Section of Pharmaceutical Design and Drug Delivery, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Section of Pharmaceutical Design and Drug Delivery, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark; Bioneer-FARMA, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Eur J Pharm Sci. 2018 Jan 15;112:112-121. doi: 10.1016/j.ejps.2017.11.007. Epub 2017 Nov 13.
Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm), immunocytochemistry and low fluxes of the paracellular flux marker [C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. K and V of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124μM and 111±16pmol·cm·min (n=3, total N=6), respectively. V and K of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01μM (n=3, total N=6) and 0.04±0.01μM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC values of inhibition, K values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.
最近，我们转染了猪肠细胞系 IPEC-J2，使其表达人 P-糖蛋白（P-gp，ABCB1）。由此产生的细胞系 iP-gp 在顶膜中高表达功能性人 P-gp，而低表达非人类三磷酸腺苷结合盒（ABC）转运体。本研究的目的是研究 iP-gp 细胞系在确定原型 P-gp 底物地高辛和罗丹明 123 的跨上皮转运动力学方面的可用性。细胞系在培养 16 天后生成紧密的单层，表现为高跨上皮电阻值（TEER>15,000Ω·cm）、免疫细胞化学和低透过率的细胞旁渗透标记物[C]-甘露醇。单层完整性不受常见溶剂二甲基亚砜（DMSO）、甲醇和乙醇在高达 2%（v/v）浓度的影响。[H]标记的地高辛和罗丹明 123 的跨上皮通量在不同的供体浓度下进行测量，并估计动力学参数。P-gp 介导的罗丹明 123 基底外侧至顶侧（B-A）通量的 K 和 V 估计值分别为 332±124μM 和 111±16pmol·cm·min（n=3，总 N=6）。由于地高辛的水溶解度低，地高辛 B-A 通量的 V 和 K 无法估计。选择性 P-gp 抑制剂佐斯奎达（LY-335979）的半最大抑制浓度（IC）分别估计为 0.05±0.01μM（n=3，总 N=6）和 0.04±0.01μM（n=3，总 N=6），用于地高辛和罗丹明 123 作为底物的转运实验。在转染的 Madin Darby 犬肾细胞（MDCK II MDR1）中测量地高辛和罗丹明 123 的双向通量，并与 iP-gp 细胞单层获得的通量进行比较。由于细胞旁途径更紧密，iP-gp 细胞中的外排比最高。总之，地高辛和罗丹明 123 均可用于获得抑制的 IC 值，仅使用罗丹明 123 可获得 K 值。观察到的紧密性、对溶剂的稳健性以及高外排比证实，iP-gp 细胞系可用作研究底物-P-gp 相互作用和调节 P-gp 功能的有用筛选工具。
