Impact of the mucin barrier on the in vitro membrane permeability of cyclic peptides

This study investigated the role of the mucin layer in cyclic peptide permeability using an in vitro assay system. We evaluated the membrane permeability of 18 cyclic peptides and reference compounds using biosimilar mucin and Caco-2/HT29 cells. The permeability of paracellular markers (FD4 and LY) and digoxin (a low molecular weight compound) remained unaffected by mucin presence when using biosimilar mucin. Conversely, cyclosporin A, a commercially available cyclic peptide drug, showed reduced permeability. In Caco-2/HT29 co-cultured cells compared with Caco-2 cells only, most compounds exhibited increased permeability due to enhanced interstitial permeability, except for cyclosporin A, which showed decreased permeability in the presence of mucin. Similarly, the permeability of LUNA18, a cyclic peptide under development, was reduced by 54.1 and 26.0 % in both Caco-2/HT29 co-cultured and HT29 cells. Furthermore, 12 of 18 cyclic peptides exhibited ≥ 2-fold decrease in membrane permeability in Caco-2/HT29 co-cultured cells. These findings suggest that current permeability evaluation methods, which do not account for the mucin layer, may overestimate the permeability of highly lipophilic cyclic peptides. The mucin layer may serve as a rate-limiting factor in cyclic peptide membrane permeability, highlighting the importance of including mucin layer permeability in cyclic peptide evaluation protocols.