Almost all small molecule drugs soon after administration become reversibly bound to blood plasma proteins, most commonly albumin, lipoproteins and α1-acid-glycoprotein. The bound drug molecules fraction is generally considered not available for interaction with their biological targets. Determining the extent of this binding is a critical phase of a drug candidate development because this parameter influences compound efficacy, dosing, clearance rate and its potential for drug interactions.
The “golden standard” method for the % plasma protein binding (%PPB) determination is the rapid equilibrium dialysis (RED).
We determine binding capabilities of drug candidates to plasma proteins by spiking test compounds at concentrations of 1 µM and 10 µM into plasma and dialyzing against buffer until equilibrium was achieved. The assay is performed in a multiple-use 96-well dialysis unit HTD96b dialyser (HTDialysis). Each individual well unit consists of 2 chambers separated by a vertically aligned dialysis membrane of predetermined pore size (MWCO 12-14 kDa). Non-diluted plasma spiked with the compound of interest is added to one chamber and the dialysis buffer to the other chamber. Free compound diffuses from the plasma chamber to the buffer chamber until equilibrium is reached. Concentrations of the compounds in plasma and buffer are determined by LC-MS, and the percentage of plasma protein bound compound is calculated. All incubations are performed in duplicates.
Whole plasma or an individual plasma protein (albumin, AGP) can be used in this assay. Plasma from different species is available, including human, mouse, dog and rat.
2 µmol of a dry compound or 10 mM DMSO stock solutions can be used.