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ADME PK

BCRP Substrate Identification assay for Screening and Regulatory Reporting Purposes

Understand the potential of your compound to be effluxed by BCRP using our BCRP substrate identification assay.

BCRP substrate identification is in our portfolio of in vitro transporter services. Cyprotex deliver consistent, high quality data for either your early stage screening projects or your later stage regulatory studies.

In vitro BCRP substrate identification

  • Transporters can have a significant impact on the absorption, distribution, metabolism and excretion of drugs. They have been shown to play a part in clinically relevant drug-drug interactions1.
  • BCRP (breast cancer resistance protein; ABCG2) is an important efflux transporter. It was originally discovered in cancer cells in vitro with a function associated with multidrug resistance. It is expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis, and placenta1. It extrudes a wide range of endogenous and exogenous substrates across biological membranes.
  • Caco-2 cells express BCRP. The EMA2 and draft FDA3 drug interaction regulatory guidelines recommend Caco-2 cells as one of the preferred methods for evaluating the role of BCRP in the efflux of new chemical entities.
  • The assay investigates bidirectional transport (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer. The study is performed with and without the selective BCRP inhibitor, fumitremorgin C, to determine if active efflux is occurring, and whether this efflux is mediated by BCRP. The assay can also investigate the effect of fumitremorgin C on unidirectional transport (available on request).
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 conjugates4.

4Chen Z et al., (2010) Int J Cancer 126(4); 841-851

Protocol

BCRP substrate identification assay protocol for screening (1 concentration) or regulatory type studies (4 concentrations & single concentration plus inhibitor)

Test Article Concentration Screening study - 10 μM (different concentrations available)
Regulatory study - 1, 10, 50 and 100 µM (different concentrations available) plus inhibition at a single substrate concentration
Assay Conditions Apical to basolateral and basolateral to apical in presence and absence of 10 µM fumitremorgin C
Number of Replicates 2
Incubation Time 120 min
Analysis Method LC-MS/MS quantification
Integrity Marker Lucifer Yellow
Data Delivery Papp
Efflux ratio for bidirectional assessment in presence and absence of fumitremorgin C

Data

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-sulphate, 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-sulphate.

Estrone 3-sulphate 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-sulphate as a BCRP substrate. Data show the mean ± standard deviation.
Figure 2
Decision tree for BCRP substrate interactions as detailed in the original ITC review1 and the draft FDA guidance3.

The decision tree for BCRP in the draft FDA Drug Interaction Guidance is based on the P-gp decision tree published in the original ITC review1. For both P-gp and BCRP, similar in vitro methodologies and criteria are used to determine if a clinical DDI study is necessary.

Q&A

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 in the ABCG2 gene have been shown to impact on the pharmacokinetics (e.g., irinotecan6, rosuvastatin7, sulfasalasine8 and topotecan9) and toxicity (e.g., gefitinib-induced diarrhea10) of marketed drugs.

The International Transporter Consortium1,11, the draft FDA guidance3 and the EMA guidance2 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’ methodology used to identify drugs as substrates and/or 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 identifying BCRP substrates. The cells are seeded on a Multiscreen™ plate (Millipore, MA, USA) and form a confluent monolayer over 20 days prior to the experiment. 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 compartment 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 then it is assumed that the cell monolayer is compromised and either n=1 is reported or 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 cytotoxic effects against the Caco-2 cells.

Three control compounds, propranolol (passive transcellular transport), atenolol (paracellular transport) and estrone 3-sulphate (BCRP substrate) are screened alongside the test compounds 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 screen.

How do I decide if a clinical study is required?

The FDA Draft Guidance for Industry (Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations, Sept 2012)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 net flux ratio is ≥ 2 and the efflux is inhibited by a BCRP inhibitor.

References

1 The International Transporter Consortium (2010) Membrane transporters in drug development.  Nat Rev Drug Disc 9; 215-236
2 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
3 Draft FDA Guidance for Industry – Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations, February 2012
4 Chen Z et al., (2010) Suppression of ABCG2 inhibits cancer cell proliferation. Int J  Cancer 126(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 Oncol 20(13); 2943-2950
6 Zhou Q et al. (2005) Pharmacogenetic profiling across the irinotecan pathway in Asian patients with cancer. Br J Clin Pharmacol 59(4); 415–424
7 Zhang W et al. (2006) Role of BCRP 421C>A polymorphism on rosuvastatin pharmacokinetics in healthy Chinese males. Clin Chim Acta 373; 99–103
8 Yamasaki Y et al. (2008) Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans Clin Pharmacol Ther 84(1); 95–103
9 Sparreboom A et al. (2005) Effect of ABCG2 genotype on the oral bioavailability of topotecan. Cancer Biol Ther 4; 650–658
10 Cusatis G et al. (2006) Pharmacogenetics of ABCG2 and adverse reactions to gefitinib. J Natl Cancer Inst 98(23); 1739–1742
11 Hillgren KM et al., (2013) Emerging transporters of clinical importance: an update from the International Transporter Consortium. Clin Pharmacol Ther 94(1); 52-63

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