Assessment of Unbound Rebastinib in Human Plasma and Serum Albumin by Equilibrium Dialysis Coupled with High-resolution Orbitrap Mass Spectrometry

Abstract

Plasma protein binding (PPB) is a critical determinant of drug pharmacokinetics, as only the unbound fraction (fᵤ) of a drug is available for tissue distribution, target engagement, and clearance. However, the interaction of rebastinib with plasma proteins has not been fully elucidated to date. In this study, the free fraction of rebastinib in human plasma and isolated human serum albumin (HSA) was determined quantitatively using rapid equilibrium dialysis (RED) combined with ultra-high-performance liquid chromatography (UHPLC) - Orbitrap high resolution mass spectrometry operated in parallel reaction monitoring (PRM) mode. The binding study was carried out at 37°C using the RED apparatus under optimised equilibrium conditions. Warfarin and verapamil were used as standards for the method validation. It was found that rebastinib has very high plasma protein binding (99.90 ± 0.01%; fu = 0.001), but the interaction with albumin was much comaratively less pronounced (87.81 ± 2.32%; fu = 0.122). Thus, albumin plays an essential role in rebastinib plasma binding, but other plasma proteins may also be involved. Overall, the developed methodology had acceptable stability, repeatability, and extraction recovery for both matrices. The extremely low unbound fraction of rebastinib implies strong protein affinity and can strongly impact its pharmacokinetic profile. As much as is feasible, the present effort endeavours to provide the first comprehensive comparative study of rebastinib interactions with plasma and HSA. It reveals valuable input data for further PBPK studies and pharmacokinetic translation.