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Phase I and II metabolism and MRP 2‐mediated export of bosentan in a MDCKII ‐ OATP1B1‐CYP3A4‐UGT1A1‐MRP2 quadruple‐transfected cell line
Author(s) -
Fahrmayr C,
König J,
Auge D,
Mieth M,
Münch K,
Segrestaa J,
Pfeifer T,
Treiber A,
Fromm MF
Publication year - 2013
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12126
Subject(s) - bosentan , multidrug resistance associated protein 2 , cell culture , transfection , cyp3a4 , intracellular , pharmacology , cell , biology , chemistry , transporter , metabolism , biochemistry , endothelin receptor , receptor , atp binding cassette transporter , cytochrome p450 , genetics , gene
Background and Purpose Hepatic 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 Approach A 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 Results Intracellular 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 R o 48–5033 and R o 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 Implications These 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.