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Research ArticleArticle

Correlation between Membrane Protein Expression Levels and Transcellular Transport Activity for Breast Cancer Resistance Protein

Houfu Liu, Liang Huang, Yi Li, Tingting Fu, Xueying Sun, Yan-Yan Zhang, Ruina Gao, Qingfang Chen, Wandong Zhang, Jasminder Sahi, Scott Summerfield and Kelly Dong
Drug Metabolism and Disposition May 2017, 45 (5) 449-456; DOI: https://doi.org/10.1124/dmd.116.074245

Abstract

Emerging evidence indicates an important role for the breast cancer resistance protein (BCRP) in limiting brain penetration of substrate drugs. While in vitro transwell assays can provide an indication of BCRP substrate potential, the predictability of these assays in relation to in vivo brain penetration is still under debate. The present study examined the correlation of BCRP membrane protein expression level and transcellular transport activity across Madin-Darby canine kidney (MDCK) II monolayers. We expressed human BCRP or murine BCRP1 in MDCKII wild-type cells using BacMam2 virus transduction. The selective P-glycoprotein (P-gp) inhibitor LY335979 (1 μM) was included in the transport medium to measure BCRP-mediated transcellular transport for P-gp and BCRP cosubstrates. The BCRP levels in membrane extracts from MDCKII-BCRP or MDCKII-Bcrp1 cells were quantified by liquid chromatography–tandem mass spectrometry. The results are summarized as follows: 1) the membrane protein expression levels correlate with the corrected efflux ratios of substrates for human BCRP and murine BCRP1 within the efflux ratios investigated; 2) we demonstrate good concordance in rank order between the BCRP and BCRP1-mediated efflux ratios for 12 drugs; and 3) we propose an approach to contextualize in vitro BCRP transport data of discovery compounds by comparing them to the in vitro and in vivo transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted compromised brain penetration for 25 discovery compounds in rodents, which were BCRP substrates but not P-gp or weak P-gp substrates. These results suggest that BCRP-expressing MDCKII cells are useful in predicting the in vivo role of BCRP in brain penetration.

Introduction

Drugs designed for brain disorders should be able to the cross the blood-brain barrier (BBB) at appropriate concentrations in order to exert their desired pharmacology. The strength of the BBB is the result of very tight junctions between endothelial cells, polarized active efflux transporters, and limited pinocytosis. The major efflux transporters expressed at the luminal side of the BBB are the ATP-binding cassette (ABC) family of transporters including P-glycoprotein [(P-gp); ABCB1] and the breast cancer resistance protein [(BCRP); ABCG2]. The prominent role of P-gp in limiting the entry of various drugs into the central nervous system (CNS) has been well documented (Schinkel et al., 1996; Doran et al., 2005; Shen and Zhang, 2010). Thus, polarized cell monolayers such as Madin-Darby canine kidney (MDCK) or Caco2 with expression of human P-gp were deployed by the pharmaceutical industry to triage drug candidates in CNS drug discovery. The predictive value of these in vitro assays was widely recognized and strong P-gp substrates were unarguably deprioritized for centrally acting compounds (Yamazaki et al., 2001; Mahar Doan et al., 2002; Feng et al., 2008; Kikuchi et al., 2013).

BCRP (ABCG2) is the second member of the G subfamily of ABC transporter proteins. BCRP is constitutively expressed in human tissues such as CNS (the luminal membrane of BBB endothelia), intestine, kidney, liver, placenta, testis, and stem cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds (Schnepf and Zolk, 2013). Since its discovery in 1998, many drugs and endogenous substances have been identified to be transported by BCRP (Doyle et al., 1998; Lee et al., 2015). Brain penetration studies in naive, Mdr1a/b−/−, Bcrp1−/−, and Mdr1a/b−/−Bcrp1−/− mice or rats clearly illustrated the restrictive effect of BCRP1 on brain disposition for substrate drugs, such as cancer therapeutics, dantrolene, and phytoestrogens (Enokizono et al., 2007; Schnepf and Zolk, 2013). Recent liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based proteomic analyses revealed that BCRP is the most abundant transporter at human BBB and shows approximately 2-fold higher expression in human than in rodent. This is in contrast to about 2- to 3-fold lower P-gp expression at the BBB in human than in rodent (Kamiie et al., 2008; Uchida et al., 2011b; Hoshi et al., 2013). In addition, BCRP expression is up-regulated in some neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis patients (Xiong et al., 2009; Jablonski et al., 2012). These data could imply a more prominent role of BCRP in brain penetration in diseased humans compared with mice and rats.

