0022-3565/99/2902-0561$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 290:561–568, 1999
Vol. 290, No. 2
Printed in U.S.A.
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)
This paper is available online at http://www.jpet.org
ABSTRACT
The blood-brain barrier (BBB) transport and metabolism of a
novel arginine-vasopressin fragment 4-9 [AVP4-9, isoelectric
point; (pI) 5 9.2] analog, that is, cationic AVP4-9 (C-AVP4-9, PI 5
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 AVP4-9 and 125I-labeled C-AVP4-9 were
0.103 and 0.330 ml/g cerebrum, respectively, and the BBB
permeation clearances were 1.47 3 1024 and 3.10 3 1024
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 35Slabeled AVP4-9 and 125I-labeled C-AVP4-9 to MBEC4 at 120 min
were 0.93 and 1.95 ml/mg protein (as the cell/medium ratios),
respectively. 35S-labeled AVP4-9 showed two-phase saturable
acid-resistant binding, and its half-saturation constants (KD)
were 3.8 nM (high affinity) and 45.7 mM (low affinity). 125I-
Arginine-vasopressin (AVP) fragment 4-9 [AVP4-9, isoelectric point; (pI) 5 9.2; Fig. 1] is a stable major metabolite of
AVP in the central nervous system (CNS; Burbach et al.,
1983, 1993). It is well documented that AVP4-9 exerts a more
potent memory-facilitative effect than AVP (Gaffori and De
Weid, 1986; De Weid et al., 1987).
Previous studies indicated that a novel cationic AVP4-9
analog (C-AVP4-9, PI 5 9.8, Fig. 1) possesses more potent
memory-facilitative activity than its parent peptide, AVP4-9
(Tanabe et al., 1997). C-AVP4-9 was designed to be converted
to the neuroactive parent peptide, AVP4-9, by the postproline
cleaving enzyme (PPCE), which was previously demonReceived for publication January 19, 1999.
labeled C-AVP4-9 showed single-phase saturable acid-resistant
binding, with a KD value of 16.4 mM. The acid-resistant binding
of 125I-labeled C-AVP4-9 was significantly dependent on temperature and medium osmolarity. The acid-resistant binding of
125
I-labeled C-AVP4-9 was inhibited by dancylcadaverine, phenylarsine oxide (endocytosis inhibitors), 2,4-dinitrophenol (a
metabolic inhibitor), and AVP4-9, 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-AVP4-9 to AVP4-9 in the
cerebral homogenate was confirmed by HPLC and mass spectrometry. The present results demonstrate that C-AVP4-9 is
transported through the BBB more effectively than AVP4-9, via
absorptive-mediated endocytosis, and that C-AVP4-9 is converted to the neuroactive parent peptide, AVP4-9, in the cerebrum.
strated to be abundant in the CNS and scarce in the plasma
(Yoshimoto et al., 1979).
To act on the CNS, drugs and neuropeptides should be
transported through the blood-brain barrier (BBB), which
consists of brain capillary endothelial cells possessing tight
intercellular junctions. With regard to BBB transport of peptides, previous studies have reported the receptor-mediated
endocytosis (RME) of peptides, such as insulin (Duffy and
Pardridge, 1987), anti-insulin receptor antibody (Pardridge
et al., 1995; Wu et al., 1998), transferrin (Fishman et al.,
1987; Skarlatos et al., 1995), antitransferrin receptor antibody (Pardridge et al., 1991; Shin et al., 1995; Walus et al.,
1996), and the absorptive-mediated endocytosis (AME) of
cationic peptides, such as E-2078 (Terasaki et al., 1989),
ABBREVIATIONS: AME, absorptive-mediated endocytosis; AVP, arginine-vasopressin; AVP4-9, AVP fragment 4-9; C-AVP4-9, cationic AVP4-9
analog; BBB, blood-brain barrier; AUC, area under the curve; CNS, central nervous system; ESI-LC-MS, electrospray ionization-liquid chromatography-mass spectrometry; MBEC4, mouse brain capillary endothelial cells immortalized by SV40 infection; pI, isoelectric point; PPCE,
postproline cleaving enzyme; RME, receptor-mediated endocytosis; TCA, trichloroacetic acid; ZPP, N-benzyloxycarbonyl-prolyl-prolinal; cell/
medium ratios, cell-to-medium concentration ratios.
561
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Accepted for publication March 29, 1999
562
Tanabe et al.
Fig. 1. The structures of AVP4-9, C-AVP4-9, and B chain. *The 35S-labeled
position. **The 125I-labeled position. The structure of the B chain is
enclosed in a solid line.
Materials and Methods
Radiolabeling of Peptides. AVP 4-9 ([pGlu 4 ,Cyt 6 ,Arg 8 ]vasopressin fragment 4-9; Sigma Chemical Co., St. Louis, MO) was
labeled with 35S. As an intermediate of 35S-labeled AVP4-9, 3-nitro2-pyridinesulfenyl-AVP4-9 was synthesized by a previously demonstrated method (Shimohigashi et al., 1992). [35S]Cysteine (Amersham Pharmacia Biotech, Buckinghamshire, UK) was used
immediately after purification to remove dithiothreitol by HPLC (see
below). 3-nitro-2-pyridinesulfenyl-AVP4-9, 16.2 ml (1 mg/ml) was reacted with 74 MBq of [35S]cysteine in 500 ml of distilled water at
room temperature for 24 h. C-AVP4-9 (American Peptide Company,
Sunnyvale, CA) was labeled with 125I by the chloramine T method
(Hunter and Greenwood, 1962; Tanabe et al., 1997). The reaction
mixtures of 35S-labeled AVP4-9 and 125I-labeled C-AVP4-9 were purified by HPLC as described below. The 35S-labeled AVP4-9 and 125Ilabeled C-AVP4-9 obtained had specific activities of 22.6 to 46.0
TBq/mmol (purity .94.4%) and 48.3 to 138.5 TBq/mmol (purity
.99.0%), respectively.
