Maria L. H. Vlaming

Physiological and pharmacological roles of ABCG2 (BCRP): recent findings in Abcg2 knockout mice.

The multidrug transporter ABCG2 (BCRP/MXR/ABCP) can actively extrude a broad range of endogenous and exogenous substrates across biological membranes. ABCG2 limits oral availability and mediates hepatobiliary and renal excretion of its substrates, and thus influences the pharmacokinetics of many drugs. Recent work, relying mainly on the use of Abcg2(-/-) mice, has revealed important contributions of ABCG2 to the blood-brain, blood-testis and blood-fetal barriers. Together, these functions indicate a primary biological role of ABCG2 in protecting the organism from a range of xenobiotics. In addition, several other physiological functions of ABCG2 have been observed, including extrusion of porphyrins and/or porphyrin conjugates from hematopoietic cells, liver and harderian gland, as well as secretion of vitamin B(2) (riboflavin) and possibly other vitamins (biotin, vitamin K) into breast milk. However, the physiological significance of these processes has been difficult to establish, indicating that there is still a lot to learn about this intriguing protein.

Carcinogen and Anticancer Drug Transport by Mrp2 in Vivo: Studies Using Mrp2 (Abcc2) Knockout Mice

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 (Abcc4) in kidney of Mrp2-/- mice were approximately 2-fold increased. After oral administration of the food-derived carcinogens [14C]PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and [14C]IQ (2-amino-3-methylimidazo[4,5-f]quinoline) plasma values were 1.9- and 1.7-fold higher in Mrp2-/- mice versus wild-type mice, respectively, demonstrating the role of Mrp2 in restricting exposure to these compounds. At a high dose of 50 mg/kg of the drug [3H]methotrexate, the plasma area under the curve for i.v. administration was 1.8-fold higher in Mrp2-/- mice (1345 ± 207 versus 734 ± 81 min ·μg/ml). No clear plasma concentration difference arose at low dose (1 mg/kg). Subsequently, Mdr1a/b/Mrp2 knockout mice were generated. Their biliary excretion of doxorubicin after i.v. administration (5 mg/kg) was 54-fold decreased (0.32 ± 0.13 versus 17.30 ± 6.59 nmol/g liver in wild type), and a role for both Mdr1a/b and Mrp2 in this process was revealed. Our results demonstrate that the Mrp2-/- mouse provides a valuable tool for studies of the impact of Mrp2 on behavior of drugs and other toxins, especially when combined with other ABC transporter knockout mice.

Multidrug Resistance Proteins 2 and 3 Provide Alternative Routes for Hepatic Excretion of Morphine-Glucuronides

Glucuronidation is a major hepatic detoxification pathway for endogenous and exogenous compounds, resulting in the intracellular formation of polar metabolites that require specialized transporters for elimination. Multidrug resistance proteins (MRPs) are expressed in the liver and can transport glucuronosyl-conjugates. Using morphine as a model aglycone, we demonstrate that morphine-3-glucuronide (M3G), the predominant metabolite, is transported in vitro by human MRP2 (ABCC2), a protein present in the apical membrane of hepatocytes. Loss of biliary M3G secretion in Mrp2(-/-) mice results in its increased sinusoidal transport that can be attributed to Mrp3. Combined loss of Mrp2 and Mrp3 leads to a substantial accumulation of M3G in the liver, from which it is transported across the sinusoidal membrane at a low rate, resulting in the prolonged presence of M3G in plasma. Our results show that murine Mrp2 and Mrp3 provide alternative routes for the excretion of a glucuronidated substrate from the liver in vivo.

Multidrug Resistance Protein 2 Is an Important Determinant of Paclitaxel Pharmacokinetics

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−/− 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 Mrp2−/− and Mdr1a/1b−/− mice was 1.3-fold higher than in wild-type mice, and in Mdr1a/1b/Mrp2−/− 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−/− mice. In contrast, P-gp dominated the direct intestinal excretion, with a minor role for Mrp2. The AUCoral 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 AUCoral 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.

Hepatic transport mechanisms of Cholyl-L-Lysyl-Fluorescein.

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(−/−), and Abcc3(−/−) mice. Human Na+-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 Km values of 4.6 ± 2.7, 3.3 ± 2.0, and 3.7 ± 1.0 μM, respectively. In Abcc2(−/−) 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(−/−) 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.

