Centre for Applied Pharmacokinetic Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom.
Drug Metab Dispos. 2010 Dec;38(12):2139-46. doi: 10.1124/dmd.110.035824. Epub 2010 Sep 16.
Accurate assignment of the concentration of victim drug/inhibitor available at the enzyme active site, both in vivo and within an in vitro incubation, is an essential requirement in rationalizing and predicting drug-drug interactions. Inhibitor accumulation within the liver, whether as a result of active transport processes or intracellular binding, may best be accounted for using hepatocytes rather than hepatic microsomes to estimate in vitro inhibitory potency. The aims of this study were to compare K(i) values determined in rat liver microsomes and freshly isolated rat hepatocytes of four cytochrome P450 (P450) inhibitors (clarithromycin, enoxacin, nelfinavir, and saquinavir) with known hepatic transporter involvement and a range of uptake (cell/medium concentration ratios 20-3000) and clearance (10-1200 μl/min/10(6) cells) properties. Inhibition studies were performed using two well established P450 probe substrates (theophylline and midazolam). Comparison of unbound K(i) values showed marked differences between the two in vitro systems for inhibition of metabolism. In two cases (clarithromycin and enoxacin, both low-clearance drugs), inhibitory potency in hepatocytes markedly exceeded that in microsomes (10- to 20-fold), and this result was consistent with their high cell/medium concentration ratios. For nelfinavir and saquinavir (high-clearance, extensively metabolized drugs), the opposite trend was seen in the K(i) values: despite very high cell/medium concentration ratios, stronger inhibition was evident within microsomal preparations. Hence, the consequences of hepatic accumulation resulting from uptake transporters vary according to the clearance of the inhibitor. This study demonstrates that transporter-enzyme interplay can result in differences in inhibitory potency between microsomes and hepatocytes and hence drug-drug interaction predictions that are not always intuitive.
准确测定酶活性部位中可用的受检药物/抑制剂的浓度，无论是在体内还是在体外孵育中，都是合理化和预测药物相互作用的必要条件。无论是由于主动转运过程还是细胞内结合，肝内抑制剂的蓄积都最好使用肝细胞而不是肝微粒体来估计体外抑制效力。本研究的目的是比较四种细胞色素 P450（CYP）抑制剂（克拉霉素、依诺沙星、奈法唑酮和沙奎那韦）的 K i 值，这些抑制剂已知与肝转运体有关，且具有广泛的摄取（细胞/介质浓度比为 20-3000）和清除率（10-1200 μl/min/10 6 细胞）特性，在大鼠肝微粒体和新鲜分离的大鼠肝细胞中进行测定，并与已知的肝转运体参与和一系列摄取（细胞/介质浓度比 20-3000）和清除率（10-1200μl/min/10 6 细胞）特性进行比较。抑制研究使用两种已建立的 CYP 探针底物（茶碱和咪达唑仑）进行。与两种体外系统相比，未结合 K i 值的比较显示出代谢抑制作用存在明显差异。在两种情况下（克拉霉素和依诺沙星，均为低清除率药物），肝细胞中的抑制效力明显高于微粒体（10-20 倍），这一结果与其高细胞/介质浓度比一致。对于奈法唑酮和沙奎那韦（高清除率，广泛代谢药物），K i 值则出现相反的趋势：尽管细胞/介质浓度比非常高，但在微粒体制剂中观察到更强的抑制作用。因此，摄取转运体引起的肝蓄积的后果因抑制剂的清除率而异。本研究表明，转运体-酶相互作用可导致微粒体和肝细胞之间的抑制效力存在差异，从而导致药物相互作用的预测并不总是直观的。
