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

The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [¹²⁵I]ebiratide (H-Met(O₂)-Glu[¹²⁵I]His-Phe-D-Lys-Phe-NH(CH₂)₈NH₂), was examined in cultured mono-layers 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 n M–1 m M). The maximal internalization capacity (B_max) at 30 min was 7.96 ± 3.27 µmol/mg of protein with a half-saturation constant (K_d) of 15.9 ± 6.4 µM. 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.