Phase I and II metabolism and MRP2‐mediated export of bosentan in a MDCKII‐OATP1B1‐CYP3A4‐UGT1A1‐MRP2 quadruple‐transfected cell line

Abstract

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-

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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).…”

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