Di Marco Annalise, Yao Dan, Laufer Ralph
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Merck Sharp and Dohme Research Laboratories, Rome, Italy.
Eur J Biochem. 2003 Sep;270(18):3768-77. doi: 10.1046/j.1432-1033.2003.03763.x.
Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.
肝微粒体制剂通常用于预测因细胞色素P450（CYP）介导的代谢受到抑制而可能在体内发生的药物相互作用。然而，游离药物（底物和抑制剂）在其肝内作用部位的浓度，这一无法直接测量的变量，可能与微粒体孵育系统中的浓度显著不同。完整细胞更能反映CYP底物和抑制剂在肝脏中所接触的环境，因此，评估一种药物在分离的肝细胞中引起CYP抑制的潜力可能是有必要的。本研究的目的是比较一系列CYP2D抑制剂在大鼠肝微粒体和肝细胞中的抑制效力。为此，我们开发了一种适用于快速分析这两种系统中CYP介导的药物相互作用的检测方法，使用放射性标记的右美沙芬，它是CYP2D家族酶的一种特征明确的探针底物。右美沙芬去甲基化在大鼠微粒体和肝细胞中表现出饱和动力学，其表观Km值和Vmax值分别为2.1与2.8 μM以及每毫克微粒体蛋白0.74 nM·min⁻¹与每毫克细胞蛋白0.11 nM·min⁻¹。奎宁、奎尼丁、吡苄明、普罗帕酮、维拉帕米、酮康唑和特非那定抑制大鼠肝微粒体和肝细胞中的右美沙芬O - 去甲基化，IC50值在低微摩尔范围内。其中一些化合物表现出双相抑制动力学，表明与不止一种CYP2D同工型相互作用。尽管在这两种系统之间未观察到抑制效力的重要差异，但大多数抑制剂，包括奎宁和奎尼丁，在肝细胞中的IC50比在微粒体中低2 - 3倍。发现奎宁和奎尼丁与细胞相关的浓度显著高于细胞外培养基中的浓度，这表明细胞内积累可能增强这些化合物的作用。对于因细胞转运而在肝脏中富集的化合物，对完整肝细胞中的CYP抑制进行研究可能是必要的。
