Human BCRP Research Articles

A fluorescence-based in vitro assay for drug interactions with breast cancer resistance protein (BCRP, ABCG2)

Purpose: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein BCRP (ABCG2). Methods: BCRP expression was verified by immunostaining and Western blots in intact porcine brain capillaries, isolated endothelial cells (PBCECs) and in MDCKII-cells over-expressing human wild-type BCRP (MDCKII-hBCRP). Transport of fluorescent mitoxantrone across cells was determined to assess a preferred transport direction. Sensitivity of cultured cells versus mitoxantrone in the absence and in the presence of transport modulators was examined at increasing concentrations of the cytostatic using the AlamarBlue™ assay. In addition, cells were incubated with mitoxantrone in the absence and presence of increasing concentrations of different compounds with the potential to interact with BCRP. Intracellular fluorescence accumulation was measured using a flow cytometer. Results: Isolated capillaries as well as 7-day old PBCECs showed expression of BCRP. Cell sensitivity to mitoxantrone significantly increased in the presence of the BCRP inhibitors KO143 and GF120918. Transport of mitoxantrone across PBCEC monolayers was directed with P app (apical to basolateral) 5.6 × 10 −6 cm s −1 and with P app (basolateral to apical) 2.8 × 10 −5 cm s −1. FACS analysis revealed a different extent of fluorescence accumulation dependent on the kind and concentration of BCRP modulating compounds. Conclusions: The mitoxantrone-based assay can be used as a rapid FACS screening system to assess drug interactions with BCRP at the blood–brain barrier and therefore represents a useful tool in drug profiling.

Co‐administration of proton pump inhibitors delays elimination of plasma methotrexate in high‐dose methotrexate therapy

To assess whether or not co-administration of proton pump inhibitors (PPIs) is a risk factor for delayed elimination of plasma methotrexate (MTX) in high-dose MTX (HDMTX) therapy for malignant diseases. To assess the effects of PPI co-administration on elimination of plasma MTX, we examined plasma MTX concentration data on 171 cycles of HDMTX therapy performed in 74 patients. We performed multiple logistic regression analysis to evaluate PPI co-administration as a risk factor. Inhibitory potencies of omeprazole, lansoprazole, rabeprazole and pantoprazole on MTX transport via breast cancer resistance protein (BCRP, ABCG2) were also investigated in an in vitro study using membrane vesicles expressing human BCRP. We identified co-administration of PPIs as a risk factor for delayed elimination (odds ratio 2.65, 95% confidence interval 1.03, 6.82) as well as renal and liver dysfunction. All four PPIs inhibited BCRP-mediated transport of MTX, with half-maximal inhibitory concentrations of 5.5-17.6 microM--considerably higher than the unbound plasma concentrations of the PPIs. Our results support previous findings suggesting that PPI co-administration is associated with delayed elimination of plasma MTX in patients with HDMTX therapy. This drug interaction, however, cannot be explained solely by the inhibitory effects of PPIs on BCRP-mediated MTX transport.

Transport of Diclofenac by Breast Cancer Resistance Protein (ABCG2) and Stimulation of Multidrug Resistance Protein 2 (ABCC2)-Mediated Drug Transport by Diclofenac and Benzbromarone

Diclofenac is an important analgesic and anti-inflammatory drug, widely used for treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Consequently, diclofenac is often used in combination regimens and undesirable drug-drug interactions may occur. Because many drug-drug interactions may occur at the level of drug transporting proteins, we studied interactions of diclofenac with apical ATP-binding cassette (ABC) multidrug efflux transporters. Using Madin-Darby canine kidney (MDCK)-II cells transfected with human P-glycoprotein (P-gp; MDR1/ABCB1), multidrug resistance protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) and murine Bcrp1, we found that diclofenac was efficiently transported by murine Bcrp1 and moderately by human BCRP but not by P-gp or MRP2. Furthermore, in Sf9-BCRP membrane vesicles diclofenac inhibited transport of methotrexate in a concentration-dependent manner. We next used MDCK-II-MRP2 cells to study interactions of diclofenac with MRP2-mediated drug transport. Diclofenac stimulated paclitaxel, docetaxel, and saquinavir transport at only 50 microM. We further found that the uricosuric drug benzbromarone stimulated MRP2 at an even lower concentration, having maximal stimulatory activity at only 2 microM. Diclofenac and benzbromarone stimulated MRP2-mediated transport of amphipathic lipophilic drugs at 10- and 250-fold lower concentrations, respectively, than reported for other MRP2 stimulators. Because these concentrations are readily achieved in patients, adverse drug-drug interactions may occur, for example, during cancer therapy, in which drug concentrations are often critical and stimulation of elimination via MRP2 may result in suboptimal chemotherapeutic drug concentrations. Moreover, stimulation of MRP2 activity in tumors may lead to increased efflux of chemotherapeutic drugs and thereby drug resistance.

Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins

Chloroacetanilide herbicides are among the most commonly used herbicides in agriculture. Several studies have demonstrated a number of them to be carcinogenic. ATP binding cassette (ABC) transporters are efflux pumps expressed in cell membranes, which form an important wall of defense against xenobiotics from different sources. We tested the interaction of the herbicides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor with human multidrug resistance transporters MDR1, MRP1, MRP2 and BCRP. A number of metabolites were studied for interaction with MRP1, MRP2 and MRP3. Transporter interactions were studied by measuring ATPase activity, inhibition of fluorescent dye efflux and vesicular transport. Also inhibition of MDR1 was monitored by measuring digoxin transport on Caco-2 monolayers and paclitaxel toxicity on K562-MDR cells. Acetochlor, alachlor, metolachlor and metazachlor showed specific interactions with MDR1. Digoxin permeability and paclitaxel cytotoxicity studies revealed that these herbicides are potent inhibitors of MDR1 that can modulate drug absorption and cause chemosensitization of cells. MRP1 was demonstrated to transport an important intermediate of the acetochlor detoxification pathway. Several specific interactions were shown when studying the interaction of chloroacetanilides with human transporter proteins. This study suggests an important role for transporter proteins in hazard prediction of agrochemicals and demonstrates how transporter interactions can be easily detected using in vitro screening methods.

In vitro transport of gimatecan (7-t-butoxyiminomethylcamptothecin) by breast cancer resistance protein, P-glycoprotein, and multidrug resistance protein 2

Lipophilic camptothecin derivatives are considered to have negligible affinity for breast cancer resistance protein (BCRP; ABCG2). Gimatecan, a new orally available 7-t-butoxyiminomethyl-substituted lipophilic camptothecin derivative, has been previously reported to be not a substrate for BCRP. Using a panel of in vitro models, we tested whether gimatecan is a substrate for BCRP as well as for P-glycoprotein (MDR1) or multidrug resistance protein 2 (MRP2; ABCC2), ATP-binding cassette drug efflux transporters involved in anticancer drug resistance, and able to affect the pharmacokinetics of substrate drugs. Cell survival, drug transport, accumulation, and efflux were studied in IGROV1 and (human BCRP overexpressing) T8 cells, Madin-Darby canine kidney II (MDCKII-WT, MDCKII-Bcrp1, MDCKII-MDR1, and MDCKII-MRP2), and LLCPK (LLCPK-WT and LLCPK-MDR1) cells. Competition with methotrexate uptake was studied in Sf9-BCRP membrane vesicles. In vitro, expression of BCRP resulted in 8- to 10-fold resistance to gimatecan. In Transwell experiments, gimatecan was transported by Bcrp1 and transport was inhibited by the BCRP/P-glycoprotein inhibitors elacridar and pantoprazole. Efflux of gimatecan from MDCKII-Bcrp1 cells was faster than in WT cells. In Sf9-BCRP membrane vesicles, gimatecan significantly inhibited BCRP-mediated transport of methotrexate. In contrast, gimatecan was not transported by MDR1 or MRP2. Gimatecan is transported by BCRP/Bcrp1 in vitro, although to a lesser extent than the camptothecin analogue topotecan. Implications of BCRP expression in the gut for the oral development of gimatecan and the interaction between gimatecan and other BCRP substrate drugs and/or inhibitors warrant further clinical investigation.

