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

使用培养的Caco-2细胞单层，我们试图研究细胞色素P-450（CYP）3A 4和P-糖蛋白（P-gp）的参与，并预测体外口服药物的药物相互作用。普罗布考是一种降脂药物，可引起环孢素A（CYP 3A 4和P-gp的底物）跨Caco-2细胞单层转运的P-gp非依赖性抑制。用各种诱导剂预处理未能增加P-gp诱导的药物挤出到Caco-2细胞单层的顶侧。Caco-2细胞的微粒体组分即使用CYP 3A 4诱导剂处理也没有显示出由CYP 3A 4介导的硝苯地平氧化。来自肠上皮和肝脏的微粒体组分显示硝苯地平氧化（平均生产率;分别为35和160 pmol/min/mg蛋白质）。我们构建了离体肠灌流系统，并检测了P-gp底物药物的吸收和分泌。P-gp抑制剂（维拉帕米、酮康唑）可增加P-gp底物地高辛向外膜侧的转运，减少其向肠腔灌流液的分泌，表明该系统可用于评价P-gp参与药物在肠道的吸收。用苯巴比妥（一种CYP 1A 1诱导剂）处理可增加肝脏中的CYP 3A 4活性（+130%），但不增加肠上皮的活性。另一种诱导剂利福平可升高肠上皮细胞中的CYP 3A 4活性（+32%），而不影响肝脏中的活性。利福平还增强了肠道中P-gp的活性。这些来自体外研究的结果表明，β-淀粉样蛋白诱导剂可能导致β-淀粉样蛋白或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出的机制需要澄清。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.