Evaluation of a Competitive Equilibrium Dialysis Approach for Assessing the Impact of Protein Binding on Clearance...

Fraction unbound (f_u) is an important consideration when characterizing the ADME properties of drug candidates. For highly bound compounds, there can be low confidence in quantifying f_u introducing uncertainty in certain parameter estimations. Specifically, predictions of clearance (CL) rely on accurate f_u values measured in plasma (f_u,p) and microsomes (f_u,mic) to scale in vitro intrinsic CL to in vivo CL. However, determining the ratio of f_u,p/f_u,mic may circumvent the need to measure discrete binding values. The purpose of this study was to evaluate a plasma-to-microsome competitive equilibrium dialysis (cED) method to determine f_u,p/f_u,mic ratio (fuR) for nine physiochemically-distinct compounds, and to investigate the impact of altering microsomal concentrations on fuR. The values of fuR were comparable to ratios calculated from discretely measured f_u,p and f_u,mic values. Furthermore, increasing microsomal concentrations increased fuR for basic and neutral compounds. When using fuR values, there was a good in vitro-in vivo correlation (IVIVC) (≤3-fold observed in vivo CL). These results suggest that the cED method used to determine fuR may be an appropriate, alternative IVIVC approach. Application of cED may extend beyond IVIVC of CL to evaluate other parameters such as species differences in protein binding and free tissue to plasma ratios.