A Preliminary In Vitro Interaction of ET‐26‐HCl With Human Serum Albumin Through Rapid Equilibrium Dialysis Under Simulated Physiological Conditions

In the present study, preliminary in vitro interaction between ET-26-HCl and human serum albumin (HSA) was investigated, and the potential competitive binding effect from its major metabolite, ET-26-acid, was evaluated. Rapid equilibrium dialysis (RED) was utilized under simulated physiological conditions (67 mM phosphate-buffered saline solution, pH 7.4, 37 °C). After a methanol-based deproteinization procedure, a validated HPLC-UV method was employed to quantify post-dialysis samples. It was found that the binding affinity of ET-26-HCl to HSA was relatively lower than that of etomidate, with a binding association constant (K) of 7.75 × 10³ M^-1 and a protein binding rate (PB%) of 54.58%, respectively. Displacement experiments using warfarin and flurbiprofen as specific site markers revealed that ET-26-HCl predominantly binds to Sudlow's Site I on HSA. Furthermore, ET-26-acid was found to exhibit no significant competitive binding effect. These findings support ET-26-HCl as a promising alternative to etomidate and warrant its further development for clinical anesthetic applications.