A framework for plasma protein binding: Comparing methods for diverse small molecules and adapting for novel...

Area of Science:

• Drug Discovery and Development
• Pharmacology
• Analytical Chemistry

Background:

• Accurate plasma protein binding (PPB) measurements are crucial for drug development.
• Understanding PPB informs therapeutic windows and pharmacokinetic/pharmacodynamic (PK/PD) relationships.

Purpose of the Study:

• To evaluate the concordance of equilibrium dialysis (ED), ultrafiltration (UF), and ultracentrifugation (UC) for PPB assessment.
• To optimize PPB methods for novel modalities like peptides and degraders.
• To provide guidance for selecting appropriate PPB methods.

Main Methods:

• Orthogonal evaluation of ED, UF, and UC across 26 small molecules.
• Method adaptations for peptides (increased incubation, solutol addition) and validation with adapted UC.
• Assessment of degraders using standard high-throughput dialysis (HTD).

Main Results:

• Strong correlation observed between the three PPB methods (R² = 0.95).
• ED data showed high agreement with literature values (R² = 0.97).
• Optimized methods for peptides and degraders yielded high confidence results (R² > 0.87 for peptides).

Conclusions:

• ED is a reliable high-throughput method, with adaptations for challenging compounds.
• Optimized methods ensure robust PPB assessment for diverse therapeutic agents.
• A decision tree aids in selecting the best PPB method based on compound characteristics.