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Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport.

ABSTRACT: Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na(+)-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid transport individually or in combination. [(3)H]-Taurocholate served as the NTCP/BSEP probe substrate. Individually, cyclosporin A and rifampin decreased taurocholate in vitro biliary clearance (Cl(biliary)) and biliary excretion index (BEI) by more than 20% in rat SCH, suggesting that these drugs primarily inhibited canalicular efflux. In contrast, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, carbamazepine, pioglitazone, and troglitazone decreased the in vitro Cl(biliary) by more than 20% with no notable change in BEI, suggesting that these drugs primarily inhibited taurocholate uptake. Cassette dosing (n=2-4 compounds per cassette) in rat SCH yielded similar findings, and results in human SCH were consistent with rat SCH. In summary, cassette dosing in SCH is a useful in vitro approach to identify compounds that inhibit the hepatic uptake and/or excretion of bile acids, which may cause DILI.

SUBMITTER: Wolf KK

PROVIDER: S-EPMC3129609 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport.

Wolf Kristina K KK Vora Sapana S Webster Lindsey O LO Generaux Grant T GT Polli Joseph W JW Brouwer Kim L R KL

Toxicology in vitro : an international journal published in association with BIBRA 20090823 1

Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na(+)-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid t ...[more]

PMID: 19706322

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Project description:PurposeProbe substrates are used routinely to assess transporter function in vitro. Administration of multiple probe substrates together as a "cocktail" in sandwich-cultured human hepatocytes (SCHH) could increase the throughput of transporter function assessment in a physiologically-relevant in vitro system. This study was designed to compare transporter function between cocktail and single agent administration in SCHH.MethodsRosuvastatin, digoxin, and metformin were selected as probe substrates of hepatic transporters OATP1B1, OATP1B3, BCRP, P-gp, and OCT1. Total accumulation (Cells+Bile) and biliary excretion index (BEI) values derived from administration of the cocktail were compared to values obtained after administration of single agents in the absence and presence of a model inhibitor, erythromycin estolate.ResultsFor rosuvastatin and metformin accumulation, the ratio of means [90% confidence interval (CI)] for cocktail to single agent administration was 100% [94%, 106%] and 90% [82%, 99%], respectively. Therefore, the cocktail and single-agent mode of administration were deemed equivalent per standard equivalence criterion of 80-120% for rosuvastatin and metformin accumulation, but not for digoxin accumulation (77% [62%, 92%]). The ratio of means [90% CI] for rosuvastatin BEI values between the two administration modes (105% [97%, 114%]) also was deemed equivalent. The ratio for digoxin BEI values between the two administration modes was 99% [78%, 120%]. In the presence of erythromycin estolate, the two administration modes were deemed equivalent for evaluation of rosuvastatin, digoxin, and metformin accumulation; the ratio of means [90% CI] was 104% [94%, 115%], 94% [82%, 105%], and 100% [88%, 111%], respectively. However, rosuvastatin and digoxin BEI values were low and quite variable in the presence of the inhibitor, so the BEI results were inconclusive.ConclusionsThese data suggest that rosuvastatin and metformin can be administered as a cocktail to evaluate the function of OATP1B1, OATP1B3, BCRP, and OCT1 in SCHH, and that digoxin may not be an ideal component of such a cocktail.

Project description:Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na(+)-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Dataset Information

Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport.

Publications

Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport.

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