Transporting Drug Safety Forward

Catherine Shaffer, Contributing Editor
The role of membrane transporters in drug-drug interactions is emerging as an increasingly important part of the overall picture of drug safety and drug metabolism. Drug metabolizing enzymes are well-characterized; previously, the U.S. Food and Drug Administration (FDA) offered detailed guidance on the design of preclinical and clinical studies to evaluate the interactions of drugs with those enzymes.
In contrast, only recently has information and guidance emerged concerning the study of transporter mediated drug-drug interactions. More than 400 membrane transporters have been identified. Those of most interest in drug development are found in the intestines, liver, kidney, and the blood-brain barrier. Transporter effects of a drug can affect the uptake or clearance of another drug. These effects are largely discovered during clinical testing.
In late February 2012, the FDA issued long-awaited draft guidance which included recommendations for in vitro and in vivo studies for drug transport, drug metabolism, and drug-drug or drug-therapeutic protein interactions. (See the sidebar in Policies & Projection for details.)
A major initiative, spearheaded by the International Transporter Consortium, seeks to provide guidance around tools and methods for characterizing transporter mediated drug interactions in vitro, at the preclinical stage, in order to better design clinical studies and identify potential safety issues at an earlier stage.

Drugs can affect transporters in many ways
There are several potential types of transporter mediated interactions that could be of concern. A compound may inhibit a transporter involved in an endogenous process. Serum chemistry markers provide an in vivo signal for this type of complication. Clinical manifestations resulting from such an interaction—hyperbilirubinemia, for example—can be an obstacle to further development.
Alternate routes of elimination or disposition of the drug can mitigate the risk of a drug interaction. For life-saving drugs, the choice may be made to tolerate a moderate drug interaction in order to achieve the desired outcome. Things can become complicated very quickly when two or more drugs interact with similar transport mechanisms.
Drugs can also be substrates or potentiators of transporters, with effects resulting from increases in transport.
“When compounds are potential perpetrators of drug interactions, an understanding of co-medications becomes even more important so that therapeutic concentrations of a novel agent will not result in subsequent elevations of co-meds that may have narrow therapeutic indices. In the case of chronic dosing, little is understood about the long-term inhibition of transporters,” says Keith Hoffmaster, PhD, a scientist for Novartis Institutes for BioMedical Research (Cambridge, Mass.).Patients with diseases that are managed by polypharmacy are at greater risk of transporter-mediated drug interactions.
Some of the most common of those are cancer, diabetes, cardiovascular disease, and infectious disease. For example, lipid-lowering therapies like statins can interact with other drugs. The immunosuppressant drug cyclosporine increases systemic exposure to all statin drugs. Rosuvastatin is a substrate for the human liver transporter, organic anion transporting polypeptide (OATP-1B1). In one study, rosuvastatin exposure was significantly increased in heart transplant recipients on an antirejection regimen that included cyclosporine. (Clin Pharmacol Ther. 2004; 76(2): 167-77)
Through a better understanding of drug-transporter interactions, it may also be possible to harness interactions such as cyclosporine/rosuvastatin to enhance therapeutic outcomes.

