The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug‐Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2

The canalicular membrane of hepatocytes contains several transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated transport processes is the vesicular transport assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a transport assay for a known or potential substrate and test small molecule inhibition of the transporters.