ASSEMBLY AND DISSOCIATION OF HUMAN INSULIN AND LYS(B28)PRO(B29)-INSULIN HEXAMERS - A COMPARISON STUDY

Purpose. Investigations into the kinetic assembly and dissociation of hexameric Lys(B28)Pro(B29)-human insulin (LysPro), a rapid-acting insu lin analog produced by the sequence inversion of amino acids at positi ons B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog. Methods. The kinetics of phenolic ligand binding to human insu lin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R(6) hexamer disruption were studied by extraction of Co(II) with EDTA. Results. Phenolic ligand binding to human insulin yielded rate c onstants for a fast and slow phase that increased with increasing liga nd concentration and are attributed to the T-6 --> T(3)R(3) and T(3)R( 3) --> R(6) transitions, respectively However, the kinetics of phenoli c ligand binding with LysPro was dominated by rates of hexamer assembl y. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit o f the LysPro analog. The extraction of Co(II) from both hexameric comp lexes by EDTA chelation is slow at pH 8.0 and highly dependent on liga nd concentration. Cobalt extraction from LysPro was pH dependent. Of t he various phenolic ligands tested, the relative affinities for bindin g to the human and LysPro hexamer are resorcinol > phenol > m-cresol. Conclusions. The extraction data support the formation of an R(6)-type LysPro hexamer under formulation conditions, i.e., in the presence of divalent metal and phenolic ligand, that is similar in nature to that observed in insulin. However, the formation kinetics of LysPro identi fy a radically different monomeric assembly process that may help expl ain the more rapid pharmacokinetics observed with the hexameric formul ation of LysPro insulin relative to human insulin.