“三明治”培养大鼠原代肝细胞模型评价P-gp介导的洛哌丁胺胆汁排泄

Evaluation of P-glycoprotein mediated in vitro loperamide biliary excretion with sandwich-cultured rat hepatocytes model

应用模型药物罗丹明123(Rh123)和"三明治"培养大鼠原代肝细胞(SCRH),建立并验证基于P-gp的胆汁排泄体外评价模型。SCRH培养5天后形成胆管,加入Rh123(10μmol.L-1),在含Ca2+和无Ca2+Hanks缓冲液中孵育,用LC-MS/MS分别测定Rh123的细胞蓄积量,计算胆汁排泄指数(BEI)和体外的内在胆汁清除率(CLbile,int)。在孵育体系中预先加入已知的P-gp抑制剂环孢素A(CyA)、tariquidar(TQD)和奎尼丁(QND)预孵育30 min,评价抑制P-gp转运体对底物胆汁排泄的影响。Rh123的BEI值为(17.8±1.3)%,平均CLbile,int值为(10.7±0.9)mL.min—1.kg—1,3个抑制剂对Rh123的胆汁清除均呈现剂量依赖性的抑制作用,表明模型构建成功。在此模型上,研究了P-gp介导的洛哌丁胺(LPAD)胆汁排泄以及P-gp抑制剂的影响。LPAD的BEI值为(12.9±1.2)%,平均CLbile,int为(6.1±0.3)mL.min—1.kg—1。CyA、TQD和QND对LPAD胆汁排泄也呈现浓度依赖的抑制作用,证实了P-gp转运体在LPAD胆管外排中的作用,与P-gp抑制剂合用可显著降低LPAD经胆汁的排泄。本文应用SCRH建立了B-Clear模型,为评价新药候选物基于转运体的胆汁排泄和相互作用提供了一个有用的体外工具。

An in vitro P-glycoprotein mediated drug biliary excretion model （B-Clear model） was developed and validated using sandwich-cultured rat hepatocytes （SCRH） and a model substrate rhodamine 123 （Rh123）. SCRH formed fimctional bile canalicular networks after 5 days of culture. Rh123 （10 pmol＇L-1） was then incubated with the SCRH in standard Ca2＋ Hanks buffer or Ca2＋-free buffer. The cumulative cell uptake and canalicular effiux of Rh123 under Ca2＋ and Ca2＋-free conditions were measured with a LC-MS/MS method. The biliary excretion index （BEI） and instinct biliary clearance （CLbile, int） were calculated. To assess the effect of known P-gp inhibitors on the effiux of Rh123, cyclosporine A （CyA）, tariquidar （TQD） or quinidine （QND） （10, 50 and 100 pmol＇L-1） was pre-incubated separately with SCRH for 30 min, then co-incubated with Rh123. The BEI and CLbil int of Rhl23 obtained from the SCRH model were （17.8 ± 1.3） % and （10.7± 0.9） mL.min-l.kg-1, respectively. All the three P-gp inhibitors showed a dose-dependent inhibition on the bile clearance of Rh123, indicating that the B-Clear model with SCRH was functional properly. The biliary excretion of loperamide （LPAD） and the role of P-gp were further investigated with this validated model. The BEI and CLbile, mt for LPAD （20 μmol.L-1） were obtained after it was incubated with SCRH for 30 min, and foundto be （12.9 ±1.2） % and （6.1± 0.3） mL＇minl＇kg 1 respectively. The dose-dependent inhibition on LPAD biliary excretion by CyA, TQD or QND confirmed the major role of P-gp in LPAD canalicular efflux. The results suggested that the B-Clear model with SCRH would be a useful tool for evaluation of P-gp mediated efflux and drug-drug interaction.