Method to screen substrates of apical sodium-dependent bile acid transporter

Area of Science:

• Pharmacology
• Biochemistry
• Drug Delivery

Background:

• The human apical sodium-dependent bile acid transporter (hASBT) is a key target for prodrug development.
• Prodrugs targeting hASBT may exhibit poor solubility or hydrolytic instability.
• Existing assay methods require NaOH for cell lysis, which can degrade compounds.

Purpose of the Study:

• To develop a novel assay method for screening hASBT-mediated uptake of compounds.
• To determine compatible cosolvent concentrations for hASBT active transport assays.
• To establish a NaOH-free cell lysis method for sample processing.

Main Methods:

• Evaluated cosolvent compatibility (DMAC, DMF, DMSO, ethanol, methanol, PEG-400, propylene glycol, dioxane) with hASBT transport assays and cytotoxicity.
• Assessed the impact of cosolvents on hASBT Michaelis-Menten kinetics.
• Developed and validated two acetonitrile-based, NaOH-free cell lysis methods, comparing them to the traditional NaOH method for sample recovery and hASBT kinetic parameter assessment.

Main Results:

• Maximum permissible cosolvent concentrations without compromising assay integrity or causing cytotoxicity were identified: 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol.
• Two NaOH-free, acetonitrile-based lysis methods demonstrated comparable sample recovery and hASBT kinetic results to the NaOH method.
• The developed assay method effectively screens for active transport using solubilizing cosolvents and avoids compound-degrading NaOH treatment.

Conclusions:

• An optimized assay method for screening hASBT-mediated compound uptake has been successfully developed.
• The assay accommodates cosolvents to enhance compound solubility and utilizes NaOH-free lysis, preventing sample degradation.
• This advancement facilitates the development of novel hASBT-targeted prodrugs with improved characteristics.