Rhodes Susan P, Otten Jennifer N, Hingorani Gary P, Hartley Dylan P, Franklin Ronald B
Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, USA.
J Pharmacol Toxicol Methods. 2011 May-Jun;63(3):223-6. doi: 10.1016/j.vascn.2010.11.002. Epub 2010 Nov 24.
The human nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) are known to regulate gene expression of the cytochrome P450 (CYP) enzymes, 3A4, 2B6, and 1A2, respectively. In conventional CYP induction studies, the activity of each CYP enzyme is assessed in a separate incubation with the appropriate marker substrate. The objective of this study was to assess, simultaneously, the induction of CYP3A4, CYP2B6, and CYP1A2 activity in cultured human hepatocytes treated with various prototypical ligands of PXR, CAR, and AhR by utilizing an optimized substrate cocktail, as well as a rapid, sensitive liquid chromatography-mass spectrometry method.
To evaluate the xenobiotic-mediated induction of hepatocellular gene expression, the prototypical inducers rifampicin (10 μM) and phenobarbital (1 mM) were used for CYP3A4, CITCO (1 μM) and artemisinin (50 μM) were used for CYP2B6, and 3-methylcholanthrene (1 μM) and omeprazole (50 μM) were utilized for induction of CYP1A2. Primary human hepatocytes were treated with each compound for 48h, followed by a 30-min incubation of the hepatocyte culture along with the addition of three marker substrates for specific CYP activity: midazolam (CYP3A4; 5 μM), bupropion (CYP2B6; 50 μM), and phenacetin (CYP1A2; 100μM). The assessment of CYP activity was performed with a rapid, sensitive liquid chromatography-tandem mass spectrometry method which simultaneously assessed activity of CYP3A4, CYP2B6, and CYP1A2 in a single 3-min method by examining the formation of the probe substrate metabolites, 1'-hydroxymidazolam, hydroxybupropion, and acetaminophen, respectively.
The average fold-induction of CYP3A4, CYP2B6, and CYP1A2 activity was comparable between the cocktail and the conventional assay.
The combination of three marker substrates in a single 30-min incubation, in addition to a rapid, sensitive LC-MS/MS method, resulted in an efficient and robust method for assessing cytochrome P450 induction as compared to the conventional methodology.
已知人类核受体孕烷X受体（PXR）、组成型雄甾烷受体（CAR）和芳烃受体（AhR）分别调节细胞色素P450（CYP）酶3A4、2B6和1A2的基因表达。在传统的CYP诱导研究中，每种CYP酶的活性是在与适当的标记底物单独孵育时进行评估的。本研究的目的是通过使用优化的底物混合物以及快速、灵敏的液相色谱-质谱法，同时评估用PXR、CAR和AhR的各种原型配体处理的培养人肝细胞中CYP3A4、CYP2B6和CYP1A2活性的诱导情况。
为了评估异生物素介导的肝细胞基因表达诱导，使用原型诱导剂利福平（10 μM）和苯巴比妥（1 mM）诱导CYP3A4，使用CITCO（1 μM）和青蒿素（50 μM）诱导CYP2B6，使用3-甲基胆蒽（1 μM）和奥美拉唑（50 μM）诱导CYP1A2。将原代人肝细胞用每种化合物处理48小时，然后将肝细胞培养物孵育30分钟，同时加入三种用于特定CYP活性的标记底物：咪达唑仑（CYP3A4；5 μM）、安非他酮（CYP2B6；50 μM）和非那西丁（CYP1A2；100 μM）。使用快速、灵敏的液相色谱-串联质谱法评估CYP活性，该方法通过分别检测探针底物代谢物1'-羟基咪达唑仑、羟基安非他酮和对乙酰氨基酚的形成，在单个3分钟的方法中同时评估CYP3A4、CYP2B6和CYP1A2的活性。
混合物法与传统检测方法相比，CYP3A4、CYP2B6和CYP1A2活性的平均诱导倍数相当。
与传统方法相比，在单个30分钟的孵育中使用三种标记底物，再结合快速、灵敏的液相色谱-串联质谱法，产生了一种有效且可靠的评估细胞色素P450诱导的方法。
