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Updated: Feb 12, 2026

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells
Published on: November 17, 2017
Approaches to measure protein binding of enzymatically unstable compounds in plasma
1Drug Metabolism & Pharmacokinetics, Vertex Pharmaceuticals, Inc. Boston, MA 02210, USA.
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.
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