Dolberg Anne M, Reichl Stephan
Institut für Pharmazeutische Technologie, Technische Universität Braunschweig , Braunschweig 38106, Germany.
Zentrum für Pharmaverfahrenstechnik, Technische Universität Braunschweig , Braunschweig 38106, Germany.
Mol Pharm. 2017 May 1;14(5):1577-1590. doi: 10.1021/acs.molpharmaceut.6b00838. Epub 2017 Mar 30.
The profound influence of ATP-binding cassette (ABC) transporters on the disposition of numerous drugs has led to increased interest in characterizing their expression profiles in various epithelial and endothelial barriers. The present work examined the presence and functional activity of five ABC efflux proteins, i.e., MRP 1-5, in freshly isolated human nasal epithelial cells and two in vitro models based on the human RPMI 2650 cell line. To evaluate the expression patterns of MRP1, MRP2, MRP3, MRP4, and MRP5 at the mRNA and protein levels in the ex vivo model and the differently cultured RPMI 2650 cells, reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and indirect immunofluorescence staining were used. The functionality of the MRP transporters in the three models was assessed using efflux experiments and accumulation assays with the respective substrates and inhibitors. The mRNA and protein expression of all selected ABC transporters was detected in excised human nasal mucosa as well as in the corresponding cell culture models. Moreover, the functional expression of the MRP transport proteins was demonstrated in the three models for the first time. Therefore, the potential impact of multidrug resistance-associated proteins 1-5 on drug disposition after intranasal administration may be taken into consideration for future developments. The specimens of human nasal turbinate exhibited slightly lower efflux capacities of MRP1, MRP3, and MRP5 in relation to the submerged and ALI-cultured RPMI 2650 cells, but showed a promising comparability to both in vitro models concerning the activity of MRP2 and MRP4. In this regard, the different RPMI 2650 cell culture models will be able to provide useful experimental data in the preclinical phase to estimate the interaction of particular efflux transporters with drug candidates for nasal application.
ATP结合盒（ABC）转运蛋白对众多药物的处置具有深远影响，这使得人们对其在各种上皮和内皮屏障中的表达谱特征的兴趣日益增加。本研究检测了5种ABC外排蛋白，即多药耐药相关蛋白（MRP）1 - 5，在新鲜分离的人鼻上皮细胞以及基于人RPMI 2650细胞系的两种体外模型中的存在情况和功能活性。为了评估MRP1、MRP2、MRP3、MRP4和MRP5在离体模型和不同培养条件下的RPMI 2650细胞中mRNA和蛋白水平的表达模式，采用了逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹分析和间接免疫荧光染色。使用外排实验以及相应底物和抑制剂的积累实验评估了这三种模型中MRP转运蛋白的功能。在所切除的人鼻黏膜以及相应的细胞培养模型中均检测到了所有选定ABC转运蛋白的mRNA和蛋白表达。此外，首次在这三种模型中证实了MRP转运蛋白的功能表达。因此，在未来的研发中，可能需要考虑多药耐药相关蛋白1 - 5对鼻内给药后药物处置的潜在影响。与浸没培养和空气 - 液体界面（ALI）培养的RPMI 2650细胞相比，人鼻甲标本中MRP1、MRP3和MRP5的外排能力略低，但在MRP2和MRP4的活性方面与两种体外模型具有良好的可比性。在这方面，不同的RPMI 2650细胞培养模型将能够在临床前阶段提供有用的实验数据，以评估特定外排转运蛋白与鼻用候选药物之间的相互作用。
