PREDICTION OF DRUG DISPOSITION BY HUMAN DRUG METABOLIZING ENZYMES

测定了12种与人NADPH-P450还原酶（NPR）共表达的重组人细胞色素P450（P450）在细菌膜（P450/NPR膜）中的药物氧化活性，并与其它重组系统和人肝微粒体的药物氧化活性进行了比较。向P450/NPR膜中添加外源性膜结合NPR可增强CYP 2C 8、CYP 2C 9、CYP 2C 19、CYP 3A 4和CYP 3A 5的催化活性;然而，在向每个P450中添加4摩尔过量的NPR后，未观察到膜中CYP 1A 1、CYP 1A 2、CYP 1B 1、CYP 2A 6、CYP 2B 6、CYP 2D 6和CYP 2 E1活性的增强。外源性纯化的人细胞色素b_5（b_5）进一步增强了CYP 2A 6、CYP 2B 6、CYP 2C 8、CYP 2 E1、CYP 3A 4和CYP 3A 5/NPR膜的催化活性。在重组体系中加入b_5可提高CYP 2C 9和CYP 2C 19的催化活性，而在P450/NPR膜中则无此作用。除CYP 2 E1/NPR膜和含有纯化的CYP 2C 8或CYP 2 E1的重组系统与b_5相比外，载脂蛋白b_5（不含血红素）加入到这两种系统中时均增强了催化活性。P450/NPR膜加b_5系统的催化活性与用共表达P450和NPR（和b_5）的昆虫细胞微粒体和/或人肝微粒体测得的活性大致相似。这些结果表明，在膜和重构系统中共表达的P450和NPR的相互作用似乎是不同的一些人CYP 2家族酶，可能是由于b_5的构象作用。含b_5的P450/NPR膜系统是预测肝微粒体P450依赖性药物氧化速率的有用模型。

Drug oxidation activities of twelve recombinant human cytochrome P450 (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5 ; however, enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR in 4-molar excess to each P450. Exogenous purified human cytochrome b_5 (b_5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b_5 in reconstitution systems but not in the P450/NPR membranes. Apo b_5 (devoid of heme) enhanced catalytic activities when added to the both systems, except for CYP2E1/NPR membranes and the reconstituted systems containing purified CYP2C8 or CYP2E1 in comparison with b_5. Catalytic activities in P450/NPR membranes plus b_5 systems were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b_5) and/or of human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes and reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b_5. P450/NPR membrane systems containing b_5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.