Despite this emerging evidence, BCRP is largely overlooked in CNS drug discovery programs due to several reasons. First, BCRP and P-gp share largely overlapping substrate specificity. These transporters cooperate to limit the brain distribution of their cosubstrates and compensate for each other when the function of one is impaired. Teasing out BCRP-specific impact on drug brain penetration in intact systems is not an easy task, in part due to the limited specificity of substrates and inhibitors. In addition, utilization of mouse or rat as the most-evaluated preclinical species to examine the role of efflux transporters at the BBB in brain penetration may underestimate and reinforce the contribution of BCRP and P-gp, respectively, due to species difference in protein expression. Second, BCRP is distinct from P-gp and multidrug resistance–associated protein since it is a half-transporter that functions as a dimer or oligomer (Poguntke et al., 2010). The structural understanding of drug binding and transport by BCRP is still incomplete. The structural and physicochemical properties governing the interaction with BCRP remain largely unexplored. This complexity poses many uncertainties for project teams in relation to knowing how to walk away from this liability. Third, the predictive value of in vitro BCRP transport assays remains controversial. BCRP was reported to play a minimal role at the BBB in vivo despite mediating transport of various compounds in vitro (Zhao et al., 2009). Another study did not demonstrate a positive relationship between in vivo and in vitro BCRP activities in brain and testis penetration for eight compounds (Enokizono et al., 2008). These knowledge gaps cast doubt on the benefit of compound screening against BCRP in CNS discovery projects.

In the present study, we aimed to examine the relationship between BCRP expression and transcellular transport activity using newly constructed human and murine BCRP-expressing MDCKII cells. We have also examined species differences in mouse and human BCRP transport activities for 12 drugs. Finally, we examined the relationship between the BCRP efflux ratio (ER) and the rodent unbound brain-to-blood (plasma) concentration ratio (Kp,uu) for 25 compounds, which were BCRP substrates but not P-gp or weak P-gp substrates.

Materials and Methods

Materials.

Axitinib and flavopiridol were purchased from Toronto Research Chemicals (Toronto). Dantrolene sodium salt, coumestrol, prazosin hydrochloride, and Ko143 were obtained from Sigma (St. Louis, MO). Daidzein was procured from J&K Chemical Ltd. (Shanghai, China); genistein was procured from Acros Organics (Thermo Fisher Scientific, Waltham, MA); erlotinib hydrochloride and sunitinib maleate were procured from BIONET (Cornwall, United Kingdom); dasatinib and gefitinib were procured from Nanjing Chemlin Chemicals Co., Ltd. (Nanjing, China); and imatinib tosylate and sorafenib tosylate were procured from Far Top Limited Co., Ltd. (Nanjing, China). LY335979 hydrochloride was purchased from Shanghai Haoyuan Chemexpress Co., Ltd. (Shanghai, China). Amprenavir was obtained from the GlaxoSmithKline compound library. Cell culture reagents were purchased from Invitrogen (Carlsbad, CA). All other reagents used were of bioanalytical grade or higher. For BCRP and BCRP1 quantification, signature peptide (SSLLDVLAAR) standard and corresponding stable isotope labeled ([13C15N]-leucine, SSLLDVL*AAR) internal standard were obtained from GL Biochem (Shanghai) Ltd. (Shanghai, China). The ProteoExtract native membrane protein extraction kit was procured from Calbiochem (Temecula, CA). The protein quantification bicinchoninic acid assay kit was purchased from Thermo Fisher Scientific. Tosylsulfonyl-phenylalanyl-chloromethyl ketone-treated trypsin from bovine pancreas was purchased from Sigma and used for protein digestion. Iodoacetamide was obtained from Sigma and dithiotrietol was purchased from Fluka (Buchs, Switzerland).

Generation of BacMam2 Virus Carrying Human BCRP and Murine Bcrp1 cDNA.

