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.