Role of the Abcg2 Transporter in Secretion into Milk of the Anthelmintic Clorsulon: Interaction with Ivermectin.

Clorsulon is a benzenesulfonamide drug that is effective in treating helminthic zoonoses such as fascioliasis. When used in combination with the macrocyclic lactone ivermectin, it provides high broad-spectrum antiparasitic efficacy. The safety and efficacy of clorsulon should be studied by considering several factors such as drug-drug interactions mediated by ATP-binding cassette (ABC) transporters due to their potential effects on the pharmacokinetics and drug secretion into milk. The aim of this work was to determine the role of ABC transporter G2 (ABCG2) in clorsulon secretion into milk and the effect of ivermectin, a known ABCG2 inhibitor, on this process. Using in vitro transepithelial assays with cells transduced with murine Abcg2 and human ABCG2, we report that clorsulon was transported in vitro by both transporter variants and that ivermectin inhibited its transport mediated by murine Abcg2 and human ABCG2. Wild-type and Abcg2-/- lactating female mice were used to carry out in vivo assays. The milk concentration and the milk-to-plasma ratio were higher in wild-type mice than in Abcg2-/- mice after clorsulon administration, showing that clorsulon is actively secreted into milk by Abcg2. The interaction of ivermectin in this process was shown after the coadministration of clorsulon and ivermectin to wild-type and Abcg2-/- lactating female mice. Treatment with ivermectin had no effect on the plasma concentrations of clorsulon, but the milk concentrations and milk-to-plasma ratios of clorsulon decreased in comparison to those with treatment without ivermectin, only in wild-type animals. Consequently, the coadministration of clorsulon and ivermectin reduces clorsulon secretion into milk due to drug-drug interactions mediated by ABCG2.

ABCG2 transporter plays a key role in the biodistribution of melatonin and its main metabolites.

The ATP-binding cassette G2 (ABCG2) is an efflux transporter expressed in the apical membrane of cells from a large number of tissues, directly affecting bioavailability, tissue accumulation, and secretion into milk of both xenobiotics and endogenous compounds. The aim of this work was to characterize the role of ABCG2 in the systemic distribution and secretion into milk of melatonin and its main metabolites, 6-hydroxymelatonin, and 6-sulfatoxymelatonin. For this purpose, we first showed that these three molecules are transported by this transporter using in vitro transepithelial assays with MDCK-II polarized cells transduced with different species variants of ABCG2. Second, we tested the in vivo effect of murine Abcg2 in the systemic distribution of melatonin and its metabolites using wild-type and Abcg2-/- mice. Our results show that after oral administration of melatonin, the plasma concentration of melatonin metabolites in Abcg2-/-  mice was between 1.5 and 6-fold higher compared to the wild-type mice. We also evaluated in these animals differences in tissue accumulation of melatonin metabolites. The most relevant differences between both types of mice were found for small intestine and kidney (>sixfold increase for 6-sulfatoxymelatonin in Abcg2-/-  mice). Finally, melatonin secretion into milk was also affected by the murine Abcg2 transporter, with a twofold higher milk concentration in wild-type compared with Abcg2-/-  lactating female mice. In addition, melatonin metabolites showed a higher milk-to-plasma ratio in wild-type mice. Overall, our results show that the ABCG2 transporter plays a critical role in the biodistribution of melatonin and its main metabolites, thereby potentially affecting their biological and therapeutic activity.

Secretion into Milk of the Main Metabolites of the Anthelmintic Albendazole Is Mediated by the ABCG2/BCRP Transporter.