Functionally Overlapping Roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the Elimination of Methotrexate and Its Main Toxic Metabolite 7-Hydroxymethotrexate In vivo

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 for Abcg2 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.

Pharmacokinetic assessment of multiple ATP-binding cassette transporters: the power of combination knockout mice.

A TP-binding cassette (ABC) multidrug transporters are cellular efflux pumps with broad and often widely overlapping substrate specificities. They can have a major impact on the pharmacokinetics and hence overall pharmacological behavior of many drugs. To study their separate roles and functional overlap, or complementarity, a collection of mice deficient in two or more ABC transporters has been generated. This review discusses recent findings obtained with these models, focusing on pharmacokinetic studies with a number of clinically relevant drugs. In addition, the characterization of these mice and some physiological aspects of ABC multidrug transporters are addressed.

Impact of Abcc2 (Mrp2) and Abcc3 (Mrp3) on the In vivo Elimination of Methotrexate and its Main Toxic Metabolite 7-hydroxymethotrexate

Purpose: ATP-binding cassette sub-family C member 2 [ABCC2; multidrug resistance–associated 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 (AUCi.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 AUCi.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 especially Abcc2;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.

P-glycoprotein (P-gp/Abcb1), Abcc2, and Abcc3 Determine the Pharmacokinetics of Etoposide

Purpose: Despite the extensive use of etoposide for the treatment of different malignant neoplasms, its main pharmacokinetic determinants are not completely defined. We aimed to study the impact of P-glycoprotein (P-gp/ABCB1) and the multidrug resistance proteins ABCC2 (MRP2) and ABCC3 (MRP3) on the pharmacokinetics of etoposide. Experimental Design: Abcb1a/1b−/−, Abcc2−/−, Abcc3−/−, Abcb1a/1b;Abcc2−/−, and Abcc2;Abcc3−/− mice were used to investigate the separate and combined impact of P-gp, Abcc2, and Abcc3 on the in vivo behavior of etoposide. Results: P-gp restricted the oral (re)uptake of unchanged etoposide, and mediated its excretion across the gut wall. In contrast, hepatobiliary excretion was almost entirely dependent on Abcc2. Yet, complete loss of Abcc2 did not result in elevated liver or plasma concentrations of etoposide. Instead, Abcc2−/− mice displayed an increased hepatic formation of etoposide glucuronide, which was secreted via Abcc3 from the liver to the blood circulation and eliminated with the urine. Combination Abcc2;Abcc3−/− mice had highly increased accumulation of etoposide glucuronide in their livers, whereas both single knockouts did not, indicating that Abcc2 and Abcc3 provide alternative pathways for the hepatic elimination of etoposide glucuronide. Conclusions: P-gp, ABCC2, and ABCC3 significantly affect the pharmacokinetics of etoposide and/or etoposide glucuronide. Variation in transporter expression or activity may explain the high variation in oral availability of etoposide (25-80%) among cancer patients. However, despite the fact that substantial variations in transporter activity can occur, we believe that cancer patients are often relatively protected from etoposide toxicity due to overlapping functions of these transporters in the elimination of etoposide. Clin Cancer Res; 16(1); 130–40

Liquid chromatography-tandem mass spectrometric assay for sorafenib and sorafenib-glucuronide in mouse plasma and liver homogenate and identification of the glucuronide metabolite.

The first bioanalytical assay for the simultaneous determination of sorafenib and sorafenib-glucuronide in mouse plasma and liver homogenate was developed and validated. In addition, the structure of the glucuronide metabolite was elucidated. The quantitative assay started with addition of isotopically labeled internal standards to a 20 microl sample volume and protein precipitation with acetonitrile, the supernatant was diluted with water and injected into the chromatographic system. A polar embedded reversed-phase column with gradient elution using formic acid in water-acetonitrile was used. The eluate was transferred into an electrospray interface with positive ionization and the analytes were detected and quantified using triple quadrupole mass spectrometry. The assay was validated in the ranges 10-5000 ng/ml for sorafenib and 1-500 ng/ml for sorafenib-glucuronide, the lowest levels of these ranges (10 and 1 ng/ml) being the lower limits of quantification (LLQ). Within day precisions were 2-8%, between day precisions 2-10% (both excluded the LLQ level of the glucuronide) and accuracies were between 89% and 106%. Both analytes were chemically stable under all relevant conditions. The assay was successfully applied in pilot in vivo pharmacokinetic studies with sorafenib in mice.