ABCG2 human cDNA was amplified from brain, placenta, and testis cDNA using polymerase chain reaction (PCR) and then cloned into pCDNA3.2TOPO. The BCRP sequence was verified in-house. Abcg2 murine cDNA clone was purchased from Origene (Rockville, MD). PCR cloning of human BCRP or murine Bcrp1 cDNA into pHTBV1mcs3 vector between BamHI and XhoI sites was verified. The BacMam plasmid pHTBV-BCRP or pHTBV-Bcrp1 was transformed into DH10Bac Escherichia coli competent cells (Invitrogen), and a single colony from a freshly streaked selective plate was picked and inoculated into 5.0 ml of medium containing the appropriate selective antibiotic. After ∼16 hour of incubation at 37°C with vigorous shaking (200 rpm), bacmid DNA was extracted. The purified BCRP or Bcrp1 bacmid DNA was transfected into Sf9 cells in 6-well dishes at 9 × 105 cells/well using Cellfectin (Invitrogen). After 3 to 4 days when the cytopathic effect (the inhibition of cell growth and lysis of cells) was evident, the culture was collected and centrifuged to pellet the cell debris. The supernatant containing the virus (P0 virus) was transferred to a sterile 15-ml screw-cap tube, and stored at 4°C in the dark. The virus stock was further amplified by infecting 200 ml of Sf9 cells (2 × 106 cells/ml) at a multiplicity of infection (MOI) ranging from 0.05 to 0.1. The P1 virus was harvested 4 days later, and titration was performed in a viral plaque assay using the BacPAK Baculovirus Rapid Titer Kit (Clontech, Palo Alto, CA). The recombinant virus was confirmed to contain the human BCRP or murine Bcrp1 gene insert by PCR using extracted virus DNA as the template and referred to as BacMam2-BCRP or BacMam2-Bcrp1.

Construction of Human BCRP- and Murine BCRP1-Expressing MDCKII Cells.

Polarized MDCKII wild-type (WT) cells were obtained from The Netherlands Cancer Institute (Amsterdam). MDCKII cells were seeded at a density of 0.1 million/transwell insert in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. BacMam2-BCRP or BacMam2-Bcrp1 at an appropriate MOI was administered to the apical side of the 24-well transwell inserts (Millipore Corporation, Bedford, MA) 24 hours post MDCKII cell seeding. The cells were cultured for another 48 hours before transport experiments.

Transport Studies across BacMam2-BCRP or BacMam2-Bcrp1 Cells.

On the day of transport experiments, donor solutions were prepared by diluting test compounds in transport medium (Dulbecco’s modified Eagle’s medium supplemented with 4500 mg/l d-glucose, l-glutamine, and 25 mM HEPES but without sodium pyruvate and phenol red, pH 7.4). The receiver solution was the transport medium. The transport of the test compounds was measured in both directions [apical to basolateral (A→B) and basolateral to apical (B→A)] for 90 minutes in the absence and presence of transporter inhibitor. The transepithelial electrical resistance was measured using a Millipore Millicell-ERS System with chopstick electrodes (Millipore Corporation). Lucifer yellow was used as a paracellular marker to determine the integrity of MDCKII monolayers and was measured by a SpectraMax Gemini Cytofluorimeter (Molecular Devices, Sunnyvale, CA) set to an excitation wavelength of 430 nm and emission wavelength of 540 nm. The exact permeability (Pexact, nm/s) and mass balance (MB) percentage for test compound across MDCKII monolayers were determined using the eqs. 1 & 2, as follows:Embedded Image(1)Embedded Image(2)where VD and VR are the donor and receiver well volumes (in ml), respectively; A is the membrane surface area (in cm2); t is the transport time (in seconds); CR(t) and CD(t) are the measured mass spectrometric response or concentration of the test compound in the receiver and donor wells at time t (90 minutes), respectively; and CD(t0) is the measured mass spectrometric response or concentration of the test compound in the initial donor solution at time 0. Equation 1 was also used to determine the membrane permeability of lucifer yellow.

The ER is defined as the permeability ratio of the basolateral-to-apical direction to the apical-to-basolateral direction. The corrected ER is the ratio of ER in the absence of inhibitor to the presence of inhibitor completely abolishing transporter activity. This is introduced to take into account any involvement of endogenous transporters in MDCKII-WT cells in transcellular transport of test compounds.

Quantification of Test Compounds in Transport Samples by LC-MS/MS.

Quantification of drugs in MDCKII transport samples was performed using the ACQUITY UPLC system (Waters, Milford, MA) coupled with the 4000 Q-Trap or API 5000 triple-quadrupole mass spectrometer (AB Sciex, Foster City, CA). The samples were processed by deproteination with the same volume of acetonitrile containing an appropriate internal standard. The chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18 or UPLC HSS T3 (50 × 2.1 mm, 1.7 µm) analytic column at 40°C, using a gradient of aqueous (solvent A: 1 mM ammonia acetate in water or 0.1% formic acid in water) and organic [solvent B: CH3CN-CH3OH with or without 0.1% formic acid (4:1, v/v)] mobile phase at a flow rate of 600 µl/min. The run time for each compound was 2.0 minutes. The key chromatographic and mass spectrometric settings were optimized to yield the best sensitivity for each test compound and are detailed in Supplemental Table 1.

Protein Quantification of Human BCRP and Murine BCRP1 by LC–MS/MS.