Albendazole (ABZ) is an anthelmintic with a broad-spectrum activity, widely used in human and veterinary medicine. ABZ is metabolized in all mammalian species to albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO2) and albendazole 2-aminosulphone (ABZSO2-NH2). ABZSO and ABZSO2 are the main metabolites detected in plasma and all three are detected in milk. The ATP-binding cassette transporter G2 (ABCG2) is an efflux transporter that is involved in the active secretion of several compounds into milk. Previous studies have reported that ABZSO was in vitro transported by ABCG2. The aim of this work is to correlate the in vitro interaction between ABCG2 and the other ABZ metabolites with their secretion into milk by this transporter. Using in vitro transepithelial assays with cells transduced with murine Abcg2 and human ABCG2, we show that ABZSO2 and ABZSO2-NH2 are in vitro substrates of both. In vivo assays carried out with wild-type and Abcg2-/- lactating female mice demonstrated that secretion into milk of these ABZ metabolites was mediated by Abcg2. Milk concentrations and milk-to-plasma ratio were higher in wild-type compared to Abcg2-/- mice for all the metabolites tested. We conclude that ABZ metabolites are undoubtedly in vitro substrates of ABCG2 and actively secreted into milk by ABCG2.

Kinetics and Optimization of the Lysine-Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.

Cocaine relapse and health-related quality of life: a 23 weeks study.

INTRODUCTION: Cocaine dependence is a disorder where relapses are frequently presented and many factors are involved. Furthermore, cocaine dependence is associated with poor health-related quality of life (HRQoL) outcomes. This study aims to explore perceived HRQoL as an indicator of drug relapse in cocaine-dependent patients (CDP). SUBJECTS AND METHODS: A longitudinal study was carried out in CDP during 23 weeks. A consecutive sampling method was applied, 39 participants composed the initial sample (mean age 35.6 years), only 15 participants completed outpatient follow-up period. CDP were assessed with psychiatric and HRQoL instruments (SCID-I, SCID-II, BDI, STAI scale and SF-36) in different points of the study. The patients were followed up, and cocaine relapses were assessed. The sample was divided according with the relapse (early vs. late relapse). Data were compared and analyzed in order to evaluate whether HRQoL measure could be related to cocaine relapse. RESULTS: There are differences in perceived HRQoL measures between CDP with/without early relapse, especially in Mental health and Social functioning dimensions (p<0.05). Furthermore, Late/relapse-patients have higher improvement of HRQoL than patients with early relapse. CONCLUSIONS: Perceived HRQoL might predict early relapse and could be a possible predictor tool of potential future relapses. More research in this field is needed.

Role of ABCG2 in transport of the mammalian lignan enterolactone and its secretion into milk in Abcg2 knockout mice.

Lignans are phytoestrogens that are metabolized by the gut microbiota to enterodiol and enterolactone, the main biologically active enterolignans. Substantial interindividual variation in plasma concentration and urinary excretion of enterolignans has been reported, this being determined, at least in part, by the intake of lignan precursors, the gut microbiota, and the host's phase 2 conjugating enzyme activity. However, the role of ATP-binding cassette (ABC) transporters in the transport and disposition of enterolactone has not been reported so far. Active transport assays using parental and Madin-Darby canine kidney epithelial cells transduced with murine and human ABCG2 showed a significant increase in apically directed translocation of enterolactone in transduced cells, which was confirmed by using the selective ABCG2 inhibitor Ko143. In addition, enterolactone also inhibited transport of the antineoplastic agent mitoxantrone as a model substrate, with inhibition percentages of almost 40% at 200 µM for human ABCG2. Furthermore, the endogenous levels in plasma and milk of enterolactone in wild-type and Abcg2((-/-)) knockout female mice were analyzed. The milk/plasma ratio decreased significantly in the Abcg2((-/-)) phenotype, as compared with the wild-type mouse group (0.4 ± 0.1 as against 6.4 ± 2.6). This paper is the first to report that enterolactone is a transported substrate and therefore most probably a competitive inhibitor of ABCG2, which suggests it has a role in the interindividual variations in the disposition of enterolactone and its secretion into milk. The inhibitory activity identified provides a solid basis for further investigation in possible food-drug interactions.

The ABCG2 protein in vitro transports the xenobiotic thiabendazole and increases the appearance of its residues in milk.

