Examining the effects of vitamin B12 conjugation on the biological activity of insulin: a molecular dynamic and in vivo oral uptake investigation

Abstract

The practical use of the vitamin B₁₂ uptake pathway to orally deliver peptides and proteins is much debated. To understand the full potential of the pathway however, a deeper understanding of the impact B₁₂ conjugation has on peptides and proteins is needed. We previously reported an orally active B₁₂ based insulin conjugate attached at LysB29 with hypoglycemic properties in STZ diabetic rats. We are exploring an alternative attachment for B₁₂ on insulin in an attempt to determine the effect B₁₂ has on the protein biological activity. We describe herein the synthesis, characterization, and purification of a new B₁₂–insulin conjugate, which is attached between the B₁₂ ribose hydroxyl group and insulin PheB1. The hypoglycemic properties resulting from oral administration (gavage) of such a conjugate in STZ diabetic rats was similar to that noted in a conjugate covalently linked at insulin LysB29, demonstrating the availability of both positions on insulin for B₁₂ attachment. A possible rationale for this result is put forward from MD simulations. We also conclude that there is a dose dependent response that can be observed for B₁₂–insulin conjugates, with doses of conjugate greater than 10⁻⁹ M necessary to observe even low levels of glucose drop.