Localization of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) in Lipid Rafts/Caveolae and Modulation of Its Activity by Cholesterol in Vitro

Breast cancer resistance protein (BCRP/ABCG2) is an active efflux pump that belongs to the ATP-binding cassette (ABC) transporter family. It is located in various tissues involved in drug absorption, distribution, and elimination and plays an important role in multidrug resistance. For P-glycoprotein, another member of the ABC transporter family, it is well established that it is at least partly located in cholesterol and sphingolipid-enriched domains of the plasma membrane called "lipid rafts" and that the composition of the membrane lipids may modulate its efflux activity. This study addressed the compartmentalization of BCRP in the plasma membrane and the influence of membrane cholesterol on the efflux activity of BCRP. As a cell model, we used the canine kidney epithelial cell line MDCKII-BCRP transfected with the cDNA encoding human BCRP and the corresponding parental cell line MDCKII. Cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) provoked a 40% decrease in BCRP activity (p < 0.01) assessed with flow cytometry (pheophorbide A efflux assay). Cholesterol repletion with MbetaCD/cholesterol-inclusion complexes restored BCRP function, and cholesterol saturation of native cells did not further enhance BCRP activity. Coimmunoprecipitation experiments indicated a physical interaction between BCRP and caveolin-1, and Western blot analysis after density gradient ultracentrifugation demonstrated that BCRP is located in detergent-resistant membranes that also contain caveolin-1. In conclusion, our results demonstrate for the first time that BCRP is located in membrane rafts and that cholesterol has impact on its efflux activity.

Effect of Breast Cancer Resistance Protein (Bcrp/Abcg2) on the Disposition of Phytoestrogens

The effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of the phytoestrogens daidzein, genistein, and coumestrol was investigated using Bcrp(-/-) mice. Expression of the genes for either mouse Bcrp or human BCRP in MDCK II cells induced apically directed transport of the three phytoestrogens, whereas their transcellular transport was identical in mock and LLC-PK1 cells expressing mouse Mdr1a. After oral administration, the plasma levels of daidzein and genistein were increased in Bcrp(-/-) mice, but only a minimal change was observed for coumestrol. At steady state, tissue-to-plasma concentration ratios of the three phytoestrogens in the brain and testis of wild-type mice were very small and similar to those of [(14)C]inulin, whereas those were significantly increased in the brain and testis of Bcrp(-/-) mice. The largest increases were observed with genistein (9.2- and 5.8-fold in the brain and testis, respectively). The distributions of genistein in the epididymis and fetus, but not the ovary, were also increased in Bcrp(-/-) mice. The Bcrp protein was localized in the luminal membrane of the endothelial cells in the testis and the body of the epididymis and in both the luminal and abluminal side of ducts in the head of the epididymis. These results suggest that Bcrp limits the oral availability and distribution into the brain and testis, epididymis, and fetus of phytoestrogens.

Involvement of Breast Cancer Resistance Protein (ABCG2) in the Biliary Excretion Mechanism of Fluoroquinolones

Fluoroquinolones are effective antibiotics for the treatment of bile duct infections. It has been shown that the biliary excretion of grepafloxacin is partly accounted for by multidrug resistance-associated protein 2 (MRP2/ABCC2), whereas neither MRP2 nor P-glycoprotein is involved in the biliary excretion of ulifloxacin. In the present study, we examined the involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion of fluoroquinolones (grepafloxacin, ulifloxacin, ciprofloxacin, and ofloxacin). In Madin-Darby canine kidney II cells expressing human BCRP or mouse Bcrp, the basal-to-apical transport of grepafloxacin and ulifloxacin was greater than that of the mock control, which was inhibited by a BCRP inhibitor, 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143). Plasma and bile concentrations of fluoroquinolones were determined in wild-type and Bcrp(-/-) mice after i.v. bolus injection. The cumulative biliary excretion of fluoroquinolones was significantly reduced in Bcrp(-/-) mice, resulting in a reduction of the biliary excretion clearances to 86, 50, 40, and 16 of the control values, for ciprofloxacin, grepafloxacin, ofloxacin, and ulifloxacin, respectively. Preinfusion of sulfobromophthalein significantly inhibited the biliary excretion of grepafloxacin in Bcrp(-/-) mice. There was no change in the tissue/plasma concentration ratios of fluoroquinolones in the liver or brain, whereas those in the kidney were increased 3.6- and 1.5-fold for ciprofloxacin and grepafloxacin, respectively, in Bcrp(-/-) mice but were unchanged for ofloxacin and ulifloxacin. The present study shows that BCRP mediates the biliary excretion of fluoroquinolones and suggests that it is also involved in the tubular secretion of ciprofloxacin and grepafloxacin.

