Species Differences in Hepatobiliary Disposition of Taurocholic Acid in Human and Rat Sandwich-Cultured Hepatocytes: Implications for Drug-Induced Liver Injury

Abstract

The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CL Bile ) versus basolateral efflux (CL BL ) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CL Bile 5 0.14 6 0.04 ml/min per g liver; CL BL 5 0.042 6 0.019 ml/min per g liver), whereas CL Bile and CL BL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CL Bile 5 0.34 6 0.07 ml/min per g liver; CL BL 5 0.26 6 0.07 ml/min per g liver).Troglitazone decreased TCA uptake, CL Bile , and CL BL ; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CL Bile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CL Bile and CL BL ) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.

Prediction of Altered Bile Acid Disposition Due to Inhibition of Multiple Transporters: An Integrated Approach Using Sandwich-Cultured Hepatocytes, Mechanistic Modeling, and Simulation

“…Using the mechanistic model, the estimated total CL Uptake of TCA was 0.63 mL/min/g liver (Table 2) and the unbound CL Uptake of TCA was 4.2 mL/min/g liver (calculated as total CL Uptake /f u,med ). This value is close to the reported unbound CL Uptake (2.2 mL/min/g liver) (Yang et al, 2015). CL BL and CL Bile were similar to values reported previously (CL BL 5 0.042 mL/min/g liver and CL Bile 5 0.14 mL/min/g liver) (Yang et al, 2015).…”

“…A model scheme incorporating linear uptake and efflux CL was adopted (Pfeifer et al, 2013c;Yang et al, 2015) and was fit to d 8 -TCA Cells 1 Bile, Cells, and incubation medium total masstime data from three individual SCHH experiments (Fig. 1A) …”

“…Cellular concentrations were calculated by dividing the mass by the estimated cellular volume of 7.4 mL/mg protein (Pfeifer et al, 2013c;Yang et al, 2015); cytosolic concentrations were calculated by dividing the mass by the estimated cytosolic volume, assuming cytosolic volume represents 70% of cellular volume (Grunberg et al, 2009). Unbound inhibitor concentrations ([I] u ) were calculated as the product of total inhibitor concentration ([I] t ) and f u .…”

“…where C t,Cells represents the total intracellular concentration, and was calculated as X Cells /V Cells ; V Cells was calculated and fixed using the protein content of each preparation and a value of 7.4 mL/mg protein (Pfeifer et al, 2013c;Yang et al, 2015); "1" and "2" refer to Ca 21 -containing (standard HBSS) and Ca 21 -free HBSS, respectively; X t,Cells represents the total amount in Cells; X t,Cells1Bile represents the total amount in Cells 1 Bile; X t,Bile represents the total amount in Bile; C t,Buffer represents the total Buffer concentration; V Buffer was set as a constant (0.3 mL); CL Uptake represents total uptake CL; CL BL represents total basolateral efflux CL; CL Bile represents total biliary CL; and K Flux represents the first-order rate constant that describes the flux from bile networks into the medium due to periodic contraction of the bile canalicular networks (Oshio and Phillips, 1981;Phillips et al, 1982;Lee et al, 2010;Pfeifer et al, 2013c). CL units (mL/min/mg protein) were converted to mL/min/g liver based on the protein content in liver tissue (90 mg protein/g liver) (Sohlenius-Sternbeck, 2006).…”

“…To overcome this limitation, mechanistic pharmacokinetic modeling coupled with data from sandwichcultured hepatocytes has been used to deconvolute the relative contribution of various clearance (CL) pathways to the disposition of rosuvastatin, mycophenolic acid, and 3 H-taurocholic acid (TCA) (Pfeifer et al, 2013c;Matsunaga et al, 2014;Yang et al, 2015). Transporters are expressed and properly localized in the sandwich-cultured hepatocyte system, which can be used to assess the function of multiple transporters (Yang et al, 2016).…”