HPLC Conditions. To quantify the unchanged 35S-labeled
AVP4-9 and 125I-labeled C-AVP4-9 in in vivo and in vitro uptake
studies, HPLC was used. For the separation, the column J’sphere
ODS-H80 (YMC Inc., Wilmington, NC) was used. In the studies
using radiolabeled peptides, elution was performed at a flow rate of
1.5 ml/min with the following linear gradient: solvent A 5 0.1%
trifluoroacetic acid; solvent B 5 acetonitrile; B in A (v/v): from 0 to
20% for 20 min for separation, from 20 to 40% for 2 min, holding 40%
for 5 min to wash the column, and from 40% to 0% for 3 min with
equilibration for 15 min before subsequent injection. The HPLC
eluates were collected automatically (0.75 ml/fraction). In the studies
using only 125I-labeled peptides, the radioactivity in each eluate was
counted using a gamma counter (Autogamma 5550; Packard Instrument Co., Meriden, CT). In the studies using dual nuclides (35S and
3
H, or 125I and 3H) or only 35S, the radioactivity was counted using a
liquid scintillation counter (LSC-5000; Beckman Coulter Inc., Fullerton, CA). In the studies using unlabeled peptides, the HPLC
elution was performed with the following linear gradient: solvent B
in A (v/v): from 0 to 5% for 15 min, from 5 to 15% for 10 min, from 15
to 40% for 5 min, holding 40% for 5 min, and from 40% to 0% for 5
min with equilibration for 10 min. The eluate was monitored by UV
absorbance at 210 nm.
In Vivo Pharmacokinetic Studies. 35S-labeled AVP4-9 (130
KBq), 125I-labeled C-AVP4-9 (1110 KBq), or 370 KBq of 125I-labeled
BSA (144 KBq/mg; NEN Life Sciences, Boston, MA) was administered i.v. into the tail vein of normal male ICR mice (6 – 8 weeks old;
CLEA Japan, Tokyo). At 1, 5, 15, 30, and 45 min after the administration, mice were sacrificed, and the plasma, cerebrum, liver, kidney, heart, and lung were obtained. The unchanged 35S-labeled
AVP4-9 and 125I-labeled C-AVP4-9 in the plasma and tissues were
quantified as follows. Ice-cold 2% phosphoric acid (33 volume for
plasma, 43 volume for cerebrum, and 103 volume for other tissues)
was added, and the mixtures were homogenized with an Ultraturrax (IKA Labortechnik, Staufen, Germany). The homogenates
were then centrifuged (14,000g, 4°C, 30 min), and 500-ml aliquots of
the supernatants were analyzed by HPLC. In the 125I-labeled BSA
studies, plasma and tissues were homogenized with a 53 volume of
30% ice-cold trichloroacetic acid (TCA). After centrifugation
(14,000g, 4°C, 30 min), the radioactivity in each pellet was measured
as unchanged 125I-labeled BSA by a gamma counter. The plasma
concentrations [Cp(t)] of unchanged labeled peptides were normalized as a percentage of the injected dose per milliliter. The Cp(t) data
were fitted to the function (1) by a nonlinear least-squares regression
analysis using a computer program, MULTI (Yamaoka et al., 1980):
C p~ t ! 5 Ae
2a t
1 Be
2b t
(1)
where A and B are the intercepts, and a and b are the rate constants
describing the fast and slow compartments of clearance from plasma,
respectively. As an index of the distribution of unchanged labeled
peptides to the tissues, the apparent tissue-to-plasma concentration
ratio (Kp,app), defined by the radioactivity of unchanged radiolabeled
peptides per gram of tissue divided by that per milliliter of plasma,
was calculated (Terasaki et al., 1984). The BBB permeation clearances (PS/Vbr) of labeled peptides were calculated using the function
(2):
C br~ t ! /C p~ t ! 5 PS/V br 3 AUC~ 0 2 t ! /C p~ t ! 1 V 0 /V br
(2)
where V0, Vbr, and Cbr(t) are the rapidly equilibrated distribution
volume of labeled peptides, the cerebrum weight, and the cerebral
concentration of labeled peptides, respectively. PS/Vbr values of labeled peptides were obtained from the initial slopes of Cbr(t)/Cp(t)
[Kp,app versus the area under the curve [AUC(0 2 t)] Cp(t) as an
integration plot (Smith et al., 1987; Kakee et al., 1996; Yang et al.,
1997)].
Capillary Depletion Study. The left and right renal artery and
vein of mice were ligated under ether anesthesia before use. 125Ilabeled BSA (370 KBq), 35S-labeled AVP4-9 (130 KBq), or 125I-labeled
C-AVP4-9 (1110 KBq) was i.v. administered into the tail vein of mice.
At 30 min after the administration, plasma and cerebrum were
obtained. The cerebra were separated into the parenchyma and the
capillary fractions by a method reported previously (Yang et al.,
1997). The unchanged 35S-labeled AVP4-9 and 125I-labeled C-AVP4-9
in the parenchyma fraction were extracted two times with a 1/103
volume of ice-cold 50% phosphoric acid and a 103 volume of ice-cold
acetonitrile. After centrifugation (3500g, 4°C, 1 h), the supernatants
were lyophilized and suspended in 1 ml of ice-cold methanol. After
further centrifugation (3500g, 4°C, 1 h), the supernatants were again
lyophilized and reconstituted with 600 ml of 0.1% trifluoroacetic acid,
and 500-ml aliquots were analyzed by HPLC. The unchanged 35Slabeled AVP4-9 and 125I-labeled C-AVP4-9 in the capillary fraction
were extracted with 600 ml of 5% phosphoric acid, and 500-ml aliquots were analyzed by HPLC. The unchanged 125I-labeled BSA in
the parenchyma and the capillary fractions were precipitated by
ice-cold 30% TCA (53 volume for the parenchyma fraction and 500 ml
for the capillary fraction), and the radioactivity in each pellet was
measured with a gamma counter.