Protein quantification of human BCRP in BacMam2-BCRP–transduced or murine BCRP1 in BacMam2-Bcrp1–transduced MDCKII membrane extracts was conducted by mass spectrometry–based targeted proteomics using validated LC-MS/MS methods. Briefly, the cells were harvested from semipermeable membranes in the transwell inserts using extraction buffer I in the ProteoExtract membrane extraction kit (Calbiochem). Total membrane proteins were isolated and digested using a previously outlined procedure (Kamiie et al., 2008; Uchida et al., 2013).

The chromatographic and spectrometric conditions for protein quantification of BCRP and BCRP1 are described subsequently. Trypsin digest (8 μl) was injected onto the column (ACQUITY UPLC HSS T3, 2.1 × 100 mm, 1.8 μm). A mobile phase consisting of water containing 0.2% acetic acid (A) and acetonitrile-methanol (1:1, v/v) containing 0.2% acetic acid (B) was employed. A flow rate of 0.5 ml/min was used with elution starting at 20% B for 0.2 minutes, followed by a linear gradient increasing to 60% B (0.2–4.5 minutes). This was followed by another steep linear gradient increasing to 95% B (4.5–4.55 minutes) and then eluting the column with 95% mobile phase B for 0.65 minutes, and re-equilibrating it at 20% B for 1.25 minutes. The MS/MS analysis of BCRP and BCRP1 was performed by monitoring the signature peptide (522.5–644.7) and the internal standard (526.8–651.7) using an optimized cone voltage and collision energy. Representative LC-MS/MS chromatograms and calibration curves of the surrogate peptide are shown in Supplemental Fig. 1.

The accuracy and precision of the assay were >80% and <20%, respectively. BCRP and BCRP1 protein expression data (picomoles per milligram) were expressed relative to the total protein content of the isolated membrane, as determined by the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). All samples were digested and measured in duplicate.

Relationship between the ER and Efflux Activity.

The relationship between the ER and the efflux activity (PSefflux) for BCRP across MDCKII monolayers was adapted from a three-compartment model (apical, cellular, and basolateral) described previously (Kalvass and Pollack, 2007):Embedded Image(3)where PSefflux is the BCRP-mediated efflux activity; PS is the passive permeability-surface area product; and ER is the permeability ratio of the basolateral-to-apical direction to the apical-to-basolateral direction. The corrected ER was also used herein to take into account any influence introduced by endogenous transporters in MDCKII-WT cells. Based on eq. 3, the PSefflux value is quantitatively proportional to ER-1 multiplied by the passive permeability.

Dantrolene is a specific BCRP substrate and was used as a reference compound in each in vitro experiment. Under the condition of the linear relationship between BCRP expression and in vitro or in vivo transcellular transport activity, the following equation is derived:Embedded Image(4)Substituting eq. 3 into eq. 4 and rearranging yield the following equation:Embedded Image(5)where ERin vivo is defined as the brain-to-plasma (blood) ratio in Bcrp-deficient animals divided by the brain-to-blood (plasma) ratio in Bcrp-competent animals at steady state. The Kp,uu value of a discovery compound can be described by the reciprocal of ERin vivo value as follows (Uchida et al., 2011a):

Embedded Image(6)

Statistical Analysis.

The permeability and ERs are presented as the mean ± S.D. of technical or experimental replicates. The protein levels are the mean values of duplicate measurements. Linear regression analysis was performed with Microsoft Excel 2007. In all cases, P < 0.05 was considered to be statistically significant.

Results

Construction of MDCKII Monolayers Expressing BCRP or BCRP1.

BacMam2 viruses carrying BCRP or Bcrp1 cDNA in Dulbecco’s modified Eagle’s medium were dosed into the apical side of the transwell inserts at a designated MOI. Transport studies were carried out 2 days after transduction of BacMam2. The transepithelial electrical resistance values of MDCKII monolayers were elevated after BacMam2 virus transduction. The extent of increase in the transepithelial electrical resistance values correlated positively with the MOI of BacMam2 (Fig. 1A). Low permeability of paracellular marker Lucifer yellow (typically, <20 nm/s) confirmed the tightness of MDCKII-Bcrp1 or MDCKII-BCRP monolayers. MDCKII-WT cells expressed endogenous canine P-gp, the transport activity of which was probed by amprenavir, a P-gp–specific substrate. The ER of amprenavir was reduced along with increasing transport of dantrolene, a specific probe substrate of BCRP1 (Fig. 1B). This was consistent with reverse correlation of canine P-gp and BCRP mRNA levels in BacMam2-BCRP–transduced MDCKII cells (data not shown). Inclusion of 1 µM of P-gp–specific inhibitor LY335979 in transport medium completely inhibited endogenous P-gp activity (Shepard et al., 2003), with no effect on BCRP1 activity in MDCKII cells (Fig. 1C). Interbatch variability in BCRP transport activity was notable (Fig. 1D). The ER of dantrolene was 15.9 ± 8.9 in 16 consecutive experiments with a coefficient of variation of 56% in MDCKII-BCRP cells. The ER of dantrolene collapsed to around unity (0.935 ± 0.307) in the presence of 0.2 µM of BCRP-specific inhibitor Ko143 in transport medium (Allen et al., 2002).

