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 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.

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.

Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response.

The present article aims to review the up-to-date information on the most recent studies of the interaction of flavonoids with ABC transporters, in particular the drug pharmacokinetic consequences of such a relationship. In addition, the modulation of the expression of the ABC transporters by flavonoids is also illustrated. Flavonoids are a large group of plant polyphenols present extensively in our daily diets and herbal products. High intake of isoflavones has been associated with a variety of beneficial effects on several common diseases. These polyphenols interact with ABC drug transporters involved in drug resistance and drug absorption, distribution and excretion. A number of studies have demonstrated inhibition of drug transporters by flavonoids. This flavonoid-ABC-transporter interaction could be beneficial for poorly absorbed drugs but could also result in severe drug intoxication, especially drugs with a narrow therapeutic window. On the other hand, flavonoids are themselves substrates of ABC transporters. These proteins can affect the oral availability and tissue distribution of these compounds, modifying their beneficial effects. The challenge is to find a suitable way to predict harmful drug-flavonoid interactions mediated by these transporters.

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.