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ebiratide (Shimura et al., 1991), histone (Pardridge et al.,
1989), and cationized albumin (Pardridge et al., 1990). In
addition, these reports demonstrated that the AME-transported peptides show a far higher maximal internalization
capacity into the brain capillary than the RME-transported
peptides.
Accordingly, it is of great interest to investigate the BBB
transport mechanism of C-AVP4-9, because C-AVP4-9 is a
cationic peptide at physiological pH.
The purpose of the present study is to evaluate the BBB
transport of C-AVP4-9 based on pharmacokinetic analysis
and clarify the BBB transport mechanism of C-AVP4-9. We
also demonstrated the conversion of C-AVP4-9 to AVP4-9 by
PPCE in the CNS.
Vol. 290
1999
BBB Transport of Cationic AVP4-9 Analog
Cell/medium ratio 5 @~
3
3
125
I or
35
S-R 2
125
3 H-R/ H-S ! /mg MBEC4 protein]/[
I or
125
I or
35
S-S
35
(3)
S-M/ m l of medium#
where 3H, 35S, and 125I are the radioactivities of unchanged [3H]inulin, 35S-labeled AVP4-9, and 125I-labeled C-AVP4-9, respectively; and
-R, -S, and -M are the acid-resistant, the acid-soluble, and the unbound fractions, respectively. The maximal internalization capacity
(Bmax) and the half-saturation constant (KD) were estimated by
MULTI (Yamaoka et al., 1980). The cell/medium ratios were statistically analyzed using the Kruskal-Wallis test and Dunnett’s test.
Enzymatic Conversion of C-AVP4-9 to AVP4-9 by Mouse Cerebral Homogenate. The mouse cerebra were homogenized with a
43 volume of metabolism assay buffer (the incubation buffer without
BSA and Complete). The PPCE activity in the mouse cerebral homogenate was determined by a method reported previously (Yoshimoto et al., 1979). After preincubation at 37°C for 10 min, C-AVP4-9
was incubated with PPCE derived from Flavobacterium meningosepticum (authentic PPCE, 0.1 U/ml; Seikagaku Corp., Tokyo, Japan) or
the mouse cerebral homogenate (20 mg cerebrum/ml, equivalent to
1.5 3 1023 U/ml) in 250 ml of metabolic assay buffer in the presence
or absence of the PPCE-specific inhibitor N-benzyloxycarbonylprolyl-prolinal (ZPP, 1 mM; Yakult Central Institute for Microbiological Research, Tokyo, Japan) at 37°C for designated times. The
metabolic reaction was stopped by adding 250 ml of 2% phosphoric
acid. After centrifugation (14,000g, 4°C, 10 min), 400-ml aliquots of
supernatants were analyzed by electrospray ionization-liquid chromatography-mass spectrometry (ESI-LC-MS) using a Finnigan TSQ
7000 triple quadrupole mass spectrometer with ESI-LC-MS interface (Thermo Quest Corp., San Jose, CA). The operating conditions
were as follows: spray voltage, 4.5 kV; sheath gas pressure, 70 psi
(nitrogen); auxiliary gas flow, 5 arbitrary units; heated capillary
temperature, 200°C. Mass spectra were acquired every 3 s with a
scanning m/z range of 50 to 1500. The other conditions were as
described for HPLC conditions. The Michaelis constant (Km) and
maximum velocity (Vmax) were also estimated by MULTI (Yamaoka
et al., 1980). The intrinsic clearance of the conversion of C-AVP4-9 to
AVP4-9 (CLint) was defined as the Vmax value divided by the Km value.
Results
In Vivo Pharmacokinetic Studies. The plasma concentration of i.v. administered 125I-labeled BSA was monoexponentially fitted (Table 1, Fig. 2). In contrast, the plasma
concentrations of i.v. administered 35S-labeled AVP4-9 and
125
I-labeled C-AVP4-9 were characterized by a biexponential
function and reached apparent steady states at 15 min after
administration (Table 1, Fig. 2). The Kp,app values of both
35
S-labeled AVP4-9 and 125I-labeled C-AVP4-9 were the highest in the kidney among the tissues examined (Figs. 3 and 4).
The Kp,app values of 125I-labeled C-AVP4-9 in the liver and
the lung were decreased compared with those of 35S-labeled
AVP4-9 (Fig. 3), whereas the Kp,app values of 125I-labeled
C-AVP4-9 in the cerebrum were increased compared with
those of 35S-labeled AVP4-9 (Fig. 4). At 45 min after the
administration, Kp,app values of 35S-labeled AVP4-9 and 125Ilabeled C-AVP4-9 in the cerebrum were 0.103 6 0.019 and
0.330 6 0.030 ml/g cerebrum, respectively (Fig. 4). The Kp,app
values of 125I-labeled BSA in the cerebrum were maintained
TABLE 1
Plasma pharmacokinetic parameters of
AVP4-9, and 125I-labeled C-AVP4-9
Parameter
A (%/ml)
a (min21)
T1⁄2a (min)
B (%/ml)
b (min21)
T1⁄2b (min)
125
125
I-labeled BSA,
35
I-labeled BSA
57.2 6 1.0
0.020 6 0.002
35.2 6 0.6
S-labeled
AVP4-9
8.2 6 0.4
0.67 6 0.18
1.03 6 0.05
0.31 6 0.22
0.06 6 0.02
11.7 6 4.3
35
S-labeled
125
I-labeled
C-AVP4-9
4.9 6 1.0
0.48 6 0.05
1.42 6 0.28
0.06 6 0.02
0.06 6 0.01
12.3 6 2.3
125
I-labeled BSA (37 pmol/mouse), 35S-labeled AVP4-9 (2.8 pmol/mouse), or 125Ilabeled C-AVP4-9 (23.0 pmol/mouse) was administered intravenously into the mice.
