Academic Unit of Clinical Pharmacology, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK.
Biopharm Drug Dispos. 2011 Sep;32(6):303-18. doi: 10.1002/bdd.760. Epub 2011 Jul 4.
The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.
“相对活性因子”（RAF）比较了重组细胞色素 P450 酶（rhCYP）和人肝微粒体蛋白中每单位的活性，而没有分离出潜在的变异源（即每毫克蛋白中的酶含量或每单位酶的活性变化）。无维数的“系统间外推因子”（ISEF）将活性差异与 CYP 丰度差异区分开来。目前缺乏用于群体体外-体内外推（IVIVE）的这种标量的详细测定方案。本研究确定了 CYP2C9 的 ISEF，并首次系统地评估了探针底物、细胞色素 b5 以及评估内在清除率（CL(int)）的方法的影响。使用 rhCYP2C9 + 还原酶 + b5 BD Supersomes™中代谢物形成的动力学值 V(max)和 K(m)推导得到 CL(int)值时，S-华法林、甲苯磺丁脲和双氯芬酸的 ISEF 值分别为 0.75±0.18、0.57±0.07 和 0.37±0.07。使用 rhCYP2C9 + 还原酶 BD Supersomes™获得的 ISEF 值变化较大，S-华法林、甲苯磺丁脲和双氯芬酸的 ISEF 值分别为 7.16±1.25、0.89±0.52 和 0.50±0.05。尽管三种探针底物从 rhCYP2C9 + 还原酶 + b5 获得的 ISEF 值在统计学上存在差异（p<0.001），但使用平均值 0.54 可使所有三种底物的预测口服清除率值与文献观察值的 1.4 倍内。为了在底物间保持相对活性的一致性，建议使用表达 b5 的重组系统，并从完整的动力学数据中计算出内在清除率，以生成 CYP2C9 ISEF。此外，由于 ISEF 已被发现对辅助蛋白的差异敏感，因此建议使用 rhCYP 系统特异性 ISEF。
