Impact of probe compound in MRP2 vesicular transport assays Heidi Kidron a,1 , Gloria Wissel a,b,1 , Nenad Manevski a,b , Marika Häkli a , Raimo A. Ketola a , Moshe Finel a , Marjo Yliperttula c , Henri Xhaard a , Arto Urtti a,⇑ a Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland b Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland c Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P.O. Box 56, University of Helsinki, 00014 Helsinki, Finland article info Article history: Received 18 January 2012 Received in revised form 21 February 2012 Accepted 21 February 2012 Available online 3 March 2012 Keywords: Drug transport ABC transporter Efflux Membrane vesicle MRP2 ABCC2 abstract MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test com- pounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7- dichlorofluorescein, leukotriene C 4 and estradiol-17-b-D-glucuronide (E 2 17bG). Five of the test com- pounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used. Ó 2012 Elsevier B.V. All rights reserved. 1. Introduction The ATP-binding cassette (ABC) transporters are a family of membrane proteins that are involved in the ATP-dependent efflux of a wide variety of endogenous compounds and drugs from the cells (Schinkel and Jonker, 2003). The ability to promote resis- tance to chemotherapy has previously been one of the main rea- sons for interest in the ABC transporters, but recently they have also been recognized to play a role in drug–drug interactions (reviewed in Marquez and Van Bambeke, 2011). Multidrug resistance associated protein 2 (MRP2, ABCC2) is a member of the ABCC family, which consists of 13 transporters in human. MRP2 is expressed mainly in the canalicular mem- brane of hepatocytes, where its physiological role is to expel polar and ionic compounds into the bile. Inhibition of MRP2 transport can lead to an accumulation of metabolites and other MRP2 sub- strates within the cell and result in hepatotoxicity. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. In addition, enhanced expres- sion of MRP2 has been observed in various cancer cells, but its involvement in multidrug resistance during cancer therapy is cur- rently unclear (reviewed in Jemnitz et al., 2010). The substrates of MRP2 include endogenous glucuronide, sulfate, and glutathione conjugates, such as leukotriene C 4 (LTC 4 ) andestradiol-17-b-D-glu- curonide (E 2 17bG), as well as different polar and anionic drugs and drug conjugates (reviewed in Jemnitz et al., 2010). Many of the MRP2 substrates have low membrane permeabil- ity, which makes them difficult to study in cell-based assays. The vesicular transport assay with inverted membrane vesicles en- ables direct interaction of the MRP2 transporter with the com- pounds added to the reaction buffer. Therefore, it is a widely used in vitro method to identify substrates, inhibitors, or modu- lators of MRP2 (Bodo et al., 2003; Chu et al., 2004; Heredi-Szabo et al., 2008, 2009; Jemnitz et al., 2010; Pedersen et al., 2008; Zelcer et al., 2003). It is possible to directly measure MRP2 med- iated transport of a radioactively labeled or fluorescent com- pound. However, for unlabeled compounds, particularly in high-throughput screening assays, it is common to indirectly 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2012.02.016 ⇑ Corresponding author. Tel.: +358 9 191 59636; fax: +358 9 191 59725. E-mail addresses: heidi.kidron@helsinki.fi (H. Kidron), gloria.wissel@helsinki.fi (G. Wissel), nenad.manevski@helsinki.fi (N. Manevski), marika.hakli@helsinki.fi (M. Häkli), raimo.ketola@helsinki.fi (R.A. Ketola), moshe.finel@helsinki.fi (M. Finel), marjo.yliperttula@helsinki.fi (M. Yliperttula), henri.xhaard@helsinki.fi (H. Xhaard), arto.urtti@helsinki.fi (A. Urtti). 1 These authors contributed equally to this work. European Journal of Pharmaceutical Sciences 46 (2012) 100–105 Contents lists available at SciVerse ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps