Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/04/11/dmd.116.069468.DC1 1521-009X/44/6/857–866$25.00 http://dx.doi.org/10.1124/dmd.116.069468 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:857–866, June 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Cysteinyl Leukotriene Receptor 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance Proteins (MRP1-4) s Mark A. Csandl, Gwenaëlle Conseil, and Susan P. C. Cole Departments of Biomedical and Molecular Sciences (M.A.C., S.P.C.C.), and Pathology and Molecular Medicine (G.C., S.P.C.C.), Division of Cancer Biology and Genetics, Queen’s University Cancer Research Institute, Kingston, ON, Canada Received January 13, 2016; accepted April 7, 2016 ABSTRACT Active efflux of both drugs and organic anion metabolites is class of antagonists showed any MRP selectivity. For E217bG Downloaded from mediated by the multidrug resistance proteins (MRPs). MRP1 uptake, LTM IC50s ranged from 1.2 to 26.9 mMandweremost (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), and MRP4 (ABCC4) have comparable for MRP1 and MRP4. The LTM rank order inhibitory partially overlapping substrate specificities and all transport 17b- potencies for E217bGversusLTC4 uptake by MRP1, and E217bG estradiol 17-(b-D-glucuronide) (E217bG). The cysteinyl leukotriene versus PGE2 uptake by MRP4, were also similar. Three of four receptor 1 (CysLT1R) antagonist MK-571 inhibits all four MRP CysLT1R-selective LTMs also stimulated MRP2 (but not MRP3) homologs, but little is known about the modulatory effects of newer transport and thus exerted a concentration-dependent biphasic leukotriene modifiers (LTMs). Here we examined the effects of effect on MRP2. The fourth CysLT1R antagonist, LY171883, only dmd.aspetjournals.org seven CysLT1R- and CysLT2R-selective LTMs on E217bG uptake stimulated MRP2 (and MRP3) transport but none of the MRPs were into MRP1–4-enriched inside-out membrane vesicles. Their effects stimulated by either CysLT2R-selectiveLTM.Weconcludethat,in on uptake of an additional physiologic solute were also measured contrast to their CysLTR selectivity, CysLTR antagonists show no for MRP1 [leukotriene C4 (LTC4)] and MRP4 [prostaglandin E2 MRP homolog selectivity, and data should be interpreted cau- (PGE2)]. The two CysLT2R-selective LTMs studied were generally tiously if obtained from LTMs in systems in which more than one more potent inhibitors than CysLT1R-selective LTMs, but neither MRP is present. at ASPET Journals on October 1, 2021 Introduction Elevated MRP1/ABCC1 levels in tumor cells cause multidrug ’ Nine proteins composing a subset of the C subfamily of the ATP- resistance, consistent with MRP1 s ability to efflux chemotherapeutic binding cassette (ABC) superfamily are known as ABCC or multidrug agents (Cole et al., 1994; Cole, 2014a). MRP1 also protects normal resistance proteins (MRPs) (Slot et al., 2011). The four “short” MRPs tissues from xenobiotics when expressed at blood-tissue interfaces have a four-domain structure with two nucleotide binding domains, each (Wijnholds et al., 1998). In addition, MRP1 mediates the efflux of many preceded by a membrane-spanning domain (MSD1/2). The five “long” organic anions, including glutathione (GSH) and glucuronide-conjugated MRPs have an additional NH2-terminally located MSD0. MSD1 and metabolites (Cole, 2014a, b). The physiologic relevance of MRP1 is MSD2 form the pore through which solutes are translocated, powered by associated with its ability to efflux GSH, as well as cysteinyl leukotriene ATP binding and hydrolysis. (CysLT) C4 (LTC4)and17b-estradiol 17-(b-D-glucuronide) (E217bG) Each MRP has its own distinct, albeit somewhat overlapping, (Jedlitschky et al., 1996; Loe et al., 1996). substrate specificity and tissue distribution, and their roles in human The substrate specificities of MRP2/ABCC2 and MRP1 are similar, (patho)physiology and pharmacology vary accordingly (Slot et al., but unlike MRP1, MRP2 is found predominantly in the liver, where it 2011; Keppler, 2011). The long MRP1–3 and the short MRP4 are the helps maintain biliary homeostasis through elimination of conjugated most pharmacologically relevant MRPs and can influence pharmacoki- metabolites (Nies and Keppler, 2007). In addition, MRP2 deficiency is netics, play important roles in tissue defense, and participate in signaling the underlying cause of Dubin-Johnson syndrome, a disorder charac- pathways (Tang et al., 2013; van der Schoor et al., 2015). terized by conjugated hyperbilirubinemia (Kartenbeck et al., 1996). MRP3/ABCC3 is most closely related to MRP1, but like MRP2 it has a narrower tissue distribution and substrate specificity. Unlike MRP1 and MRP2, MRP3 does not transport GSH and only poorly transports This work was supported by the Canadian Institutes of Health Research [MOP- LTC and other GSH conjugates although it shares an ability to transport 106513, MOP-133584]. M.A.C. was the recipient of the Eldon Boyd Fellowship 4 b from Queen’s