Modulation of human BCRP (ABCG2) activity by anti-HIV drugs

The safety and effectiveness of highly active antiretroviral therapy (HAART) is challenged by viral resistance to antiretrovirals and the frequent occurrence of drug interactions which may limit the access of these drugs to the target sites. In particular, drug distribution and elimination may be modified by active efflux transporters. While P-glycoprotein is well evaluated in this regard, the interaction of antiretrovirals with the ABC transporter BCRP (ABCG2) is far from being elucidated. The aim of this study was therefore to investigate the influence of all important anti-HIV drugs on BCRP activity in vitro in one assay to allow unrestricted comparison of the results. BCRP inhibition was assessed by an increase in pheophorbide A accumulation in MDCKII-BCRP cells and compared with the corresponding parental cell line MDCKII lacking human BCRP. According to the IC(50) estimation, the rank order for BCRP inhibition was lopinavir > nelfinavir > delavirdine > efavirenz > saquinavir > atazanavir > amprenavir > abacavir. Whereas nevirapine and zidovudine exerted weak inhibition, the inhibitory potency for ritonavir and tipranavir could not be estimated due to their low solubility and all other tested compounds (indinavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir and zalcitabine) were devoid of an effect. Taken together, our study demonstrates significant inhibition of BCRP by many anti-HIV drugs. These results suggest that inhibition of BCRP might contribute to drug-drug interactions observed during HAART in vivo and possibly also the superior effectiveness of combination antiretroviral therapy.

Effects of the Flavonoid Chrysin on Nitrofurantoin Pharmacokinetics in Rats: Potential Involvement of ABCG2

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter, important in drug disposition and in the development of multidrug resistance in cancer. Flavonoids, a large class of natural compounds widely present in the diet and herbal products, have been shown in vitro to be BCRP inhibitors. The flavonoid chrysin is a potent inhibitor of BCRP, inhibiting the efflux of mitoxantrone with an IC(50) of 0.39 microM in BCRP-overexpressing human MCF-7 breast cancer cells. The purpose of this study was to investigate the potential pharmacokinetic interactions between chrysin and nitrofurantoin (a specific BCRP substrate) in rats. In Madin-Darby canine kidney cells expressing human BCRP or murine Bcrp1, the polarized transport of nitrofurantoin was effectively inhibited by chrysin at concentrations of 20 and 100 microM. Compared with the vehicle-treated group, p.o. coadministration of chrysin (200 mg/kg) significantly increased the area under the curve (AUC) and C(max) of nitrofurantoin (10 mg/kg) by 1.76- (p < 0.01) and 1.72-fold (p < 0.05), respectively. When nitrofurantoin (2 mg/kg) was given i.v., administration of chrysin (50 mg/kg i.p.) significantly increased the AUC of nitrofurantoin (123 +/- 34.0 versus 91.5 +/- 18.0 microg/ml x min in controls, p < 0.05). Moreover, the cumulative hepatobiliary excretion of nitrofurantoin (1.5 mg/kg i.v.) was significantly decreased by approximately 75% at the end of 120 min after the coadministration of chrysin (50 mg/kg i.p.). Taken together, these results indicate that the flavonoid chrysin significantly inhibits nitrofurantoin transport mediated by human BCRP and murine Bcrp1. Bcrp1 inhibition by chrysin is likely one potential mechanism for the observed chrysin-nitrofurantoin pharmacokinetic interactions in rats.

