Determination of free fraction (fu) of compounds by flux dialysis using the rapid equilibrium dialysis (RED) device: establishing empirical permeability for rapid estimation

Precise evaluation of plasma protein binding (fu) is critical for predicting the pharmacokinetic behaviour of new chemical entities.In this study, we describe a Rapid Equilibrium Dialysis (RED)-based Flux Dialysis approach for estimating plasma protein binding(fu) utilising pre-equilibration diffusion data.The empirical membrane permeability (Pmem) of the RED system was established using reference compounds spanning a wide range of binding affinities, including quinidine, imipramine, fluconazole, and sertraline.The averaged Pmem value was subsequently applied to determine fu for test compounds such as Efavirenz, mifepristone, and warfarin, representing diverse physicochemical and binding properties.The current approach demonstrates immediate applicability for low- to moderately bound compounds, where reliable flux data can be obtained within a shorter timeframe.While early-phase diffusion provides useful kinetic information, accurate fu estimation for highly bound compounds requires extended sampling to obtain reliable flux measurements.Fu values obtained from the flux-based model showed close agreement with literature-reported equilibrium dialysis data, supporting the robustness and predictive validity of the approach.The results demonstrate proof-of-concept feasibility of a flux-based approach using RED device, while highlighting limitations related to signal sensitivity and assumptions associated with empirical permeability.