Method to screen substrates of apical sodium-dependent bile acid transporter.
Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.
Rais
Rana
R
School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, Maryland 21201, USA.
Gonzalez
Pablo M
PM
Zheng
Xiaowan
X
Wring
Stephen A
SA
Polli
James E
JE
D000081:Acetamides / Q000378:metabolism / Q000494:pharmacology; D000097:Acetonitriles / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D004121:Dimethyl Sulfoxide / Q000378:metabolism / Q000494:pharmacology; D004126:Dimethylformamide / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism / Q000494:pharmacology; D012972:Sodium Hydroxide / Q000378:metabolism; D013379:Substrate Specificity
D000081:Acetamides; D000097:Acetonitriles; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; D011092:Polyethylene Glycols; D012972:Sodium Hydroxide; D004126:Dimethylformamide; C013959:dimethylacetamide; D004121:Dimethyl Sulfoxide; C032159:acetonitrile