Vesicular transport assays

"Inside-out" membrane vesicles containing ABC transporters are utilized in the vesicular transport assay to determine the IC₅₀ of the compound. The standard vesicular transport assay is an indirect inhibitory assay, performed with cold test compound. The transport of the fluorescent or radiolabeled probe substrate is measured in the presence of the test article and IC₅₀ is defined as the concentration inhibiting the transport of the probe substrate by 50%.

Should radiolabeled form of the investigated compound or adequate analytical methods (LC/MS, HPLC) be available, the vesicular transport assay may be performed in a direct format without the probe substrate and may identify substrate nature of the test article. Direct vesicular transport assay is a low throughput assay. It is suitable for low permeability test compounds as high permeability compounds may escape from the vesicles through the lipid bilayer. Standard assays are always recommended before running the direct measurements.

To learn more about the development of direct vesicular transport assays please click here.

Direct Vesicular Transport assay is organized into two basic Parts (Part 1 and 2), and one optional Part (Part 3) to evaluate potential drug-drug interactions. The launch of the given Part(s) depend(s) on the success of the previous Part(s).

Part 1 - Pilot experiments to determine feasibility of testing -The vesicular transport of the test compound is determined using membranes containing the selected efflux transporter and using control membranes. The expected outcome of Part 1 is the determination of whether or not the effective efflux transporter mediated vesicular uptake of the cold or isotope-labeled compound into the inside out vesicles can be measured.

Part 2 - Detailed characterization of the transport: Part 2 will be initiated if the effective transport of the test drug in Part 1 can be detected. The aim of Part 2 is to optimize the incubation parameters. Time dependence and concentration dependence will be measured to determine the K_M and V_max.

Part 3 - Optional Part for characterizing potential drug-drug interactions: The IC₅₀s of selected known transporter-interacting drugs will be determined in incubations of the test compound with transporter expressing membranes as established in Part 2.

For more details on the scientific background of vesicular transport assays please click here.