Vesicle- and Hepatocyte-Based Assays for Identification of Drug Candidates Inhibiting BSEP Function

Transporters play a crucial role in the uptake of endo- and exogenous molecules in hepatocytes and efflux into the bile. The bile salt export pump (BSEP; ABCB11) is of major importance for efflux of bile salts to the bile and BSEP inhibition frequently provokes drug-induced cholestasis. This chapter describes two assays to determine inhibition of BSEP-mediated bile salt excretion. The first assay uses inside-out membrane vesicles, prepared from BSEP-transfected cell lines. The cholestasis potential of compounds can be determined by specifically investigating the ability to inhibit BSEP-mediated uptake of tauro-nor-THCA-24-DBD, a fluorescent bile salt derivative. For the second assay, relative accumulation of tauro-nor-THCA-24-DBD in sandwich-cultured hepatocytes, which represents a more biorelevant in vitro system, is investigated. Through incubation with standard or Ca²⁺/Mg²⁺-free buffer, the substrate signal can be determined in the cells and bile or the cells alone, respectively. Performing this assay in the presence and absence of potentially interfering compounds of interest enables exploration of the relative effect of these compounds on biliary excretion of the probe substrate.