Structure-Permeation Relations of Met-enkephalin Peptide Analogues on Absorption and Secretion Mechanisms in Caco-2 Monolayers

Abstract

Due to the low effective permeabilities of peptides at many absorption sites, their structure-permeation relations are of high interest. In this work structure-permeation relations of Met-enkephalin analogues are presented using confluent Caco-2 cells as an in vitro permeation model. Four model peptides (Met-enkephalin, [D-Ala²]Met-enkephalin, [D-Ala²]Met-enkephalinamide, and metkephamid) were tested in terms of permeability, lipophilicity, charge, and molecular size. Permeability coefficients (P_eff) across Caco-2 cells were low, 3.3×10^-8 to 9.5×10^-8 cm s^-1, and were similar to typical paracellular markers. No correlation of permeability and the log(apparent octanol/buffer partition coefficient) was observed. A 40-fold increase of the permeability of metkephamid in the presence of 10mM EDTA suggested a significant contribution of paracellular transport. Independent support for this conclusion was obtained by visualizing the pathway of the fluorescein isocyanate isomer I 1–metkephamid by confocal laser scanning microscopy (CLSM). The fluorophore-labeled peptide was observed in the intercellular space only. Metkephamid permeabilities were found to be direction-specific. Permeabilities from basolateral to apical (b-to-a) were significantly higher (ca. 4-fold) than in the opposite (a-to-b) direction. The addition of verapamil equalized the permeabilities in the a-to-b and b-to-a directions, suggesting the involvement of a P-glycoprotein-mediated secretion mechanism. Similar observations were obtained with [D-Ala²]- Met-enkephalinamide, but not with Met-enkephalin and [D-Ala²]Metenkephalin. In contrast to the other analogues, metkephamid and [D-Ala²]Met-enkephalinamide are positively charged at neutral pH, as demonstrated by their isoelectric points (pl=8.6 for [D-Ala²]Metenkephalinamide and metkephamid and 5.3 for [D-Ala²]Met-enkephalin and Met-enkephalin). The data is in agreement with the literature showing that most compounds secreted by the P-glycoprotein transporter carry a positive charge.