Thiabendazole (TBZ) is a broad-spectrum anthelmintic and fungicide used in humans, animals, and agricultural commodities. TBZ residues are present in crops and animal products, including milk, posing a risk to food safety and public health. ABCG2 is a membrane transporter which affects bioavailability and milk secretion of xenobiotics. Therefore, the aim of this work was to characterize the role of ABCG2 in the in vitro transport and secretion into milk of 5-hydroxythiabendazole (5OH-TBZ), the main TBZ metabolite. Using MDCK-II polarized cells transduced with several species variants of ABCG2, we first demonstrated that 5OH-TBZ is efficiently in vitro transported by ABCG2. Subsequently, using Abcg2 knockout mice, we demonstrated that 5OH-TBZ secretion into milk was affected by Abcg2, with a more than 2-fold higher milk concentration and milk to plasma ratio in wild-type mice compared to their Abcg2-/- counterpart.

The bovine ATP-binding cassette transporter ABCG2 Tyr581Ser single-nucleotide polymorphism increases milk secretion of the fluoroquinolone danofloxacin.

The bovine adenosine triphosphate-binding cassette transporter G2 (ABCG2/breast cancer resistance protein) polymorphism Tyr581Ser (Y581S) has recently been shown to increase in vitro transepithelial transport of antibiotics. Since this transporter has been extensively related to the active secretion of drugs into milk, the potential in vivo effect of this polymorphism on secretion of xenobiotics in livestock could have striking consequences for milk production, the dairy industry, and public health. Our purpose was to study the in vivo effect of this polymorphism on the secretion of danofloxacin, a widely used veterinary antibiotic, into milk. Danofloxacin (1.25 mg/kg) was administered to six Y/Y 581 homozygous and six Y/S 581 heterozygous lactating cows, and plasma and milk samples were collected and analyzed by high-performance liquid chromatography. No differences were found in the pharmacokinetic parameters of danofloxacin in plasma between the two groups of animals. In contrast, Y/S heterozygous cows showed a 2-fold increase in danofloxacin levels in milk. In addition, the pharmacokinetic elimination parameters, mean residence time and elimination half-life, were significantly lower in the milk of the animals carrying the Y/S polymorphism. These in vivo results are in agreement with our previously published in vitro data, which showed a greater capacity of the S581 variant in accumulation assays, and demonstrate, for the first time, an important effect of the Y581S single-nucleotide polymorphism on antibiotic secretion into cow milk. These findings could be extended to other ABCG2 substrates, and may be relevant for the treatment of mastitis and for the design of accurate and novel strategies to handle milk residues.

Coadministration of ivermectin and abamectin affects milk pharmacokinetics of the antiparasitic clorsulon in Assaf sheep.

In veterinary field, drug exposure during milk production in dairy cattle is considered a major health problem which concerns dairy consumers. The induced expression of the ABC transporter G2 (ABCG2) in the mammary gland during lactation plays a significant role in the active secretion of many compounds into milk. The main objective of this study was to determine the involvement of ABCG2 in the secretion into milk of the antiparasitic clorsulon in sheep as well as the possible effect of the coadministration of model ABCG2 inhibitors such as macrocyclic lactones on this process. Cells transduced with the ovine variant of ABCG2 were used to carry out in vitro transepithelial transport assays in which we showed that clorsulon is a substrate of the ovine transporter. In addition, ivermectin and abamectin significantly inhibited clorsulon transport mediated by ovine ABCG2. In vivo interactions were studied in Assaf sheep after coadministration of clorsulon (in DMSO, 2 mg/kg, s.c.) with ivermectin (Ivomec®, 0.2 mg/kg, s.c.) or abamectin (in DMSO, 0.2 mg/kg, s.c.). After ivermectin and abamectin treatment, no relevant statistically significant differences in plasma levels of clorsulon were reported between the experimental groups since there were no differences in the area under the plasma concentration-curve (AUC) between clorsulon treatment alone and coadministration with macrocyclic lactones. With regard to milk, total amount of clorsulon, as percentage of dose excreted, did not show statistically significant differences when macrocyclic lactones were coadministered. However, the AUC for clorsulon significantly decreased (p < 0.05) after coadministration with ivermectin (15.15 ± 3.17 µg h/mL) and abamectin (15.30 ± 3.25 µg h/mL) compared to control group (20.73 ± 4.97 µg h/mL). Moreover, milk parameters such as half-life (T1/2) and mean residence time (MRT) were significantly lower (p < 0.05) after coadministration of macrocyclic lactones. This research shows that the milk pharmacokinetics of clorsulon is affected by the coadministration of ABCG2 inhibitors, reducing drug persistence in milk.

