Method to screen substrates of apical @entity1372 -dependent XXXX transporter.

@entity1 @entity7362 @entity2771 @entity137 @entity1042 @entity9104 @entity235 @entity1372 @entity1626 @entity9105 @entity3733 @entity9106 @entity6442 @entity6741 @entity6674 @entity6246 @entity21514 @entity1068 @entity968
@entity1 apical @entity1372 -dependent @entity6741 transporter ( @entity21514 ) is a potential prodrug target under study. Development of prodrugs that target @entity21514 may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids @entity6674 for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for @entity21514 uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an @entity21514 active transport assay. The second objective was to develop a @entity6674 -free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: @entity9105 ( @entity9106 ), @entity6442 ( @entity9104 ), @entity3733 ( @entity3733 ), @entity1068 , @entity1042 , @entity968 , @entity2771 , and @entity6246 . Initial studies included @entity7362 flux studies across @entity21514 -Madin-Darby @entity235 kidney monolayers using up to 10% cosolvent, as well as @entity137 studies. The effect of selected cosolvent concentrations on the @entity21514 Michaelis-Menten kinetic parameters was evaluated. Additionally, two @entity1626 -based cell lysis methods that do not use @entity6674 were evaluated in terms of percent sample recovery and @entity21514 kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for @entity21514 uptake studies, without compromising assay results or causing @entity137 , are 1% @entity9106 , 1% @entity9104 , 2.5% @entity3733 , 2.5% @entity1042 , and 2.5% @entity1068 . Additionally, both @entity6674 -free, @entity1626 -based cell lysis methods provided similar recovery and @entity21514 results, compared to @entity6674 method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid @entity6674 sample treatment, which can otherwise degrade compound.