Fig. 1.
Fig. 1.

Construction of MDCKII monolayers expressing human BCRP and murine BCRP1. (A) Transepithelial electrical resistance (TEER) values of MDCKII-WT monolayers after transduction of varying MOI of BacMam2-Bcrp1 (murine) and BacMam2-BCRP (human) viruses (n = 12). (B) ERs of dantrolene and amprenavir (n = 3, donor concentration 1 µM) across MDCKII-WT monolayers transduced with varying MOIs of BacMam2-Bcrp1 viruses. (C) Effect of different concentrations of P-gp–specific inhibitor LY335979 on apical-to-basolateral (gray bar) and basolateral-to-apical (black bar) permeability of dantrolene (n = 3, donor concentration 1 µM) across BacMam2-Bcrp1 transduced MDCKII-WT monolayers. (D) ER of dantrolene (donor concentration 3 µM) from 16 consecutive transport experiments across BacMam2-BCRP transduced MDCKII-WT monolayers in the absence or presence of 0.2 µM of Ko143; mean ± S.D. is indicated near the horizontal solid line.

Correlation between BCRP Membrane Protein Concentration and Transcellular Transport Activity across MDCKII Monolayers.

The BCRP and BCRP1 protein levels in membrane extracts of MDCKII cells transduced by BacMam2 viruses were quantified by a validated LC-MS/MS method. While the multiple surrogate peptides for BCRP and BCRP1 were observed to have different absolute quantitative values, these correlated strongly (data not shown; R2 typically greater than 0.9). The difference could be the result of differential digestion efficiency of signature peptides, unexpected post-translational modifications of signature peptides, or inaccurate quantification of synthetic signature peptides. We report herein, protein expression levels measured by a surrogate peptide consistently providing the highest protein quantification results, which may more accurately represent the true protein abundance, as reported previously (Balogh et al., 2013; Peng et al., 2015).

MDCKII cells with different levels of protein expression of BCRP or BCRP1 were generated by titrating the MOI of BacMam2-BCRP or BacMam2-Bcrp1 virus in transwell apical compartments. A linear correlation between mRNA level and protein expression for BCRP was observed for the initial five MOI investigated. Higher BacMam2-BCRP or BacMam2-Bcrp1 virus load led to reduction in mRNA level (data not shown). This might be due to interference in the transcription and translation pathway of BCRP by high BacMam2 virus load, and therefore these specific data were not included in the following correlation analysis.

Dantrolene, coumestrol, daidzein, and genistein are BCRP-specific substrates (Enokizono et al., 2007; Fuchs et al., 2014). Transport of these four compounds across MDCKII monolayers with different levels of BCRP expression was examined (Table 1; Supplemental Table 2). The ERs of coumestrol, daidzein, and genistein were collapsed to 0.5 or below in the presence of BCRP inhibitor Ko143, indicating their transcellular transport across MDCKII monolayers was impacted by endogenous canine transporters. Thus, the corrected ER was used herein to represent BCRP-mediated transport activity. Three micromolar donor concentrations were used in this investigation. A plot of the human BCRP levels in membrane extracts versus in vitro corrected ERs in BacMam2-BCRP–transduced MDCKII cells showed a significant correlation for dantrolene (R2 = 0.89, P < 0.05) and genistein (R2 = 0.85, P < 0.05) and a moderate correlation for coumestrol (R2 = 0.64, P = 0.1) and daidzein (R2 = 0.64, P = 0.1) (Fig. 2).

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TABLE 1

Protein expression of BCRP in membrane extracts and corrected ERs of dantrolene, coumestrol, daidzein, and genistein across MDCKII monolayers transduced with varying titers of BacMam2-BCRP viruses

Fig. 2.
Fig. 2.

Correlation of BCRP expression level in membrane extracts of BCRP-expressing MDCKII cells with in vitro corrected ERs of dantrolene, coumestrol, daidzein, and genistein. ERs are presented as mean ± S.D. from three individual transwell assays. Protein levels are the mean value of duplicate measurements.

