In an era in which pharmaceutical companies feel pressure to control costs while at the same time take new measures to ensure drug safety, the issue of how to predict drug-drug interactions (DDIs) effectively is a hot topic. Increasingly, pharma companies are turning to companies that provide specialized assistance by offering sensitive and specific assays for a drug and its important metabolites. Specifically, there is an increased focus on the examination of transporter-mediated DDIs.

The FDA has supported this shift; in its September 2006 draft Guidance for Industry, Drug Interaction Studies–Study Design, Data Analysis, and Implications for Dosing and Labeling, FDA indicated a focus on transporter-mediated DDIs, specifically ones that involve P-glycoprotein (P-gp). Scientists have developed increasingly specific assays for particular applications, including P-gp testing. One indication of the speed at which the drug transporter field is changing is the fact that, nearly four years later, this draft guidance has not been finalized. Instead, we are expecting a “new draft” guidance on the topic later this year.

Since 1997, FDA has endorsed in vitro studies of new drug candidates through the use of liver enzyme assays to test for possible DDIs involving drug metabolism. Such interactions occur when two or more administered drugs abruptly alter each other’s metabolism, which may lead to toxicity and adverse reactions in patients. This initiative has been motivated not only by concerns for drug safety, but also to help put promising new drug candidates on a fast-track to drug approval as part of the FDA Critical Path Initiative. FDA’s current view continues to be that the metabolism of an investigational new drug (IND) 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

Now the study of transporter-mediated DDIs is front and center as a key component to the future of in vitro, early-stage drug safety testing. The International Transporter Consortium, a group of experts from industry, academia and the FDA, recently published a white paper summarizing the state of the art in the drug transporter field.² This group proposed seven specific transporters, interactions with which should be tested in vitro by drug developers, a recommendation that was subsequently endorsed by the FDA Pharmaceutical Science and Clinical Pharmacology Advisory Committee. A number of DDIs related to transporters have been documented, resulting in alterations in the absorption, distribution and/or elimination of the drugs involved. Of the various transporters, including efflux transporters such as P-gp and uptake transporters such as the organic anion transporting polypeptide (OATP) family, P-gp is the best understood and was singled out in the 2006 FDA draft guidance. It is generally accepted that co-administration of drugs that interact with this transporter (as a substrate, inhibitor, or inducer) can result in DDIs that affect the pharmacokinetics, pharmacodynamics or toxicity of the co-administered drugs.³

The recent focus on drug transporters has spurred research into improved preclinical in vitro testing methods, and 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 format specified as “definitive” by FDA for identifying drugs that interact with membrane transporters as either a substrate or an inhibitor.⁴ 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 DDIs.

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 relatively quick preparation time.⁵ However, some characteristics of this model are problematic, including the fact that it is a canine cell line that expresses canine P-gp in addition to human P-gp. As a result, it can be difficult to determine the relative contributions of canine and human P-gp. The MDR1-MDCK cell-based bidirectional transport assay is still considered very useful in spite of its shortcomings.

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 characterizedin the late 1980sas an in vitro model for predicting the absorption of orally administered drugs,⁶ the Caco-2 cell monolayer is one of the models designated in the draft guidance as appropriate for transporter-based drug-drug interaction studies.⁷

In addition to P-gp, Caco-2 cells express BCRP and MRP-2, two other clinically important human drug transporters. With this cell line, it is difficult 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, few if any transporter-specific inhibitors are currently available.

A series of custom-engineered Caco-2 cell lines, each missing one key transporter, would provide 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/fail feedback, but more specific information than is currently available. It would also enable quicker and better decision-making 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).⁸ The table and figure below illustrate the use of this Caco-2-derived cell line, called CPT-B1. An efflux ratio (ER) substantially greater than 1 in control Caco-2 cells indicates that a compound is a substrate of an efflux transporter, but provides no information as to which transporter is involved. An efflux ratio at least twofold lower in the BCRP-knockdown cell line (i.e., a relative efflux ratio >2) indicates the involvement of BCRP. Testing a drug candidate with this cell line, in parallel with control cells, provides specific data indicating whether or not it is a substrate of BCRP.

Systematic comparison of efflux ratios in control cells vs. knockdown cells can identify the transporter(s) involved in efflux of a compound. Caco-2 cells in which P-gp and MRP-2 expression were targeted (one at a time) were used recently to confirm the mechanism of an unexpected drug-drug interaction involving ximelagatran and erythromycin.⁹

Further development of cell lines that each lack a specific transporter will offer drug developers tools for determining transporter-related DDIs. 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, 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 generate definitive results with significantly reduced testing times. Outsourcing partners that focus on testing drug transporters will continue to add value by offering pharma companies specialized knowledge and advanced technologies capable of providing the right mix of cost-effectiveness and quality data.

References

1. U.S. Food and Drug Administration. Guidance for Industry, “Drug Interaction Studies–Study Design, Data Analysis, and Implications for Dosing and Labeling," September 2006, Clinical Pharmacology, p. 1.
2. Giacomini, K.M., et al. “Membrane transporters in drug development” Nature Reviews Drug Discovery, March 2010; 9(3):215-36.
3. U.S. Food and Drug Administration. Guidance for Industry, “Drug Interaction Studies–Study Design, Data Analysis, and Implications for Dosing and Labeling," September 2006,Clinical Pharmacology,pp. 1, 4, 38.
4. ibid., p. 39.
5. Borchardt, R.T., et al. “Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa?” Pharmaceutical Research, June 2002; 19(6):773-9.
6. Hidalgo, I.J., Raub, T.J., Borchardt, R.T. “Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability” Gastroenterology. 96, 736-49, 1989.
7. U.S. Food and Drug Administration. Guidance for Industry, “Drug Interaction Studies–Study Design, Data Analysis, and Implications for Dosing and Labeling," September 2006, Clinical Pharmacology, p. 39.
8. Zhang, W., et al. “Silencing the breast cancer resistance proteinexpression and function in Caco-2 cells using lentiviral vector-based short hairpin RNA” Drug Metabolism and Disposition, April 2009; 37(4):737-44.
9. Darnell, M., et al. “Investigation of the involvement of P-glycoprotein and multidrug resistance-associated protein 2 in the efflux of ximelagatran and its metabolites by using short hairpin RNA knockdown in Caco-2 cells” Drug Metabolism and Disposition, March 2010; 38(3):491-7.

Dr. Bode has worked in the pharmaceutical industry, including the DMPK field, in various capacities for many years. He can be reached at cbode@absorption.com. Absorption Systems is a contract research organization specializing in preclinical ADME studies.