Addressing the Accuracy of Plasma Protein Binding Measurement for Highly Bound Compounds Using the Dilution Method

Currently, regulatory guidelines recommend using 0.01 as the lower limit of plasma fraction unbound (f_u) for prediction of drug-drug interactions (DDI) to err on the conservative side. One way to increase experimental f_u of highly bound compounds is to dilute the plasma. With the dilution method, a diluted f_u, or f_u,d, of ≥ 0.01 can be achieved by adjusting the dilution factor. The undiluted f_u can be calculated from f_u,d and be used for DDI prediction. In this study, the dilution method was evaluated, and the results showed that it gave similar f_u values as those determined using the pre-saturation method without plasma dilution. The dilution method enables generation of accurate f_u values and alignment with the regulatory recommendation of reportable f_u values of ≥ 0.01 for DDI prediction. We recommend using the dilution method to bridge the regulatory recommended f_u limit of 0.01 for DDI prediction and the pre-saturation or equivalent methods for definitive plasma protein binding studies. As the pharmaceutical industry continues to generate high quality PPB data, regulatory agencies will gain confidence in the accuracy of f_u measurements for highly bound compounds, and the f_u lower limit may no longer be needed in the future.