Sensitive and specific assays for a drug and its important metabolites are critical to the study of drug-drug interactions.
Transporter-mediated drug-drug interactions are a key component to the future of in vitro, early-stage drug safety testing. Interaction occurs when two or more administered drugs abruptly alter each other’s metabolism and excretion, which may lead to adverse reactions and toxicities. The current view of the US Food and Drug Administration (FDA) is that the metabolism of an investigational new drug should be defined during drug development and that its interactions with other drugs should be explored as part of an adequate assessment of its safety and efficacy.1 In its September, 2006 draft Guidance for Industry, “Drug Interaction Studies–Study Design, Data Analysis, and Implications for Dosing and Labeling”, FDA indicated a focus on transporter-mediated drug-drug interactions, specifically ones that involve P-glycoprotein (P-gp). Scientists have developed increasingly specific assays for particular applications, including P-gp testing.
Since 1997, FDA has endorsed in vitro studies of new drug compounds by use of liver enzyme assays to test for possible drug-drug interactions. The program was implemented not only for drug safety concerns, but to help put promising new drug candidates on a fast-track to drug approval as part of the FDA Critical Path Initiative. The recent focus on drug transporters necessitates additional research into preclinical in vitro testing methods, since P-gp is only one of many transporters that potentially interact with drugs. Companies are searching for better ways to determine the safety of new drug candidates by characterizing a variety of drug transporters and developing efficient assays around them.
The monolayer bidirectional transport assay is the definitive format specified by FDA for identifying drugs that interact with membrane transporters as either a substrate or an inhibitor.2 This assay format is also a valuable platform from which new tools are being developed to study transporters as a potentially life-saving application in terms of the ability to predict clinical drug interactions.
Very few cell lines will form polarized, tight monolayers in culture, a necessity for the monolayer bidirectional transport assay format. Two that do are MDR1-MDCK and Caco-2.
MDR1-MDCK cells offer both high expression of human P-gp and quick preparation time.3 However, MDR1-MDCK is a canine cell line that expresses canine P-gp in addition to human P-gp. As a result, FDA recommends that wild-type (non-transfected) MDCK cells be tested in parallel to determine the relative contributions of canine and human P-gp. Researchers running these tests in parallel double the amount of work and costs associated with the analysis. The MDR1-MDCK cell-based bidirectional transport assay is still considered very useful, but the shortcomings must be considered.
Table 1: BCRP function knocked down in a Caco-2 derived cell line. (Source: Absorption Systems L.P.) | |||
Caco-2 Cell Type |
Control |
BCRP Knockdown Clone D |
|
Passage # |
8 |
10 |
|
TEER: transepithelial electrical resistance |
507 |
556 |
|
Passive diffusion, Papp (x10-6) |
Atenolol |
0.23 |
0.42 |
Propranolol |
22.36 |
23.23 |
|
P-gp transport, Papp (x10-6) |
Digoxin Efflux Ratio |
24.59 |
21.53 |
BCRP transport, Papp (x10-6) |
Estrone-3-sulfate Efflux Ratio |
28.51 |
2.61 |
Digoxin: P-gp substrate Features of Clone D: |
The cell line most commonly associated with the bidirectional transport assay is Caco-2, a cell line of human origin that expresses several different transporters in the appropriate subcellular locations. Initially characterized as an in vitro model for predicting the absorption of orally-administered drugs in the late 1980s,4 the Caco-2 cell monolayer was one of the assays designated in the draft Guidance as appropriate for transporter-based drug-drug interaction studies.5
In addition to P-gp, Caco-2 cells express BCRP and MRP-2, two other common human drug transporters. In this cell line, scientists are unable to tell with certainty which of these three transporters is responsible for efflux of any given test compound. This problem would not be considered a disadvantage if there were chemical inhibitors specific for one transporter compared to another. Different inhibitors could be added, one at a time, to determine which transporter was involved. However, no transporter-specific inhibitors are currently available.
|
A series of custom-engineered Caco-2 cell lines that are each missing one key transporter would enable increased specificity for bidirectional transporter assays. Using the process of elimination, one could systematically deduce which transporter is interacting with the test compound. This approach to a testing format could not only provide pass or fail feedback, but more specific information than is currently available. It would also enable quicker and better decision-making abilities for new drug sponsors.
A Caco-2 cell line has been engineered to reduce the expression of the drug efflux transporter, Breast Cancer Resistance Protein (BCRP). Table 1 and Figure 1 show, at the functional and protein levels, respectively, the degree to which BCRP expression has been knocked down in this Caco-2-derived cell line. Testing a drug compound with this cell line will provide specific data indicating whether the compound is a substrate or inhibitor of the BCRP transporter protein, or does not interact with BCRP at all.
Further development of cell lines that each lack a specific transporter will offer drug designers tools for determining transporter-related drug-drug interactions. By testing the transport of compounds in each of the derivative cell lines compared with the parental cell line, it will be possible to deduce the specific transporter or transporters involved in the uptake and/or efflux of that compound. Specificity at this level enables drug sponsors to more easily design clinical trials, to determine whether or not subsequent in vivo studies are even required, or decide if the compound in development should be dropped altogether.
In the foreseeable future, drug transporters will still be tested in vitro, but in new ways that will significantly reduce testing times.
About the Author
Dr. Hidalgo is the co-recipient of the 2007 SBS PolyPops Foundation Award for his work in the discovery of Caco-2 cells as a model of human intestinal mucosa.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 2, February, 2008, pp. 34-35.
Filed Under: Drug Discovery