Structure-Internalization Relationship for Adsorptive-Mediated Endocytosis of Basic Peptides at the Blood-Brain Barrier1

  1. Ikumi Tamai1,
  2. Yoshimichi Sai1,
  3. Hiroyuki Kobayashi1,
  4. Makoto Kamata2,
  5. Tateaki Wakamiya2,2 and
  6. Akira Tsuji1
  1. 1Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, 13–1 Takara-machi, Kanazawa 920 (I.T., Y.S., H.K., A.T.) and 2Department of Chemistry, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan (M.K., T.W.)

    Abstract

    For the purpose of the brain delivery of peptides, the structural specificity of adsorptive-mediated endocytosis at the blood-brain barrier was studied by measuring transport of a newly synthesized basic peptide 001-C8, H-MeTyr-Arg-MeArg-d-Leu-NH(CH2)8NH2, using primary cultured bovine brain capillary endothelial cells. The apparent uptake of [125I]001-C8 increased time-dependently and reached a steady-state at 60 min. The steady-state uptake of [125I]001-C8 was temperature and concentration dependent and was significantly decreased in the presence of dansylcadaverine, protamine or poly-l-lysine. Uptakes of peptides modified by 1,8-octanediamine, 1,5-pentanediamine, 1,2-ethanediamine or ethylamide and peptides with a free carboxyl terminal were significantly higher than, and similar to, that of [3H]PEG900, respectively. The half-saturation constants and the maximal uptake capacities of these peptides were in the ranges of 0.2 to 134 μM and 1.1 to 408 pmol/mg protein, respectively. These values were correlated with the basicity of the molecules. In conclusion, not the number of constituent amino acids of peptides, but rather the C-terminal structure and the basicity of the molecules are the most important determinants for the uptake by the adsorptive-mediated endocytosis system at the blood brain barrier.

    Footnotes

    • Send reprint requests to: Prof. Akira Tsuji, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, 13–1 Takaramachi, Kanazawa 920, Japan.

    • ↵1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan and by a grant from the Japan Health Sciences Foundation, Drug Innovation Project.

    • ↵2 Current address: Faculty of Science and Technology, Kinki University, Higashi-osaka, Osaka 577, Japan.

    • Abbreviations:
      AME
      adsorptive-mediated endocytosis
      BBB
      blood-brain barrier
      BCEC
      brain capillary endothelial cells
      PEG
      polyethylene glycol
      Kd
      half-saturation constant
      HPLC
      high-performance liquid chromatography
      • Received April 22, 1996.
      • Accepted September 3, 1996.
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    1. JPET January 1, 1997 vol. 280 no. 1 410-415
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