Rebeca Real

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.

Fluoroquinolone Efflux Mediated by ABC Transporters

Quinolones and fluoroquinolones are broad spectrum bactericidal drugs, which are widely used in both human and veterinary medicine. These drugs can quite easily enter cells and are often used to treat intracellular pathogens. Some fluoroquinolones have been reported to undergo efflux, which could explain their low bioavailability. There is a growing need to understand resistance mechanisms to quinolones, involving for instance mutations or the action of efflux pumps. Several members of the ATP-binding cassette (ABC) drug efflux transporter family (MDR, MRP, ABCG2) significantly affect the pharmacokinetic disposition of quinolones. Active secretory mechanisms common to all fluoroquinolones have been suggested, as well as competition between fluoroquinolones at transporter sites. For grepafloxacin and its metabolites, MRP2 has been demonstrated to mediate biliary excretion. However, MDR1 is responsible for grepafloxacin intestinal secretion. Recently it has been shown that ciprofloxacin and enrofloxacin are efficiently transported ABCG2 substrates which are actively secreted into milk. It appears that multiple ABC transporters contribute to the overall secretion of fluoroquinolones. The objective of this work is to review the recent advances in insights into ABC transporters and their effects on fluoroquinolone disposition and resistance including data on drug secretion into milk.

In vitro and in vivo interaction of moxidectin with BCRP/ABCG2.

The study characterizes the interaction between BCRP/ABCG2 and moxidectin by means of cellular transport, and pharmacokinetic studies in Bcrp1 (-/-) and wild-type mice. Milbemycin moxidectin ([(3)H]-moxidectin) was tested for its ability to be transported across MCDK-II epithelial monolayer cultures transfected with BCRP. In a second approach, accumulation assays by BCRP-expressing Xenopus laevis oocytes were carried out. Finally, pharmacokinetic studies were performed in order to establish the role of the transporter in milk secretion and tissue distribution. The efflux was negligible in polarized cells but moxidectin was efficiently transported in BCRP-expressing X. laevis oocytes. The transport was blocked by an acridone derivative, a novel BCRP inhibitor. Moxidectin secretion into breast milk was decreased in Bcrp1-knockout mice and the milk to plasma ratio was 2-fold higher in wild-type mice after i.v. administration. Drug accumulation in intestinal content, bile, and intestine was higher in wild-type mice but the plasma concentration was not different. Moxidectin is identified as a BCRP substrate since its Bcrp1-mediated secretion into breast milk and the involvement of Bcrp1 in intestinal and bile secretion has been demonstrated. This interaction has pharmacokinetic and toxicological consequences. The most important toxicological consequences of the interaction between BCRP and moxidectin may be related with the presence of drug residues in milk.

Involvement of breast cancer resistance protein (BCRP/ABCG2) in the secretion of danofloxacin into milk: interaction with ivermectin

Danofloxacin, a veterinary fluoroquinolone antimicrobial drug, is actively secreted into milk by an as yet unknown mechanism. One of the main determinants of active drug secretion into milk is the transporter (BCRP/ABCG2). The main purpose was to determine whether danofloxacin is an in vitro substrate for Bcrp1/BCRP and to assess its involvement in danofloxacin secretion into milk. In addition, the role of potential drug-drug interactions in this process was assessed using ivermectin. Danofloxacin was transported in vitro by Bcrp1/BCRP, and ivermectin efficiently blocked this transport. Experiments with Bcrp1(-/-) mice showed no evidence of the involvement of Bcrp1 in plasma pharmacokinetics of danofloxacin. However, the milk concentration and milk-to-plasma ratio of danofloxacin were almost twofold higher in wild-type compared with Bcrp1(-/-) mice. The in vivo interaction with ivermectin was studied in sheep after co-administration of danofloxacin (1.25 mg/kg, i.m.) and ivermectin (0.2 mg/kg, s.c.). Ivermectin had no significant effect on the plasma levels of danofloxacin but significantly decreased danofloxacin concentrations in milk by almost 40%. Concomitant administration of multiple drugs, often used in veterinary therapy, may not only affect their pharmacological activity but also their secretion into milk, because of potential drug-drug interactions mediated by BCRP.

Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models1

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.

Milk secretion of nitrofurantoin, as a specific BCRP/ABCG2 substrate, in assaf sheep: modulation by isoflavones.

Studies on residues in milk used for human consumption have increased due to health concerns and priority interest in the control of potentially risky drugs. The protein BCRP/ABCG2, present in the mammary epithelia, actively extrudes drugs into milk and can be modulated by isoflavones. Nitrofurantoin is a specific BCRP substrate which is actively excreted into milk by this transporter. In this research, we studied nitrofurantoin transport into milk in four experimental groups: G1-calves fed forage with isoflavones; G2-calves fed forage with isoflavones and administered exogenous genistein and daidzein; G3-calves fed forage without isoflavones; G4-calves fed forage without isoflavones and administered exogenous genistein and daidzein. Results show increased levels of nitrofurantoin in milk from calves without isoflavones (G3) and decreased nitrofurantoin residues in milk when isoflavones were present, either by forage (G1 and G2) or by exogenous administration (G4). The values of C(max) in milk were significantly lower in those groups with isoflavones in forage (G1, G2). Plasma levels were low and unmodified among the groups. Inter-individual variation was high. All these results seem to point to a feasible control of drug secretion into milk through isoflavones in the diet when the drug is a good BCRP/ABCG2 substrate.

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.

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.

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.

Novel in vitro systems for prediction of veterinary drug residues in ovine milk and dairy products

A new in vitro tool was developed for the identification of veterinary substrates of the main drug transporter in the mammary gland. These drugs have a much higher chance of being concentrated into ovine milk and thus should be detectable in dairy products. Complementarily, a cell model for the identification of compounds that can inhibit the secretion of drugs into ovine milk, and thus reduce milk residues, was also generated. The ATP-binding cassette transporter G2 (ABCG2) is responsible for the concentration of its substrates into milk. The need to predict potential drug residues in ruminant milk has prompted the development of in vitro cell models over-expressing ABCG2 for these species to detect veterinary drugs that interact with this transporter. Using these models, several substrates for bovine and caprine ABCG2 have been found, and differences in activity between species have been reported. However, despite being of great toxicological relevance, no suitable in vitro model to predict substrates of ovine ABCG2 was available. New MDCKII and MEF3.8 cell models over-expressing ovine ABCG2 were generated for the identification of substrates and inhibitors of ovine ABCG2. Five widely used veterinary antibiotics (marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin and difloxacin) were discovered as new substrates of ovine ABCG2. These results were confirmed for the bovine transporter and its Y581S variant using previously generated cell models. In addition, the avermectin doramectin was described as a new inhibitor of ruminant ABCG2. This new rapid assay to identify veterinary drugs that can be concentrated into ovine milk will potentially improve detection and monitoring of veterinary drug residues in ovine milk and dairy products. Graphical Abstract