title: Transport mechanism of a new behaviorally highly potent adrenocorticotropic hormone (ACTH) analog, ebiratide, through the blood-brain barrier.
abstract: The binding and internalization of a novel adrenocorticotropic hormone (ACTH) analog having a potent neuromodulating effect, ebiratide (H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2), by isolated bovine brain capillaries, were examined. Metabolism of [5-125I-His]ebiratide occurred during a 30-min incubation with bovine brain capillaries at 37 degrees C. In the presence of 20 mM EDTA, added to inhibit this metabolism, the medium, after 30 min of incubation, contained 82.3 +/- 0.5% of the unchanged ebiratide. The total binding and acid-resistant binding of [125I]ebiratide increased with time and reached an equilibrium at about 15 min. The total binding and acid-resistant binding at 30 min (as the cell/medium ratios corrected with [14C]sucrose) were 13.07 +/- 0.86 and 5.00 +/- 0.18 microliters/mg of protein, respectively. The acid-resistant binding showed significant dependence on temperature and medium osmolarity. The [125I]ebiratide binding was significantly inhibited by dansylcadaverine, an endocytosis inhibitor. The saturable acid-resistant binding was obtained by the addition of unlabeled ebiratide (100 nM-5 mM), and the maximal internalization capacity (Bmax) at 30 min was 144.2 pmol/mg of protein, with the half-saturation constant (KD) of 62.1 microM. The nonsaturable acid-resistant binding [cell/medium ratio in the presence of the unlabeled compound (1 mM or more)] was 2.2 microliters/mg of protein. The acid-resistant binding was significantly inhibited by human ACTH, poly-L-lysine, protamine and E-2078, a basic peptide, but was not inhibited by poly-L-glutamate, insulin or transferrin. These results demonstrate that ebiratide is transported through the blood-brain barrier via a basic peptide-specific absorptive-mediated endocytosis.
authors.affiliation: Pharma Research Laboratories, Hoechst Japan Limited, Saitama.
authors.forename: T
authors.initials: T
authors.lastname: Shimura
authors.forename: S
authors.initials: S
authors.lastname: Tabata
authors.forename: T
authors.initials: T
authors.lastname: Ohnishi
authors.forename: T
authors.initials: T
authors.lastname: Terasaki
authors.forename: A
authors.initials: A
authors.lastname: Tsuji
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier; D001921:Brain / Q000098:blood supply / Q000378:metabolism; D002196:Capillaries / Q000378:metabolism; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D004705:Endocytosis; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013696:Temperature
chemical_list: D010446:Peptide Fragments; D011480:Protease Inhibitors; D000324:Adrenocorticotropic Hormone; D004492:Edetic Acid; C057139:ebiratide