Published OnlineFirst December 8, 2009; DOI: 10.1158/1535-7163.MCT-09-0668 3350 Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo Maria L. H. Vlaming,1 Anita van Esch,1 Zeliha Pala,3 and Abcg2 together mediate the rapid elimination of Els Wagenaar,1 Koen van de Wetering,1 MTX and 7OH-MTX after i.v. administration and can Olaf van Tellingen,2 and Alfred H. Schinkel1 to a large extent compensate for each other's absence. This may explain why it is still comparatively safe to Divisions of 1Molecular Biology and 2Clinical Chemistry, The use a toxic drug such as MTX in the clinic, as the risk Netherlands Cancer Institute, Amsterdam, the Netherlands; of highly increased toxicity due to dysfunctioning of and 3Department of Pharmacology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey ABCC2, ABCC3, or ABCG2 alone is limited. Neverthe- less, cotreatment with possible inhibitors of ABCC2, ABCC3, and ABCG2 should be done with utmost cau- Abstract tion when treating patients with methotrexate. [Mol The multidrug transporters ABCC2, ABCC3, and ABCG2 Cancer Ther 2009;8(12):3350–9] can eliminate potentially toxic compounds from the body and have overlapping substrate specificities. To in- Introduction vestigate the overlapping functions of Abcc2, Abcc3, and Abcg2 in vivo, we generated and characterized The ATP-binding cassette (ABC) transporters ABCC2, Abcc3;Abcg2−/− and Abcc2;Abcc3;Abcg2−/− mice. We ABCC3, and ABCG2 are transmembrane proteins that are subsequently analyzed the relative impact of these involved in the export of potentially toxic endogenous transport proteins on the pharmacokinetics of the anti- and exogenous compounds from the cell. ABCC2 and cancer drug methotrexate (MTX) and its main, toxic, ABCG2 are expressed in the apical cell membrane of hepa- metabolite 7-hydroxymethotrexate (7OH-MTX) after i.v. tocytes and epithelial cells of small intestine and kidney, administration of MTX (50 mg/kg). Whereas in single pumping their substrates into bile, feces, and urine. ABCC3 is expressed basolaterally in hepatocytes and intestinal epi- and double knockout mice, the plasma and liver concen- – trations of MTX and 7OH-MTX decreased rapidly after thelial cells and pumps its substrates toward the blood (1 MTX administration, in the Abcc2;Abcc3;Abcg2−/− 5). Due to their sites of expression, these ABC transporters mice, they remained very high. One hour after adminis- are involved in the elimination of potential toxins from the tration, 67% of the MTX dose was still present in livers body, thereby protecting the organism from these toxins. of Abcc2;Abcc3;Abcg2−/− mice as MTX or 7OH-MTX ABCC2, ABCC3, and ABCG2 have very broad substrate versus 7% in wild-type, showing dramatic liver accumu- specificities. They can influence the pharmacokinetics of a lation of these toxic compounds when Abcc2, Abcc3, wide range of (anticancer) drugs and carcinogens, as well as potentially toxic endogenous compounds such as bile and Abcg2 were all absent. Furthermore, the urinary – and fecal excretion of the nephrotoxic metabolite 7OH- salts, bilirubin, or porphyrins (1 6). The overlap in substrate MTX were increased 27- and 7-fold, respectively, in specificity of these three proteins is relatively large. They Abcc2;Abcc3;Abcg2−/− mice. Thus, Abcc2, Abcc3, can, for example, all transport the anticancer drugs etopo- side and methotrexate (MTX), as well as a range of glucuro- nide conjugates of drugs and endogenous compounds (1–9). The widely used anticancer and antirheumatic drug MTX Received 7/21/09; revised 10/15/09; accepted 10/27/09; published is transported by many ABC transporters such as ABCB1, OnlineFirst 12/8/09. ABCC1-5, and ABCG2 in vitro (1,6,7,10,11).Using − − Grant support: NKI 2003-2940 from the Dutch Cancer Society. Z. Pala Abcc2;Abcg2 / mice, we have shown recently that Abcc2 was supported by a grant of the Scientific and Technological Research Council of Turkey. K. van de Wetering was supported by ZonMw TOP and Abcg2 are the main determinants for the biliary excre- grant 40-00812-98-07-028. tion of MTX and its main toxic metabolite 7-hydroxymetho- The costs of publication of this article were defrayed in part by the trexate (7OH-MTX) after i.v. administration of MTX (50 payment of page charges. This article must therefore be hereby marked −/− advertisement in accordance with 18 U.S.C. Section 1734 solely to mg/kg; ref. 12). Furthermore, using Abcc2 and Abcc2; −/− indicate this fact. Abcc3 mice, we found that when Abcc2 was absent (up- Note: Supplementary material for this article is available at Molecular regulated), Abcc3 in the liver caused increased sinusoidal Cancer Therapeutics Online (http://mct.aacrjournals.org/). efflux of MTX and 7OH-MTX from liver (where 7OH- Requests for reprints: Alfred H. Schinkel, Division of Molecular Biology, MTX is primarily formed) to blood, leading to increased uri- The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX −/− Amsterdam, the Netherlands. Phone: 31-20-5122046; Fax: 011-31-20- nary excretion of both compounds (13). Using Abcc3 6691383. E-mail: [email protected] mice, Kitamura et al. (14) recently showed that Abcc3 in- Copyright © 2009 American Association for Cancer Research. creases the systemic exposure to [3H]MTX after oral admin- doi:10.1158/1535-7163.MCT-09-0668 istration or continuous infusion in mice by mediating Mol Cancer Ther 2009;8(12). December 2009 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst December 8, 2009; DOI: 10.1158/1535-7163.MCT-09-0668 Molecular Cancer Therapeutics 3351 basolateral efflux in liver and duodenum. They found no ments Australia Pty. Ltd. MRPr1 and M4I-80 were kind gifts − − difference between wild-type and Abcc4 / mice in the oral of Dr. G.L. Scheffer (Free University Hospital, Amsterdam, plasma pharmacokinetics of [3H]MTX. Also, in ABCC2- the Netherlands). A polyclonal antibody against mouse deficient rats, it was shown that the biliary excretion of Abcc2 (25) was kindly provided by Prof. Dr. J.-M. Fritschy MTX was only 10% of the biliary MTX excretion in wild- (University of Zürich, Zürich, Switzerland). Real-time PCR type rats (15). Combining these literature data, there seem primers were from QIAGEN. to be important and overlapping functions of especially Western Analysis Abcc2, Abcc3, and Abcg2 in the pharmacokinetics of Crude membrane fractions from tissues were prepared as MTX and 7OH-MTX. Also, in humans, it was shown that described (23, 26). Western blotting was done as described specific mutations or polymorphisms in ABCC2 or ABCG2 (27). Equal protein loading was confirmed by Ponceau S could influence the pharmacokinetics of MTX, which was staining of the membranes after transfer. Abcc1 (Mrp1), in some cases associated with increased toxicity or efficacy Abcc2 (Mrp2), and Abcc4 (Mrp4) were detected with mono- in patients with loss-of-function mutations (16–20). No clonal antibodies MRPr1 (dilution, 1:1,000), a polyclonal anti- correlations between ABCC3 polymorphisms and MTX murine Abcc2 antibody (dilution, 1:1,000), and M4I-80 (di- pharmacokinetics or toxicity have been reported yet. lution, 1:400), respectively. Bound primary antibodies were Although quite extensive in vivo studies on the influence detected by incubating the blot with horseradish peroxi- of MTX (and 7OH-MTX) have been done already, the rela- dase–labeledrabbitanti-ratIgG(1:1,000;DAKO)orgoat tive effect of the different ABC transporters is unclear. When anti-rabbit IgG (1:1,000; DAKO). Densitometric analysis using single (and even double) knockout mice, the effects of was done using the TINA 2.09 software program (Raytest). the deleted gene(s) might be underestimated because other Real-time PCR Analysis transporters may compensate for their loss. For example, we RNA isolation, cDNA synthesis, and real-time PCR anal- did not find an effect of Abcc3 on MTX pharmacokinetics ysis on livers of female mice (n = 3) were done as described − − after i.v. bolus administration when we analyzed Abcc3 / (28). mice, but when Abcc2 was additionally deleted, a clear ef- Histologic, Clinical-Chemical, and Hematologic fect of Abcc3 expression was found (13). The same was true Analysis for the effect of Abcg2 on 7OH-MTX pharmacokinetics, Histologic analysis of tissues (n = 5, male and female), which only became apparent in the absence of Abcc2 (12). standard clinical chemistry analyses on serum (twice within In the present study, we describe the generation and char- atimespanof1.5y,n =5–6), and standard hematologic − − − − acterization of Abcc3;Abcg2 / and Abcc2;Abcc3;Abcg2 / analysis of male and female mice (n = 6) were done as de- mice. These mice were subsequently used to further unravel scribed (21). the overlapping and possibly compensatory functions of Plasma and Tissue Pharmacokinetic Experiments − − Abcc2, Abcc3, and Abcg2 in determining the pharmacoki- MTX was administered to female wild-type, Abcc3;Abcg2 / − − netics of MTX and 7OH-MTX in vivo.Weshowherethat and Abcc2;Abcc3;Abcg2 / mice (n =3–13) by injecting 5 μL/g these three transporters are the main determinants of the body weight of a 10 mg/mL MTX solution in saline into the rapid elimination of MTX and 7OH-MTX from the liver tail vein. Animals were killed by terminal bleeding through and plasma. Other ABC transporters seem less important cardiac puncture under methoxyflurane anesthesia at 7.5, in these processes. 15, 30, 60, or 120 min after MTX administration, and organs were removed at each time point. Fecal and Urinary MTX Excretion Experiment − − − − Materials and Methods Female wild-type, Abcc3;Abcg2 / and Abcc2;Abcc3;Abcg2 / Animals mice (n =5–9) were individually housed in Ruco Type M/1 Mice were housed and handled according to institution- stainless steel metabolic cages. They were allowed 24 h to al guidelines complying with Dutch legislation.