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-Ala2Met-enkephalin, D-Ala2Met-enkephalinamide, and metkephamid) were tested in terms of permeability, lipophilicity, charge, and molecular size. Permeability coefficients (Peff) 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-Ala2Metenkephalin. In contrast to the other analogues, metkephamid and D-Ala2Met-enkephalinamide are positively charged at neutral pH, as demonstrated by their isoelectric points (pl=8.6 for D-Ala2Metenkephalinamide and metkephamid and 5.3 for D-Ala2Met-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.
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‐Ala2Met‐enkephalin, D‐Ala2Met‐enkephalinamide, and metkephamid) were tested in terms of permeability, lipophilicity, charge, and molecular size. Permeability coefficients (Peff) 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 10 mM 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‐Ala2Met‐enkephalin. In contrast to the other analogues, metkephamid and D‐Ala2Met‐enkephalinamide are positively charged at neutral pH, as demonstrated by their isoelectric points (pI = 8.6 for D‐Ala2Met‐enkephalinamide and metkephamid and 5.3 for D‐Ala2Met‐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.