The anthelmintic triclabendazole and its metabolites inhibit the membrane transporter ABCG2/BCRP.

ABCG2/BCRP is an ATP-binding cassette transporter that extrudes compounds from cells in the intestine, liver, kidney, and other organs, such as the mammary gland, affecting pharmacokinetics and milk secretion of antibiotics, anticancer drugs, and other compounds and mediating drug-drug interactions. In addition, ABCG2 expression in cancer cells may directly cause resistance by active efflux of anticancer drugs. The development of ABCG2 modulators is critical in order to improve drug pharmacokinetic properties, reduce milk secretion of xenotoxins, and/or increase the effective intracellular concentrations of substrates. Our purpose was to determine whether the anthelmintic triclabendazole (TCBZ) and its main plasma metabolites triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO(2)) inhibit ABCG2 activity. ATPase assays using human ABCG2-enriched membranes demonstrated a clear ABCG2 inhibition exerted by these compounds. Mitoxantrone accumulation assays using murine Abcg2- and human ABCG2-transduced MDCK-II cells confirmed that TCBZSO and TCBZSO(2) are ABCG2 inhibitors, reaching inhibitory potencies between 40 and 55% for a concentration range from 5 to 25 µM. Transepithelial transport assays of ABCG2 substrates in the presence of both TCBZ metabolites at 15 µM showed very efficient inhibition of the Abcg2/ABCG2-mediated transport of the antibacterial agents nitrofurantoin and danofloxacin. TCBZSO administration also inhibited nitrofurantoin Abcg2-mediated secretion into milk by more than 2-fold and increased plasma levels of the sulfonamide sulfasalazine by more than 1.5-fold in mice. These results support the potential role of TCBZSO and TCBZSO(2) as ABCG2 inhibitors to participate in drug interactions and modulate ABCG2-mediated pharmacokinetic processes.

Ivermectin inhibits ovine ABCG2-mediated in vitro transport of meloxicam and reduces its secretion into milk in sheep.

The ATP-binding cassette transporter G2 (ABCG2) is an efflux protein involved in the bioavailability and secretion into milk of several compounds including anti-inflammatory drugs. The aim of this work was to determine the effect in sheep of an ABCG2 inhibitor, such as the macrocyclic lactone ivermectin, on the secretion into milk of meloxicam, a non-steroidal anti-inflammatory drug widely used in veterinary medicine, and recently reported as an ABCG2 substrate. In vitro meloxicam transport assays in ovine ABCG2-transduced cells have shown that ivermectin is an efficient inhibitor of in vitro transport of meloxicam mediated by ovine ABCG2, with a 75% inhibition in the transport ratio (24.85 ± 4.62 in controls vs 6.31 ± 1.37 in presence of ivermectin). In addition, the role of ovine ABCG2 in secretion into milk of meloxicam was corroborated using Assaf lactating sheep coadministered with ivermectin. Animals were administered subcutaneously with meloxicam (0.5 mg/kg) with or without ivermectin (0.2 mg/kg). No difference in plasma pharmacokinetic parameters was found between treatments. In the case of milk, a significant reduction in the area under concentration-time curve (AUC) (3.92 ± 0.66 vs 2.26 ± 1.52 µg·h/mL) and the AUC milk-to-plasma ratio (0.17 ± 0.03 vs 0.09 ± 0.06) was reported for ivermectin-treated animals compared to controls.

Bioavailability of the glucuronide and sulfate conjugates of genistein and daidzein in breast cancer resistance protein 1 knockout mice.

The dietary polyphenols genistein and daidzein are potent effectors of biological processes. The plasma profile of both isoflavones is governed by the presence of phase II conjugates, mainly glucuronides and sulfates. Breast cancer resistance protein (ABCG2/BCRP) interacts with genistein and daidzein, which are among the natural substrates of the transporter and competitively inhibit ABCG2-mediated drug efflux. ABCG2/BCRP can also transport glucuronide and sulfate conjugates. In this study, we analyzed the plasma levels of aglycones and derived conjugated metabolites, glucuronides, and sulfates, after intragastric administration of these isoflavones to wild-type and Bcrp1(-/-) knockout mice. The results show that overall plasmatic profile is mainly governed by sulfate and glucuronide derivatives, the concentration of which was significantly increased (7- to 10-fold) in Bcrp1(-/-) mice. The total AUC h nM (0-180 min), as the sum of aglycones, glucuronides, and sulfates, was 901 ± 207 in wild-type mice versus 4988 ± 508 in Bcrp1(-/-) mice after genistein administration (50 mg/kg b.wt.); 584.3 ± 90 in wild-type mice versus 4012 ± 612 in Bcrp1(-/-) after daidzein administration (50 mg/kg); and 926 ± 140 in wild-type mice versus 5174 ± 696 in Bcrp1(-/-) after genistein+daidzein administration (25 + 25 mg/kg). Therefore, our results indicate a direct and conclusive Bcrp1 efflux action on phase II metabolites of these isoflavones in vivo and suggest a possible novel concept for ABCG2/BCRP as part of metabolism-driven efflux transport of these conjugates.

The ABC membrane transporter ABCG2 prevents access of FAAH inhibitor URB937 to the central nervous system.

The O-arylcarbamate URB937 is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the deactivation of the endocannabinoid anandamide. URB937 is unique among FAAH inhibitors in that is actively extruded from the central nervous system (CNS), and therefore increases anandamide levels exclusively in peripheral tissues. Despite its limited distribution, URB937 exhibits marked analgesic properties in rodent models of pain. Pharmacological evidence suggests that the extrusion of URB937 from the CNS may be mediated by the ABC membrane transporter ABCG2 (also called Breast cancer resistance protein, BCRP). In the present study, we show that URB937 is a substrate for both mouse and human orthologues of ABCG2. The relative transport ratios for URB937 in Madin-Darby canine kidney (MDCKII) cells monolayers over-expressing either mouse Abcg2 or human ABCG2 were significantly higher compared to parental monolayers (13.6 and 13.1 vs. 1.5, respectively). Accumulation of the compound in the luminal/apical side was prevented by co-administration of the selective ABCG2 inhibitor, Ko-143. In vivo studies in mice showed that URB937 (25 mg kg(-1)) readily entered the brain and spinal cord of Abcg2-deficient mice following intraperitoneal administration, whereas the same dose of drug remained restricted to peripheral tissues in wild-type mice. By identifying ABCG2 as a transport mechanism responsible for the extrusion of URB937 from the CNS, the present results should facilitate the rational design of novel peripherally restricted FAAH inhibitors.

Role of the Abcg2 transporter in plasma levels and tissue accumulation of the anti-inflammatory tolfenamic acid in mice.

The Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter that is expressed in the apical membrane of cells from relevant tissues involved in drug pharmacokinetics such as liver, intestine, kidney, testis, brain and mammary gland, among others. Tolfenamic acid is an anti-inflammatory drug used as an analgesic and antipyretic in humans and animals. Recently, tolfenamic acid has been repurposed as an antitumoral drug and for use in chronic human diseases such as Alzheimer. The aim of this work was to study whether tolfenamic acid is an in vitro Abcg2 substrate, and to investigate the potential role of Abcg2 in plasma exposure, secretion into milk and tissue accumulation of this drug. Using in vitro transepithelial assays with cells transduced with Abcg2, we showed that tolfenamic acid is an in vitro substrate of Abcg2. The in vivo effect of this transporter was tested using wild-type and Abcg2-/- mice, showing that after oral and intravenous administration of tolfenamic acid, its area under the plasma concentration-time curve in Abcg2-/- mice was between 1.7 and 1.8-fold higher compared to wild-type mice. Abcg2-/- mice also showed higher liver and testis accumulation of tolfenamic acid after intravenous administration. In this study, we demonstrate that tolfenamic acid is transported in vitro by Abcg2 and that its plasma levels as well as its tissue distribution are affected by Abcg2, with potential pharmacological and toxicological consequences.

Role of eprinomectin as inhibitor of the ruminant ABCG2 transporter: Effects on plasma distribution of danofloxacin and meloxicam in sheep.

Therapeutic outcome results of the coadministration of several drugs in veterinary medicine is affected by, among others, the relationship between drugs and ATP-binding cassette (ABC) transporters, such as ABCG2. ABCG2 is an efflux protein involved in the bioavailability and milk secretion of drugs. The aim of this work was to determine the role of eprinomectin, a macrocyclic lactone (ML) member of avermectin class, as inhibitor of ABCG2. The experiments were carried out through in vitro inhibition assays based on mitoxantrone accumulation and transport assays in ovine ABCG2 transduced cells using the antimicrobial drug danofloxacin and the anti-inflammatory drug meloxicam, both widely used in veterinary medicine and well known ABCG2 substrates. The inhibition results obtained showed that eprinomectin was an efficient in vitro ABCG2 inhibitor, tested in mitoxantrone accumulation assays. In addition, this ML decreased ovine ABCG2-mediated transport of danofloxacin and meloxicam. To evaluate the role of eprinomectin in systemic exposure of drugs, pharmacokinetic assays based on subcutaneous coadministration of eprinomectin with danofloxacin (1.25 mg/kg) or meloxicam (0.5 mg/kg) in sheep were performed obtaining a significant increase of systemic exposure of these drugs. Especially relevant was the increase of the systemic concentration of meloxicam, since coadministration with eprinomectin increased significantly the plasma concentration of meloxicam, obtaining an increase of AUC (0-72 h) value of more than 40%.

Analysis of the interaction between tryptophan-related compounds and ATP-binding cassette transporter G2 (ABCG2) using targeted metabolomics.

ATP-binding cassette transporter G2 (ABCG2) is involved in the secretion of several compounds in milk. The in vitro and in vivo interactions between tryptophan-related compounds and ABCG2 were investigated. The tryptophan metabolome was determined by liquid chromatography-tandem mass spectrometry in milk and plasma from wild-type and Abcg2-/- mice as well as dairy cows carrying the ABCG2 Y581S polymorphism (Y/S) and noncarrier animals (Y/Y). The milk-to-plasma ratios of tryptophan, kynurenic acid, kynurenine, anthranilic acid, and xanthurenic acid were higher in wild-type mice than in Abcg2-/- mice. The ratio was 2-fold higher in Y/S than in Y/Y cows for kynurenine. In vitro transport assays confirmed that some of these compounds were in vitro substrates of the transporter and validated the differences observed between the two variants of the bovine protein. These findings show that the secretion of metabolites belonging to the kynurenine pathway into milk is mediated by ABCG2.

In vivo inhibition of BCRP/ABCG2 mediated transport of nitrofurantoin by the isoflavones genistein and daidzein: a comparative study in Bcrp1 (-/-) mice.

