Characterization of the Interaction between Human α ₁ -Acid Glycoprotein and Apremilast Enantiomers: HPLC, Isothermal Titration Calorimetry, Ultracentrifugation, and Docking Studies

The interactions between human α₁-acid glycoprotein (AGP) and the enantiomers of apremilast (APR) were investigated by using a combination of HPLC, isothermal titration calorimetry (ITC), ultracentrifugation, and molecular docking. HPLC analyses on a chiral AGP column revealed enantioselective binding, with S-APR showing slightly higher stability for AGP compared with R-APR. However, the stability difference is sufficient for the baseline separation of APR enantiomers. ITC experiments confirmed the spontaneous and exothermic nature of the binding, with similarly high binding constants (log K > 5) for both enantiomers but a more exothermic enthalpy for the S-form. Ultracentrifugation studies further supported the high stability of the AGP-APR complexes, with the S-enantiomer again demonstrating marginally stronger binding. Molecular docking revealed that S-APR formed more direct interactions, including additional hydrogen bonds and π-π interactions with AGP compared to R-APR, corroborating the experimental findings. This enhanced interaction also contributes to the chiral separation. Comparison of the protein binding data suggests that AGP plays a significant role in APR transport. Altogether, the multianalytical approach provided detailed insight into the enantioselective binding of APR to AGP, which may contribute to understanding its pharmacokinetic behavior and therapeutic action.