An Experimental Approach To Evaluate the Impact ofImpaired Transport Function on Hepatobiliary Drug Disposition UsingMrp2-Deficient TR– Rat Sandwich-Cultured Hepatocytesin Combination with Bcrp Knockdown

摘要

Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR^–) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1–10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR^– status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretionof RSV in vivo in rats. Relative to WT rat SCH, marginalmean BEI (%) of RSV in TR^– rat SCH decreased by28.6 (95% CI: 5.8–51.3). Ad-siBcrp decreased marginal meanBEI (%) of RSV by 13.3 (7.5–9.1) relative to SCH infected withadenoviral vectors expressing a nontargeting shRNA (Ad-siNT). TheBEI of RSV was almost ablated in TR^– rat SCH withBcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT ratSCH (45.4 ± 6.6%). These results demonstrated the feasibilityof Bcrp knockdown in TR^– rat SCH as an invitro system to assess the impact of impaired Bcrp and Mrp2function. At MOI of 5, viral infection had minimal effects on RSVtotal accumulation, but significantly decreased marginal mean taurocholatetotal accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0–12.8)and 7.5 (3.7–11.3), respectively, relative to noninfected SCH.These findings may be due to off-target effects on hepatic bile acidtransporters, even though no changes in protein expression levelsof the hepatic bile acid transporters were observed. This study establisheda strategy for optimization of the knockdown system, and demonstratedthe potential use of RNAi in SCH as an in vitro toolto predict altered hepatobiliary drug disposition when canaliculartransporters are impaired.