February 15, 2008 (Vol. 28, No. 4)

New Tool Sheds Light on the Role of BCRP Protein in Drug-Drug Interactions

Researchers must now take a serious look at drug transporter proteins because of a 2006 FDA draft guidance document: Guidance for Industry, Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling.

In the document, the FDA said its current view is that a new drug’s interactions with other drugs should be explored as part of an adequate assessment of safety and effectiveness. New drug sponsors must now provide in vitro data on metabolism and drug-drug interactions involving either metabolic enzymes or transporters for all NDAs and BLAs.
The role transporters play in drug-drug interactions is critical because of the profound effect they have on the absorption of drugs and their distribution within the body. Although P-glycoprotein (P-gp) is the best understood, there are other drug transporters that need to be evaluated as well.

One of these proteins is the so-called breast cancer resistance protein (BCRP), which is particularly important in the small intestine and blood-brain barrier. Like P-gp, BCRP is an efflux transporter that can reduce the effectiveness of a drug that is a substrate or cause toxicity when inhibited by a coadministered drug. 

In humans, high BCRP gene expression may be linked to chemotherapeutic drug resistance in cancer. This article discusses how shRNA interference can be used to create stable BCRP knockdown in Caco-2 cells for the purpose of evaluating the importance of the protein in drug efflux. The development of a stable Caco-2 cell line modified to down-regulate BCRP expression will provide a novel tool to study BCRP-mediated drug resistance as well as pharmacokinetic drug-drug interactions.

Reducing Expression

As a member of the ATP-binding cassette (ABC) transporter superfamily, high levels of BCRP enhance drug efflux, which can result in excessive resistance to many different cancer drugs. A common approach to overcoming this drug resistance is to block BCRP-mediated transport with potent chemical inhibitors. Unfortunately, success has been modest because of toxicity problems and unfavorable pharmacokinetic interactions.

Although a recent study has demonstrated a reduction in BCRP expression in Caco-2 cells using RNAi technology, the reduction is only temporary when RNAi is created in vitro, as full expression is restored only a few days after transfection. Moreover, this technique is limited to easily transfected cells and by a lack of information on the stability of the knockdown phenotype.

A Novel Approach

Absorption Systems took the existing method of BCRP downregulation using in vitro RNAi and improved upon it by transducing Caco-2 cells with viruses carrying expression cassettes encoded with synthetic shRNAs for BCRP. More specifically, these viral vector-based shRNA lentiviral transduction particles were used to infect and integrate five shRNA/BCRP constructs into Caco-2 cells. 

Both the BCRP knockdown and control cells were grown on Transwell® inserts to form monolayers under conditions normally employed for growing Caco-2 cell cultures. After three weeks in culture, bidirectional transport experiments were conducted using a solution containing 10 µM propranolol, 10 µM atenolol, and 5 µM estrone-3-sulfate (E3S) for two hours. All samples were analyzed by LC/MS/MS.

The results showed that there was a significant reduction in the expression of BCRP mRNA and protein in cells that were transfected with the RNAi-BCRP lentivirus vectors in comparison to cells transfected with a noncoding shRNA vector. RT-PCR and Western blot (Figures 1 and 2) results showed that expression of human BCRP mRNA and protein in all five shRNA/BCRP clone cells were reduced by as much as 75% and 50%, respectively, compared with control cells.

Figures 1 and 2

We demonstrated that the reduced expression of BCRP mRNA is stable through at least 20 cell passages in shRNA/BCRP clone cells. The results also showed that the expression of b-actin mRNA was constant from cell passage 5 to 20 (Figure 3). The duration of gene downregulation is therefore much better than using transient transfection of RNAi.

Figure 3

As a functional consequence, knockdown of BCRP by shRNA reduced the efflux ratio of the BCRP substrate E3S by 96% when compared to the control cells. As the Table shows, all shRNA/BCRP clones demonstrated a significant reduction in the efflux ratio of E3S (range 0.91–14.42) compared to the control Caco-2 cells (25.58). The percentage of inhibition for E3S transport in these five BCRP-knockdown clone cells ranged from 43% to 96%. The transcellular transport of reference compounds when compared with shRNA/BCRP clone cells showed minor differences between them (Table).


This study showed that sequence-specific BCRP-gene knockdown in Caco-2 cells can be successfully established using shRNA lentiviral-transduction particles. In addition, lentiviral shRNA were shown to effectively reduce BCRP-gene expression and function in BCRP-knockdown cells. Utilizing these transporter-specific knockdown cells instead of the less-specific pharmacologic inhibitors currently used is a novel and effective laboratory tool to evaluate the role of BCRP in drug and nutrient efflux.

Helping Bring New Drugs to Market

The effort to understand the role of transporters in drug-drug interactions continues to attract attention in the scientific community. As more knowledge is gathered on the subject, there is the potential to develop better tools that will provide drug companies with precise and accurate transporter-interaction data for a variety of different transporters and applications. Increased specificity for transporter analysis enables faster pass/fail decisions in drug development.
Through new guidances for the industry, the FDA encourages drug companies to produce better, safer drugs on faster timelines. The technology used to screen potential new drug candidates will advance in parallel.

Transporters are a key component in drug development. As more information about them is uncovered, there will be quicker, more specific and accurate screening methods available to help significantly improve decision making during the drug discovery process and to, ultimately, bring new drugs to market.

Ismael Hidalgo, Ph.D., is chief
scientist at Absorption Systems. Web: www.absorption.com. E-mail: [email protected].

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