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.

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.

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.

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.

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.

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.

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.

An altered tissue distribution of flaxseed lignans and their metabolites in Abcg2 knockout mice.

Lignans are dietary polyphenols, which are metabolized by gut microbiota into the phytoestrogenic metabolites enterolignans, mainly enterolactone and enterodiol. Breast Cancer Resistance Protein (BCRP/ABCG2) is an efflux transporter that affects the plasma and milk secretion of several drugs and natural compounds. We hypothesized here that Abcg2 could influence the levels of lignans and their derived metabolites in target tissues. Consequently, we aimed to evaluate the role of Abcg2 in the tissue distribution of these compounds. We used Abcg2-/- knockout and wild-type male mice fed with a lignan-enriched diet for one week and analysed their plasma, small intestine, colon, liver, kidneys and testicles. High levels of lignans as well as enterolignans and their glucuronide and sulfate conjugates in the small intestine and colon were detected, with higher concentrations of the conjugates in the wild-type compared with Abcg2-/- mice. Particularly relevant was the detection of 24-fold and 8-fold higher concentrations of enterolactone-sulfate and enterolactone-glucuronide, respectively, in the kidney of Abcg2-/- compared with wild-type mice. In conclusion, our study showed that lignans and their derived metabolites were in vivo substrates of Abcg2, which affected their plasma and tissue levels. These results highlight the role of Abcg2 in influencing the health-beneficial properties of dietary lignans.

ABCG2/BCRP interaction with the sea grass Thalassia testudinum.

BACKGROUND: The aqueous ethanolic extract from leaves of the marine plant Thalassia testudinum has shown antioxidant, cytoprotective, and neuroprotective properties. The chemical composition of this extract, rich in polyphenols, could interfere with active transport of drugs out of the cell and circumvent the phenomenon of multidrug resistance (MDR). The extract can act as an MDR modulator through its interaction with efflux transporters. The ABCG2/BCRP has been shown to confer MDR acting in tumor cells. METHODS: To evaluate the interaction of ABCG2/BCRP with the extract, studies in cells overexpressing human BCRP transporter and its murine ortholog Bcrp1 were performed. RESULTS AND CONCLUSIONS: T. testudinum extract could be included as MDR modulator, as interaction with ABCG2/BCRP has been shown through flow cytometry and MTT assays. The cells overexpressing ABCG2/BCRP in the presence of the extract (25-150 µg/mL) decreased the survival rates of the anti-tumoral mitoxantrone. Our results support its inclusion as a possible MDR modulator against tumor cells that overexpress ABCG2/BCRP.

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.

Effects of triclabendazole on secretion of danofloxacin and moxidectin into the milk of sheep: role of triclabendazole metabolites as inhibitors of the ruminant ABCG2 transporter.

ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) mediates drug-drug interactions that affect the secretion of drugs into milk. The aims of this study were: (1) to determine whether the major plasma metabolites of the flukicide triclabendazole (TCBZ), triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO2), inhibit ovine and bovine ABCG2 and its Y581S variant in vitro, and (2) to examine whether coadministration of TCBZ with the ABCG2 substrates danofloxacin (a fluoroquinolone) and moxidectin (a milbemycin) affects the secretion of these drugs into the milk of sheep. TCBZSO and TCBZSO2 inhibited ruminant ABCG2 in vitro by reversing the reduced mitoxantrone accumulation and reducing basal to apical transport of nitrofurantoin in cells transduced with bovine variants (S581 and Y581) and the ovine variant of ABCG2. Coadministration of TCBZ with moxidectin or danofloxacin to sheep resulted in significantly reduced levels of moxidectin, but not danofloxacin, in the milk of TCBZ-treated sheep compared to sheep administered moxidectin or danofloxacin alone. The milk area under concentration time curve (AUC 0-48 h) was 2.99±1.41 µg h/mL in the group treated with TCBZ and moxidectin, and 7.75±3.58 µg h/mL in the group treated with moxidectin alone. The AUC (0-48 h) milk/plasma ratio was 37% lower in the group treated with TCBZ and moxidectin (7.34±1.51) than in the group treated with moxidectin alone (11.68±3.61). TCBZ metabolites appear to inhibit ruminant ABCG2 and affect the secretion of ABCG2 substrates into milk of sheep.

The gut microbiota ellagic acid-derived metabolite urolithin A and its sulfate conjugate are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP).

The breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter that can affect the pharmacological and toxicological properties of many molecules. Urolithins, metabolites produced by the gut microbiota from ellagic acid (EA) and ellagitannins, have been acknowledged with in vivo anti-inflammatory and cancer chemopreventive properties. This study evaluated whether urolithins (Uro-A, -B, -C, and -D) and their main phase II metabolites Uro-A sulfate, Uro-A glucuronide, and Uro-B glucuronide as well as their precursor EA were substrates for ABCG2/BCRP. Parental and Bcrp1-transduced MDCKII cells were used for active transport assays. Uro-A and, to a lesser extent, Uro-A sulfate showed a significant increase in apically directed translocation in Bcrp1-transduced cells. Bcrp1 did not show affinity for the rest of the tested compounds. Data were confirmed for murine, human, bovine, and ovine BCRP-transduced subclones as well as with the use of the selective BCRP inhibitor Ko143. The transport inhibition by Uro-A was analyzed by flow cytometry compared to Ko143 using the antineoplastic agent mitoxantrone as a model substrate. Results showed that Uro-A was able to inhibit mitoxantrone transport in a dose-dependent manner. This study reports for the first time that Uro-A and its sulfate conjugate are ABCG2/BCRP substrates. The results suggest that physiologically relevant concentrations of these gut microbiota-derived metabolites could modulate ABCG2/BCRP-mediated transport processes and mechanisms of cancer drug resistance. Further in vivo investigations are warranted.

Inhibition of ABCG2/BCRP transporter by soy isoflavones genistein and daidzein: effect on plasma and milk levels of danofloxacin in sheep.

Danofloxacin is a synthetic fluoroquinolone antibacterial agent and a substrate for ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). This protein actively extrudes drugs from cells in the intestine, liver, kidney, and other organs, such as the mammary gland. The purpose of this study was to determine whether genistein and daidzein, isoflavones present in soy and known inhibitors of ABCG2, could diminish danofloxacin secretion into milk. The results obtained from BCRP-transduced MDCK-II cells (Mardin-Darby canine kidney) showed that both isoflavones efficiently inhibited the in vitro transport of the drug. In addition, danofloxacin transport into milk was studied in Assaf sheep. The experimental design with ewes (n = 18) included ewes fed with standard forage, soy-enriched forage for 15 days prior to the experiment or standard forage paired with orally administered exogenous genistein and daidzein. The danofloxacin levels in the milk of ewes in the soy-enriched diet group were decreased. The area under concentration-time curve AUC (0-24 h) was 9.3 ± 4.6 vs. 16.58 ± 4.44 µgh/mL in the standard forage or control group. The plasma levels of danofloxacin were unmodified. The AUC (0-24 h) milk/plasma ratio decreased by over 50% in the soy-enriched diet group, compared to the control group (4.90 ± 2.65 vs. 9.58 ± 2.17). Exogenous administration of isoflavones did not modify danofloxacin secretion into milk. This study showed that milk excretion of a specific substrate of BCRP, such as danofloxacin, can be diminished by the presence of isoflavones in the diet.

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.

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.

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.

Differential inhibition of murine Bcrp1/Abcg2 and human BCRP/ABCG2 by the mycotoxin fumitremorgin C.

Breast Cancer Resistance Protein (ABCG2/BCRP) is an ATP-binding cassette transporter expressed in absorptive and excretory organs whose main physiological role is protection of cells against xenobiotics. In addition, ABCG2/BCRP expression has been linked to cellular resistance to anticancer drugs due to the acquisition of a multidrug resistance phenotype. Fumitremorgin C (FTC) is a mycotoxin described as a potent ABCG2/BCRP inhibitor that reverses multidrug resistance. However, little is known about its species-specificity. This issue is scientifically relevant since FTC is widely used to evaluate the in vitro role of BCRP. We compared the FTC-mediated inhibition of human BCRP and its murine orthologue, overexpressed in two independent cell lines, MDCKII and MEF3.8 transduced cell lines. Accumulation experiments, using mitoxantrone and chlorine e6 as substrates, revealed that although FTC inhibits both Bcrp1 and BCRP, the human transporter is more potently inhibited, resulting in significantly lower IC(50) values. Transcellular transport of known Bcrp1/BCRP substrates, such as nitrofurantoin and mitoxantrone, was completely inhibited by FTC 1muM in human BCRP-transduced cells but only moderately in murine Bcrp1-transduced cells. Finally, cytotoxicity assays using mitoxantrone and topotecan as substrates revealed that the EC(90) values for FTC were always significantly lower in human BCRP-transduced cells. Altogether, these results indicate that human BCRP is more sensitive to inhibition by FTC than murine Bcrp1. This differential inhibition could have a great impact on the use of in vitro models of toxicity and pharmacological interaction for drug discovery and development involving FTC as Bcrp1/BCRP inhibitor.

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.