Researchers must now take a serious look at drug transporter proteins because of a 2006 FDA draft guidance document: Guidance for Industry, Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling.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