New Methodology for Determining Plasma Protein Binding Kinetics Using an Enzyme Reporter Assay Coupling with High-Resolution Mass Spectrometry.

Abstract

Determination of drug binding kinetics in plasma is important yet extremely challenging. Accordingly, we introduce "dynamic free fraction" as a new binding parameter describing drug-protein binding kinetics. We demonstrate theoretically and experimentally that the dynamic free fraction can be determined by coupling the drug binding assay with a reporter enzyme in combination with high-resolution mass spectrometry measuring the relative initial steady-state rates of enzymatic reactions in the absence and presence of matrix proteins. This novel and simple methodology circumvents a long-standing challenge inherent in existing methods for determining binding kinetics constants, such as kon and koff, and enables assessment of the impact of protein binding kinetics on pharmaceutical properties of drugs. As demonstrated with nine model drugs, the predicted liver extraction ratio, a measure of efficiency of drug removal by the liver, correlates significantly better to the observed extraction ratio when using the dynamic free fraction (fD) in place of the unbound fraction (fu) of the drug in plasma. Similarly, the in vivo hepatic clearance of these drugs, a measure of liver drug elimination, is highly comparable to the clearance values calculated with the dynamic free fraction (fD), which is markedly better than those calculated with the unbound fraction (fu). In contrast to the prevailing view, these results indicate that protein binding kinetics is an important pharmacokinetic property of a drug. As plasma protein binding is one of the most important drug properties, this new methodology may represent a breakthrough and could have a real impact on the field.