title: An experimental approach to evaluate the impact of impaired transport function on hepatobiliary drug disposition using Mrp2-deficient TR- rat sandwich-cultured hepatocytes in combination with Bcrp knockdown.
abstract: 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 excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR(-) rat SCH decreased by 28.6 (95% CI: 5.8-51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5-9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR(-) rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR(-) rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total 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 acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired.
authors.affiliation: Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, §Curriculum in Toxicology, and ⊥Department of Biostatistics, UNC Gillings School of Public Health, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, 27599-7569, United States.
authors.forename: Kyunghee
authors.initials: K
authors.lastname: Yang
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Pfeifer
authors.forename: Rhiannon N
authors.initials: RN
authors.lastname: Hardwick
authors.forename: Wei
authors.initials: W
authors.lastname: Yue
authors.forename: Paul W
authors.initials: PW
authors.lastname: Stewart
authors.forename: Kim L R
authors.initials: KL
authors.lastname: Brouwer
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001659:Biliary Tract / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D002478:Cells, Cultured; D003902:Detergents / Q000494:pharmacology; D005464:Fluorobenzenes / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008297:Male; D011743:Pyrimidines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000068718:Rosuvastatin Calcium; D013449:Sulfonamides / Q000494:pharmacology*; D013656:Taurocholic Acid / Q000494:pharmacology*
chemical_list: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; C489249:Abcg2 protein, rat; D003902:Detergents; D005464:Fluorobenzenes; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013449:Sulfonamides; D013656:Taurocholic Acid; D000068718:Rosuvastatin Calcium