Involvement of Breast Cancer Resistance Protein (BCRP/ABCG2) in the Biliary Excretion and Intestinal Efflux of Troglitazone Sulfate, the Major Metabolite of Troglitazone with a Cholestatic Effect

Troglitazone sulfate (TGZS) is the major metabolite of troglitazone (TGZ), an antidiabetic agent, and thought to be a cause of the cholestasis induced by TGZ. The aim of the present study is to elucidate the involvement of breast cancer resistance protein (BCRP/ABCG2) in the hepatic disposition of TGZS. The basal-to-apical transport of TGZS was enhanced in organic anion transporting polypeptide 1B1-expressing Madin-Darby canine kidney II cells by infection of recombinant adenovirus harboring human BCRP and mouse Bcrp cDNA. TGZS was given to wild-type and Bcrp (-/-) mice by constant infusion. Biliary excretion is the predominant elimination pathway of TGZS in wild-type mice, and the biliary excretion clearance of TGZS with regard to the hepatic concentration was reduced to 30% of the control in Bcrp (-/-) mice. However, plasma and hepatic concentrations were unchanged, suggesting induction of compensatory mechanisms in Bcrp (-/-) mice for the elimination of TGZS. Involvement of BCRP in the intestinal efflux transport of TGZS was examined using everted sacs. The mucosal efflux clearance of TGZS showed only a slight reduction (15% reduction) in Bcrp (-/-) mice. Our results suggest that BCRP plays a major role in the biliary excretion but a minor role in the intestinal transport of TGZS.

Lack of Interactions between Breast Cancer Resistance Protein (BCRP/ABCG2) and Selected Antiepileptic Agents

Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood-brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined. To study substrate affinity of tested AEDs to BCRP, transport experiments were performed in epithelial BCRP-expressing MDCKII-BCRP and MDCKII-parent cell lines cultured on microporous membrane. For detection of inhibitory potency of AEDs to BCRP, accumulation assays were carried out in MEF3.8-BCRP cells with known BCRP substrates, BODIPY FL prazosin and mitoxantrone. No obvious interactions of tested AEDs with BCRP transporter were observed. Therefore these drugs in relevant therapeutic concentrations are neither substrates nor inhibitors of BCRP. Based on our in vitro data we can conclude that resistance to treatment with the tested AEDs probably is not caused by the overexpression of BCRP in the BBB of epileptogenic brain tumors.

BacMam recombinant baculovirus in transporter expression: A study of BCRP and OATP1B1

Human BCRP and OATP1B1 have recently been identified as important transporters in the absorption, distribution, and elimination of clinically significant drugs. In this report, we illustrate the use of modified baculoviruses, termed BacMam viruses for the expression of functional BCRP and OATP1B1 in mammalian cells. We show a variety of host cells efficiently transduced to express BCRP including HEK 293, LLC-PK, and U-2 OS, where protein levels on the cell-surface were modulated by titrating different amounts of viral inoculum. In addition, using the BODIPY-prazosin efflux assay and the BacMam reagent we illustrate inhibition of BCRP activity with GF120918 or Fumitremorgin C. Furthermore, we present data demonstrating simultaneous expression of BCRP and OATP1B1 in BacMam transduced mammalian cells by simply adding viral inoculum of each transporter. Thus these results indicate that BacMam mediated gene delivery provides a novel and efficient research tool for the investigation of single or multiple transporters in vitro.

BREAST CANCER RESISTANCE PROTEIN (BCRP/ABCG2) TRANSPORTS FLUOROQUINOLONE ANTIBIOTICS AND AFFECTS THEIR ORAL AVAILABILITY, PHARMACOKINETICS, AND MILK SECRETION

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds, including their secretion into the milk. The purpose of this study was to determine whether three widely used fluoroquinolone antibiotics (ciprofloxacin, ofloxacin, and norfloxacin) are substrates of Bcrp1/BCRP and to investigate the possible role of this transporter in the in vivo pharmacokinetic profile of these compounds and their secretion into the milk. Using polarized cell lines, we found that ciprofloxacin, ofloxacin, and norfloxacin are transported by mouse Bcrp1 and human BCRP. In vivo pharmacokinetic studies showed that the ciprofloxacin plasma concentration was more than 2-fold increased in Bcrp1(-/-) compared with wild-type mice (1.77 +/- 0.73 versus 0.85 +/- 0.39 microg/ml, p < 0.01) after oral administration of ciprofloxacin (10 mg/kg). The area under the plasma concentration-time curve in Bcrp1(-/-) mice was 1.5-fold higher than that in wild-type mice (48.63 +/- 5.66 versus 33.10 +/- 4.68 min x microg/ml, p < 0.05) after i.v. administration (10 mg/kg). The milk concentration and milk/plasma ratio of ciprofloxacin were 2-fold higher in wild-type than in Bcrp1(-/-) lactating mice. We conclude that Bcrp1 is one of the determinants for the bioavailability of fluoroquinolones and their secretion into the milk.

