Characterization of Taurocholic Acid Binding With Insulin for Potential Oral Formulation Using Different Methods

Abstract

In diabetes management, oral formulation of insulin (INS) has the potential to improve safety, convenience, and patient-centered care compared to subcutaneous injections. However, its bioavailability remains limited, necessitating improved delivery strategies. Recent clinical trials indicate that taurocholic acid (TCA) can enhance the bioavailability of oral INS as an absorption enhancer. In this work, electrospray ionization mass spectrometry (ESI-MS) analysis revealed the formation of 1:1–1:4 INS–TCA complexes. MS/MS was used to explore the fragmentation pathway of complex ions and confirm binding stability in the gas phase. Circular dichroism spectra showed no clear conformational change in INS upon TCA binding, even though TCA enhanced INS's structural stability. Using Taylor dispersion analysis (TDA), we determined the diffusion coefficient and hydrodynamic radius of INS and its complexes. TCA binding was observed to increase INS size in both the 1:1 and 1:2 INS–TCA complexes. The binding constant of INS and TCA (1.3 × 10³ L/mol) with approximately five binding sites was obtained via pressure-assisted capillary electrophoresis frontal analysis. Molecular docking simulations indicated that TCA binds to external binding sites on the INS B chain (near Ser-B9, Glu-B13, and Phe-B24 residues), consistent with ESI-MS and TDA results. These findings suggest that TCA binding may enhance INS absorption and increase the bioavailability of oral INS therapy.