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 (fu) for prediction of drug-drug interactions (DDI) to err on the conservative side. One way to increase experimental fu of highly bound compounds is to dilute the plasma. With the dilution method, a diluted fu, or fu,d, of ≥ 0.01 can be achieved by adjusting the dilution factor. The undiluted fu can be calculated from fu,d and be used for DDI prediction. In this study, the dilution method was evaluated, and the results showed that it gave similar fu values as those determined using the pre-saturation method without plasma dilution. The dilution method enables generation of accurate fu values and alignment with the regulatory recommendation of reportable fu values of ≥ 0.01 for DDI prediction. We recommend using the dilution method to bridge the regulatory recommended fu 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 fu measurements for highly bound compounds, and the fu lower limit may no longer be needed in the future.