In Vitro

ATPase Assay - A membrane assay that indirectly measures activity of the transporter of interest. The transport of substrates for ABC transporters requires ATP hydrolysis. ATP hydrolysis results in the release of inorganic phosphate which can be measured using simple colorimetric analysis.

Membrane Vesicle Assay - Inverted plasma membrane vesicles have been used to study transport. Cell lines used to prepare membrane vesicles include drug selected cells, transfected cells, and baculovirus infected insect cells. The activity of ABC transporters have been studied using this type of assay. As the membranes are inverted, influx rather than efflux is used to measure transporter activity. Using this system, detailed kinetic experiments can be performed.

Cell Lines - Polarized cell line assays where the flux of compound from apical to basolateral and basolateral to apical are commonly used to study transport and permeability. Cell lines used in this type of assay include Caco-2 (human epithelial colorectal adenocarcinoma cells) and MDCK (Madin-Darby canine kidney cells) cells. The expression of transporters in Caco-2 cells is comparable to that observed in the small intestine. In contrast, endogenous expression of transporters in MDCK cells is low. In addition, as MDCK cells are derived from dog, transporters expressed will be of canine origin.

Transfected Cell Lines - Transfected cell lines contain recombinant transporters that are either stably or transiently expressed. Transfections can be single or double and can include efflux and/or uptake transporters. Cell lines used for transfection include MDCK, LLC-PK1, HEK 293, or CHO cells. An example of a commonly used transfected cell line is MDR1 transfected MDCK cells for the study of P-gp.

Primary Cells - Primary cells in some cases can be isolated from intact tissue and will contain the full complement of transporters present in the tissue of interest. Primary cells adapt to culture conditions quickly and transporter expression can change. An example of a primary cell assay is a brain microvessel endothelial cell assay. Properties of primary cells in culture must be understood prior to utilizing primary cell assays.

Hepatocyte in Sandwich Culture - A hepatocyte sandwich culture assay involves culturing hepatocytes between two layers of gelled collagen. Hepatocytes in this configuration have the ability to form bile canaliculi and have a full complement of hepatic transporters on sinusoidal and canalicular membranes. Thus, this type of assay has been used to investigate biliary clearance of drugs.

Knockout models and naturally occurring transporter deficient animal models are useful tools to understand the in vivo contribution of transporters. An example of the use of knockout animal studies to investigate the role of transporters on in vivo disposition is the use of Mdr 1a~y~ mice to demonstrate the role of P-gp in limiting brain exposure. Despite the advantages of using in vivo systems to investigate transporters, there are limitations to these models. Alterations in the expression of other transporters and enzymes not being studied can occur in knockout animals. In addition, the level of expression of transporters can sometimes differ between animals and humans. These factors need to be considered when interpreting the results from studies using knockout animals.

The incorporation of the impact of transporters into physiologically based pharmacokinetic models is still in its infancy. Although the necessary theory is available, insufficient information is available on the expression of transporters in various tissues and their maximum capacity to transport various substrates in humans.

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