The ATP-binding-cassette (ABC) transporter multidrug resistance protein (MRP) 2 (ABCC2) forms a natural barrier and efflux system for various (conjugates of) drugs, other xenotoxins, and endogenous compounds. To obtain insight in the pharmacological and physiological functions of Mrp2, we generated Mrp2 knockout mice, which were viable and fertile but suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin monoglucuronides into bile. The mice also had an 80-fold decreased biliary glutathione excretion and a 63% reduced bile flow. Levels of Mrp3 (Abcc3) in liver and Mrp4 The multidrug transporter MRP2 (ABCC2, cMOAT), a member of the ATP-binding-cassette (ABC) superfamily, confers resistance to a range of anticancer drugs (Borst and Oude Elferink, 2002;Chan et al., 2004). The protein is mainly present in the apical membranes of polarized cells in liver, small intestine, and kidney and mediates active transport of both endogenous and xenobiotic compounds to bile, urine, or feces (Chan et al., 2004). MRP2 is functionally deficient in patients with the Dubin-Johnson syndrome (Zimniak, 1993), in TR Ϫ rats (Jansen et al., 1985), and Eisai hyperbilirubinemic rats (EHBRs) (Hosokawa et al., 1992), which all show impaired secretion of bilirubin glucuronides into the bile and as a consequence suffer from conjugated hyperbilirubinemia (Jansen et al., 1985; Hosokawa et al., 1992;Zimniak, 1993). The mutant rat strains also show substantially reduced biliary excretion of glutathione and glutathione conjugates (Paulusma et al., 1999).Besides its role in transport of endogenous compounds, MRP2 plays an important role in the transport of various 1 These authors contributed equally to this work. Article, publication date, and citation information can be found at

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Purpose: ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX). Experimental Design: We generated and characterized Abcc2;Abcg2 -/-mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Results: Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2 -/-and Abcc2 -/-mice, respectively, and 3.3-fold increased in Abcc2;Abcg2 -/-mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2-/-mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2 -/-mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2 -/-and Abcc2;Abcg2 -/-mice, respectively. This indicates that Abcc2 compensates forAbcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2 -/-mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX. Conclusions: Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.

Purpose: P-glycoprotein (P-gp; ABCB1) efficiently transports lipophilic amphipathic drugs, including the widely used anticancer drug paclitaxel (Taxol). We found previously that human multidrug resistance protein 2 (MRP2; ABCC2) also transports paclitaxel in vitro, and although we expected that paclitaxel pharmacokinetics would be dominated by P-gp, the effect of Mrp2 was tested in vivo. Experimental Design: We generated and characterized Mdr1a/1b/Mrp2 À/À mice, allowing assessment of the distinct roles of Mrp2 and Mdr1a/1b P-gp in paclitaxel pharmacokinetics. Results: Surprisingly, the effect of Mrp2 on i.v. administration of paclitaxel was as great as that of P-gp. The area under plasma concentration-time curve (AUC) i.v. in both Mrp2À/À and Mdr1a/ 1b À/À mice was 1.3-fold higher than in wild-type mice, and in Mdr1a/1b/Mrp2 À/À mice, a 1.7-fold increase was found. In spite of this similar effect, Mrp2 and P-gp had mostly complementary functions in paclitaxel elimination. Mrp2 dominated the hepatobiliary excretion, which was reduced by 80% in Mrp2 À/À mice. In contrast, P-gp dominated the direct intestinal excretion, with a minor role for Mrp2. The AUC oral of paclitaxel was 8.5-fold increased by Mdr1a/1b deficiency but not affected by Mrp2 deficiency. However, in the absence of Mdr1a/1b P-gp, additional Mrp2 deficiency increased the AUC oral another 1.7-fold. Conclusions:Thus far, Mrp2 was thought to mainly affect organic anionic drugs in vivo. Our data show that Mrp2 can also be a major determinant of the pharmacokinetic behavior of highly lipophilic anticancer drugs, even in the presence of other efficient transporters.Variation in MRP2 activity might thus directly affect the effective exposure to paclitaxel, on i.v. administration, but also on oral administration, especially when P-gp activity is inhibited.

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Purpose: ATP-binding cassette sub-family C member 2 [ABCC2; multidrug resistanceâ ssociated protein 2 (MRP2)] and ABCC3 (MRP3) mediate the elimination of toxic compounds, such as drugs and carcinogens, and have a large overlap in substrate specificity.We investigated the roles of Abcc2 and Abcc3 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) in vivo. Experimental Design: Abcc2;Abcc3 -/-mice were generated, characterized, and used to investigate possibly overlapping or complementary roles of Abcc2 and Abcc3 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Results: Abcc2;Abcc3 -/-mice were viable and fertile. In Abcc2 -/-mice, the plasma area under the curve (AUC i.v. ) for MTX was 2.0-fold increased compared with wild type, leading to 1.6-fold increased urinary excretion, which was not seen in Abcc2;Abcc3 -/-mice. Biliary excretion of MTX was 3.7-fold reduced in Abcc2 -/-but unchanged in Abcc2;Abcc3 -/-mice. The plasma AUC i.v. s of 7OH-MTX were 6.0-fold and 4.3-fold increased in Abcc2 -/-and Abcc2;Abcc3 -/-mice, respectively, leading to increased urinary excretion. The biliary excretion of 7OH-MTX was 5.8-fold reduced in Abcc2 -/-but unchanged in Abcc2;Abcc3 -/-mice. 7OH-MTX accumulated substantially in the liver of Abcc2 -/-and especiallyAbcc2;Abcc3 -/-mice. Conclusions: Abcc2 is important for (biliary) excretion of MTX and its toxic metabolite 7OH-MTX.When Abcc2 is absent, Abcc3 transports MTX and 7OH-MTX back from the liver into the circulation, leading to increased plasma levels and urinary excretion. Variation in ABCC2 and/or ABCC3 activity may therefore have profound effects on the elimination and severity of toxicity of MTX and 7OH-MTX after MTX treatment of patients.The multidrug resistance proteins (MRP) ATP-binding cassette sub-family C member 2 (ABCC2; MRP2) and ABCC3 (MRP3) are members of the ATP-binding cassette (ABC) transporter super-family. ABCC2 is present in the apical membranes of hepatocytes and the epithelial cells of the small intestine and kidney, and is involved in the elimination of both endogenous and exogenous compounds from the body (1). ABCC3 is also found in the liver, kidney, and small intestine as well as in the adrenal glands and pancreas. In contrast to ABCC2, ABCC3 localizes to the basolateral membrane of polarized cells (2). Both transporters are expressed in various tumors and can transport a range of (anticancer) drugs (1, 2). There is a large overlap in the substrate specificity of ABCC2 and ABCC3. They can, for example, both transport bilirubin glucuronides (3 -5) and, in patients with the Dubin-Johnson syndrome, who have functionally deficient ABCC2 (6, 7), both plasma bilirubin glucuronide levels and ABCC3 protein levels are increased. It has been speculated that these two findings are related: ABCC3 up-regulation in the absence of ABCC2 would allow increased basolateral efflux of bilirubin glucuronides from the liver (7). Because Abcc2 and Abcc3 are both invol...

Cholyl-L-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(Ϫ/Ϫ), and Abcc3(Ϫ/Ϫ) mice. Human Na ϩ -dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with K m values of 4.6 Ϯ 2.7, 3.3 Ϯ 2.0, and 3.7 Ϯ 1.0 M, respectively. In Abcc2(Ϫ/Ϫ) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(Ϫ/Ϫ) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function.