Using Counter Equilibrium Dialysis (CED) to Increase Confidence in the Measurement of Free Fraction for Challenging Compounds

The confidence in fraction unbound (ƒ_u) using equilibrium dialysis (ED) is often questioned (e.g., highly bound, labile compounds) due to uncertainty in whether true equilibrium is achieved. Different methods have been developed to increase confidence in ƒ_u measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒ_u measurement can still suffer due to non-specific binding and inter-run variations introduced during equilibrium and analysis. To address this concern, we introduce an orthogonal approach called counter equilibrium dialysis (CED) in which non-labeled and isotope-labeled compounds are dosed counter-directionally in rapid equilibrium dialysis (RED). ƒ_u values of both non-labeled and labeled compounds are measured simultaneously in the same run. These tactics not only minimize non-specific binding and inter-run variability but also enable the confirmation of true equilibrium. If equilibrium is reached in both dialysis directions, the ƒ_u for the non-labeled compound and the labeled compound will converge. The refined methodology was extensively tested with various compounds of diverse physicochemical properties and plasma binding characteristics. Our results demonstrated that, by using the CED method, ƒ_u values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds.