PHYSICOCHEMICAL BASIS FOR THE RAPID TIME-ACTION OF LYS(B28)PRO(B29)-INSULIN - DISSOCIATION OF A PROTEIN-LIGAND COMPLEX

The rate-limiting step for the absorption of insulin solutions after s ubcutaneous injection is considered to be the dissociation of self-ass ociated hexamers to monomers. To accelerate this absorption process, i nsulin analogues have been designed that possess full biological activ ity and yet have greatly diminished tendencies to self-associate. Sedi mentation velocity and static light scattering results show that the p resence of zinc and phenolic ligands (m-cresol and/or phenol) cause on e such insulin analogue, Lys(B28)Pro(B29)-human insulin (LysPro), to a ssociate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamic s. The dissociation of the stabilized hexameric analogue has been stud ied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hy pothesized to be due to altered subunit packing within the hexamer. Ev idence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, re ported here, has been interpreted with respect to the crystal structur e results. In addition, the phenolic ligand binding differences betwee n LysPro and insulin have been compared using isothermal titrating cal orimetry and visible absorption spectroscopy of cobalt-containing hexa mers. These studies establish that rapid-acting insulin analogues of t his type can be stabilized in solution via the formation of hexamer co mplexes with altered dissociation properties.