Characterization of Polarized Expression of Point- or Deletion-Mutated Human BCRP/ABCG2 in LLC-PK1 Cells

In polarized cells, such as hepatocytes and intestinal epithelial cells, transporters are localized on the apical or basolateral membranes and play important roles in the vectorial transport of their substrates. In the current study, we have aimed to clarify the mechanism for the cellular sorting of human breast cancer resistance protein (BCRP/ABCG2), which is expressed on the apical membrane of many tissues and functions as an efflux transporter. After the expression vector, including wild type or mutants of human BCRP cDNA, was transfected into LLC-PK1 cells, immunohistochemical staining and Western blot analyses were performed to characterize the cellular localization and the status of BCRP, respectively. The transfected cDNA product of wild-type BCRP was expressed on the apical membrane in LLC-PK1 cells. Glycosylation consensus sequences-disrupted mutants showed the apical localization as the wild type, whereas the apical-selective expression disappeared when disulfide bonds could not be formed. Furthermore, examination of the localization of deletion mutants of human BCRP emphasized the importance of some peptide sequences. The region between the N-terminal and ATP-binding cassette and proximal C-terminal region, both of which are well conserved in various animal species, were found to be significant for proper localization. These results suggest that, although the presence of N-glycan does not affect the localization of BCRP, disulfide bonds and some peptide sequences in both the N- and C-terminals are necessary for the apical expression of BCRP.

Sex-Dependent Expression and Activity of the ATP-Binding Cassette Transporter Breast Cancer Resistance Protein (BCRP/ABCG2) in Liver

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter present in the liver and other tissues that affects the pharmacological behavior of many compounds. To assess the possible role of BCRP in sex-dependent pharmacokinetics, we studied the in vivo disposition of several murine Bcrp1 substrates in male and female wild-type and Bcrp1 knockout mice. After oral administration of the antibiotic nitrofurantoin, the area under the plasma concentration-time curve in wild-type female mice was approximately 2-fold higher than in wild-type male mice. Moreover, after i.v. administration of nitrofurantoin, the antiulcerative cimetidine, the anticancer drug topotecan, and the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the plasma levels in wild-type female mice were all significantly higher than those in wild-type male mice. Analysis of the expression of murine Bcrp1 in several pharmacokinetically important tissues showed that only the hepatic Bcrp1 expression was higher in male mice compared with female mice. In line with this difference, the hepatobiliary excretion for nitrofurantoin and PhIP was, respectively, 9-fold higher and approximately 2-fold higher in male compared with female wild-type mice. No significant sex differences were observed in plasma levels or hepatobiliary excretion for any of the tested compounds in Bcrp1-/- mice, indicating that Bcrp1 was the main cause of the sex difference in wild-type mice. Analysis of hepatic expression of human BCRP also indicated a higher expression in men compared with women. In conclusion, sex-dependent expression of BCRP/Bcrp1 in the liver may be a cause of sex-specific variability in the pharmacokinetics of BCRP substrates, with potential impact on the clinical-therapeutic applications and toxicity risks of drugs.

The Breast Cancer Resistance Protein (BCRP/ABCG2) Affects Pharmacokinetics, Hepatobiliary Excretion, and Milk Secretion of the Antibiotic Nitrofurantoin

Nitrofurantoin is a commonly used urinary tract antibiotic prescribed to lactating woman. It is actively transported into human and rat milk by an unknown mechanism. Our group has demonstrated an important role of the breast cancer resistance protein (BCRP/ABCG2) in the secretion of xenotoxins into the milk. This ATP-binding cassette drug efflux transporter extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds. We investigated whether Bcrp1 is involved in the pharmacokinetic profile of nitrofurantoin and its active secretion into the milk. Using polarized cell lines, we found that nitrofurantoin is efficiently transported by murine Bcrp1 and human BCRP. After oral administration of 10 mg/kg nitrofurantoin, the area under the plasma concentrationtime curve in Bcrp1 knockout mice was almost 4-fold higher than in wild-type mice (148.8 +/- 30.4 versus 37.5 +/- 6.8 min x microg/ml); and after i.v. administration (5 mg/kg), 2-fold higher (139.2 +/- 22.0 versus 73.9 +/- 9.0 min x microg/ml). Hepatobiliary excretion of nitrofurantoin was almost abolished in Bcrp1 knockout mice (9.6 +/- 3.2 versus 0.2 +/- 0.1% in wild-type and Bcrp1 knockout mice, respectively). The milk-to-plasma ratio of nitrofurantoin was almost 80 times higher in wild-type compared with Bcrp1 knockout lactating female mice (45.7 +/- 16.2 versus 0.6 +/- 0.1). Nitrofurantoin elimination via milk was quantitatively even higher than hepatobiliary elimination. We conclude that Bcrp1 is an important determinant for the bioavailability of nitrofurantoin and the main mechanism involved in its hepatobiliary excretion and secretion into the milk.