PURPOSE: The aim of this study was to determine in vivo inhibition by the isoflavones genistein and daidzein of nitrofurantoin (NTF), a well-known substrate of the ABC transporter BCRP/ABCG2. METHODS: MDCKII cells and their human BCRP- and murine Bcrp1-transduced subclones were used to establish inhibition in transepithelial transport assays. Bcrp1(-/-) and wild-type mice were coadministered with nitrofurantoin (20 mg/kg) and a mixture of genistein (100 mg/kg) and daidzein (100 mg/kg). RESULTS: Transepithelial NFT transport was inhibited by the isoflavones. Plasma concentration of NTF at 30 min was 1.7-fold higher (p ≤ 0.05) in wild-type mice after isoflavone administration. AUC values were not significantly different. BCRP/ABCG2-mediated secretion into milk was inhibited since milk/plasma ratios were lower in wild-type mice with isoflavones (7.1 ± 4.2 vs 4.2 ± 1.6, p ≤ 0.05). NTF bile levels were significantly decreased by isoflavone administration in wild-type animals (8.8 ± 3.4 µg/ml with isoflavones vs 3.7 ± 3.3 µg/ml without isoflavones). CONCLUSION: Our data showed that in vivo interaction of high doses of soy isoflavones with BCRP substrates may affect plasma levels but the main effect occurs in specific target organs, in our case, liver and mammary glands.

Abcg2 transporter affects plasma, milk and tissue levels of meloxicam.

ATP-binding cassette (ABCG2) is an efflux transporter that extrudes xenotoxins from cells in liver, intestine, mammary gland, brain and other organs, affecting the pharmacokinetics, brain accumulation and secretion into milk of several compounds, including antitumoral, antimicrobial and anti-inflammatory drugs. The aim of this study was to investigate whether the widely used anti-inflammatory drug meloxicam is an Abcg2 sustrate, and how this transporter affects its systemic distribution. Using polarized ABCG2-transduced cell lines, we found that meloxicam is efficiently transported by murine Abcg2 and human ABCG2. After oral administration of meloxicam, the area under the plasma concentration-time curve in Abcg2-/- mice was 2-fold higher than in wild type mice (146.06 ± 10.57 µg·h/ml versus 73.80 ± 10.00 µg·h/ml). Differences in meloxicam distribution were reported for several tissues after oral and intravenous administration, with a 20-fold higher concentration in the brain of Abcg2-/- after oral administration. Meloxicam secretion into milk was also affected by the transporter, with a 2-fold higher milk-to-plasma ratio in wild-type compared with Abcg2-/- lactating female mice after oral and intravenous administration. We conclude that Abcg2 is an important determinant of the plasma and brain distribution of meloxicam and is clearly involved in its secretion into milk.

Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

Transporters in the Mammary Gland-Contribution to Presence of Nutrients and Drugs into Milk.

A large number of nutrients and bioactive ingredients found in milk play an important role in the nourishment of breast-fed infants and dairy consumers. Some of these ingredients include physiologically relevant compounds such as vitamins, peptides, neuroactive compounds and hormones. Conversely, milk may contain substances-drugs, pesticides, carcinogens, environmental pollutants-which have undesirable effects on health. The transfer of these compounds into milk is unavoidably linked to the function of transport proteins. Expression of transporters belonging to the ATP-binding cassette (ABC-) and Solute Carrier (SLC-) superfamilies varies with the lactation stages of the mammary gland. In particular, Organic Anion Transporting Polypeptides 1A2 (OATP1A2) and 2B1 (OATP2B1), Organic Cation Transporter 1 (OCT1), Novel Organic Cation Transporter 1 (OCTN1), Concentrative Nucleoside Transporters 1, 2 and 3 (CNT1, CNT2 and CNT3), Peptide Transporter 2 (PEPT2), Sodium-dependent Vitamin C Transporter 2 (SVCT2), Multidrug Resistance-associated Protein 5 (ABCC5) and Breast Cancer Resistance Protein (ABCG2) are highly induced during lactation. This review will focus on these transporters overexpressed during lactation and their role in the transfer of products into the milk, including both beneficial and harmful compounds. Furthermore, additional factors, such as regulation, polymorphisms or drug-drug interactions will be described.