We also investigated the relationship between BCRP1 protein expression levels in membrane extract versus in vitro corrected ERs in BacMam2-Bcrp1–transduced MDCKII cells. A strong linear correlation between protein level and in vitro corrected ER was observed for dantrolene (R2 = 0.83, P < 0.05), daidzein (R2 = 0.79, P < 0.05), genistein (R2 = 0.99, P < 0.01), and prazosin (R2 = 0.94, P < 0.01) (Fig. 3; Table 2; Supplemental Table 3). The P-gp–specific inhibitor LY335979 was included in the transport medium with prazosin, a known P-gp and BCRP cosubstrate (Zhou et al., 2009). According to eq. 3, these results indicated a linear correlation between expression level and transcellular transport activity for BCRP (PSefflux) within the ERs investigated.

Fig. 3.
Fig. 3.

Correlation of BCRP1 protein expression level in membrane extracts of BCRP1-expressing MDCKII cells with in vitro corrected ERs of dantrolene, daidzein, genistein, and prosozin. ERs are presented as mean ± S.D. from three individual transwell assays. Protein levels are the mean value of duplicate measurements.

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TABLE 2

Protein expression of BCRP1 in membrane extracts and corrected ERs of dantrolene, daidzein, genistein, and prazosin across MDCKII monolayers transduced with varying titers of BacMam2-Bcrp1 viruses

Correlation between BCRP and BCRP1 ERs across MDCKII Monolayers.

The in vitro BCRP1 and BCRP ERs were assessed using a set of drugs including P-gp and BCRP cosubstrates across MDCKII monolayers. Endogenous canine P-gp activity was inhibited by including LY335979 (1 µM) in transport medium. To avoid data differences due to any interbatch variation in BCRP expression, the 12 drugs were evaluated in a single experiment. One micromolar donor concentration was used for all drugs. All of the selected drugs were identified to be murine and human BCRP substrates. The ERs for these drugs were greater than 2 and collapsed to around or below unity in the presence of the BCRP-specific inhibitor Ko143 (Tables 3 and 4). When the impact of BCRP on directional transport was ranked across all tested drugs, considerable overlap between human and mouse was observed. The corrected ERs of BCRP1 and BCRP were within 2-fold for all drugs except flavopiridol, which had 2.2-fold higher efflux in human (Fig. 4).

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TABLE 3

Permeability and ERs of drugs across MDCKII monolayers transduced with BacMam2-BCRP viruses

Permeability across MDCKII-BCRP was determined with 1 µM of donor concentration and 90 minutes of transport time. One micromolar of LY335979 was included in the transport medium. Data are presented as the mean ± S.D. from three individual transwell assays.

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TABLE 4

Permeability and ERs of drugs across MDCKII monolayers transduced with BacMam2-Bcrp1 viruses

Permeability across MDCKII-Bcrp1 was determined with 1 µM of donor concentration and 90 minutes of transport time. One micromolar of LY335979 was included in the transport medium. Data are presented as the mean ± S.D. from three individual transwell assays.

Fig. 4.
Fig. 4.

Correlation of corrected ERs in transwell assays using MDCKII-Bcrp1 and MDCKII-BCRP monolayers for 12 drugs. The y-axis is the human BCRP-corrected ER and the x-axis is the murine BCRP1-corrected ER. Data are shown as mean ± S.D. (n = 3). Dashed lines are the line of 2-fold deviation from unity.

Discussion

Screening for BCRP substrates in CNS drug discovery is mainly issue driven and this typically takes place when a discovery compound with high passive permeability has a low Kp,uu value and is not a strong P-gp substrate (Di et al., 2013; Poirier et al., 2014). A challenge facing project teams is how to contextualize the in vitro MDCKII-BCRP transport data. To predict transporter-mediated disposition at the BBB, physiologically based pharmacokinetic approaches use in vitro data from over-expressing cell lines and scale these to the in vivo data by correcting the differences in transporter protein expression via in vitro/in vivo extrapolation. This approach requires making two assumptions: 1) passive permeability is similar across epithelial (MDCK) and endothelial (BBB) membranes; and 2) there is a linear correlation between expression and activity of the transporter protein (Kumar et al., 2015). Available evidence supports the hypothesis that there is no substantial difference in passive permeability across epithelial and endothelial membranes (Uchida et al., 2011a; Liu et al., 2014). The current study aims to examine the validity of the second assumption for BCRP, using MDCKII-BCRP and MDCKII-Bcrp1 monolayers. Our data demonstrate a linear correlation between BCRP membrane protein expression and BCRP-mediated transcellular transport activity across MDCKII monolayers, within the ERs investigated. This implies that it is feasible to predict BCRP-mediated in vivo impact on brain penetration using a BCRP-expressing MDCKII transwell system. This was previously exemplified by P-gp substrates that showed transcellular transport activity was proportional to the transporter expression levels (Tachibana et al., 2010). Successful reconstruction of in vivo brain distribution of P-gp substrates has been based on the BBB transporter protein concentration, in vitro intrinsic transport activity, and in vitro transporter expression in mice and monkey (Uchida et al., 2011a, 2014).