University. S.P.C.C. is Canada Research Chair in Cancer Biology E217 G (Kool et al., 1999). Hepatic MRP3 has a role in the disposition and Bracken Chair in Genetics and Molecular Medicine. of certain drugs and/or their metabolites (van de Wetering et al., 2007). dx.doi.org/ 10.1124/dmd.116.069468. The substrate specificity of the short MRP4/ABCC4 overlaps to a s This article has supplemental material available at dmd.aspetjournals.org. lesser extent with the long MRP1–3. Thus MRP4 is involved in the ABBREVIATIONS: ABC, ATP-binding cassette; CysLT, cysteinyl leukotriene; CysLTR, CysLT receptor; E217bG, 17b-estradiol 17-(b-D- glucuronide); GSH, glutathione; HEK, human embryonic kidney; HRPase, horseradish peroxidase; LTC4, leukotriene C4; LTM, leukotriene modifier; mAb, monoclonal antibody; MRP, multidrug resistance protein; PGE2, prostaglandin E2. 857 858 Csandl et al. tissue distribution and elimination of antimetabolites used to treat The development of antagonists or leukotriene modifiers (LTMs) that viral and neoplastic diseases (Schuetz et al., 1999; Slot et al., 2011; Park can distinguish between CysLT1R and CysLT2R raises the possibility et al., 2014). It is also distinct in its ability to efflux prostanoids that, unlike MK-571, one or more of them might also be selective for [e.g., prostaglandin E2 (PGE2)] and cyclic nucleotides (Reid et al., 2003; one of the MRP homologs. The goal of the present study was to test this Lin et al., 2008; Jin et al., 2014). However, like MRP1–3, MRP4 can idea by comparing the effects of seven CysLT1R-selective, CysLT2R- transport E217bG. selective, and non-CysLTR-selective LTMs on organic anion transport There has been a long-standing interest in identifying small-molecule by MRP1–4. inhibitors of MRP1-mediated drug efflux in human tumors (Boumendjel et al., 2005; Cole, 2014a). This interest extended to other MRPs as their roles in drug distribution and elimination have been elucidated. MK-571 Materials and Methods was originally developed as a CysLT receptor (CysLTR) antagonist to Materials. AMP, ATP, and E217bG were from Sigma-Aldrich (Oakville, ON, treat asthma (Jones et al., 1989). However, it also sensitizes tumor cells Canada). LTC4 was from Calbiochem (San Diego, CA). Dulbecco’s modified expressing MRP1, and inhibits MRP1-mediated transport of organic Eagle’s medium (DMEM), OptiMEM, and Lipofectamine 2000 were from Gibco/ Life Technologies (Burlington, ON) and protease inhibitors were from Roche anions including LTC4 (Gekeler et al., 1995; Cole, 2014a). MK-571 also modulates transport by other MRPs (Keppler, 2011), other ABC (Mississauga, ON). MK-571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phe- transporters (Matsson et al., 2009), the solute carrier OATP1B3/ nyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) (Fig. 1A), montelukast (1-[[[(1R)-1-[3-(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3- SLCO1B3 (Letschert et al., 2005) and flavonol conjugation (Barrington [2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]-methyl]-cyclopropaneacetic acid)) et al., 2015). Despite its well documented lack of selectivity, MK-571 is (Fig. 1A), pranlukast (N-[4-oxo-2-(1H-tetrazol-5-yl)-4H-1-benzopyran-8-yl]-4-(4- Downloaded from still the most widely used small-molecule MRP inhibitor today. phenylbutoxy)-benzamide) (Fig. 1A), LY171883 (1-[2-hydroxy-3-propyl-4-[4- The CysLTs LTC4 and LTD4 act on two classes of G protein-coupled (1H-tetrazol-5-yl)butoxy]phenyl]-ethanone) (Fig. 1A), BAY-u9773 (4-[[(1R, CysLTRs (Haeggström and Funk, 2011). CysLT1R has a prominent role 2E,4E,6Z,9Z)-1-[(1S)-4-carboxy-1-hydroxybutyl]-2,4,6,9-pentadecatetraenyl] in the pathogenesis of asthma, and is selectively targeted by the thio]-benzoic acid) (Fig. 1C), and PGE2 were from Cayman Chemical (Ann experimental agents MK-571 and LY171883, and the clinically used Arbor, MI). HAMI 3379 (3-[[(3-carboxycyclohexyl)amino]carbonyl]-4-[3-[4- [4-(cyclohexyloxy)butoxy]phenyl]propoxy]-benzoic acid) (Fig. 1B) and BayCysLT2 montelukast and pranlukast (Fig. 1A) (Lynch et al., 1999; Sarau et al., dmd.aspetjournals.org (3-[[(3-carboxycyclohexyl)amino]carbonyl]-4-[3-[4-(4-phenoxybutoxy)phenyl] 1999) . The role of CysLT2R in human health and disease is less well propoxy]-benzoic acid) (Fig. 1B) were kind gifts from Dr. Colin Funk understood. It is found in immune cells and the vasculature of several 3 21 (Queen’s University, Kingston, ON). [6,7- H(N)]E217bG (42 Ci mmol ), tissues, including brain and heart (Heise et al., 2000; Nothacker 3 21 3 [14,15,19,20- H(N)]LTC4 (166.3 Ci mmol ), and [5,6,8,11,12,14,15- H(N)] et al., 2000). Two experimental CysLT2R-selective inhibitors are 21 PGE2 (153.7 Ci mmol ) were from PerkinElmer Life Sciences (Boston, MA). available, HAMI 3379 and BayCysLT2 (Fig. 1B) (Wunder et al., Murine monoclonal antibody (mAb) M2Ι-4 (anti-MRP2) (ALX-801-015) and rat 2010; Ni et al., 2011). mAb M4Ι-10 (anti-MRP4) (ALX-801-038) were from Enzo (Farmingdale, NY).