PREDICTION OF DRUG INTERACTION IN VITRO -FOCUSING ON CYTOCHROME P-450 3A AND P-GLYCOPROTEIN-

利用培养的Caco-2细胞单层，我们试图研究细胞色素P-450 (CYP) 3A4和p -糖蛋白（P-gp）的参与，并预测口服药物在体外的药物相互作用。Probucol是一种降脂药物，可引起环孢素a （CYP3A4和P-gp的底物）跨Caco-2细胞单层转运的P-gp非依赖性抑制。各种诱导剂预处理均未能增强p- gp诱导的药物向Caco-2细胞单层顶端侧的挤压。Caco-2细胞微粒体部分即使用CYP诱导剂处理，也没有表现出由CYP3A4介导的硝苯地平氧化。因此，我们使用完整的大鼠进行了离体实验。来自肠上皮和肝脏的微粒体显示硝苯地平氧化（平均生成速率分别为35和160 pmol/min/mg蛋白）。构建离体肠道灌注系统，检测P-gp底物药物的吸收和分泌。P-gp抑制剂（维拉帕米，酮康唑）增加了P-gp底物地高辛向外膜侧的转运，并减少了向腔内灌注的分泌，表明该系统可用于评估P-gp在肠道药物吸收中的作用。用苯巴比妥（一种cypp诱导剂）治疗，肝脏中CYP3A4活性增加（+ 130%），但肠上皮中没有。另一种诱导剂利福平提高肠上皮CYP3A4活性（+ 32%），但不影响肝脏活性。利福平还能提高肠道P-gp的活性。这些离体研究结果表明，CYP诱导剂可能引起CYP或P-gp活性的组织特异性诱导。组织特异性诱导药物代谢或药物挤压的机制有待阐明。这可能有助于了解药物相互作用的机制。

Using cultured Caco-2 cell monolayers, we tried to investigate the involvement of cytochrome P-450 (CYP) 3A4 and P-glycoprotein (P-gp) and to predict drug interaction of orally administered agents in vitro. Probucol, a lipid-lowering drug, caused P-gp-independent suppression of cyclosporine A (a substrate of CYP3A4 and P-gp) transport across Caco-2 cell monolayers. Pretreatment with various inducers failed to augment the P-gp-induced drug extrusion into the apical side of Caco-2 cell monolayers. Microsome fraction from Caco-2 cells even treated with CYP inducers did not demonstrate oxidization of nifedipine which is mediated by CYP3A4.We thus performed ex vivo experiments using intact rats. Microsome fractions from the intestinal epithelium and liver showed oxidation of nifedipine (averaged production rate ; 35 and 160 pmol/min/mg protein, respectively). We constructed ex vivo intestinal perfusion system and examined the absorption and secretion of P-gp substrate drug. P-gp inhibitor (verapamil, ketoconazole) increased the transport of digoxin, a P-gp substrate, to the adventitial side, and decreased the secretion into the luminal perfusate, indicating that this system can be used to assess the involvement of P-gp in the drug absorption at the intestinal tract. Treatment with phenobarbital, a CYP inducer, increased CYP3A4 activity in the liver (+ 130 %), but not intestinal epithelium. Another inducer, rifampicin, elevated CYP3A4 activity in the intestinal epithelium (+ 32 %) without affecting the activity in the liver. Rifampicin also enhanced the P-gp activity in the intestine. These results from ex vivo study suggest that CYP inducers may cause tissue-specific induction of CYP or P-gp activity. The mechanism of tissue-specific induction of drug metabolism or drug extrusion needs to be clarified. It may be helpful to understand the mechanism of drug interaction.