Dantrolene is a specific BCRP substrate with well-characterized BBB transport profiles in preclinical species. We use this reference drug as an indicator of BCRP competency in the transwell system. A review of the literature showed that the in vivo brain penetration ERs of dantrolene in Bcrp1−/− to WT mice and rats were 6.30 and 3.30 at steady state, respectively, and the mean Bcrp expressions at the mouse, rat, and human BBBs were 3.37, 4.15, and 7.15 fmol/µg⋅protein, respectively (Table 5). By averaging the Bcrp expression and in vivo brain penetration ERs in mice and rats and assuming no species differences in BCRP transport activity, we predicted the human ERin vivo, Dan to be 9.1 and the unbound brain-to-plasma concentration ratio to be 0.11 (Table 5). The predicted human BCRP ERin vivo of dantrolene is useful to contextualize the BCRP in vitro transport data for discovery compounds. A Kp,uu value greater than 0.3 is typically used as a starting point of acceptable brain penetration in CNS drug discovery programs. Based on eq. 5 and 6, this would require (ERin vitro, test compound − 1)/(ERin vitro, Dan − 1) < (1/0.3 − 1)/(9.1 − 1) = 0.29 in MDCKII-BCRP transport studies. Conversely, (ERin vitro, test compound − 1)/(ERin vitro, Dan − 1) > 0.29 would predict Kp,uu values smaller than 0.3 and raise a red flag for BCRP-mediated compromise in brain penetration at the human BBB. Verification of the strength of our predictions requires the clinical ER of dantrolene. This will now be possible because the positron emission tomography BCRP probe [13N]dantrolene has been successfully synthesized (Kumata et al., 2012).

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TABLE 5

Prediction of the in vivo BCRP ER of dantrolene in human based on the in vivo BCRP ER of dantrolene in preclinical species and BCRP expression at BBB

To verify this contextualization approach, we applied this method to 22 proprietary discovery compounds and three phytoestrogens including coumestrol, daidzein, and genistein, which are BCRP substrates but not P-gp or weak P-gp substrates (Supplemental Table 4). We observed good correlation of restricted brain penetration with benchmarked ERin vivo values in rodents (Fig. 5). Compromised brain penetration (Kp,uu < 0.3) is correctly predicted when (ERin vitro, test compound − 1)/(ERin vitro, Dan − 1) is greater than 0.6 (quadrant I, where 0.6 is derived from the mean ERin vivo of dantrolene in rat and mouse). Some compounds show greater restriction in brain penetration than expected (quadrant IV), likely due to additional BBB efflux mechanisms or species differences in BCRP or P-gp transport. In our experience, it is not unusual for a compound to have limited brain penetration (e.g., Kp,uu < 0.3), even though it is not a P-gp or BCRP substrate and has high passive permeability. It is possible that members of the multidrug resistance–associated protein family of transporters are involved, or some other yet-to-be-identified efflux pathways. This highlights the gap in our understanding of the very intricate BBB. Empirically, an ER of BCRP greater than 5 was used as a red flag in brain penetration impairment. However, this empirical approach does not consider interbatch BCRP expression variability and may only be applied in our laboratory. The proposed contextualization approach has been improved by addressing this issue and may be transferable to other laboratories.

Fig. 5.
Fig. 5.

Relationship between BCRP ERs and rat/mouse Kp,uu for coumestrol, daidzein, genistein, and 22 proprietary discovery compounds. All compounds are BCRP substrates with ER > 2 across MDCKII-BCRP monolayers and ER < 1.8 or apical-to-basolateral permeability ratios in the presence to absence the P-gp inhibitor GF120918 (2 micromolar) < 1.8 across MDCKII-MDR1 monolayers. Proprietary discovery compounds have passive permeability > 250 nm/s except for two compounds, which have passive permeability = 71 nm/s. Compromised brain penetration (Kp,uu < 0.3) is predicted when (ERin vitro, test compound −1)/(ERin vitro, Dan −1) > 0.6 (eqs. 5 and 6; Table 5). Mean in vivo ER of 4.8 in rat and mouse was used. Quandrant I: Kp,uu < 0.3 and (ERin vitro, test compound −1)/(ERin vitro, Dan −1) > 0.6; quadrant II: Kp,uu > 0.3 and (ERin vitro, test compound −1)/(ERin vitro, Dan −1) > 0.6; quadrant III: Kp,uu > 0.3 and (ERin vitro, test compound −1)/(ERin vitro, Dan −1) < 0.6; quadrant IV: Kp,uu < 0.3 and (ERin vitro, test compound −1)/(ERin vitro, Dan −1) < 0.6.

