title: Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells.
abstract: The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [125I]ebiratide (H-Met(O2)-Glu[125I]His-Phe-D-Lys-Phe-NH(CH2)2NH2), was examined in cultured monolayers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [125I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [125I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [125I]ebiratide. The acid-resistant binding of [125I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM-1 mM). The maximal internalization capacity (Bmax) at 30 min was 7.96 +/- 3.27 pmol/mg of protein with a half-saturation constant (Kd) of 15.9 +/- 6.4 microM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.
authors.affiliation: Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Terasaki
authors.forename: S
authors.initials: S
authors.lastname: Takakuwa
authors.forename: A
authors.initials: A
authors.lastname: Saheki
authors.forename: S
authors.initials: S
authors.lastname: Moritani
authors.forename: T
authors.initials: T
authors.lastname: Shimura
authors.forename: S
authors.initials: S
authors.lastname: Tabata
authors.forename: A
authors.initials: A
authors.lastname: Tsuji
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D007457:Iodine Radioisotopes; D010446:Peptide Fragments / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D000324:Adrenocorticotropic Hormone; C057139:ebiratide