title: 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 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 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 intercallular 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]Met-enkephalin. 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]Met-enkephalinamide 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.
authors.affiliation: Department of Pharmacy, Swiss Federal Institute of Technology Zurich, Switzerland.
authors.forename: V B
authors.initials: VB
authors.lastname: Lang
authors.forename: P
authors.initials: P
authors.lastname: Langguth
authors.forename: C
authors.initials: C
authors.lastname: Ottiger
authors.forename: H
authors.initials: H
authors.lastname: Wunderli-Allenspach
authors.forename: D
authors.initials: D
authors.lastname: Rognan
authors.forename: B
authors.initials: B
authors.lastname: Rothen-Rutishauser
authors.forename: J C
authors.initials: JC
authors.lastname: Perriard
authors.forename: S
authors.initials: S
authors.lastname: Lang
authors.forename: J
authors.initials: J
authors.lastname: Biber
authors.forename: H P
authors.initials: HP
authors.lastname: Merkle
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D001692:Biological Transport; D018938:Caco-2 Cells; D017097:Electric Impedance; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007408:Intestinal Absorption / Q000502:physiology; D007526:Isoelectric Point; D018613:Microscopy, Confocal; D015394:Molecular Structure; D010539:Permeability; D013329:Structure-Activity Relationship
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004744:Enkephalin, Methionine; C022118:enkephalinamide-Met, Ala(2)-; C013801:enkephalin-Met, Ala(2)-; C028208:metkephamid