Approaches to measure protein binding of enzymatically unstable compounds in plasma.

Abstract

To develop approaches to measure plasma protein binding (PPB) of enzymatically unstable compounds. Bis-para-nitrophenyl phosphate (BNPP) was used to inhibit enzyme activity and stabilize two model compounds (diltiazem and oseltamivir) that are subject to enzyme-catalyzed hydrolysis in plasma. Protein binding of the compounds in BNPP-treated rat plasma was measured using equilibrium dialysis or ultrafiltration. PPB measurement of unstable compounds was improved by using enzyme inhibitor to stabilize the compounds in plasma during the assay. The effect of BNPP concentration on drug-protein binding appeared to be compound dependent. Given the compound's nonspecific binding to the assay device can be accounted for in the unbound fraction measurement, ultrafiltration can be a viable alternative or complementary approach for PPB assay of unstable compounds while minimizing the potential impact of enzyme inhibitor on drug-protein binding.