BCRP (breast cancer resistance protein; ABCG2) is an important efflux transporter. It is expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis, and placenta1.
The ITC1, the EMA guideline2 and the FDA guidance3 recommend investigating BCRP due to the clinical importance of BCRP in the absorption and disposition of drugs. Furthermore, clinically relevant genetic polymorphisms of ABCG2 have been shown to have an impact on the pharmacokinetics and toxicity of marketed drugs, and underlie the ethnic differences in exposure between Japanese and Caucasian populations for some substrate drugs.
Caco-2 cells express BCRP. The EMA2 and FDA3 regulatory guidelines recommend polarized Caco-2 cell monolayers as one of the preferred methods for evaluating the role of BCRP in the efflux of new chemical entities.
The assay investigates bidirectional transport across the cell monolayer in the presence and absence of the selective BCRP reference inhibitor, fumitremorgin C, to determine if active efflux is occurring, and whether this efflux is mediated by BCRP.
Where Caco-2 cell assays indicate a compound has inherently low passive permeability, then BCRP membrane vesicles can be used as an alternate in vitro test system to identify BCRP substrates.
ABCG2 is a high-capacity efflux transporter with wide substrate specificity recognizing large, hydrophobic molecules of either negative or positive charge, organic anions, and sulfate conjugates.
4Chen Z et al., (2010) Int J Cancer126(4); 841-851
BCRP substrate identification assay protocol for screening (1 concentration) or regulatory type studies (4 concentrations & 2 concentrations plus inhibitor)
Test Article Concentration
Screening study - 10 μM plus/minus inhibitor (different concentrations available)
Regulatory study - 1, 10, 50 and 100 µM (different concentrations available) plus inhibition at two substrate concentrations (1 and 10 µM)
Apical to basolateral and basolateral to apical in presence and absence of 10 µM fumitremorgin C
Number of Replicates
2 (screening) or 3 (regulatory)
120 min (screening) or 90 min (regulatory)
Efflux ratio in presence and absence of fumitremorgin C
Data from Cyprotex's BCRP substrate identification assay
The expression and functional activity of BCRP in our Caco-2 cells were determined. Relative mRNA expression levels (relative to housekeeping gene) of the main transporters were analyzed by qRT-PCR. BCRP mRNA was expressed in the cells and had comparable relative expression levels with MDR1 (0.041 ± 0.011 for BCRP and 0.047 ± 0.014 for MDR1). Functional activity of BCRP was determined by investigating the inhibition of the BCRP substrate, estrone 3-sulfate, by a number of BCRP and P-gp inhibitors.
Figure 1 Graph showing effect of the selective BCRP inhibitor, fumitremorgin C (FTC) and the selective P-gp inhibitor, verapamil, on the efflux of the BCRP substrate, estrone 3-sulfate.
Estrone 3-sulfate efflux was not inhibited by the P-gp inhibitor, verapamil, but was inhibited by the selective BCRP inhibitor, fumitremorgin C (FTC), showing selectivity of estrone 3-sulfate as a BCRP substrate. Data show the mean ± standard deviation.
Question and answers on BCRP substrate identification assay
Why is it important to identify substrates of BCRP?
Breast cancer resistance protein (BCRP; ABCG2) is an ATP-binding cassette drug efflux transporter which is apically expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis and placenta1.
Interactions with respect to BCRP have been shown to be responsible for clinical drug-drug interactions. For example, the BCRP inhibitor GF120918 inhibits the transport of the BCRP substrate topotecan resulting in a significant increase in plasma AUC5.
In addition, clinically relevant genetic polymorphisms of ABCG2 have been shown to impact on the pharmacokinetics (e.g., rosuvastatin6,7, atorvastatin7, sulfasalasine8 and topotecan9) and toxicity (e.g., gefitinib-induced diarrhea10) of marketed drugs, and underlie the ethnic differences in exposure observed for drugs such as rosuvastatin between Japanese and Caucasian populations7,11.
The International Transporter Consortium1, the EMA guideline2 and the FDA guidance3 recommend investigating BCRP due to BCRP’s clinical importance in the absorption and disposition of drugs.
Please provide an overview of Cyprotex's BCRP substrate identification assay.
