Human BCRP Research Articles

In vitro screening model for compound interactions with human and dairy animal BCRP orthologs.

Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood-milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis-Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an invitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.

Applicability of MDR1 Overexpressing Abcb1KO-MDCKII Cell Lines for Investigating In Vitro Species Differences and Brain Penetration Prediction.

Implementing the 3R initiative to reduce animal experiments in brain penetration prediction for CNS-targeting drugs requires more predictive in vitro and in silico models. However, animal studies are still indispensable to obtaining brain concentration and determining the prediction performance of in vitro models. To reveal species differences and provide reliable data for IVIVE, in vitro models are required. Systems overexpressing MDR1 and BCRP are widely used to predict BBB penetration, highlighting the impact of the in vitro system on predictive performance. In this study, endogenous Abcb1 knock-out MDCKII cells overexpressing MDR1 of human, mouse, rat or cynomolgus monkey origin were used. Good correlations between ERs of 83 drugs determined in each cell line suggest limited species specificities. All cell lines differentiated CNS-penetrating compounds based on ERs with high efficiency and sensitivity. The correlation between in vivo and predicted Kp,uu,brain was the highest using total ER of human MDR1 and BCRP and optimized scaling factors. MDR1 interactors were tested on all MDR1 orthologs using digoxin and quinidine as substrates. We found several examples of inhibition dependent on either substrate or transporter abundance. In summary, this assay system has the potential for early-stage brain penetration screening. IC50 comparison between orthologs is complex; correlation with transporter abundance data is not necessarily proportional and requires the understanding of modes of transporter inhibition.

Studies on the interaction of five triazole fungicides with human renal transporters in cells

The widespread use of triazole fungicides in agricultural production poses a potential risk to human health. This study investigates the interaction of five triazole fungicides, i.e., tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole with human renal transporters, including OAT1, OAT3, OCT2, OCTN1, OCTN2, MATE1, MATE2-K, MRP2, MDR1, and BCRP, using transgenic cell models. For uptake transporters, triticonazole was the substrate of OAT1 and OAT3 and the inhibitor of OCT2. Tebuconazole and penconazole inhibited OCTN2 (100 μM), while tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole inhibited MATE1 (100 μM). Tebuconazole and hexaconazole inhibited MATE2-K (100 μM). All five triazole fungicides were not substrates or strong inhibitors of MRP2, MDR1, and BCRP efflux transporters. Penconazole inhibited OCT2 with IC50 = 1.12 μM. Penconazole and uniconazole inhibited MATE1 with IC50 = 0.94 μM and 0.87 μM. Tebuconazole and hexaconazole inhibited MATE2-K with IC50 = 0.96 μM and 1.04 μM, indicating that triazole fungicides may inhibit renal drug transporter activity at low concentrations. Triticonazole was transported by OAT1 and OAT3, and the Km values of triticonazole were 5.81 ± 1.75 and 47.35 ± 14.27, respectively. Tebuconazole and uniconazole were transported by OAT3, and the Km values of tebuconazole and uniconazole were 30.28 ± 7.18 and 87.61 ± 31.70, respectively, which may induce nephrotoxicity.

Maternal and Fetal Exposure to (-)-Δ9-tetrahydrocannabinol and Its Major Metabolites in Pregnant Mice Is Differentially Impacted by P-glycoprotein and Breast Cancer Resistance Protein.

(-)-Δ9-tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis. 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are respectively the active and nonactive circulating metabolites of THC in humans. While previous animal studies reported that THC could be a substrate of mouse P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), we have shown, in vitro, that only THC-COOH is a weak substrate of human BCRP, but not of P-gp. To confirm these findings and to investigate the role of P-gp and/or Bcrp in the maternal-fetal disposition of THC and its metabolites, we administrated 3 mg/kg of THC retro-orbitally to FVB wild-type (WT), P-gp -/-, Bcrp -/-, or P-gp-/- /Bcrp-/- pregnant mice on gestation day 18 and estimated the area under the concentration-time curve (AUC) of the cannabinoids in the maternal plasma, maternal brain, placenta, and fetus, as well as the tissue/maternal plasma AUC geometric mean ratios (GMRs) using a pooled data bootstrap approach. We found that the dose-normalized maternal plasma AUCs of THC in P-gp-/- and P-gp-/- /Bcrp-/- mice, and the placenta-to-maternal plasma AUC GMR of THC in Bcrp-/- mice were 279%, 271%, and 167% of those in WT mice, respectively. Surprisingly, the tissue-to-maternal plasma AUC GMRs of THC and its major metabolites in the maternal brain, placenta, or fetus in P-gp -/-, Bcrp -/- or P-gp-/- /Bcrp-/- mice were 28-78% of those in WT mice. This study revealed that P-gp and Bcrp do not play a role in limiting maternal brain and fetal exposure to THC and its major metabolites in pregnant mice. SIGNIFICANCE STATEMENT: This study systematically investigated whether P-gp and/or Bcrp in pregnant mice can alter the disposition of THC, 11-OH-THC, and THC-COOH. Surprisingly, except for Bcrp, which limits placental (but not fetal) exposure to THC, we found that P-gp-/- , Bcrp-/- , and/or P-gp-/- /Bcrp-/- significantly decreased exposure to THC and/or its metabolites in maternal brain, placenta, or fetus. The mechanistic basis for this decrease is unclear and needs further investigation. If replicated in humans, P-gp- or BCRP-based drug-cannabinoid interactions are not of concern.