in vitro methods mimic in vivo processes
Currently available in vitro methods for studying transporters include ATPase assays, membrane vesicle assays, and cell-based assays such as transfected cell lines or primary hepatocyte suspensions or cultures.
According to Stephen Ferguson, PhD, a senior staff scientist in ADME/Tox at Life Technologies Inc., tools for studying transporters can be classified into two major categories: specific transporter assays and “clearance” assays, where the net effect of multiple transport processes on drug clearance can be modeled in a physiologically relevant system.
In order to adequately characterize transporter mediated interactions, it is necessary to have tools that include “integrated” whole systems, as well as tools that isolate the behavior of individual transporters.
According to Ferguson, the set of tools needed is similar to those used to study metabolic effects of cytochrome P450s. However, he says, “Unlike P450s, we’re well behind where we need to be to assess in vivo potential of these processes.”
Stephen Wright, PhD, a professor in the Department of Physiology at the University of Arizona, studies transporter interactions of the kidney. His group is interested in the mechanism of renal secretion of organic electrolytes. A large proportion of pharmacological compounds are anionic or cationic in nature, and thus fall into the category of electrolytes. The kidney protects the body from toxins by excreting xenobiotic compound, and its tissues are rich in transporters tailored for that function.
Wright’s lab uses cultured renal cells to stably transfect cloned human transport proteins, and uses radiolabeled substrates or substrates with fluorescent properties. At the tissue level, they work with isolated, intact renal proximal tubules to study electrolyte secretion in the native epithelium.
“We’re teaming up with computational chemists who use powerful analytical computational tools to compare biological activity to the intrinsic structural chemical properties of those compounds,” Wright says.
Wright has used these methods to study multidrug resistance-associated protein 2 (MRP2) in the epithelium of the eye, concluding that calcein, dichlorofluorescein, and doxorubicin accumulated inside the cells in greater quantities in the presence of an MRP2 inhibitor. (J Pharmacol Exp Ther. 329: 479-85.)
Wright has also published studies relating to organic anion transporter 3 (OAT3), organic cation transporter 2 (OCT2) and multidrug and toxin extruder 1 (MATE1).
Kim Brouwer, PharmD, PhD, a professor at the University of North Carolina’s Eschelman School of Pharmacy, Division of Pharmacotherapy and Experimental Therapeutics, was an innovator of the sandwich-cultured hepatocyte. Suspended primary hepatocytes lose their polarity upon isolation, making it difficult to assess efflux. Culturing hepatocytes in a sandwich configuration between two layers of collagen restores that polarity. Compared to suspended hepatocytes, sandwich-cultured hepatocytes more closely model in vivo biliary clearance. Brouwer’s sandwich-cultured hepatocyte system is called B-Clear, and she co-founded the company Qualyst Inc., a UNC spin-off company, around the technology.
In her UNC laboratory, Brouwer studies hepatotoxicity, specifically the role of bile acid transport proteins.
Bile acids are important in the maintenance of hepatocyte function. Polymorphisms in bile acid transport proteins that decrease transport of those acids can cause serious liver injury. Brouwer recently published results elucidating the mechanisms determining differences in systemic exposure of two very similar active drug metabolites.
Brouwer says that hepatocytes are in short supply because they are obtained from donors. “We’re looking at further development of the sandwich-cultured hepatocyte model, including stem cell-derived hepatocytes,” Brouwer says.

Working together for drug clearance
BD Biosciences, a segment of BD (Franklin Lakes, N.J.) provides tools for studying the two major drug transporter protein families typically involved in drug-interactions: ABC transporters for drug efflux and the SLC transporter family, which are important for drug uptake.
“An area of great interest is the interplay between drug transport and metabolism, and how transporters contribute to the overall clearance of a drug. The uptake and efflux transporters on the sinusoidal (blood) and biliary membranes of the liver can regulate the concentration of the drug in the liver cells, which can ultimately affect the rate of drug clearance from the blood,” says Chris Patten, PhD, a scientist with BD Biosciences, Discovery Labware.
Technology advances in fields such as engineering and analytical chemistry, will also help improve knowledge of transporter mediated drug interactions. “We’re seeing advances in the imaging area,” Brouwer says. Those advances may improve understanding of intracellular drug exposure. Up until about 10 years ago, it was frequently assumed that drugs diffused passively in and out of the blood stream. It is now known that membrane transporters may facilitate those movements. This means plasma levels of drug may not be reflective of every tissue in the body.
These advances, as well as the FDA draft guidance, indicate that transporters are now getting the attention they deserve.
About the Author
Catherine Shaffer is a freelance science writer specializing in biotechnology and related disciplines with a background in laboratory research in the pharmaceutical industry.