Bidirectional transport studies are one of the preferred ‘industry standard’ methodologies used to identify drugs as substrates of BCRP and are the current recommended approach indicated by the regulatory authorities. The Caco-2 cell line is a commonly used in vitro model for identifying BCRP substrates. The cells are seeded on a multiwell-insert plate and form a confluent monolayer over 18-22 days prior to the experiment (screening or regulatory assays are conducted on 20 or 18-22 days post-seeding, respectively). The test article is then added to either the apical or basolateral side of a confluent monolayer of the cells and permeability is measured by monitoring its appearance on the opposite side of the membrane using LC-MS/MS.
The permeability coefficient (Papp) is calculated from the following equation:
Where dQ/dt is the rate of permeation of the drug across the cells, C0 is the donor concentration at time zero and A is the area of the cell monolayer.
An efflux ratio is calculated from the mean apical to basolateral (A-B) Papp data and basolateral to apical (B-A) Papp data.
The permeability is assessed in the presence and absence of a selective BCRP inhibitor to confirm the test article is a substrate of BCRP.
How do you know if the cells have formed a confluent monolayer?
Transepithelial electrical resistance (TEER) measurement is used to determine tight-junction formation between cells. In addition, lucifer yellow, a membrane integrity marker, is co-incubated with the test compound at the start of the experiment. If the Papp of the lucifer yellow exceeds 1 x 10-6 cm/s in one well, but the derived Papp result for the test compound or positive control substrate in that well is qualitatively similar to that determined in the remaining replicate well(s) (within the lucifer yellow threshold) then, based upon the scientific judgement of the responsible scientist, the cell monolayer may be considered acceptable. If this is not the case, then the result from the affected monolayer is excluded. For regulatory studies, if lucifer yellow Papp values are above the threshold in all replicate wells for a particular test compound concentration, then the data for that concentration is excluded with a comment that toxicity or inherent fluorescence of the test compound is assumed. No further experiments are performed. For screening studies, if one replicate well is excluded, then an n=1 result is reported, or the compound may be re-tested. If both replicates are affected then the compound is re-screened. If both lucifer yellow Papp values fail for the same compound on two separate occasions then it is assumed that the compound exhibits either cytotoxic effects against the Caco-2 cells or inherent fluorescence.
A positive control substrate (estrone 3-sulfate) is evaluated alongside the test compound in the presence and absence of fumitremorgin C.
How and why is the % recovery calculated?
The % recovery can be useful in interpreting the Caco-2 data. If the recovery is very low, this may indicate problems with binding of the compound to the plate or accumulation of the compound in the cell monolayer. However, poor solubility is the most common reason for unexpected recoveries in the Caco-2 test system.
How do I decide if a clinical study is required?
The FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020)3 and The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (2012)2 recommend that all investigational drugs are evaluated in vitro to determine if they are BCRP substrates or inhibitors. For BCRP substrates, the draft FDA guidance recommends that there should be an assessment of nonclinical and clinical information to determine if an in vivo DDI study is warranted if the efflux ratio is ≥ 2 and the efflux is reduced towards unity by a BCRP reference inhibitor.
1 The International Transporter Consortium (2010) Membrane transporters in drug development. Nat Rev Drug Disc9; 215-236 2 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012) 3 FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) 4 Chen Z et al., (2010) Suppression of ABCG2 inhibits cancer cell proliferation. Int J Cancer126(4); 841-851 5 Kruijtzer CM et al., (2002) Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol20(13); 2943-2950 6 Zhang W et al. (2006) Role of BCRP 421C>A polymorphism on rosuvastatin pharmacokinetics in healthy Chinese males. Clin Chim Acta373; 99–103 7 Birmingham BK et al. (2015) Impact of ABCG2 and SLCO1B1 polymorphisms on pharmacokinetics of rosuvastatin, atorvastatin and simvastatin acid in Caucasian and Asian subjects: a class effect? Eur J Clin Pharmacol71(3); 341-355 8 Yamasaki Y et al. (2008) Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans Clin Pharmacol Ther84(1); 95-103 9 Sparreboom A et al. (2005) Effect of ABCG2 genotype on the oral bioavailability of topotecan. Cancer Biol Ther4; 650–658 10 Cusatis G et al. (2006) Pharmacogenetics of ABCG2 and adverse reactions to gefitinib. J Natl Cancer Inst98(23); 1739–1742 11 Birmingham BK et al., (2015) Rosuvastatin pharmacokinetics and pharmacogenetics in Caucasian and Asian subjects residing in the United States. Eur J Clin Pharmacol71(3); 329-340
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