羟基化、2种O-脱甲基化、1种N-脱甲基化、1种O-脱乙基化和2种亚砜化）表示，这些反应主要由CYP（CYP 3A 4、CYP 2C 9、CYP 1A 2和CYP 2D 6）的各种组合催化（>或= 20%），其特征在于表观Km值范围较宽（12-820 μ M）。简而言之，将用单个rCYP测量的反应速率相对于天然人肝微粒体中相应rCYP的标称比含量进行归一化。然后，将每个rmb的标准化率相加，得到”总标准化率”（TNR），并且每个rmb的标准化率表示为TNR的百分比（% TNR）。最后，% TNR与抑制（在α型选择性化学抑制剂存在下的抑制百分比;% I）和用天然人肝微粒体获得的单变量回归分析（r>或= 0.63; P<或= 0.05; N>或= 10个不同的肝）数据相关。因此，通过整合所有三个数据集（r、% TNR和% I）来指定药物的反应表型。
With the increased availability of human liver tissue, recombinant (cDNA-expressed) cytochrome P450 proteins (rCYPs), and knowledge of the human CYP pool (eg immunoquantitated levels of each CYP form in native liver microsomes), it is now possible to carry out in vitro" CYP reaction phenotyping" in an integrated manner. Reaction phenotyping allows one to identify which CYP form (s) is (are) involved in the metabolism of a given drug, using a combination of data obtained with native human liver microsomes and rCYP proteins. The following describes how one can attempt to integrate such data. A total of ten drugs are included in the analysis, represented by twelve reactions (six hydroxylations, two O-demethylations, one N-demethylation, one O-deethylation, and two sulfoxidations) that are largely catalyzed (> or= 20%) by various combinations of CYPs (CYP3A4, CYP2C9, CYP1A2, and CYP2D6), and characterized by a wide range of apparent Km values (12-820 microM). Briefly, reaction rates measured with individual rCYPs are normalized with respect to the nominal specific content of the corresponding CYP in native human liver microsomes. In turn, the normalized rates for each rCYP are summed, yielding a" total normalized rate"(TNR), and the normalized rate for each rCYP is expressed as a percent of the TNR (% TNR). Finally,% TNR is related to inhibition (percent inhibition in the presence of CYP form selective chemical inhibitors;% I) and univariate regression analysis (r> or= 0.63; P< or= 0.05; N> or= 10 different livers) data obtained with native human liver microsomes. Therefore, the reaction phenotype of a drug is assigned by integrating all three data sets (r,% TNR, and% I).