Brimer C, Dalton J T, Zhu Z, Schuetz J, Yasuda K, Vanin E, Relling M V, Lu Y, Schuetz E G
Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Pharm Res. 2000 Jul;17(7):803-10. doi: 10.1023/a:1007599923694.
To develop model polarized cell systems expressing cytochrome P4503A4. NADPH P450 reductase, and P-glycoprotein (Pgp).
LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp, the product of the multidrug resistance gene (MDR1), were transfected stably using either a mammalian neomycin selectable expression vector (CYP3A4-Neo) or an episomal vector based on Epstein-Barr virus (CYP3A4-Hygro). These CYP3A4 expressing cells were compared with LLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4 vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase (AdRed) transduction. Cells were characterized for expression of CYP3A4 protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays were performed to determine whether infection with recombinant Ad3A4 +/- AdRed affected Pgp function.
The rank order of optimal CYP3A4 expression and activities in LLC-PKI and L-MDR1 cells from highest to lowest was cells cotransduced with Ad3A4 plus AdRed >> Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo. Similarly, coexpression of Ad3A4 plus AdRed led to enhanced CYP3A4 mediated metabolism in Caco-2 cells over cells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolite in the medium. In microsomes from Caco-2 and LLC-PK1 cells, each co-transduced with Ad3A4/AdRed, Vmax values for testosterone 6beta-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg, respectively. For either Caco-2 or LLC-MDR1 cells, TEER values and the rate of apical to basal and basal to apical transport of vinblastine or digoxin were similar in cells with and without Ad3A4/Red transduction.
Polarized cellular systems coexpressing Ad3A4, AdRed, and the MDR1/Pgp transporter were developed and characterized. The results document the utility of these polarized model systems for simultaneous drug transport/drug metabolism studies. Since the experimental approach can be adapted to study the interplay of multiple enzyme/ transporting systems, it may find significant application as a screening tool for the pharmaceutical industry and as a more basic research tool to study the kinetics of intestinal drug bioavailability.
构建表达细胞色素P4503A4、NADPH细胞色素P450还原酶和P-糖蛋白（Pgp）的极化细胞模型系统。
使用哺乳动物新霉素选择表达载体（CYP3A4-Neo）或基于爱泼斯坦-巴尔病毒的附加型载体（CYP3A4-Hygro），稳定转染稳定表达多药耐药基因（MDR1）产物Pgp的LLC-PK1和衍生的L-MDR1细胞。将这些表达CYP3A4的细胞与用腺病毒-3A4载体（Ad3A4）转导的LLC-PK1、L-MDR1或Caco-2细胞进行比较，转导过程中或不进行腺病毒-细胞色素P450还原酶（AdRed）的同时转导。对细胞进行CYP3A4蛋白表达以及CYP3A4介导的咪达唑仑和睾酮代谢的表征。进行膜完整性分析和药物转运试验，以确定重组Ad3A4 +/- AdRed感染是否影响Pgp功能。
LLC-PKI和L-MDR1细胞中最佳CYP3A4表达和活性从高到低的排序为：与Ad3A4加AdRed共转导的细胞>> Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo。同样，与单独使用Ad3A4的细胞相比，Ad3A4加AdRed的共表达导致Caco-2细胞中CYP3A4介导的代谢增强。用咪达唑仑孵育表达Ad3A4/AdRed的Transwell培养细胞，可在培养基中轻松检测到代谢产物。在分别与Ad3A4/AdRed共转导的Caco-2和LLC-PK1细胞的微粒体中，睾酮6β-羟化酶活性的Vmax值分别为414至1350 pmoles/分钟/毫克。对于Caco-2或LLC-MDR1细胞，在有或没有Ad3A4/Red转导的细胞中，TEER值以及长春碱或地高辛从顶端到基底和从基底到顶端的转运速率相似。
构建并表征了共表达Ad3A4、AdRed和MDR1/Pgp转运蛋白的极化细胞系统。结果证明了这些极化模型系统在同时进行药物转运/药物代谢研究中的实用性。由于该实验方法可用于研究多种酶/转运系统之间的相互作用，因此它可能作为制药行业的筛选工具以及研究肠道药物生物利用度动力学的更基础研究工具具有重要应用价值。
