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

Aim: To develop approaches to measure plasma protein binding (PPB) of enzymatically unstable compounds. Methodology: 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. Conclusion: 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.