Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT)

被引:40
作者
Balakrishnan, A [1 ]
Sussman, DJ [1 ]
Polli, JE [1 ]
机构
[1] Univ Maryland, Sch Pharm, Dept Pharmaceut Sci, Baltimore, MD 21201 USA
关键词
ASBT; bile acid; cell culture; prodrug; transporter;
D O I
10.1007/s11095-005-5274-8
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Purpose. 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. Methods. 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. Results. 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 23.0 x 10(-6) cm/sec and 2.60 x 10(-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. Conclusions. Results indicate that the developed hASBT-MDCK system is a competent, high-expression, stable assay for hASBT transport and inhibition studies.
引用
收藏
页码:1269 / U13
页数:13
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