Biliary Drug Excretion and Drug-drug Interactions in Sandwich Cultured Human Hepatocytes Predicted from Inverted Membrane Vesicles and Targeted Proteomics
(English)Manuscript (preprint) (Other academic)
The ABC multidrug resistance proteins in the canalicular membrane are important contributors to the pharmacokinetic properties of drugs and endogenous compounds. In this study, a new approach for determining the individual contribution from three ABC transporters to the biliary clearance was investigated. First, the inhibition of P-glycoprotein (Pgp/ABCB1), Breast Cancer Resistance Protein (BCRP/ABCG2) and Multidrug-Resistance Associated Protein 2 (MRP2/ABCC2) was investigated in inverted membrane vesicles from HEK293 cells overexpressing each of the ABC proteins. Inhibition profiles of prototypic substrate transport were obtained for 24 compounds and compared with results obtained in alternative expression systems. A common substrate for the three transporters was found in estradiol 17β-glucuronide (E17G). The contribution of each transporter to the E17G biliary efflux was investigated in HEK vesicles and in sandwich cultured human hepatocytes (SCHH), using nine inhibitors with different specificity towards each of the ABC-proteins. After quantification of transport kinetics and protein expression, the maximal transport activity (MTA) of each of the transporters was calculated and their contribution to the biliary clearance of E17G was predicted. The results show a good correlation between HEK-MRP2 and Sf9-MRP2 vesicle data. However, up to 40-fold lower IC50-values were obtained for Pgp and BCRP in the HEK vesicles compared to cellular expression systems. The physiologically more relevant SCHH were found to identify ABC inhibitors observed in the vesicle system that lost their ABC inhibition in this more complex model. SCHH therefore contribute with important additional information impossible to assess in vesicular systems. Finally, the MTA and subsequent of biliary clearance determinations were used to predict the absolute biliary excretion of E17G in SCHH, which was predicted with a prediction accuracy of 90%.
IdentifiersURN: urn:nbn:se:uu:diva-204199OAI: oai:DiVA.org:uu-204199DiVA: diva2:637918