The plasma protein binding capability of drug substances represents an important assay parameter in drug discovery and development. For very strong plasma protein binding molecules, however, the free fraction in plasma fu is very small and therefore difficult to determine with standard methods. To solve this problem, the EScalate equilibrium shift in vitro assay was developed. Escalating concentrations of plasma were found to shift the binding equilibrium in solution between the test item and immobilized human serum albumin. Following liquid chromatography coupled to mass spectrometry analysis of the samples, the test compound's fu in plasma is calculated with a 2-dimensional fitting procedure. Comparability of EScalate assay results was demonstrated for 4 extensively studied small molecule drugs (carbamazepine, desipramine, pyrimethamine, and warfarin) as well as for liraglutide, a fatty acid-conjugated peptide drug with very strong plasma protein binding. The results were in good agreement with published data. A free fraction of 0.51% was determined for liraglutide. Our results confirm the compound's very strong plasma protein binding properties in a novel and robust assay system.
Keywords: ADME; albumin; in vitro models; mathematical model; metabolic clearance; peptides; protein binding.
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