Uptake transporters in the basolateral membrane of hepatocytes are important for the hepatobiliary elimination of drugs. Further, since drug-metabolizing enzymes are located intracellularly, uptake into hepatocytes is a prerequisite for their subsequent metabolism. Therefore, alteration of uptake transporter function (e.g., by concomitantly administered drugs or due to functional consequences of genetic variations, leading to reduced transport function) may result in a change in drug pharmacokinetics. In this review, we focus on the hepatocellularly expressed members of the OATP and OCT family, their impact on transport-mediated drug-drug interactions, and on the functional consequences of variations in genes encoding these transporters.

BACKGROUND AND PURPOSEThe coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. EXPERIMENTAL APPROACHUsing monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single-(OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. KEY RESULTSIntracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. CONCLUSIONS AND IMPLICATIONSEzetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition. AbbreviationsBSP, bromosulphophthalein; MRP2, multidrug resistance protein 2; OATP1B1, organic anion transporting polypeptide 1B1; UGT1A1, UDP-glucuronosyltransferase 1A1

The human organic anion transporting polypeptide 1B3 (OATP1B3), located in the basolateral membrane of hepatocytes, mediates the uptake of endogenous substrates such as taurocholate and drugs from blood into hepatocytes. The transport activity of OATP1B3 is influenced by positively charged amino acids, which are facing the central pore. Molecular modeling was performed to select conserved positively charged amino acids, which may influence transport activity and anchoring of OATP1B3 in the plasma membrane. The modeling revealed that Lys361 faces the pore, and Lys399 is oriented to the plasma membrane. Therefore, the mutants L361ϾA, L361ϾR, L399ϾA, and L399ϾR were generated using sitedirected mutagenesis to investigate the impact of the positive charges on transport activity and anchoring in the membrane. Transport kinetic analyses for the substrates sulfobromophthalein and taurocholate showed a loss of function for the L361ϾA mutant, whereas the transport activity was maintained by the L361ϾR mutant, indicating that the positive charge at position 361 is important for transport activity of OATP1B3. Comparative modeling with OATP1A2 and OATP2B1 revealed that the pore size around this lysine residue is larger in OATP1A2 and smaller in OATP2B1 compared with OATP1B3, which could be related to the respective substrate spectra. Cell surface expression of L399ϾA and L399ϾR was decreased to 16 and 72% compared with wild-type OATP1B3 (p Ͻ 0.001), respectively, indicating that the positive charge of lysine at position 399 is necessary for an unimpaired cell surface expression. Furthermore, we provide a summary of amino acids, which influence the transport activity of OATP1B3.

BACKGROUND AND PURPOSEHepatic uptake (e.g. by OATP1B1), phase I and II metabolism (e.g. by CYP3A4, UGT1A1) and subsequent biliary excretion (e.g. by MRP2) are key determinants for the pharmacokinetics of numerous drugs. However, stably transfected cell models for the simultaneous investigation of transport and phase I and II metabolism of drugs are lacking. EXPERIMENTAL APPROACHA newly established quadruple-transfected MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 cell line was used to investigate metabolism and transcellular transport of the endothelin receptor antagonist bosentan. KEY RESULTSIntracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) was significantly lower in all cell lines expressing MRP2 compared to cell lines lacking this transporter (P < 0.001). Accordingly, considerably higher amounts of bosentan equivalents were detectable in the apical compartments of cell lines with MRP2 expression (P < 0.001). HPLC and LC-MS measurements revealed that mainly unchanged bosentan accumulated in intracellular and apical compartments. Furthermore, the phase I metabolites Ro 48-5033 and Ro 47-8634 were detected intracellularly in cell lines expressing CYP3A4. Additionally, a direct glucuronide of bosentan could be identified intracellularly in cell lines expressing UGT1A1 and in the apical compartments of cell lines expressing UGT1A1 and MRP2. CONCLUSIONS AND IMPLICATIONSThese in vitro data indicate that bosentan is a substrate of UGT1A1. Moreover, the efflux transporter MRP2 mediates export of bosentan and most likely also of bosentan glucuronide in the cell system. Taken together, cell lines simultaneously expressing transport proteins and metabolizing enzymes represent additional useful tools for the investigation of the interplay of transport and metabolism of drugs. AbbreviationsCYP3A4, cytochrome P450 enzyme 3A4; Mrp2/MRP2, rodent/human multidrug resistance protein 2; OATP1B1, organic anion transporting polypeptide 1B1; Ro 47-