Optimization of Incubation Time for Accurate Protein Binding Assessment in Biological Matrices Using Equilibrium Dialysis.

Abstract

INTRODUCTION: Accurate % unbound estimation in protein binding assays in drug discovery is critical for understanding Pharmacokinetics (PK), Pharmacodynamics (PD), and safety assessments. Short incubation times (2-6 hours) in Equilibrium Dialysis (ED) compromise the determination of the % unbound. Incubation times were evaluated in ED to obtain reliable estimates of % unbound across diverse biological matrices and compound types. METHODS: A time-course of 2-24 hours for 17 marketed compounds with varying physicochemical properties was conducted using RED and HTD devices across human plasma, tumor cell lines, 10% FBS, and rodent tissues to determine % unbound values. RESULTS: Compounds exhibited unbound fractions ranging from 70% and generally correlated with LogD. Incubation times <6 hours underestimated % unbound (up to 6-fold), particularly for highly bound compounds. Equilibrium was typically reached by 16-24 hours in all matrices. A <20% increase was exhibited in % unbound beyond 16 hours (except lapatinib). HTD yielded a marginally faster equilibrium than RED. DISCUSSION: Short incubation times (2-6 hours) in equilibrium dialysis often underestimate the % unbound, particularly for highly bound compounds. Extending incubation to ≥16 hours achieved equilibrium across plasma, tissues, and cell lines, yielding more reliable and consistent % unbound data. This standardized approach improves the PK/PD assessment and compound prioritization in early drug discovery. CONCLUSION: The usual 2-6 hours of incubation in ED underestimates % unbound and may be inadequate for compound prioritization. We recommend ≥16 hours of incubation for protein binding assessment in early drug discovery.

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