The effect of BCRP inhibition and its measurement in vitro
BCRP (Breast Cancer Resistance Protein/ABCG2) is expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis and placenta1.
Inhibition of BCRP has shown to be responsible for several clinical drug-drug interactions involving specific statin common co-medications such as rosuvastatin and atorvastatin, resulting in their increased absorption and subsequent exposure (up to 2 fold increase in AUC)2,3.
The International Transporter Consortium1, the FDA guidance4 and the EMA guidance5 recommend investigating BCRP due to BCRP's clinical importance in the absorption and disposition of drugs.
Cyprotex use Caco-2 cells to identify BCRP inhibitors using a range of test inhibitor concentrations in the presence of the probe substrate estrone-3-sulfate, a good surrogate for the clinically relevant BCRP substrate rosuvastatin. This method conforms with the recommended methods within the International Transporter Consortium white paper1, the FDA interactions guidance4 and the EMA drug interactions guideline5.
In vitro inhibition studies are recommended to investigate whether the investigational drug inhibits any of the transporters known to be involved in clinically relevant in vivo drug interactions.
5The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
BCRP inhibition assay protocol
1 μM [3H]-estrone 3-sulfate (surrogate in vitro probe for clinically relevant BCRP substrate rosuvastatin12)
Test Article Concentrations
Seven point IC50 (triplicate wells)
Unidirectional (basolateral to apical)
Inhibitor Preincubation Time
Liquid scintillation counting
IC50 (derived from corrected B-A Papp)
Data from Cyprotex's BCRP Inhibition assay
A set of known BCRP inhibitors were investigated in Cyprotex's BCRP Inhibition assay using estrone-3-sulfate as substrate.
Mean IC50 ± Standard deviation (µM, n=3)
Novobiocin (positive control)
2.06 ± 0.884
0.250 ± 0.0540
11.0 ± 0.737
0.581 ± 0.165
Table 1: Inhibition of BCRP-mediated estrone 3-sulfate transport by literature inhibitors.
Question and answers on BCRP inhibition
Why is it important to investigate BCRP inhibition?
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.
Inhibition of intestinal BCRP has shown to be responsible for clinical drug-drug interactions. For example, the BCRP inhibitor fostamatinib inhibits the transport of the BCRP substrate rosuvastatin resulting in a clinically significant increase in AUC due to increased absorption3.
In addition, clinically relevant genetic polymorphisms of ABCG2 have been shown to impact on the pharmacokinetics (e.g., irinotecan6, rosuvastatin7,8, atorvastatin8, sulfasalasine9 and topotecan10) and toxicity (e.g., gefitinib-induced diarrhoea11) of marketed drugs.
The International Transporter Consortium1, the FDA guidance4 and the EMA guideline5 recommend investigating BCRP due to BCRP’s clinical importance in the absorption and disposition of drugs.
Please provide an overview of Cyprotex's BCRP Inhibition assay.
Unidirectional (B-A) transport studies are the preferred ‘industry standard’ methodology used to identify drugs as inhibitors 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 investigating BCRP inhibition. The cells are seeded on a multiwell-insert plate and form a confluent monolayer over 18-22 days prior to the experiment. The BCRP substrate, [3H]-estrone 3-sulfate, is then added to the 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 liquid scintillation counting. The permeability is assessed in the presence and absence of the test compound to investigate BCRP inhibition.
How is the IC50 determined from the unidirectional Caco-2 data?
The apparent permeability coefficient (Papp) of estrone 3-sulfate in the absence and presence of test compound is calculated from the following equation:
Where dQ/dt is the rate of permeation of the drug across the cells, C0 is the donor compartment concentration at time zero and A is the area of the cell monolayer.
The determined Papp is then converted to percentage control transport activity as follows:
% control activity = ([(Papp (+test compound) - Papp (passive)) / (Papp (vehicle control) - Papp (passive))] x 100)
Where, Papp (+test compound) is the Papp of [3H]-estrone 3-sulfate in the presence of test compound Papp (vehicle control) is the Papp of [3H]-estrone 3-sulfate in the absence of test compound Papp (passive) is the Papp of [3H]-estrone 3-sulfate in the presence of the highest concentration of positive control inhibitor
Percentage control transport activity values are then plotted against test compound/positive control inhibitor concentration and subsequently fitted to calculate an IC50 value (concentration which produces 50% inhibition of vehicle control transport activity) using a four parameter logistic equation.
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)4 and The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (2012)5 recommend that investigational drugs are evaluated in vitro to determine if they are BCRP substrates or inhibitors. For BCRP inhibition, the FDA guidance recommends that a clinical drug-drug interactions trial with a BCRP substrate (for example, rosuvastatin) should be performed if [Igut]/IC50 (or Ki) ≥ 10, where [Igut] represents the theoretical intestinal concentration (dose of the inhibitor (in mol)/250mL).
How did you decide on the incubation conditions for BCRP inhibition?
The incubation conditions have been fully characterised for our chosen BCRP substrate, estrone 3-sulfate, based on time linearity and chosen substrate concentration being approximately ten-times lower than the reported Km previously determined in membrane vesicles13.
What controls do you include in Cyprotex's BCRP inhibition assay?
We evaluate the positive control inhibitor novobiocin for Cyprotex’s BCRP inhibition assay. The integrity of the monolayers throughout the experiment is checked by monitoring lucifer yellow permeation using fluorimetric analysis.
At what stage should I perform an IC50 evaluation?
As intestinal BCRP-mediated DDIs are important for the absorption of statin common co-meds, establishing the inhibitory potential (IC50) of your compound versus BCRP during preclinical candidate selection may assist in shortlisting based on favourable DDI risk assessment profile. Such knowledge during preclinical or early clinical development will help towards clinical trial design and therefore patient recruitment. The FDA guidance4 and the EMA guideline5 on drug interactions provides recommendations on whether a clinical drug interaction study is necessary based on the IC50 or Ki value..
1 The International Transporter Consortium (2010) Membrane transporters in drug development. Nat Rev Drug Disc9(3); 215–236 2 Elsby R et al., (2012) Understanding the critical disposition pathways of statins to assess drug-drug interaction risk during drug development: It's not just about OATP1B1. Clinical Pharmacology & Therapeutics92(5); 584-598 3 Elsby R et al., (2016) Solitary inhibition of the Breast Cancer Resistance Protein efflux transporter results in a clinically significant drug-drug interaction with rosuvastatin by causing up to a 2-fold increase in statin exposure. Drug Metab Dispos44(3); 398-408 4FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) 5 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012) 6 Zhou Q et al. (2005) Pharmacogenetic profiling across the irinotecan pathway in Asian patients with cancer. Br J Clin Pharmacol59(4); 415–424 7 Zhang W et al. (2006) Role of BCRP 421C>A polymorphism on rosuvastatin pharmacokinetics in healthy Chinese males. Clin Chim Acta373(1-2); 99–103 8 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 9 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 10 Sparreboom A et al. (2005) Effect of ABCG2 genotype on the oral bioavailability of topotecan. Cancer Biol Ther4(6); 650–658 11 Cusatis G et al. (2006) Pharmacogenetics of ABCG2 and adverse reactions to gefitinib. J Natl Cancer Inst98(23); 1739–1742 12Jones H et al., (2017) Estrone 3-sulfate as a surrogate Breast Cancer Resistance Protein (BCRP) in vitro probe substrate for assessing drug-drug interaction risk with rosuvastatin. View our poster from ISSX 13 Elsby R et al., (2011) Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions. Xenobiotica41(9); 764-783
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