Hepatobiliary Disposition of Troglitazone and Metabolites in Rat and Human Sandwich-Cultured Hepatocytes: Use of Monte Carlo Simulations to Assess the Impact of Changes in Biliary Excretion on Troglitazone Sulfate Accumulation

Abstract

This study examined the hepatobiliary disposition of troglitazone (TGZ) and metabolites [TGZ sulfate (TS), TGZ glucuronide (TG), and TGZ quinone (TQ)] over time in rat and human sandwich-cultured hepatocytes (SCH). Cells were incubated with TGZ; samples were analyzed for TGZ and metabolites by liquid chromatography-tandem mass spectrometry. SCH mimicked the disposition of TGZ/metabolites in vivo in rats and humans; TGZ was metabolized primarily to TS and to a lesser extent to TG and TQ. In human SCH, the biliary excretion index (BEI) was negligible for TGZ and TQ, ϳ16% for TS, and ϳ43% for TG over the incubation period; in rat SCH, the BEI for TS and TG was ϳ13 and ϳ41%, respectively. Hepatocyte accumulation of TS was extensive, with intracellular concentrations ranging from 132 to 222 M in rat SCH; intracellular TGZ concentrations ranged from 7.22 to 47.7 M. In human SCH, intracellular TS and TGZ concentrations ranged from 136 to 160 M and from 49.4 to 84.7 M, respectively. Pharmacokinetic modeling and Monte Carlo simulations were used to evaluate the impact of modulating the biliary excretion rate constant (K bile ) for TS on TS accumulation in hepatocytes and medium. Simulations demonstrated that intracellular concentrations of TS may increase up to 3.1-and 5.7-fold when biliary excretion of TS was decreased 2-and 10-fold, respectively. It is important to note that altered hepatobiliary transport and the extent of hepatocyte exposure may not always be evident based on medium concentrations (analogous to systemic exposure in vivo). Pharmacokinetic modeling/simulation with data from SCH is a useful approach to examine the impact of altered hepatobiliary transport on hepatocyte accumulation of drug/metabolites.

Models to Predict Unbound Intracellular Drug Concentrations in the Presence of Transporters

“…Enzyme kinetic modeling has predicted drug concentrations across cell monolayers in both apical-to-basolateral (A-B) and basolateral-to-apical (B-A) directions. Another example is a study wherein hepatobiliary disposition of troglitazone and its metabolites was evaluated in human sandwich-layered hepatocytes (Lee et al, 2010). Pharmacokinetic modeling and simulations based on hepatocyte data indicated that intracellular drug concentrations would increase if biliary excretion decreased.…”

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“…6 However, cholestasis may leave residual effects; for example, mitochondrial function might be inhibited secondarily after accumulation of bile acids in the intracellular space. 7,8 In turn, malfunctioning mitochondria produce less adenosine triphosphate, which in turn further impairs adenosine triphosphate-dependent BSEP functions. 9 Therefore, drug-induced and/or bile acid-induced dual inhibition of mitochondrial and BSEP functions might result in prolonged and severe drug-induced cholestasis, as in the case presented here.…”

Hepatocellular Exposure of Troglitazone Metabolites in Rat Sandwich-Cultured Hepatocytes Lacking Bcrp and Mrp2: Interplay between Formation and Excretion

“…TGZ concentrations of 1 and 10 mM were selected for investigation because they yielded unbound concentrations of TGZ in the S9 fraction that were similar to the unbound concentrations in hepatocytes. Intracellular total concentrations of TGZ ranged from 100 to 250 mM after a 30-minute incubation of WT and TR -rat SCH with 10 mM TGZ (Lee et al, 2010a). Assuming that the intracellular unbound fraction (f u ) is equal to the plasma f u of 0.000921 (Izumi et al, 1996), intracellular concentrations of unbound TGZ would be in the range of 0.09-0.23 mM.…”

“…LC-MS/MS Analysis. TGZ and generated metabolites were analyzed by LC-MS/MS as described previously (Lee et al, 2010a). Briefly, the medium and cells or cells+bile lysate samples were centrifuged at 12,000g for 10 minutes at 4°C, and the supernatant was diluted 1:6 (v/v) with 79%:21% (v/v) methanol/ water containing the internal standard (ethyl warfarin).…”

“…Previously published data suggested that TS is excreted into bile predominantly via breast cancer resistance protein (Bcrp) and multidrug resistance-associated protein 2 (Mrp2) (Kostrubsky et al, 2001;Enokizono et al, 2007). Extensive hepatic accumulation of TS (Kawai et al, 1997;Lee et al, 2010a), coupled with the finding that TS potently inhibits bile acid transporters (e.g., BSEP, MRP4), led to the hypothesis that TS is primarily responsible for altered bile acid disposition and subsequent hepatotoxicity; impaired function of Bcrp and Mrp2 (e.g., attributable to underlying disease, genetic variations, or drug-drug interactions) would increase hepatocellular TS accumulation and enhance inhibition of bile acid transport (Fig. 1).…”