Assembly and dissociation of human insulin and LysB28ProB29-insulin hexamers: a comparison study.
OBJECTIVE
Investigations into the kinetic assembly and dissociation of hexameric LysB28ProB29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions 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 insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R6 hexamer disruption were studied by extraction of Co(II) with EDTA.


RESULTS
Phenolic ligand binding to human insulin yielded rate constants for a fast and slow phase that increased with increasing ligand concentration and are attributed to the T6 --> T3R3 and T3R3 --> R6 transitions, respectively. However, the kinetics of phenolic ligand binding with LysPro was dominated by rates of hexamer assembly. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit of the LysPro analog. The extraction of Co(II) from both hexameric complexes by EDTA chelation is slow at pH 8.0 and highly dependent on ligand concentration. Cobalt extraction from LysPro was pH dependent. Of the various phenolic ligands tested, the relative affinities for binding to the human and LysPro hexamer are resorcinol > phenol > m-cresol.


CONCLUSIONS
The extraction data support the formation of an R6-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 identify a radically different monomeric assembly process that may help explain the more rapid pharmacokinetics observed with the hexameric formulation of LysPro insulin relative to human insulin.
Birnbaum
D T
DT
Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Kilcomons
M A
MA
DeFelippis
M R
MR
Beals
J M
JM
D003035:Cobalt / Q000737:chemistry; D004492:Edetic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D061268:Insulin Lispro; D008024:Ligands; D046911:Macromolecular Substances; D010636:Phenols / Q000737:chemistry
D007328:Insulin; D061268:Insulin Lispro; D008024:Ligands; D046911:Macromolecular Substances; D010636:Phenols; D003035:Cobalt; D004492:Edetic Acid