Vesicular transport assay

"Inside-out" membrane vesicles containing ABC transporters are applied in the vesicular transport assay to determine the IC₅₀ of the compound. After incubating the test article with the inverted membranes, the membranes are then separated from the drug-containing buffer and washed before measuring their contents for transported compound.
Radiolabeled reporter substrates are used for measuring transported substrate by liquid scintillation counting. The new PREDIVEZ Vesicular Transport Kits were developed for fluorescent reporter substrates.
The standard vesicular transport assay is an indirect inhibitory assay, performed with cold test articles. This assay provides information on any interaction between the ABC transporter and the test article. The transport of the reporter substrate is measured in the presence of the test article (typically in 7 concentrations) and IC₅₀ is defined as the concentration inhibiting the transport of the reporter 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 reporter substrate and may identify substrate nature of the test article. Direct vesicular transport assay is 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 with the test compound.

The project based on the 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 - Assay Development: 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 at two incubation time points and at two concentrations of the test drug. The expected outcome of Part 1 is the determination of whether or not the effective efflux transporter mediated vesicular uptake of the isotope-labeled compound into the inside out vesicles can be measured.

Part 2 - Detailed characterisation of the transport: Part 2 will be started if the effective transport of the test drug in Part 1 can be detected. The aim of the Part 2 is to optimize the incubation parameters. Time dependence of transport at 8 time points will determine the length of the first, linear phase of transport. Concentration dependence will be measured to determine the K_M and V_max. The corresponding reporter substrates and reference inhibitors of the standard assays (Table 1) are used to confirm the transporter-test drug interaction.

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

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