The plasma concentration profile of 125I-labeled BSA was analyzed by a monoexponential function, whereas 35S-labeled AVP4-9 and 125I-labeled C-AVP4-9 data were
analyzed by a biexponential function. Each value represents the mean 6 S.E. of
three or four mice.
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In Vitro Internalization Studies Using MBEC4. MBEC4 cells
(Tatsuta et al., 1992) were used. Monolayers of MBEC4 cells were
maintained in Dulbecco’s modified Eagle’s medium containing 10%
FBS in 5% CO2/95% air at 37°C. The internalization of 35S-labeled
AVP4-9 and 125I-labeled C-AVP4-9 into MBEC4 was examined by a
method reported previously (Terasaki et al., 1992) with minor modifications. Briefly, monolayers of MBEC4 cells cultured in 24-well
dishes were washed five times with 1 ml of the incubation buffer [122
mM NaCl, 3 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 0.4 mM
K2HPO4, 1.4 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, 0.1%
BSA, 1 tablet/50 ml Complete (a protease inhibitor cocktail; Boehringer Mannheim, Mannheim, Germany), pH 7.4, 300 mOsM] at
37°C. They were then preincubated in 200 ml of incubation buffer at
37°C for 30 min. In the uptake studies, except for the studies of
concentration dependencies, the uptakes were initiated by adding
200 ml of incubation buffer containing 35S-labeled AVP4-9 (18.5 KBq)
or 125I-labeled C-AVP4-9 (18.5 KBq), and 92.5 KBq of [3H]inulin (13.5
GBq/mmol, NEN Life Sciences) to MBEC4. [3H]Inulin was used as
the extracellular space marker. The effects of medium osmolarity on
the uptakes of 35S-labeled AVP4-9 and 125I-labeled C-AVP4-9 were
examined with hypertonic buffer (incubation buffer with 1.2 M sucrose, 1600 mOsM) substituted for the incubation buffer. The effects
of endocytosis inhibitors, metabolic inhibitor, and several peptides on
the uptake of 125I-labeled C-AVP4-9 were examined in the presence of
0.5 mM danylcadaverine (Sigma), 0.1 mM phenylarsine oxide (Aldrich
Chemical Co., Milwaukee, WI), 1 mM 2,4-dinitrophenol (Wako Pure
Chemical Industries, Osaka, Japan), 0.5 mM V1 vasopressin receptor
antagonist ([(b-mercapto-b,b-cyclopentamethylene propionic acid)1,Omethyl-Tyr2,Arg8]vasopressin; Peninsula Laboratories, Belmont, CA),
0.5 mM V2 vasopressin receptor antagonist ([adamantaneacetyl1,Oethyl-D-Tyr2,Val4,aminobutyryl6,Arg8,9]-vasopressin; Sigma), 0.5 mM
AVP4-9, 0.3 mM poly(L-lysine) (Sigma), 0.3 mM protamine (Wako Pure
Chemical Industries), or 0.3 mM poly(L-glutamic acid) (Sigma). In the
studies of the concentration dependencies, various concentrations of
mixtures of 35S-labeled AVP4-9 and unlabeled AVP4-9 or 125I-labeled
C-AVP4-9 and unlabeled C-AVP4-9 were added with [3H]inulin (92.5
KBq) to MBEC4 after preincubation. At designated times after incubation, the incubation supernatants (unbound fraction) were recovered,
and the cells were washed seven times with 1 ml of ice-cold incubation
buffer. An acid wash technique was then used to remove the labeled
peptide binding to the cell surface and to evaluate the amounts of
labeled peptides internalized into MBEC4. The cells were incubated
with 1 ml of ice-cold acetate-barbital buffer (28 mM CH3COONa, 120
mM NaCl, 20 mM barbital, pH 3.0, 320 mOsM) for 10 min. The buffer
was then recovered (acid-soluble fraction), and the cells were subsequently washed three additional times with 1 ml of acetate-barbital
buffer. The cells remaining were obtained as the acid-resistant fraction
and solubilized with 50 ml of NaOH (0.5 N) and 200 ml of 2% Triton
X-100. Aliquots of unbound (100 ml), acid-soluble (500 ml), and solubilized acid-resistant (200 ml) fractions were analyzed by HPLC. The
protein assay of the acid-resistant fraction was carried out with BCA
Protein Assay Reagent (Pierce, Rockford, IL). In accord with Terasaki et
al. (1989, 1992), the data on the acid-resistant binding are expressed as
the cell-to-medium concentration ratios (cell/medium ratios), corrected
for an extracellular space using [3H]inulin, as follows:
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Tanabe et al.
Vol. 290
at 0.01 ml/g cerebrum (Fig. 4). By the integration plots, the
BBB permeation clearances of 125I-labeled BSA, 35S-labeled
AVP4-9, and 125I-labeled C-AVP4-9 were estimated to be
6.26 3 1026 6 1.26 3 1025, 1.47 6 0.09 3 1024, and 3.10 6
0.35 3 1024 ml/min/g cerebrum, respectively (Fig. 5).
Capillary Depletion Study. At 30 min after the i.v. administration of 125I-labeled BSA, 35S-labeled AVP4-9, and
125
I-labeled C-AVP4-9 to the mice whose renal vessels were
ligated, the distributions of labeled peptides in the capillary
and the parenchyma fractions of cerebrum were examined.
The plasma concentrations of 125I-labeled BSA, 35S-labeled
AVP4-9, and 125I-labeled C-AVP4-9 in mice whose renal vessels were ligated were 0.9, 10, and 3 times greater than those
in the normal mice, respectively (Fig. 2). The unchanged 35Slabeled AVP4-9and 125I-labeled C-AVP4-9 in the parenchyma
fraction, quantified by HPLC, were 10.4 6 1.2% and 0.63 6
0.06% of total recovered radioactivities, respectively. As
shown in Table 2, the Kp,app value of 125I-labeled BSA in the
parenchyma fraction was 1.04 6 0.13 3 1022 ml/g cerebrum.
