Balakrishnan Anand, Sussman Daniel J, Polli James E
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, USA.
Pharm Res. 2005 Aug;22(8):1269-80. doi: 10.1007/s11095-005-5274-8. Epub 2005 Aug 3.
The human apical sodium-dependent bile acid transporter (hASBT) represents a potential target for prodrug design to increase oral drug absorption. Unfortunately, available monolayer cell culture models do not reliably express hASBT, and nonpolarized cells only allow for uptake assessment, which limits prodrug development efforts. The objective of this study was to develop and characterize a stably transfected hASBT-MDCK cell line.
cDNA encoding hASBT was cloned into pcDNA3.1-V5-polyHis-B to generate an expression plasmid that was then transfected into MDCK-II cells. Clonal populations were chosen based on high hASBT activity and monolayer integrity. Western blot confirmed the expression of the recombinant hASBT; functionality was characterized using taurocholic acid.
In the selected clone, hASBT-mediated taurocholate permeability across hASBT-MDCK monolayers was almost 25-fold higher with sodium, than without sodium where hASBT is not functional. In the presence of sodium, taurocholate and mannitol permeabilities were 23.0x10(-6) cm/sec and 2.60x10(-6) cm/s, respectively, indicating high hASBT functionality and monolayer integrity. hASBT-MDCK monolayer properties were stable over 6 months and demonstrated low within-day variability. Taurocholate uptake and inhibition kinetic parameters from hASBT-MDCK were similar to those obtained from hASBT-COS7 model, confirming hASBT functionality in hASBT-MDCK.
Results indicate that the developed hASBT-MDCK system is a competent, high-expression, stable assay for hASBT transport and inhibition studies.
人顶端钠依赖性胆汁酸转运体（hASBT）是前体药物设计以提高口服药物吸收的潜在靶点。遗憾的是，现有的单层细胞培养模型不能可靠地表达hASBT，并且非极化细胞仅允许进行摄取评估，这限制了前体药物的开发工作。本研究的目的是开发并表征一种稳定转染的hASBT-MDCK细胞系。
将编码hASBT的cDNA克隆到pcDNA3.1-V5-polyHis-B中以产生表达质粒，然后将其转染到MDCK-II细胞中。基于高hASBT活性和单层完整性选择克隆群体。蛋白质免疫印迹法证实了重组hASBT的表达；使用牛磺胆酸对其功能进行了表征。
在所选克隆中，hASBT介导的牛磺胆酸盐跨hASBT-MDCK单层的通透性在有钠存在时比无钠时（此时hASBT无功能）高近25倍。在有钠存在的情况下，牛磺胆酸盐和甘露醇的通透性分别为23.0×10⁻⁶ cm/秒和2.60×10⁻⁶ cm/秒，表明hASBT具有高功能和单层完整性。hASBT-MDCK单层特性在6个月内保持稳定，并且日内变异性低。来自hASBT-MDCK的牛磺胆酸盐摄取和抑制动力学参数与从hASBT-COS7模型获得的参数相似，证实了hASBT在hASBT-MDCK中的功能。
结果表明，所开发的hASBT-MDCK系统是用于hASBT转运和抑制研究的一种有效、高表达且稳定的检测方法。
