Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation

Amino Acids. 2012 Dec;43(6):2431-41. doi: 10.1007/s00726-012-1322-y. Epub 2012 Jun 7.

Abstract

The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH(2)), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H(2)O(2)); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH(2) derived from this study may be developed as a promising candidate for oral administration.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caco-2 Cells
  • Enzyme Stability
  • Hemin / analogs & derivatives*
  • Hemin / chemical synthesis
  • Hemin / chemistry
  • Hemin / metabolism
  • Humans
  • Intestinal Mucosa / chemistry
  • Intestinal Mucosa / metabolism*
  • Methylation
  • Mice
  • Oligopeptides / chemical synthesis
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism*
  • Permeability
  • Peroxidase / chemistry
  • Peroxidase / metabolism*
  • Substrate Specificity

Substances

  • Oligopeptides
  • deuterohemin-alanyl-histidyl-threonyl-valyl-glutamyl-lysine
  • Hemin
  • Peroxidase