Transport and Permeation Properties of Dapivirine: Understanding Potential Drug-Drug Interactions.

The dapivirine (DPV) vaginal ring was developed by the nonprofit International Partnership for Microbicides (IPM) for reducing the risk of HIV infection. A clinical study (IPM 028) showed that concomitant use of the DPV ring and miconazole (MIC) altered DPV pharmacokinetic profile. In this work, we investigated whether or not DPV transport and permeation contributed to the observed DPV-MIC interaction. Our study evaluated the interaction between DPV and several transporters that are highly expressed in the human female reproductive tract, including MRP1, MRP4, P-gp, BCRP, and ENT1, using vesicular and cellular systems. We also evaluated the impact of DPV/MIC on cellular tight junctions by monitoring transepithelial electrical resistance with the Ussing chamber. Lastly, we evaluated the effect of MIC on DPV permeability across human cervical tissue. Our findings showed that DPV was not a substrate of MRP1, MRP4, P-gp, BCRP, or ENT1 transporters. Additionally, DPV did not inhibit the activity of these transporters. DPV, MIC, and their combination also did not disrupt cellular tight junctions. MIC did not affect DPV tissue permeability but significantly reduced DPV tissue levels. Therefore, our results suggest that the DPV-MIC interaction is not due to these five transporters, altered tight junction integrity, or altered tissue permeability.

Elucidation of clearance mechanism of TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor: does a species difference in excretion routes exist between humans and animals?

TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor, is reportedly excreted predominantly through urinary excretion in an unchanged form in humans, with partial biliary excretion also possible. However, the clearance mechanisms remain unclear. The aim of this study was to investigate the clearance mechanisms in humans and to assess species differences in the excretion routes. TP0463518 was not metabolized in rat, dog, or human hepatocytes. TP0463518 is a substrate for human BCRP, OATP1B1, OATP1B3, and OAT3, suggesting that renal uptake by OAT3 is probably the predominant clearance route, with hepatic uptake by OATP1B1 and OATP1B3 contributing partially to clearance in humans. A species difference in excretion routes was observed. The unchanged urinary excretion rates in humans, male rats, female rats, dogs, and monkeys were 80.7%, 0.1%, 40.9%, 15.2%, and 72.6%, respectively. Urinary excretion was predominant in humans and monkeys, while only biliary excretion was observed in male rats. Uptake studies using hepatocytes showed that the hepatic uptake clearance in rats was 13.6-fold higher than that in humans. Therefore, not only reabsorption via renal tubules, but also hepatic uptake seems to be involved in the species differences in excretion routes between rats and humans.

Evaluation of potential herb-drug interactions based on the effect of Suxiao Jiuxin Pill on CYP450 enzymes and transporters

