The effect of multiple N-methylation on intestinal permeability of cyclic hexapeptides

Oded Ovadia, Sarit Greenberg, Jayanta Chatterjee, Burkhardt Laufer, Florian Opperer, Horst Kessler*, Chaim Gilon, Amnon Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

121 Scopus citations


Recent progress in peptide synthesis simplified the synthesis of multiple N-methylation of peptides. To evaluate how multiple N-methylation affects the bioavailability of peptides, a poly alanine cyclic hexapeptide library (n = 54), varying in the number of N-methyl (N-Me) groups (1-5 groups) and their position, was synthesized. The peptides were evaluated for their intestinal permeability in vitro using the Caco-2 model. Further evaluation of the transport route of chosen analogues was performed using rat excised viable intestinal tissue, a novel colorimetric liposomal model and the parallel artificial membrane permeability assay (PAMPA). While most members were found to have poor permeability (permeability coefficient, Papp < 1 × 10-6 cm/s, lower than mannitol, the marker for paracellular permeability), 10 analogues were found to have high Caco-2 permeability, (P app > 1 × 10-5 cm/s, similar to testosterone, a marker of transcellular permeability). No correlation was found between the number of N-methylated groups and the enhanced permeability. However, 9/10 permeable peptides in the Caco-2 model included an N-Me placed adjacently to the d-Ala position. While the exact transport route was not fully characterized, the data suggests a facilitated diffusion. It can be concluded that multiple N-methylation of peptides may improve intestinal permeability, and therefore can be utilized in the design of orally available peptide-based therapeutics.

Original languageAmerican English
Pages (from-to)479-487
Number of pages9
JournalMolecular Pharmaceutics
Issue number2
StatePublished - 4 Apr 2011


  • N-methylation
  • cyclization
  • intestinal permeability
  • peptides


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