On a simpler scale, we are able to manipulate the in vitro system to minimize impact of other transporters since the endogenous canine transporters expressed on MDCKII cells may confound the transport data of P-gp and BCRP cosubstrates. MDCKII-WT cells express canine P-gp but not BCRP. In addition, endogenous P-gp expression can be impacted by BacMam virus load (Liu et al., 2015). In previous studies, the BCRP-expressing in vitro transwell system was not found to be predictive of in vivo transport activity (Enokizono et al., 2008; Zhao et al., 2009), likely because P-gp/BCRP cosubstrates were included and the activity of endogenous P-gp was ignored. In this study, inclusion of P-gp–specific inhibitor LY335979 in transport medium inhibited endogenous P-gp activity. Quantitative proteomics combined with transport studies in this relatively pure BCRP-functioning in vitro system may be a very useful method to predict the fraction of drug transported by BCRP in vivo across the BBB. This parameter is critical when predicting the Kp,uu value of a drug and the significance of transporters in determining brain penetration of a drug (Prasad and Unadkat, 2015). In theory, the additive effect of P-gp and Bcrp on the brain-to-plasma ratio for cosubstrates can be reconstructed by their respective contribution to the net efflux at the BBB (Kodaira et al., 2010).

The mouse BCRP1 and human BCRP amino acid sequences are 81% identical and 86% homologous, with very high homology in the ATP binding region (Doyle and Ross, 2003). Despite broad use of Bcrp1 knockout mice in exploring the effect of this transporter on drug disposition, little is known about differences in the kinetics of BCRP-mediated drug transport between human and mouse. Use of newly introduced BCRP-expressing MDCKII monolayers without endogenous canine P-gp activity allows us to examine species differences in murine and human BCRP-mediated transcellular transport. To control interbatch variability in BCRP expression, 12 drugs were evaluated simultaneously in a single experiment. Good concordance of murine and human BCRP ERs across MDCKII monolayers was observed. A further comparative study with a range of chemotypes using these systems is required. Meaningful comparison can only be obtained by close monitoring of BCRP expression among different batches of MDCKII cells.

In summary, we have constructed human BCRP- and murine BCRP1-expressing MDCKII cells by BacMam2 virus transduction. Inclusion of P-gp–specific inhibitor LY335979 in the test system allowed us to create a relatively clean system to investigate BCRP-mediated transcellular transport across MDCKII monolayers. A linear correlation of membrane protein expression levels with transcellular transport activity for BCRP was observed within the ERs investigated. We proposed an approach to contextualize in vitro transport data of discovery compounds by benchmarking the in vitro transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted impaired brain penetration for compounds, which are BCRP substrates but not P-gp or weak P-gp substrates.

Acknowledgments

The authors acknowledge Fei Wang and Dan Li for bioanalytical support. The authors also thank Sijie Lu, Xinyan Lu, and Yang Li for providing technical help on transport experiments. Finally, the authors appreciate Maciej Zamek-Gliszczynski and Joseph Polli for scientific input and helpful discussions.

Authorship Contributions

Participated in research design: Liu, Sahi, Summerfield, Dong.

Conducted experiments: Liu, Huang, Li, Sun, Gao.

Contributed new reagents or analytic tools: Chen.

Performed data analysis: Liu, Huang, Li, Y.-Y. Zhang.

Wrote or contributed to the writing of the manuscript: Liu, Fu, Y.-Y. Zhang, W. Zhang, Sahi, Summerfield, Dong.

Footnotes

Abbreviations

ABC
ATP-binding cassette
BBB
blood-brain barrier
BCRP
breast cancer resistance protein
CNS
central nervous system
ER
efflux ratio
Kp,uu
unbound brain-to-blood (plasma) concentration ratio
LC-MS/MS
liquid chromatography–tandem mass spectrometry
MDCK
Madin-Darby canine kidney
MOI
multiplicity of infection
PCR
polymerase chain reaction
P-gp
P-glycoprotein
PS
permeability surface
WT
wild type

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Correlation between BCRP Expression and Transport Activity

Houfu Liu, Liang Huang, Yi Li, Tingting Fu, Xueying Sun, Yan-Yan Zhang, Ruina Gao, Qingfang Chen, Wandong Zhang, Jasminder Sahi, Scott Summerfield and Kelly Dong
Drug Metabolism and Disposition May 1, 2017, 45 (5) 449-456; DOI: https://doi.org/10.1124/dmd.116.074245
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