[Evaluation of P-glycoprotein mediated in vitro loperamide biliary excretion with sandwich-cultured rat hepatocytes model].
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 functional bile canalicular networks after 5 days of culture. Rh123 (10 micromol x L(-1)) was then incubated with the SCRH in standard Ca+ Hanks buffer or Ca(2+)-free buffer. The cumulative cell uptake and canalicular efflux of Rh123 under Ca2+ and Ca(2+)-free conditions were measured with a LC-MS/MS method. The biliary excretion index (BEI) and instinct biliary clearance (CL(bile, int)) were calculated. To assess the effect of known P-gp inhibitors on the efflux of Rh123, cyclosporine A (CyA), tariquidar (TQD) or quinidine (QND) (10, 50 and 100 micromol x L(-1)) was pre-incubated separately with SCRH for 30 min, then co-incubated with Rh123. The BEI and CL(bile, int) of Rh123 obtained from the SCRH model were (17.8 +/- 1.3) % and (10.7 +/- 0.9) mL x min(-1) x 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 CL(bile, int) for LPAD (20 micromol x L(-1)) were obtained after it was incubated with SCRH for 30 min, and found to be (12.9 +/- 1.2)% and (6.1 +/- 0.3) mL x min(-1) x 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.
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China.
Guo-Lin
GL
Shen
Xiao-Mei
XM
Zhuang
Mei
M
Yuan
Han-Xiong
HX
Sun
Hua
H
Li
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors*; D000818:Animals; D001659:Biliary Tract / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D016572:Cyclosporine / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008139:Loperamide / Q000378:metabolism*; D008297:Male; D011802:Quinidine / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020112:Rhodamine 123 / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011804:Quinolines; D020112:Rhodamine 123; D008139:Loperamide; D016572:Cyclosporine; D011802:Quinidine; C402343:tariquidar