caa a22 4500 477125581 CHVBK 20180405111648.0 cr unu---uuuuu 170330e19970101xx s 000 0 eng 10.1023/A:1012095115151 doi (NATIONALLICENCE)springer-10.1023/A:1012095115151 Assembly and Dissociation of Human Insulin and LysB28ProB29-Insulin Hexamers: A Comparison Study [Elektronische Daten] [Duane Birnbaum, Maureen Kilcomons, Michael DeFelippis, John Beals] Purpose. 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. Plenum Publishing Corporation, 1997 LysB28ProB29-human insulin nationallicence insulin nationallicence cobalt insulin hexamers nationallicence hexameric association nationallicence hexameric dissociation nationallicence Birnbaum Duane Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, 46285, Indianapolis, Indiana aut Kilcomons Maureen Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, 46285, Indianapolis, Indiana aut DeFelippis Michael Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, 46285, Indianapolis, Indiana aut Beals John Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Drop Code 3848, 46285, Indianapolis, Indiana aut Pharmaceutical Research Kluwer Academic Publishers-Plenum Publishers 14/1(1997-01-01), 25-36 0724-8741 14:1<25 1997 14 11095 https://doi.org/10.1023/A:1012095115151 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1012095115151 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Birnbaum Duane Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, 46285, Indianapolis, Indiana aut NATIONALLICENCE 700 1- Kilcomons Maureen Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, 46285, Indianapolis, Indiana aut NATIONALLICENCE 700 1- DeFelippis Michael Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, 46285, Indianapolis, Indiana aut NATIONALLICENCE 700 1- Beals John Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Drop Code 3848, 46285, Indianapolis, Indiana aut NATIONALLICENCE 773 0- Pharmaceutical Research Kluwer Academic Publishers-Plenum Publishers 14/1(1997-01-01), 25-36 0724-8741 14:1<25 1997 14 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer