Vol. 290, Issue 2, 561-568, August 1999

In Vivo and In Vitro Evidence of Blood-Brain Barrier Transport of a Novel Cationic Arginine-Vasopressin Fragment 4-9 Analog

Shuichi Tanabe, Yasuyuki Shimohigashi, Yasuhisa Nakayama, Takashi Makino, Tsugumi Fujita, Takeru Nose, Gozoh Tsujimoto, Teruo Yokokura, Mikihiko Naito, Takashi Tsuruo and Tetsuya Terasaki

Yakult Central Institute for Microbiological Research, Kunitachi-shi, Tokyo, Japan (S.T., T.M., T.Y.); Laboratory of Structure-Function Biochemistry, Department of Molecular Chemistry, Graduate School of Science, Kyushu University, Fukuoka, Japan (Y.S., T.F., T.N.); Division of Molecular Cell Pharmacology, National Children's Medical Research Center, Setagaya-ku, Tokyo, Japan (Y.N., G.T.); Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan (M.N., T. Tsuruo); and Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (T. Terasaki)

The blood-brain barrier (BBB) transport and metabolism of a novel arginine-vasopressin fragment 4-9 [AVP_4-9, isoelectric point; (pI) = 9.2] analog, that is, cationic AVP_4-9 (C-AVP_4-9, PI = 9.8), were examined in vivo and in vitro. At 45 min after an i.v. administration to mice, the cerebrum-to-plasma concentration ratios of ³⁵S-labeled AVP_4-9 and ¹²⁵I-labeled C-AVP_4-9 were 0.103 and 0.330 ml/g cerebrum, respectively, and the BBB permeation clearances were 1.47 × 10⁴ and 3.10 × 10⁴ ml/min/g cerebrum, respectively. In the in vitro study using mouse brain capillary endothelial cells immortalized by SV40 infection (MBEC4), the acid-resistant binding values of ³⁵S-labeled AVP_4-9 and ¹²⁵I-labeled C-AVP_4-9 to MBEC4 at 120 min were 0.93 and 1.95 µl/mg protein (as the cell/medium ratios), respectively. ³⁵S-labeled AVP_4-9 showed two-phase saturable acid-resistant binding, and its half-saturation constants (K_D) were 3.8 nM (high affinity) and 45.7 µM (low affinity). ¹²⁵I-labeled C-AVP_4-9 showed single-phase saturable acid-resistant binding, with a K_D value of 16.4 µM. The acid-resistant binding of ¹²⁵I-labeled C-AVP_4-9 was significantly dependent on temperature and medium osmolarity. The acid-resistant binding of ¹²⁵I-labeled C-AVP_4-9 was inhibited by dancylcadaverine, phenylarsine oxide (endocytosis inhibitors), 2,4-dinitrophenol (a metabolic inhibitor), and AVP_4-9, poly(L-lysine), and protamine (cationic substances), but not by poly(L-glutamic acid) (an anionic peptide) and the V₁ and V₂ vasopressin receptor antagonists. In addition, the conversion of C-AVP_4-9 to AVP_4-9 in the cerebral homogenate was confirmed by HPLC and mass spectrometry. The present results demonstrate that C-AVP_4-9 is transported through the BBB more effectively than AVP_4-9, via absorptive-mediated endocytosis, and that C-AVP_4-9 is converted to the neuroactive parent peptide, AVP_4-9, in the cerebrum.