McGinnity Dermot F, Berry Amanda J, Kenny Jane R, Grime Ken, Riley Robert J
Department of Physical & Metabolic Science, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK.
Drug Metab Dispos. 2006 Aug;34(8):1291-300. doi: 10.1124/dmd.106.009969. Epub 2006 May 5.
Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters kinact and KI) which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4'-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1'-hydroxylation in a time- and concentration-dependent manner. Fluoxetine also inhibited CYP2C19-dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLMs, and recombinant CYP2C19 (KI 0.4 microM and kinact 0.5 min(-1)). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, a proprietary AstraZeneca compound; KI 30 microM and kinact 0.02 min(-1)) effectively abolished CYP2C9 activity over 24 h at low (micromolar) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time-dependent inhibitors, which may have dramatic inhibitory effects on P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate.
培养的原代人肝细胞通常用于通过酶活性终点来评估细胞色素P450（P450）诱导情况。然而，其他过程可能会混淆数据解读。为此，评估了该系统中时间依赖性P450抑制的影响。采用底物盒方法，在与原型抑制剂替尼酸（CYP2C9）、红霉素、醋竹桃霉素和氟西汀（CYP3A4）孵育后测定P450活性。利用肝细胞中酶失活的动力学分析来描述这些时间依赖性抑制剂的作用，并推导抑制参数kinact和KI，其通常与使用重组P450和人肝微粒体（HLM）得到的值高度一致。替尼酸选择性抑制CYP2C9依赖性双氯芬酸4'-羟化活性，红霉素、醋竹桃霉素和氟西汀以时间和浓度依赖性方式抑制CYP3A4依赖性咪达唑仑1'-羟化。氟西汀还以时间和浓度依赖性方式在肝细胞、HLM和重组CYP2C19中抑制CYP2C19依赖性S-美芬妥因4'-羟化（KI 0.4 microM，kinact 0.5 min(-1)）。正如预期的那样，氟西汀对肝细胞中CYP2D6的作用与强效但可逆的抑制作用一致。一种非常弱的时间依赖性CYP2C9抑制剂（AZ1，阿斯利康公司的专利化合物；KI 30 microM，kinact 0.02 min(-1)）在原代培养的人肝细胞中低（微摩尔）浓度下24小时内有效消除了CYP2C9活性。这项工作表明，在用代谢稳定、弱时间依赖性抑制剂进行酶诱导研究的解读时需谨慎小心，这类抑制剂可能对该系统中的P450活性产生显著抑制作用。因此，除了酶活性外，还应测量mRNA和/或蛋白质水平，以全面评估候选药物的P450诱导潜力。
