Enhancing effect of chemically conjugated deoxycholic acid on permeability of calcitonin in Caco-2 cells

Abstract

One approach for enhancing intestinal absorption of therapeutic peptides is chemical conjugation to bile acids. In this study, recombinant salmon calcitonin (sCT) was chemically modified by covalent attachment of deoxycholic acid (DOCA). Three different sCT-DOCA conjugates, namely, sCTmono-DOCA, sCT-di-DOCA, and sCT-tri-DOCA, were prepared and characterized for their structural and biological properties. The permeability of these conjugates in the gastrointestinal tract was evaluated using Caco-2 cell monolayers to determine their potential as an oral dosage form. The conjugates were isolated by reversed-phase fast protein liquid chromatography (FPLC) and confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Circular dichroism spectra in 50% trifluoroethanol aqueous condition showed the ordered secondary structure of sCT-DOCA. The biological activities of sCT-DOCA conjugates were evaluated by cyclic AMP assay using T-47D cells, and the mean EC 50 values of sCT, sCT-mono-DOCA, and sCT-di-DOCA were 0.034, 0.076, and 0.46 nM, respectively. The transport experiments using the Caco-2 cell monolayer showed that the permeability of sCT-DOCA conjugates in buffer was not altered from the native sCT. However, the permeability of sCT-DOCA conjugates was increased up to 2.5 times that of the native sCT when sCT-DOCA was formulated in 1% dimethylsulfoxide (DMSO) solution. The conjugated DOCA of sCT-DOCA significantly enhanced the absorption of sCT-DOCA in the intestinal membrane when sCT-DOCA conjugates were completely solubilized by DMSO. In conclusion, this study proposes that therapeutic peptides that have poor absorption profiles could potentially be developed into orally active drugs by conjugation with DOCA in the formulation with appropriate solubilizing agents. Drug Dev. Res. 64:129-135, 2005.

Novel Delivery Technologies for Protein and Peptide Therapeutics

“…Conjugation of caproic acid with insulin was reported to increase its hydrophobicity and increased uptake from gut [94]. Deoxycholic acid conjugate recombinant salmon calcitonin and heparin have shown enhanced permeability in CaCo-2 cell monolayers [95,96]. Conjugates of proteins and peptides with polymers or small hydrophobic molecules might impart totally different physico-chemical properties, and poses some advantages over native macromolecule.…”