Venkatakrishnan K, von Moltke L L, Greenblatt D J
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, New England Medical Center Hospital, Boston, Massachusetts, USA.
J Pharm Sci. 1998 Jul;87(7):845-53. doi: 10.1021/js970435t.
The relative catalytic activities of CYP2C9 and CYP2C19 in human liver microsomes has been determined using the approach of relative activity factors (RAFs). Tolbutamide methylhydroxylation and S-mephenytoin 4'-hydroxylation were used as measures of CYP2C9 and CYP2C19 activity, respectively. The kinetics of these reactions were studied in human liver microsomes, in microsomes from human lymphoblastoid cells, and in insect cells expressing CYP2C9 and CYP2C19. RAFs were calculated as the ratio of Vmax (reaction velocity at saturating substrate concentrations) in human liver microsomes of the isoform-specific index reaction divided by the Vmax of the reaction catalyzed by the cDNA expressed isoform. RAFs were also determined for SUPERMIX, a commercially available mixture of cDNA expressed human drug metabolizing CYPs formulated to achieve a balance of enzyme activities similar to that found in human liver microsomes. Lymphoblast RAF2C9 in human liver microsomes ranged from 54 to 145 pmol CYP/mg protein (mean value: 87), while a value of 251 pmol CYP/mg protein was obtained for SUPERMIX. Insect cell RAF2C9 in human liver microsomes ranged from 1.6 to 143 pmol CYP/mg protein (mean value: 49), while a value of 201 pmol CYP/mg protein was obtained for SUPERMIX. Both lymphoblast and insect cell RAF2C19 in human liver microsomes ranged from 4 to 45 pmol CYP/mg protein (mean values: 29 and 28, respectively), while a value of 29 pmol CYP/mg protein was obtained for SUPERMIX. The nature of the cDNA expression system used had no effect on the kinetic parameters of CYP2C9 as a tolbutamide methylhydroxylase, or of CYP2C19 as a S-mephenytoin hydroxylase. However insect cell expressed CYP2C19 (which includes oxidoreductase) had substantially greater activity as a tolbutamide methylhydroxylase when compared to lymphoblast expressed CYP2C19. The ratio of mean lymphoblast-determined RAF2C9 to RAF2C19 in human livers was 3.0 (range 1.6-17.9; n = 10), while this ratio for SUPERMIX was 8.6. The ratio of mean insect cell-determined RAF2C9 to RAF2C19 in human livers was 1.7 (range 0.04-16.2; n = 10), while this ratio for SUPERMIX was 7.0. Neither ratio is in agreement with the 20:1 ratio of immunoquantified levels of CYP2C9 and 2C19 in human liver microsomes reported in previous studies. SUPERMIX may contain catalytically active CYP2C9 in levels higher than those in human liver microsomes.
采用相对活性因子（RAF）方法测定了人肝微粒体中CYP2C9和CYP2C19的相对催化活性。分别以甲苯磺丁脲甲基羟化和S-美芬妥因4'-羟化作为CYP2C9和CYP2C19活性的指标。在人肝微粒体、人淋巴母细胞系微粒体以及表达CYP2C9和CYP2C19的昆虫细胞中研究了这些反应的动力学。RAF计算为同工型特异性指标反应在人肝微粒体中的Vmax（底物浓度饱和时的反应速度）除以由cDNA表达同工型催化的反应的Vmax。还测定了SUPERMIX（一种市售的cDNA表达的人药物代谢CYP混合物，其配制目的是实现类似于人肝微粒体中发现的酶活性平衡）的RAF。人肝微粒体中淋巴母细胞RAF2C9范围为54至145 pmol CYP/mg蛋白（平均值：87），而SUPERMIX的值为251 pmol CYP/mg蛋白。人肝微粒体中昆虫细胞RAF2C9范围为1.6至143 pmol CYP/mg蛋白（平均值：49），而SUPERMIX的值为201 pmol CYP/mg蛋白。人肝微粒体中淋巴母细胞和昆虫细胞RAF2C19均范围为4至45 pmol CYP/mg蛋白（平均值分别为29和28），而SUPERMIX的值为29 pmol CYP/mg蛋白。所使用的cDNA表达系统的性质对作为甲苯磺丁脲甲基羟化酶的CYP2C9或作为S-美芬妥因羟化酶的CYP2C19的动力学参数没有影响。然而，与淋巴母细胞表达的CYP2C19相比，昆虫细胞表达的CYP2C19（包括氧化还原酶）作为甲苯磺丁脲甲基羟化酶具有更高的活性。人肝脏中由淋巴母细胞测定的RAF2C9与RAF2C19的平均比值为3.0（范围1.6 - 17.9；n = 10），而SUPERMIX的该比值为8.6。人肝脏中由昆虫细胞测定的RAF2C9与RAF2C19的平均比值为1.7（范围0.04 - 16.2；n = 10），而SUPERMIX的该比值为7.0。这两个比值均与先前研究报道的人肝微粒体中CYP2C9和2C19免疫定量水平的20:1比值不一致。SUPERMIX中催化活性CYP2C9的含量可能高于人肝微粒体中的含量。
