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. Graphical Abstract