The challenge of accurately measuring drug permeability

In our seventh NanoBio Lab, Erin Gallagher, a predoctoral candidate from the lab of professor Peter Searson, demonstrated the use of a cell permeability assay as a means of modeling drug diffusion through the blood brain barrier (BBB) endothelium. Assays such as this one enable us to better understand the cellular processes that govern what drug molecules are able to cross the BBB and the role of efflux pumps and transporters. Development of more accurate in vitro models is a highly valuable avenue of research, as it will allow for rational drug design to more effectively treat diseases such as Alzheimer’s, Parkinson’s and mood disorders with potentially fewer side effects.

The blood brain barrier (BBB) presents a challenge for delivery of drug molecules to the central nervous system, as many of the mechanisms it employs were evolved specifically to prevent introduction of dangerous substances into the central nervous system. Understanding the mechanisms by which various substances are able to cross the BBB will allow for more rational design of future generations of drug molecules and carrier systems.

The blood brain barrier (BBB) presents a challenge for delivery of drug molecules to the central nervous system, as many of the mechanisms it employs were evolved specifically to prevent introduction of dangerous substances into the central nervous system. Understanding the mechanisms by which various substances are able to cross the BBB will allow for more rational design of future generations of drug molecules and carrier systems.

For the assay, canine kidney cells (MDCK II) were seeded on transwells in a 24 well plate, 24 hours prior to the assay to allow the cells to form a confluent endothelial layer with functional tight junctions. When cells have formed a confluent endothelial layer, ion movement must occur through the cells themselves instead of through the much higher resistivity tight junctions. As a result, the overall resistivity measured is higher than for non-confluent cells, for which ions are able to simply diffuse through the transwell. Therefore, assessment of the integrity of the endothelial layer was done to measure the conductivity through the layer of cells.

Following assessment of the endothelial layer integrity, we ran a permeability assay for the fluorescent molecule Lucifer Yellow (LY) to determine its apparent permeability as a model for drugs diffusing across the BBB. Utilizing a standard concentration curve of LY fluorescence, the amount of LY that diffused through the layer was determined at specific time points to imply apparent permeability. For more typical non-fluorescent drug molecules, high performance liquid chromatography (HPLC) can be used to measure the amount of drug having diffused.

As a tool, assays modeling the blood brain barrier are indispensible to the pharmaceutical industry, but finding a model system that effectively reproduces in vivo conditions for less expensive, high throughput in vitro testing is a challenge. Permeability models, such as the one used in this lab, also allow development of novel strategies for moving drugs across the BBB. These strategies include molecular engineering of drug molecules to take advantage of cellular active transport mechanisms or peptide engineering that facilitates vesicle transport across the endothelium.

David Wilson is a first year PhD student in biomedical engineering working in the drug delivery laboratory of associate professor Jordan Green in biomedical engineering.

Image Citation:  Wong, A. D., Ye, M., Levy, A. F., Rothstein, J. D., Bergles, D. E., & Searson, P. C. (2013). The blood-brain barrier: an engineering perspective. Frontiers in Neuroengineering, 6(August), 1–22. doi:10.3389/fneng.2013.00007

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