The Kp, app values of 125I-labeled BSA, 35S-labeled AVP4-9,
and 125I-labeled C-AVP4-9 in the parenchyma fraction were
35, 68, and 22 times higher than those in the capillary fraction, respectively.
Time Courses of Acid-Resistant Binding of 35S-Labeled
AVP4-9 and 125I-Labeled C-AVP4-9 to MBEC4. Unchanged
35
S-labeled AVP4-9 and 125I-labeled C-AVP4-9 in the acidresistant fraction at 60 min, quantified by HPLC, were
6.48 6 0.32 and 68.7 6 5.6% of total recovered radioactivities
respectively. The cell/medium ratios of [3H]inulin coexisting
with 35S-labeled AVP4-9 (Fig. 6A) or 125I-labeled C-AVP4-9
(Fig. 6B) were increased in a slightly time-dependent manner. The acid-resistant binding of 35S-labeled AVP4-9 increased with time and reached apparent equilibrium at 30
Fig. 3. Apparent tissue-to-plasma concentration ratios of 125I-labeled
BSA, 35S-labeled AVP4-9, and 125I-labeled C-AVP4-9 after the i.v. administration. 125I-labeled BSA (37 pmol/mouse), 35S-labeled AVP4-9 (2.8 pmol/
mouse), or 125I-labeled C-AVP4-9 (23.0 pmol/mouse) was administered i.v.
into the normal mice. The unchanged 125I-labeled BSA (F), 35S-labeled
AVP4-9 (‚), and 125I-labeled C-AVP4-9 (E) in the liver (A), kidney (B),
heart (C), and lung (D) were analyzed by HPLC or the TCA precipitation
method. The tissue distributions are expressed as the apparent tissueto-plasma concentration ratios (Kp,app). The Kp,app was defined by the
concentration of unchanged radio-labeled peptides in the tissues divided
by the plasma concentration of each peptide demonstrated in Fig. 2. Each
point represents the mean 6 S.E. of three or four mice.
min (Fig. 6A). The acid-resistant binding of 125I-labeled
C-AVP4-9 increased time-dependently (Fig. 6B). The acidresistant binding of 35S-labeled AVP4-9 and 125I-labeled
C-AVP4-9 at 120 min were 0.93 6 0.07 and 1.95 6 0.20 ml/mg
protein (as the cell/medium ratios), respectively.
Concentration Dependencies of Acid-Resistant
Binding of 35S-Labeled AVP4-9 and 125I-Labeled
C-AVP4-9. In the studies of concentration dependencies, 35Slabeled AVP4-9 showed two-phase saturable acid-resistant
binding (Fig. 7). The Bmax values of 35S-labeled AVP4-9 for
the high- and low-affinity sites of MBEC4 were estimated to
be 0.72 6 0.63 pmol/mg protein and 26.5 6 6.0 pmol/mg
protein, respectively. The KD values of 35S-labeled AVP4-9 for
the high- and low-affinity sites of MBEC4 were estimated to
be 3.81 6 3.46 nM and 45.7 6 11.4 mM, respectively. In
contrast, 125I-labeled C-AVP4-9 showed single-phase saturable acid-resistant binding (Fig. 7). The Bmax and KD values of
125
I-labeled C-AVP4-9 for MBEC4 were estimated to be
14.7 6 4.1 pmol/mg protein and 16.4 6 5.0 mM, respectively.
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Fig. 2. Plasma concentration profiles of 125I-labeled BSA, 35S-labeled
AVP4-9, and 125I-labeled C-AVP4-9. 125I-labeled BSA (37 pmol/mouse),
35
S-labeled AVP4-9 (2.8 pmol/mouse), or 125I-labeled C-AVP4-9 (23.0 pmol/
mouse) was administered i.v. into normal mice or mice whose renal
vessels were ligated. The unchanged 35S-labeled AVP4-9 and 125I-labeled
C-AVP4-9 were extracted from plasma with 2% phosphoric acid and quantified by HPLC. The unchanged 125I-labeled BSA in plasma was precipitated with 30% TCA, and radioactivity was counted. The 125I-labeled
BSA (M) data in the normal mice were fitted to a monoexponential
function, whereas 35S-labeled AVP4-9 (‚) and 125I-labeled C-AVP4-9 (E)
data in the normal mice were fitted to a biexponential function. The
plasma concentrations of the unchanged 125I-labeled BSA (f), 35S-labeled
AVP4-9 (Œ), and 125I-labeled C-AVP4-9 (F) at 30 min after administration
into the mice whose renal vessels are also plotted. Each point represents
the mean 6 S.E. of three or four mice.
1999
565
Enzymatic Conversion of C-AVP4-9 to AVP4-9.
C-AVP4-9 was converted by authentic PPCE and the mouse
cerebral homogenate to the metabolites eluted at 12.0 and
13.3 min (Fig. 8A) or 13.3 min (Fig. 8C) in the HPLC. The
retention times of authentic B chain (arginyl-histidinyl-proline; Fig. 1), AVP4-9, and C-AVP4-9 were 12.0, 13.3, and 23.0
min, respectively, in the HPLC conditions used here. The
C-AVP4-9 metabolites produced by the authentic PPCE and
the mouse cerebral homogenate eluted at 13.3 min in the
HPLC (Fig. 8, A and C) showed the same m/z as protonated
authentic AVP4-9 ([M 1 H] 5 775) and doubly protonated
AVP4-9 ([M 1 2H]21 5 338) by ESI-LC-MS. Based on the
agreements of retention time in the HPLC and the mass
spectrometric behavior, C-AVP4-9 metabolites eluted at 13.3
min in Fig. 8, A and C, were identified as AVP4-9. ZZP (1 mM)
completely inhibited the conversion of C-AVP4-9 to AVP4-9 by
the authentic PPCE (Fig. 8B). That concentration of ZPP also
significantly (p , .001 by Wilcoxon’s test) inhibited 49.3 6
0.3% (mean 6 S.E., n 5 4) of C-AVP4-9 conversion to AVP4-9
by the cerebral homogenate (Fig. 8D). The authentic PPCE
and the mouse cerebral homogenate showed saturable conversion rates of C-AVP4-9 to AVP4-9 with the CLint values
2.47 6 0.24 (Fig. 9A) and 2.10 6 0.14 ml/U/min (Fig. 9B),
respectively.
