STRUCTURE-PERMEATION RELATIONS OF MET-ENKEPHALIN PEPTIDE ANALOGS ON ABSORPTION AND SECRETION MECHANISMS IN CACO-2 MONOLAYERS

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 a re presented using confluent Caco-2 cells as an in vitro permeation mo del. Four model peptides (Met-enkephalin, [D-Ala(2)]Met-enkephalin, [D -Ala(2)]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 x 10(-8) to 9.5 x 10(-8) cm s(-1), and were similar to typical paracellular markers. No correlation of permeability and the log(apparent octanol/buffer par tition coefficient) was observed. A 40-fold increase of the permeabili ty of metkephamid in the presence of 10 mM EDTA suggested a significan t 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 microscop y (CLSM). The fluorophore-labeled peptide was observed in the intercel lular space only. Metkephamid permeabilities were found to be directio n-specific, Permeabilities from basolateral to apical (b-to-a) were si gnificantly higher (ca. 4-fold) than in the opposite (a-to-b) directio n. The addition of verapamil equalized the permeabilities in the a-to- b and b-to-a directions, suggesting the involvement of a P-glycoprotei n-mediated secretion mechanism. Similar observations were obtained wit h [D-Ala(2)]-Met-enkephalinamide but not with Met-enkephalin and [D-Al a(2)]Met-enkephalin. In contrast to the other analogues, metkephamid a nd [D-Ala(2)]Met-enkephalinamide are positively charged at neutral pH, as demonstrated by their isoelectric points (pi 8.6 for [D-Ala(2)]Met -enkephalinamide and metkephamid and 5.3 for [D-Ala(2)]Met-enkephalin and Met-enkephalin). The data is in agreement with the literature show ing that most compounds secreted by the P-glycoprotein transporter car ry a positive charge.