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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

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Abstract

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 nM–1 mM). 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.

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Authors and Affiliations

  1. Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, 920, Japan

    Tetsuya Terasaki, Susumu Takakuwa, Akira Saheki & Akira Tsuji

  2. First Department of Nursing, Fukui Prefectural College, Obakakecho, Fukui, 910, Japan

    Shuzo Moritani

  3. Pharma Research Laboratories, Hoechst Japan Limited, Kawagoe Saitama, 350, Japan

    Takesada Shimura & Shigeru Tabata

Authors
  1. Tetsuya Terasaki
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  2. Susumu Takakuwa
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  4. Shuzo Moritani
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  5. Takesada Shimura
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  6. Shigeru Tabata
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  7. Akira Tsuji
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Terasaki, T., Takakuwa, S., Saheki, A. et al. 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. Pharm Res 9, 529–534 (1992). https://doi.org/10.1023/A:1015848531603

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