Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse

Abstract

Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent inhibition of bile salt transport.

Keywords: ABCB11; BSEP; Bile salt export pump; Bile salts; DILI; Drug-induced liver injury; Hepatocytes; Species difference.

Fig. 1

The disappearance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in human hepatocyte incubations. Human hepatocytes (0.25 million cells/mL) were incubated with CA or CDCA (1 μM) at 37°C for specified time points (N = 3).

Fig. 2

Effects of incubation time on the formation and transport of glycocholic acid (GCA) (left panel) and chenodeoxycholic acid (GCDCA) (right panel) in human hepatocytes. Human hepatocytes (0.25 million cells/mL) were incubated with CA or CDCA (100 μM) at 37 °C for various time points (N = 3). The amount of GCA or GCDCA in extracellular medium was used for the transport activity, and the sum of the amounts in both hepatocyte pellet and extracellular medium was used for the formation activity.

Fig. 3

Effects of concentrations of cholic acid (CA) (right panel) and chenodeoxycholic acid (CDCA) (left panel) on the formation and transport of glycocholic acid (GCA) and chenodeoxycholic acid (GCDCA) in human hepatocytes. Human hepatocytes (0.25 million cells/mL) were incubated with CA or CDCA at various concentrations at 37 °C for 1 h (N = 3). The amount of GCA or GCDCA in extracellular medium was used for the transport activity, and the sum of the amounts in both hepatocyte pellet and extracellular medium was used for the formation activity.

Fig. 4

Effects of BSEP inhibitors on bile salt transport activity in human hepatocytes in suspension. Furosemide was used as negative control. Human hepatocytes (0.25 million cells/mL) were incubated with CA (10 μM) at 37°C for 1 h (N = 3). The amount of GCA or GCDCA in extracellular medium was used for the transport activity.

Fig. 5

Inhibition of troglitazone (left panel) and glimepiride (right panel) on bile salt transport activity in mouse, rat, dog, monkey, and human hepatocytes. Glycocholic acid (GCA) was measured for rat, human, and monkey, and taurocholic acid (TCA) was used for mouse and dog (N = 3).

Fig. 6

Plots of log (BSEP activity AUC) against log (normalized ROS/ATP ratio) (a) and log (ATP activity AUC) (b). The cut-off value is indicated in dash line of each axis. The category of each drug group is labeled with colors.