Ethnopharmacology relevanceSuxiao jiuxin pill (SJP) is a Chinese medical drug with anti-inflammatory, anti-apoptotic, and vasodilatory function. It is widely used in combination with other drugs for the treatment of coronary heart disease (CHD) and angina. Nevertheless, the effect of SJP on Cytochrome P450 (CYP450) enzymes and transporters’ activity related to drug metabolism is rarely studied. ObjectiveThe aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. Materials and methodsHuman primary hepatocytes were used in present study. Probe substrates of CYP450 enzymes were incubated in human liver microsomes (HLMs) with and without SJP while IC50 values were calculated. The inhibitory effect of SJP on the activity of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. The inducing effect of SJP on the activity of CYP1A2, 2B6 and 3A4 was accessed. The inhibition of SJP on human OATP1B1 was investigated through cell-based assay. The inhibition of SJP on human MDR1 and BCRP was also estimated by means of the vesicles assay. ResultsThe results showed that the SJP under the concentration of 1000 μg/mL could inhibit the activity of CYP1A2, 2B6, 2C19, and 3A4, with IC50 values of 189.7, 308.2, 331.2 and 805.7 μg/mL, respectively. There was no inhibitory effect found in the other 3 liver drug enzyme subtypes. In addition, SJP showed no induction effect on CYP1A2, 2B6 and 3A4, however it had a significant inhibitory effect on human-derived OATP1B1 at the concentration of 100 and 1000 μg/mL, with the IC50 value of 21.9 μg/mL. Simultaneously, the SJP inhibited BCRP at high concentration of 1000 μg/mL but did not affect human MDR1. ConclusionsBased on these research results above, it is suggested that the SJP can affect some of the CYP450 enzymes and transporters' activity. When used in combination with related conventional drugs, potential herb-drug interactions should be considered.

In vitro cytochrome P450- and transporter-mediated drug interaction potential of 6β-hydroxy-21-desacetyl deflazacort-A major human metabolite of deflazacort.

6β‐Hydroxy‐21‐desacetyl deflazacort (6β‐OH‐21‐desDFZ) is a major circulating but not biologically active metabolite of deflazacort (DFZ). In vitro studies were performed to evaluate cytochrome P450 (CYP)‐ and transporter‐mediated drug interaction potentials of 6β‐OH‐21‐desDFZ. Up to 50 µM, the highest soluble concentration in the test system, 6β‐OH‐21‐desDFZ weakly inhibited (IC50 > 50 µM) the enzyme activity of CYPs 1A2, 2B6, 2C8, 2C9, and 2D6, while moderately inhibiting CYP2C19 and CYP3A4 with IC50 values of approximately 50 and 35 μM, respectively. The inhibition was neither time‐dependent nor metabolism‐based. Incubation of up to 50 µM 6β‐OH‐21‐desDFZ with plated cryopreserved human hepatocytes for 48 h resulted in no meaningful concentration‐dependent induction of either mRNA levels or enzyme activity of CYP1A2, CYP2B6, or CYP3A4. In transporter inhibition assays, 6β‐OH‐21‐desDFZ, up to 50 µM, did not show interaction with human OAT1, OAT3, and OCT2 transporters. It weakly inhibited (IC50 > 50 µM) human MATE1, MATE2‐K, and OCT1 transporter activity, and moderately inhibited human MDR1, OATP1B1, and OATP1B3 transporter activity with IC50 values of 19.81 μM, 37.62 μM, and 42.22 μM, respectively. 14C‐6β‐OH‐21‐desDFZ was biosynthesized using bacterial biotransformation and the subsequent study showed that 6β‐OH‐21‐desDFZ was not a substrate for human BCRP, MDR1, MATE1, MATE2‐K, OAT1, OATP1B1, OATP1B3, and OCT2 transporters, but appeared to be an in vitro substrate for the human OAT3 uptake transporter. At plasma concentrations of 6β‐OH‐21‐desDFZ seen in the clinic, CYP‐ and transporter‐mediated drug–drug interactions are not expected following administration of a therapeutic dose of DFZ in Duchenne muscular dystrophy (DMD) patients.

Interactions between Oroxylin A with the solute carrier transporters and ATP-binding cassette transporters: Drug transporters profile for this flavonoid

Oroxylin A is a flavonoid monomer extracted from Scutellaria baicalensis Georgi with neuroprotective, anti-tumor activity and many other biological functions. However, the interaction between Oroxylin A and the drug transporters has not been clearly reported. The purpose of this study is to investigate the interaction between Oroxylin A and the solute carrier transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2K), and ATP-binding cassette transporters (BCRP, MDR1). The HEK293 cell lines (HEK293-OATP1B1, HEK293-OATP1B3, HEK293-OAT1, HEK293-OAT3, HEK293-OCT2, HEK293-MATE1, and HEK293-MATE2K) that stably expressing previous listed human-derived transporters were employed to evaluate the solute carrier transporters. Vesicles expressing human BCRP and MDR1 transporters was employed to research ATP-binding cassette transporters. Our work suggested that Oroxylin A was a substrate of OATP1B1, OATP1B3, but not a substrate of the other transporters in the concentration range of our study. Oroxylin A shows concentration-dependent inhibition of OATP1B1, OAT1, OAT3 and BCRP transportation with the half-inhibitory concentration (IC50) of 7.03, 0.961, 0.112 μM, and 0.477 μM, respectively. No inhibitory effects on the transport activities of other transporters were observed for Oroxylin A. Drug transporters profile of Oroxylin A was first confirmed by our work, which provides important information for its pharmacokinetics, pharmacodynamics, and drug-drug interactions studies.

