January 2019

Considerations for the Conduct of In Vitro Drug Transporter Assays

Author:  Greg Loewen, Andrea Wolff, Michael Millhollen
Posted:  11 January 2019

Not only can drug transporters affect the absorption and excretion of drugs, they can be involved in pharmacokinetic-based drug-drug interactions (DDI), impacting distribution into tissues as well as the overall exposure of the drug. As a result, regulatory bodies such as the FDA, EMA and PMDA recommend studying the potential of NCEs to be inhibitors or substrates of drug transporters in vitro in order to better understand the ADME and DDI potential of drugs and help determine if clinical studies should be performed. The design and scope of in vitro transporter studies can vary depending on the physicochemical properties and major route(s) of excretion of the drug and should be considered when designing and performing in vitro studies that will support a new drug application (NDA) or decisions to run clinical DDI studies.

P-gp and BCRP test systems:

Selection of the correct test systems is important. For instance, the most common test system used to study P-gp and BCRP in vitro are polarized cell monolayers expressing the transporter of interest that are cultured on a transwell plate. Cell lines include MDCKII, LLC-PK1 or Caco-2. Other test systems are also available, such as membrane vesicles. Each test system has its advantages and disadvantages and should be selected based on the scope of the study (e.g., what questions are you trying to answer?), properties of the drug and the regulatory agencies to which the data will be submitted. In fact, the EMA recommends the use of two systems when evaluating P-gp inhibition. Watch our recent webinar “Understanding P-gp and BCRP Inhibition Assay Design and Outcomes” for a comparison of test systems and further details.

The effect of the physiochemical properties of the test compound:

To determine if a compound is a substrate of an SLC (uptake) transporter, the accumulation of the compound inside cells over-expressing the transporter is measured.  To determine if a compound is a substrate of an ABC (efflux) transporter, permeability across a monolayer of cells expressing the transporter is typically measured. Physiochemical and other properties of the drug may result in non-specific binding, chemical instability, partitioning into cell membranes or organelles, or cell toxicity, all of which can complicate interpretation of the data. Indeed, the 2017 guidance on DDI from the FDA includes evaluation of recovery, stability, non-specific binding and cell toxicity when investigating a drug as a transporter substrate in vitro. Watch our recent webinar “Challenges & Solutions in Today’s In Vitro Transporter Research Landscape” for more information.

Inclusion of an inhibitor in transporter substrate experiments:

To confirm transporter substrate potential, a substrate experiment should be performed in the presence of a known transporter inhibitor. Inclusion of the inhibitor in the initial experiment rather than repeating the experiment a second time with the inhibitor will reduce the cost and timeline. Additionally, the FDA recommends evaluating at least two inhibitors when the in vitro system expresses multiple transporters. Since all currently available in vitro cell-based transporter test system express multiple transporters, two inhibitors should be included in substrate experiments.

Incubation times and test article concentrations in transporter substrate experiments:

Lastly, while a single time point or single substrate concentration may be adequate for screening purposes, it may not be sufficient to fully assess the potential of a compound as a transporter substrate. Concentration- and time-dependency cannot be evaluated from a single substrate concentration and single time point approach and may result in an inaccurate interpretation of substrate potential. High substrate concentrations can saturate the transporter, while low concentrations may make it difficult to detect transport due to limits of quantitation. The EMA guidance on DDI recommends that the substrate concentrations should be relevant to the site of transport and four substrate concentrations should be evaluated spanning a 100-fold concentration range, while the FDA recommends multiple concentrations covering the range of clinically relevant concentrations. Similarly, multiple incubation times should be evaluated to ensure that optimal experimental conditions are evaluated. Collectively, the data should be critically reviewed to ensure that the optimal conditions are used to evaluate substrate potential. It is important that the scope of the study is comprehensive to evaluate substrate potential.

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