Discussion
Fig. 5. BBB permeation clearances of 125I-labeled BSA, 35S-labeled
AVP4-9, and 125I-labeled C-AVP4-9. The Kp,app values of 125I-labeled BSA
(F), 35S-labeled AVP4-9 (‚), and 125I-labeled C-AVP4-9 (E) shown in Fig. 4
were plotted against the indicated AUC(0 2 t)/Cp(t) values to estimate the
BBB permeation clearances of each labeled peptide (integration plot).
Each point represents the mean 6 S.E. of three or four mice.
Effect of Temperature, Osmolarity, Endocytosis Inhibitors, Metabolic Inhibitor, and Several Peptides on
Acid-Resistant Binding of 125L-Labeled C-AVP4-9. As
shown in Table 3, the incubation of 125I-labeled C-AVP4-9
with MBEC4 at 4°C for 60 min resulted in a decrease in the
acid-resistant binding to 10.4 6 1.2% of the control value.
When the osmolarity of the incubation buffer was increased
from 300 to 1600 mOsM, the acid-resistant binding of 125Ilabeled C-AVP4-9 decreased significantly (Table 3). The acidresistant binding of 125I-labeled C-AVP4-9 to MBEC4 was
significantly diminished by dancylcadaverine, phenylarsine
oxide, and 2,4-dinitrophenol (Table 3); markedly inhibited by
AVP4-9, poly(L-lysine), and protamine (Table 4); and unaffected by poly(L-glutamic acid), V1 vasopressin receptor antagonist, and V2 vasopressin receptor antagonist (Table 4).
It has been demonstrated that cationic peptides are effectively transported through the BBB and reach the parenchyma of the CNS via AME (Pardridge et al., 1991; Terasaki
et al., 1992). To enhance the BBB permeability of AVP4-9
(PI 5 9.2, Fig. 1), we combined the peptide composed of
cationic amino acids, that is, a B-chain (Fig. 1), with the free
cysteine residue of AVP4-9 and obtained C-AVP4-9 (PI 5 9.8,
Fig. 1). C-AVP4-9 was also designed to be converted to AVP4-9
in the CNS by PPCE, which is abundant in the CNS (Yoshimoto et al., 1979). It was previously demonstrated that
C-AVP4-9 facilitates the memory of rodents more effectively
than AVP4-9 (Tanabe et al., 1997). In the present study, the
BBB transport of C-AVP4-9 and its conversion to AVP4-9 in
the CNS were evaluated.
The Kp,app values of 125I-labeled C-AVP4-9 in the liver and
the lung were lower compared with those of 35S-labeled
AVP4-9 (Fig. 3), whereas the Kp,app values of 125I-labeled
C-AVP4-9 in the cerebrum were clearly higher compared with
those of 35S-labeled AVP4-9 (Fig. 4). These results suggest
that the cationization of AVP4-9 decreases the peripheral
distribution of modified molecule and increases the distribution of the molecule to the whole cerebrum, including the
parenchyma, capillary, and plasma. To evaluate the BBB
transport of neuroactive peptides, however, it is necessary to
evaluate the distribution of peptides to the parenchyma of
the cerebrum. Hence, we separated the cerebrum of the mice
administered with the labeled peptides into the capillary and
the parenchyma fractions by the capillary depletion method
(Yang et al., 1997) and evaluated the distributions of labeled
peptides to both fractions. We have demonstrated previously
that the alkaline phosphatase activity and Kp,app value of i.v.
administered [125I]-labeled low-density lipoprotein (markers
for the capillary) in the parenchyma fraction, which express
contamination of the capillary in the parenchyma fraction,
were about 10% of those in the whole cerebrum (Yang et al.,
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 4. Apparent cerebrum-to-plasma concentration ratios of 125I-labeled
BSA, 35S-labeled AVP4-9, and 125I-labeled C-AVP4-9. 125I-labeled BSA (37
pmol/mouse), 35S-labeled AVP4-9 (2.8 pmol/mouse), or 125I-labeled
C-AVP4-9 (23.0 pmol/mouse) was administered i.v. into the normal mice.
The Kp,app values of 125I-labeled BSA (F), 35S-labeled AVP4-9 (‚), and
125
I-labeled C-AVP4-9 (E) in the cerebrum were determined as described
in the legend to Fig. 3. Each point represents the mean 6 S.E. of three or
four mice.
BBB Transport of Cationic AVP4-9 Analog
566
Tanabe et al.
Vol. 290
TABLE 2
Apparent cerebrum-to-plasma concentration ratios of
125
I-labeled BSA, [35S]AVP4-9, and
125
I-labeled C-AVP4-9
Kp,app
125
I-labeled BSA
35
S-labeled AVP4-9
125
I-labeled C-AVP4-9
ml/g fraction
Parenchyma
Capillary
1.04 3 1022 6 0.13 3 1022
2.98 3 1024 6 0.17 3 1024
1.14 3 1021 6 0.16 3 1021
1.67 3 1023 6 0.61 3 1023
4.06 3 1021 6 0.36 3 1021
1.86 3 1022 6 0.67 3 1022
125
I-labeled BSA (37 pmol/mouse), [35S]AVP4-9 (2.6 pmol/mouse), or 125I-labeled C-AVP4-9 (8.0 pmol/mouse) was administered i.v. into mice whose renal vessels were
ligated. The cerebra were obtained at 30 min after the injection and separated into parenchyma and capillary fractions by the capillary depletion technique. Each value
represents the mean 6 S.E. of three or four mice.