Human efflux transport of testosterone, epitestosterone and other androgen glucuronides

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 μM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 μM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.

Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC–MS/MS proteomics. The fractional contribution of individual transporters (ft) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5′-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.

Characterization of Novel Mechanisms of Efflux Transport of the Glucuronides of Testosterone and its Metabolites: a Quantitative Proteomics‐based IVIVE Study and Relevance to Enterohepatic Recirculation of Androgens

Testosterone (T) is primarily eliminated via glucuronidation upon oral administration. As a result, testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are produced as the key circulating metabolites, which are excreted in urine and bile. The goal of this study was to characterize the in vivo fractional contribution of individual hepatic transporters (ft) in the elimination of the glucuronides of T and its metabolites. We first performed vesicular transport screening assay with recombinant human MRP2, MRP3, MRP4, MDR1, and BCRP to identify the transporters that are involved in the efflux of TG, AG, EtioG and DHTG. The transport kinetic analyses were then carried out for the transporters that exhibited activity in the initial screening. The inside‐out vesicle ratio was determined by 5′‐nucleotidase activity assay. We next quantified the protein abundances of these efflux transporters in vesicles, human liver, and intestine by quantitative LC‐MS/MS proteomics using a validated surrogate peptide‐based approach. The ft for each transporter was estimated by proteomics‐based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and by correction for the inside‐out vesicles. The transport screening assays revealed that TG, AG, EtioG, and DHTG were primarily effluxed by MRP2 and MRP3 with differential contribution of individual transporters. EtioG and DHTG were transported by MDR1 and BCRP, respectively, but at much lower rates than by MRPs. MRP2 and MRP3 exhibited higher intrinsic clearance for TG, followed by DHTG, AG, and EtioG (Figure 1). The Km value for EtioG transport by MRP3 was about 3 to 20‐fold lower than for the other glucuronides. After integrating the transporter abundance data from human tissues, the ft of MRP2 and MRP3 in efflux of TG, EtioG, and DHTG in liver was calculated to be around 0.6 and 0.4, respectively. For AG, MRP3 played a major role (68%) to its efflux in liver [corroborates with our in vivo clinical data (Basit et al., CTS, 11, 2018)]. In conclusion, our results for the first‐time demonstrated that MRP2 and MRP3 are the major transporters that are involved in the efflux of T glucuronide conjugates. Important role of MRP2 in transport of TG, EtioG, and DHTG indicates the potential importance of enterohepatic recirculation in regulating the circulating T, Etio, and DHT after deconjugation in the gut. With this respect, our unpublished data suggest efficient deconjugation of these glucuronides by human relevant β‐glucuronidases. Characterization of the efflux mechanisms of glucuronides of T and DHT is important for predicting the androgen disposition and interindividual variability, including drug‐androgen interaction in humans. The mechanistic transport data can be extrapolated (using quantitative proteomics) to other androgen relevant tissues, such as kidney, prostate, testis and placenta.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Impact of Endogenous Breast Cancer Resistance Protein on Human P-Glycoprotein-Mediated Transport Assays Using LLC-PK1 Cells Transfected With Human P-Glycoprotein

Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates.

Evaluation of Drug Transport in MDCKII-Wild Type, MDCKII-MDR1, MDCKII-BCRP and Caco-2 Cell Lines.

