Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, Missouri 64108, United States.
Mol Pharm. 2012 Jul 2;9(7):1877-86. doi: 10.1021/mp200487h. Epub 2012 Jun 19.
The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drug of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 μM naringin and 3 μM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and Western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The Vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be 10- and 3-fold lower in MMC as compared to MDCK-WT and MDCK-MDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT, indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined activities of CYP3A4 and P-gp. Transport of cortisol increased 5-fold in the presence of naringin in MMC and doubled in MDCK-MDR1. Cortisol transport in MMC was significantly lower than that in MDCK-WT in the presence of naringin. The permeability increased 3-fold in the presence of morphine, which is a weaker inhibitor of CYP3A4. Formation of 6β-hydroxy cortisol was found to decrease in the presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes toward drug-drug interactions.
本研究旨在表征和利用同时表达 P-糖蛋白和 CYP3A4 的 MDCK 细胞系作为评估药物-草药和药物滥用相互作用的体外模型。通过表达和活性研究开发并表征了同时表达 P-糖蛋白和 CYP3A4（MMC）的 MDCK 细胞系。进行了 200μM 皮质醇的细胞转运研究，以确定它们的联合活性。该研究在 MDCK-WT、MDCK-MDR1 和 MMC 细胞系中进行。在 50μM 柚皮苷和 3μM 吗啡存在的情况下也进行了类似的研究。通过 HPLC 分析样品以检测药物及其 CYP3A4 代谢物。PCR、qPCR 和 Western blot 研究证实了转染细胞中蛋白质的增强表达。Vivid CYP3A4 测定和酮康唑抑制研究进一步证实了活性蛋白的存在。与 MDCK-WT 和 MDCK-MDR1 相比，皮质醇的顶端到基底转运在 MMC 中分别降低了 10 倍和 3 倍。与 MDCK-WT 相比，MMC 中形成的代谢物更多，表明 CYP3A4 的表达增强。由于 CYP3A4 和 P-糖蛋白的联合活性，在 MMC 细胞系中观察到最高量的皮质醇代谢物形成。在 MMC 中，皮质醇的转运在柚皮苷存在的情况下增加了 5 倍，在 MDCK-MDR1 中增加了 2 倍。在 MMC 中，皮质醇的转运明显低于 MDCK-WT 在柚皮苷存在的情况下。在吗啡存在的情况下，通透性增加了 3 倍，吗啡是 CYP3A4 的较弱抑制剂。在吗啡和柚皮苷存在的情况下，发现 6β-羟基皮质醇的形成减少。这种新的模型细胞系具有增强的 CYP3A4 和 P-糖蛋白水平，加上较短的培养时间，可以作为研究药物相互作用的宝贵模型。该细胞系还可用于研究外排转运蛋白和代谢酶对药物相互作用的联合贡献。