TRANSPORT OF ANTHELMINTIC BENZIMIDAZOLE DRUGS BY BREAST CANCER RESISTANCE PROTEIN (BCRP/ABCG2)

Methylcarbamate benzimidazoles [albendazole (ABZ), fenbendazole (FBZ), and their respective sulfoxide derivatives, albendazole sulfoxide (ABZSO) and oxfendazole (OXF)] are therapeutically important anthelmintic agents with low bioavailability. We studied their in vitro interaction with the apical ATP-binding cassette (ABC) drug efflux transporters, breast cancer resistance protein (BCRP/ABCG2), P-glycoprotein (ABCB1), and MRP2 (ABCC2) using MDCKII cells transduced with human MDR1, MRP2, and BCRP, and murine Bcrp1 cDNAs. These ABC drug efflux transporters extrude a wide range of xenotoxins from cells in intestine, liver, and other organs, thus affecting the bioavailability of many compounds. In transport experiments, ABZSO and OXF were transported efficiently by murine Bcrp1 and moderately by human BCRP, but not by MDR1 or MRP2. ABZ and FBZ were not found to be Bcrp1, MRP2, or P-glycoprotein substrates in vitro. OXF was found to be a good BCRP/Bcrp1 inhibitor, with somewhat higher potency in the MDCKII-BCRP cell line. The latter results were confirmed by flow cytometry experiments demonstrating inhibition by OXF of murine Bcrp1- and human BCRP-mediated mitoxantrone transport. Further studies of interactions between OXF and known BCRP/Bcrp1 substrates will be of interest. The use of efficacious BCRP/Bcrp1 inhibitors might increase the extent and duration of systemic exposure to ABZSO and OXF, with possible therapeutically beneficial effects in extra-intestinal infections.

Human breast cancer resistance protein: interactions with steroid drugs, hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine.

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells, mediating drug resistance and affecting the pharmacological behavior of many compounds. To study the interaction of human wild-type BCRP with steroid drugs, hormones, and the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), we expressed human BCRP in the murine MEF3.8 fibroblast cell line, which lacks Mdr1a/1b P-glycoprotein and Mrp1, and in the polarized epithelial MDCKII cell line. We show that PhIP was efficiently transported by human BCRP in MDCKII-BCRP cells, as was found previously for murine Bcrp1. Furthermore, we show that six out of nine glucocorticoid drugs, corticosterone, and digoxin increased the accumulation of mitoxantrone in the MEF3.8-BCRP cell line, indicating inhibition of BCRP. In contrast, aldosterone and ursodeoxycholic acid had no significant effect on BCRP. The four most efficiently reversing glucocorticoid drugs (beclomethasone, 6alpha-methylprednisolone, dexamethasone, and triamcinolone) and 17beta-estradiol showed a significantly reduced BCRP-mediated transepithelial transport of PhIP by MDCKII-BCRP cells, with the highest reduction of PhIP transport ratio for beclomethasone (from 25.0 +/- 1.1 to 2.7 +/- 0.0). None of the tested endogenous steroids or synthetic glucocorticoids or digoxin, however, were transported substrates of BCRP. We also identified the H(2)-receptor antagonist drug cimetidine as a novel efficiently transported substrate for human BCRP and mouse Bcrp1. The generated BCRP-expressing cell lines thus provide valuable tools to study pharmacological and toxicological interactions mediated by BCRP and to identify new BCRP substrates.

Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions.

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.