Drug transporters function as gatekeepers and modulate drug access into body and various tissues. Thus, a thorough and precise understanding of transporter liability for compound uptake and efflux is critical during drug development. In the present study, we assessed the apparent permeability (Papp) and compared efflux ratio of various compounds in stably transfected Madin-Darby Canine Kidney (MDCKII) cells overexpressing human P-gp (MDCKII-MDR1), human BCRP (MDCKII-BCRP), wild-type (MDCKII-WT), and Caco-2 cell monolayers. We observed that quinidine, a substrate for MDR1 transporter, showed efflux ratio (Papp B-A/ Papp A-B) of 838 in MDCKII-MDR1 cells which plummeted to 14 in presence of verapamil, a known inhibitor of MDR1. With MDCKII-WT cells, Papp of quinidine dropped from 2 to 1, in the presence of verapamil. Caco-2 cells showed a diminutive decrease in efflux ratio of quinidine from 2.5 to 1.6 by verapamil. Prazosin and dantrolene were evaluated in MDCKII-BCRP cells and were found to have 80-fold higher efflux ratio compared to MDCKII-WT cells. In Caco-2 cells, prazosin and dantrolene showed efflux ratio of 4 and 2, respectively. Rhodamine-123, a fluorogenic probe substrate of MDR1 showed an efflux ratio of 4 in Caco-2 cells and BCRP substrate estrone-3-sulphate showed an efflux ratio of 7. In presence of BCRP inhibitor fumitremorgin-c, the efflux ratio of estrone-3-sulfate dropped to 1 in Caco-2 cells. The very high efflux ratios of MDR1 and BCRP substrates in transfected MDCKII cells clearly demonstrate the potential usefulness of these models to provide more definitive data to evaluate the transporter involvement compared to Caco-2 or MDCKII-WT cells.

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E1-G), estradiol-3- and 17-glucuronides (E2-3G and E2-17G), as well as estriol-3- and 16-glucuronides (E3-3G and E3-16G) are found in human plasma and urine. Unlike E2-17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E1-G, E2-3G, E3-3G, E3-16G and estrone-3-sulfate (E1-S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E1-S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E1-G and E2-3G, were still transported by BCRP at 10-fold higher rates than E1-S. BCRP also transported E3-16G at higher rates than the studied MRPs, while it transported E3-3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E1-G, E2-3G, E3-3G and E3-16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high Km values, between 180 and 790 μM. MRP3 transported all the tested glucuronides at rather similar rates, at Km values below 20 μM, but lower Vmax values than other transporters. In the case of E3-3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E3-16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans.

TKI combination therapy: strategy to enhance dasatinib uptake by inhibiting Pgp- and BCRP-mediated efflux.

The overexpression of efflux transporters, especially P-glycoprotein (Pgp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2), represents an important mechanism of multidrug resistance (MDR). Tyrosine kinase inhibitors (TKIs), a novel group of target-specific anticancer drugs, have recently been found to interact with Pgp and BCRP and to serve as both substrates and inhibitors. Considering their dual role, we anticipate that combination TKI therapy may represent a promising strategy to reverse efflux transporter mediated TKI resistance. Presently, investigations on these interactions are very limited. To fill the literature gap, dasatinib was used as the model drug and the effects of various TKIs on Pgp- and BCRP- mediated dasatinib efflux were evaluated. Cell uptake studies were performed using LLC-PK1 and MDCK-II cells along with their subclones that were transfected with human Pgp and BCRP, respectively. Among the 14 TKIs screened, nine TKIs greatly inhibited Pgp-mediated dasatinib efflux at 50μm. Further concentration dependent studies showed that imatinib, nilotinib and pazopanib were potent Pgp inhibitors with IC50 values of 2.42, 6.11 and 8.06μm, respectively. Additionally, 50μm of five TKIs greatly increased dasatinib accumulation through BCRP inhibition. Concentration dependent studies revealed that imatinib, erlotinib, nilotinib, axitinib and pazopanib were potent BCRP inhibitors with IC50 values of 0.94, 2.23, 2.50, 6.89 and 10.4μm, respectively. Our findings point to potential combinations of TKIs that could enhance intracellular concentrations of the targeted TKI, overcome MDR and improve TKI efficacy. Further in vivo studies are warranted to confirm the efflux transporter-mediated TKI-TKI interaction. Copyright © 2016 John Wiley & Sons, Ltd.

ABCG2/BCRP interaction with the sea grass Thalassia testudinum.

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. To evaluate the interaction of ABCG2/BCRP with the extract, studies in cells overexpressing human BCRP transporter and its murine ortholog Bcrp1 were performed. 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.

Multiple ABC Transporters Efflux Baicalin.

