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

Br J Pharmacol. 2013 May;169(1):21-33. doi: 10.1111/bph.12126.


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 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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antihypertensive Agents / metabolism
  • Biological Transport
  • Bosentan
  • Chromatography, High Pressure Liquid
  • Chromatography, Liquid / methods
  • Cytochrome P-450 CYP3A / genetics
  • Cytochrome P-450 CYP3A / metabolism*
  • Dogs
  • Endothelin Receptor Antagonists
  • Glucuronides / metabolism
  • Glucuronosyltransferase / genetics
  • Glucuronosyltransferase / metabolism*
  • Humans
  • Liver-Specific Organic Anion Transporter 1
  • Madin Darby Canine Kidney Cells
  • Mass Spectrometry / methods
  • Multidrug Resistance-Associated Proteins / genetics
  • Multidrug Resistance-Associated Proteins / metabolism*
  • Organic Anion Transporters / genetics
  • Organic Anion Transporters / metabolism*
  • Pyrimidines / metabolism
  • Sulfonamides / metabolism*
  • Transfection


  • Antihypertensive Agents
  • Endothelin Receptor Antagonists
  • Glucuronides
  • Liver-Specific Organic Anion Transporter 1
  • Multidrug Resistance-Associated Proteins
  • Organic Anion Transporters
  • Pyrimidines
  • Ro 47-8634
  • Ro 48-5033
  • SLCO1B1 protein, human
  • Sulfonamides
  • multidrug resistance-associated protein 2
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • UGT1A1 enzyme
  • Glucuronosyltransferase
  • Bosentan