TABLE 3
Effects of temperature, osmolarity, endocytosis inhibitors, and
metabolic inhibitor on the acid-resistant binding of 125I-labeled CAVP4-9 to MBEC4
Condition
Acid-Resistant Binding
Control
Low temperature (4°C)
Hypertonic (1600 mOsM)
Dancylcadaverine (0.5 mM)
2,4-Dinitrophenol (1 mM)
Controla
Phenylarsine oxide (0.1 mM)a
100 6 7.4
10.4 6 1.2c
53.4 6 14.9b
74.3 6 3.7b
26.0 6 3.9c
100 6 14.6
44.4 6 8.0b
% of control
TABLE 4
Effects of peptides on the acid-resistant binding of
AVP4-9 to MBEC4
Peptide
125
I-labeled C-
Acid-Resistant Bindinga
% of control
Control
AVP4-9 (0.5 mM)
V1 antagonist (0.5 mM)
V2 antagonist (0.5 mM)
Poly(L-lysine) (0.3 mM)
Protamine (0.3 mM)
Poly(L-glutamic acid) (0.3 mM)
100 6 5.5
36.3 6 3.4b
111 6 1.3
103 6 10.6
66.1 6 6.7a
69.6 6 5.0a
87.3 6 3.7
After preincubation for 30 min, 125I-labeled C-AVP4-9 and [3H]inulin were incubated with or without the indicated peptides at 37°C for 60 min. Each value represents the percentage of acid-resistant binding of 125I-labeled C-AVP4-9 (as cell/
medium ratios) versus the control value (mean 6 S.E. of three or four separate
experiments).
a
Significant inhibition (p , .05).
b
Significant inhibition (p , .001).
Fig. 7. Concentration dependencies of acid-resistant binding of 35S-labeled AVP4-9 and 125I-labeled C-AVP4-9 to MBEC4. Various concentrations of mixtures of 35S-labeled AVP4-9 and unlabeled AVP4-9 or 125Ilabeled C-AVP4-9 and unlabeled C-AVP4-9 were incubated with MBEC4 in
the presence of [3H]inulin (34.3 mM) at 37°C for 60 min. The acidresistant bindings of unchanged 35S-labeled AVP4-9 (F) and 125I-labeled
C-AVP4-9 (E) are expressed as the cell/medium ratios. Each point represents the mean 6 S.E. of three or four separate experiments.
1997). In the capillary depletion studies, we ligated the renal
vessels of mice before the i.v. administration of 125I-labeled
BSA, 35S-labeled AVP4-9, or 125I-labeled C-AVP4-9 to elimi-
nate the distribution of these labeled peptides to the kidney,
the tissue where 35S-labeled AVP4-9 and 125I-labeled
C-AVP4-9 showed the highest Kp,app value among the tissues
examined (Figs. 3 and 4), and to elevate the plasma concentrations and the cerebral distributions of the labeled peptides. With the ligation treatments, the plasma concentrations of 35S-labeled AVP4-9 and 125I-labeled C-AVP4-9 at 30
min were 10 times and 3 times higher than those in the
normal mice, respectively (Fig. 2), and this treatment enabled us to quantify the unchanged 35S-labeled AVP4-9 and
125
I-labeled C-AVP4-9 in the parenchyma and capillary fractions of the cerebrum by HPLC. Because it is widely accepted
that BSA remains in the plasma in the cerebrum (Triguero et
al., 1990), the plasma volume of the cerebral parenchyma
fraction was estimated to be 1.04 6 0.13 3 1022 ml/g cerebrum (Kp,app of 125I-labeled BSA in the parenchyma fraction)
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 6. Time courses of acid-resistant binding of [3H]inulin, 35S-labeled
AVP4-9, and 125I-labeled C-AVP4-9 to MBEC4. 35S-labeled AVP4-9 (4.1 nM)
or 125I-labeled C-AVP4-9 (0.7 nM) was incubated with MBEC4 in the
presence of [3H]inulin (34.3 mM) at 37°C for the indicated time. After the
incubation, unbound, acid-soluble, and acid-resistant fractions were recovered, and the unchanged [3H]inulin, 35S-labeled AVP4-9, and 125Ilabeled C-AVP4-9 in each fraction was quantified by HPLC. The acidresistant bindings of 35S-labeled AVP4-9 (E in A), 125I-labeled C-AVP4-9 (E
in B), and [3H]inulin (F) are expressed as the cell/medium ratios. Each
point represents the mean 6 S.E. of three or four separate experiments.
After preincubation for 30 min, 125I-labeled C-AVP4-9 and [3H]inulin were incubated under the indicated conditions at 4° or 37°C for 60 min. Each value represents
the percentage of acid-resistant binding of 125I-labeled C-AVP4-9 (as cell/medium
ratios) versus the control value (mean 6 S.E. of three or four separate experiments).
a
Because phenylarsine oxide was dissolved in 0.1% dimethyl sulfoxide, the control experiment was performed in the presence of 0.1% dimethyl sulfoxide.
b
Significant inhibition (p , .05).
c
Significant inhibition (p , .001).
1999
BBB Transport of Cationic AVP4-9 Analog
567
Fig. 8. Enzymatic conversion of C-AVP4-9 to AVP4-9. C-AVP4-9 (150 mM)
was incubated with the authentic PPCE (0.1 U/ml, A and B) or mouse
cerebral homogenate (20 mg cerebrum/ml equivalent to 1.5 3 1023 U/ml,
C and D) at 37°C for 20 min or 6 h, respectively. The inhibition experiments were carried out in the presence of 1 mM ZPP in the same
conditions (B and D). The metabolic reactions were stopped by the addition of 2% phosphoric acid. After the centrifugation the supernatants
were analyzed by HPLC.