Baicalein, the aglycone formed by hydrolysis of baicalin in the intestine, is well absorbed by passive diffusion but subjected to extensive intestinal glucuronidation. Efflux of baicalin, the low passive permeability glucuronide of baicalein from enterocytes, likely depends on a carrier-mediated transport. The present study was designed to explore potential drug-herb interaction by investigating the inhibitory effect of baicalin on the transport of reporter substrates by transporters and to identify the transporters responsible for the efflux of baicalin from enterocytes and hepatocytes. The interaction of baicalin with specific ABC transporters was studied using membranes from cells overexpressing human BCRP, MDR1, MRP2, MRP3 and MRP4. Baicalin was tested for its potential to inhibit vesicular transport by these transporters. The transport of baicalin by the selected transporters was also investigated. Transport by BCRP, MRP3 and MRP4 was inhibited by baicalin with an IC50 of 3.41 ± 1.83 μM, 14.01 ± 2.51 μM and 14.39 ± 5.69 μM respectively. Inhibition of MDR1 (IC50 = 94.84 ± 31.10 μM) and MRP2 (IC50 = 210.13 ± 110.49 μM) was less potent. MRP2 and BCRP are the apical transporters of baicalin that may mediate luminal efflux in enterocytes and biliary efflux in hepatocytes. The basolateral efflux of baicalin is likely mediated by MRP3 and MRP4 both in enterocytes and hepatocytes. Via inhibition of transport by ABC transporters, baicalin could interfere with the absorption and disposition of drugs.

Genistein Reduces Glyburide Efflux by the Human Placental BCRP Transporter: Transcriptional Regulation and Direct Inhibition

The hypoglycemic drug glyburide is routinely used in the treatment of gestational diabetes. Fetal exposure to glyburide is typically low because the BCRP/ABCG2 transporter on placental syncytiotrophoblasts prevents its maternal‐to‐fetal transfer. The purpose of this study was to determine the effect of the dietary soy isoflavone genistein on the transcriptional regulation and direct inhibition of BCRP. For the regulation studies, human choriocarcinoma BeWo placental cells were incubated with genistein (0‐10 µM). After 48 h exposure to genistein, placental BeWo cells exhibited a 40% reduction in BCRP mRNA and protein. Down‐regulation of BCRP led to a 50% increase in the accumulation of the BCRP substrate, Hoechst 33342 in BeWo cells treated with genistein. Further, down‐regulation of BCRP by genistein was prevented by removal of serum from the media. In addition to modulating the expression of BCRP, genistein also competitively inhibited its transport in HEK cells overexpressing the wild‐type (WT) or functionally‐reduced BCRP variant (C421A). As expected, C421A‐BCRP cells had 10% greater accumulation of 3H‐glyburide than WT‐BCRP cells. Genistein increased the accumulation of 3H‐glyburide in both WT‐ and C421A‐BCRP cells by 50%. Taken together, pregnant women expressing the common loss‐of‐function C421A‐BCRP variant and/or consume genistein through a soy diet may be at an elevated risk for fetal exposure to glyburide and subsequent neonatal hypoglycemia. Support: ES020522, ES005022, ES007148, AFPE and PhRMA Predoctoral Fellowships.

The antiepileptic drug lamotrigine is a substrate of mouse and human breast cancer resistance protein (ABCG2)

Resistance to antiepileptic drugs (AEDs) is the major problem in the treatment of epilepsy. One hypothesis to explain AED resistance suggests that seizure-induced overexpression of efflux transporters at the blood–brain barrier (BBB) restricts AEDs to reach their brain targets. Various studies examined whether AEDs are substrates of P-glycoprotein (Pgp; MDR1; ABCB1), whereas information about the potential role of breast cancer resistance protein (BCRP; ABCG2) is scanty. We used a highly sensitive in vitro assay (concentration equilibrium transport assay; CETA) with MDCKII cells transduced with murine Bcrp1 or human BCRP to evaluate whether AEDs are substrates of this major efflux transporter. Six of 7 AEDs examined, namely phenytoin, phenobarbital, carbamazepine, levetiracetam, topiramate, and valproate, were not transported by Bcrp at therapeutic concentrations, whereas lamotrigine exhibited a marked asymmetric, Bcrp-mediated transport in the CETA, which could be almost completely inhibited with the Bcrp inhibitor Ko143. Significant but less marked transport of lamotrigine was determined in MDCK cells transfected with human BCRP. Lamotrigine is also a substrate of human Pgp, so that this drug is the first AED that has been identified as a dual substrate of the two major human efflux transporters at the BBB. Previous in vivo studies have demonstrated a synergistic or cooperative role of Pgp and Bcrp in the efflux of dual substrates at the BBB, so that transport of lamotrigine by Pgp and BCRP may be an important mechanism of pharmacoresistance in epilepsy patients in whom both transporters are overexpressed.