Dynamic Free Fraction: Concept, Methodology and Utility in DMPK

As dictated by the free drug theory (FDT), fraction of unbound drug in plasma (fu,p) is routinely measured to rationalize pharmacological readouts such as drug potency and hepatic clearance. There is an increasing body of evidence contradicting the FDT when fu,p is applied, and one prominent example is the systematic under-prediction of hepatic clearance for highly bound compounds. We reason that fu,p is a static measure of drug binding extent and it does not capture drug protein binding dynamics. As a result, we have introduced the "dynamic free fraction" (fD) as a new binding parameter describing drug protein binding affinity/dynamics that can be indirectly determined by coupling the drug binding assay with a reporter enzyme in combination with high-resolution mass spectrometry, circumventing a long-standing challenge inherent in determining drug binding kinetics constants such as kon and koff. Using a large group of diverse drugs representing both CYPs and OATP transporter substrates, we demonstrated that the well-stirred model incorporating with fD correctly predicted both hepatic clearance and liver extraction ratio without apparent systematic bias, which is markedly better than those predicted with fu,p. The results suggest that dynamic free fraction (fD) as a measure of protein binding affinity is a key determinant in hepatic clearance, which is contrary to the currently held view.