title: Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin.
abstract: OBJECTIVE
  Basal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements.
  
  
  METHODS
  Self-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection.
  
  
  RESULTS
  With depletion of phenol, insulin degludec and another fatty diacid-insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T(3)R(3) conformation in presence of phenol and zinc, changing to T(6) with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly.
  
  
  CONCLUSIONS
  Insulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.
authors.affiliation: Diabetes Research Unit, Novo Nordisk, Novo Nordisk Park, 2760, Måløv, Denmark. ibjo@novonordisk.com
authors.forename: Ib
authors.initials: I
authors.lastname: Jonassen
authors.forename: Svend
authors.initials: S
authors.lastname: Havelund
authors.forename: Thomas
authors.initials: T
authors.lastname: Hoeg-Jensen
authors.forename: Dorte Bjerre
authors.initials: DB
authors.lastname: Steensgaard
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Wahlund
authors.forename: Ulla
authors.initials: U
authors.lastname: Ribel
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005227:Fatty Acids / Q000737:chemistry; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000378:metabolism; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D007004:Hypoglycemic Agents; D007328:Insulin; D012709:Serum Albumin