Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple‐transfected MDCK‐OATP1B1‐UGT1A1‐MRP2 cells

Abstract

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

Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3–Overexpressing Human Embryonic Kidney 293 Cells

“…The SULT293 cells have an advantage over other methods/ tools (such as membrane vesicles and monolayer cells overexpressing a transporter) because drug sulfates (usually lacking in commercial availability) were not required for experimentation as the metabolites are generated from the dosed drug by the cells. In addition, the SULT293 cells were free of the concerns raised in transporter identification studies using membrane vesicle or monolayer cells (Fahrmayr et al, 2012). First, drug sulfates poorly cross cellular membranes by passive diffusion.…”

“…First, drug sulfates poorly cross cellular membranes by passive diffusion. Use of polarized monolayers (expressing a transporter) with administration of the sulfate can be problematic because the sulfate may not enter the cells (Fahrmayr et al, 2012). Second, studies with inside-out vesicles are time consuming and challenging (Fahrmayr et al, 2012).…”

Characterization of Chrysin Glucuronidation in UGT1A1-Overexpressing HeLa Cells: Elucidating the Transporters Responsible for Efflux of Glucuronide

“…Evaluation of glucuronide transport would be seriously complicated by simultaneous transport of other types of metabolites (e.g., sulfates). It was noteworthy that in addition to the transfected HeLa cells, evaluation of glucuronide transport can be well performed using the monolayer cells cooverexpressing UGT enzyme and transporters (e.g., triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells) (Fahrmayr et al, 2012).…”

Transporters and Drug-Drug Interactions: Important Determinants of Drug Disposition and Effects

“…Double-or multiple-transfected cell lines (based on polarized grown MDCKII cells) with the parallel expression of one or more uptake transporters and export pumps can be used for the analysis of vectorial transport processes (Cui et al, 2005;Kopplow et al, 2005;Nies et al, 2008a). Recently the establishment and characterization of a triple-transfected cell line expressing the uptake transporter OATP1B1, the phase II enzyme UGT1A1, and the apically localized export pump MRP2 (Fahrmayr et al, 2012) and of a quadruple-transfected cell line expressing, in addition, the phase I enzyme CYP3A4 (Fahrmayr et al, 2013) has been described. Furthermore, transporter-metabolism interplay has been investigated using organ perfusion systems or studies with Caco-2 cell monolayers (Benet et al, 2003;Pang et al, 2009).…”