(Table 2). The Kp,app values of 35S-labeled AVP4-9 and 125Ilabeled C-AVP4-9 in the parenchyma fraction were markedly
higher than the plasma volume in the parenchyma fraction
and Kp,app values of each labeled peptide in the capillary
fractions (Table 2), suggesting that almost all of these labeled
peptides are distributed to the parenchyma in the whole
cerebrum. Therefore, the Kp,app values of 35S-labeled AVP4-9
and 125I-labeled C-AVP4-9 in the whole cerebrum demonstrated in Fig. 4 nearly indicate the Kp,app value of these
labeled peptides in the cerebral parenchyma, and we evaluated the BBB permeation clearances of 35S-labeled AVP4-9
and 125I-labeled C-AVP4-9 with the Kp,app values shown in
Fig. 4 by the integration plot method (Smith et al., 1987;
Kakee et al., 1996; Yang et al., 1997). With the integration
plotting, the BBB permeation clearances of 35S-labeled
AVP4-9 and 125I-labeled C-AVP4-9 were estimated to be
1.47 6 0.09 3 1024 and 3.10 6 0.35 3 1024 ml/min/g cerebrum, respectively (Fig. 5), suggesting that C-AVP4-9 is
transported through the BBB to the cerebral parenchyma
more effectively than AVP4-9.
Monolayers of MBEC4 cells were used in in vitro internalization studies. Because the acid wash treatment removes
the labeled peptides binding to the cell surface, the acidresistant binding of the labeled peptides expresses the internalized labeled peptides into the cells (Terasaki et al., 1989,
1992). The slight time dependence of acid-resistant bindings
of extracellular space marker, [3H]inulin, coexisting with
35
S-labeled AVP4-9 or 125I-labeled C-AVP4-9, to MBEC4 (Fig. 6)
may be ascribed to the fluid-phase endocytosis of MBEC4
(Terasaki et al., 1992). From 10 min after the incubation, the
acid-resistant binding of 125I-labeled C-AVP4-9 was higher
than that of 35S-labeled AVP4-9 (Fig. 6), suggesting that
C-AVP4-9 is internalized into the brain capillary endothelial
cells more effectively than AVP4-9. The KD values of RMEtransported peptides reported previously were as follows:
atrial natriuretic factor (400 pM, using cultured bovine brain
capillary endothelial cells; Smith et al., 1988), transferrin
(5.6 nM, isolated human brain capillaries; Pardridge et al.,
1987), insulin (2.9 nM, isolated human brain capillaries;
Frank et al., 1986), and leptin (5.1 nM, isolated human brain
capillaries; Golden et al., 1997). Although the KD values of
AVP4-9 for the low-affinity sites (45.7 mM) and of C-AVP4-9
(16.4 mM) were in good agreement with the values of E-2078
(4.62 mM, using isolated bovine brain capillaries; Terasaki et
al., 1989), ebiratide (15.9 mM, cultured bovine brain capillary
endothelial cells; Terasaki et al., 1992; 62.1 mM, isolated
bovine brain capillaries, Shimura et al., 1991), and histone
(15.2 mM, isolated bovine brain capillaries, Pardridge et al.,
1989), which are internalized into the BBB via AME. In light
of the agreements of KD values of AVP4-9 (low affinity) and
C-AVP4-9 with the values of AME-transported peptides in the
previous reports and the basicities of both peptides, it is
suggested that C-AVP4-9 and a part of AVP4-9 are internalized into the BBB via AME.
To more precisely elucidate the mechanism underlying the
internalization of C-AVP4-9 into the BBB, we examined the
acid-resistant binding of 125I-labeled C-AVP4-9 in several
conditions. The osmolarity dependence of the acid-resistant
binding of 125I-labeled C-AVP4-9 to MBEC4 (Table 3) suggests that C-AVP4-9 is significantly internalized into
MBEC4. As shown in Table 3, the acid-resistant binding of
125
I-labeled C-AVP4-9 to MBEC4 was significantly inhibited
by lower temperature, dancylcadaverine (an endocytosis inhibitor and a suppressor of the coated pit formation; Haigler
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 9. Enzymatic conversion rate of C-AVP4-9 to AVP4-9. Various concentrations of C-AVP4-9 were incubated with authentic PPCE (0.1 U/ml, A) or
mouse cerebral homogenate (20 mg cerebrum/ml equivalent to 1.5 3 1023
U/ml, B) at 37°C for 1 or 30 min, respectively. The metabolic reactions
were stopped by the addition of 2% phosphoric acid. After centrifugation,
the supernatants were analyzed by HPLC. Each point represents the
mean 6 S.E. of three separate experiments.
568
Tanabe et al.
Acknowledgments
We thank Drs. Hiroshi Nagata and Ken-ichi Hosoya for their
valuable discussions, and Dr. Wei-Xing Yang for the expert guidance
and assistance in the capillary depletion study.
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et al., 1980), phenylarsine oxide (an endocytosis inhibitor and
a denaturant of the SH group in the cell membrane; Knutson
et al., 1983), and 2,4-dinitrophenol (a metabolic inhibitor and
an uncoupler of phosphorylation). In addition, as shown in
Table 4, the cationic peptides [poly(L-lysine), protamine, and
AVP4-9] significantly inhibited the acid-resistant binding of
125
I-labeled C-AVP4-9 to MBEC4, whereas an anionic peptide
[poly(L-glutamic acid)] and the V1 and V2 vasopressin receptor antagonists had no effect on the acid-resistant binding.
These results suggest that C-AVP4-9 is internalized into the
BBB via the energy-dependent AME by its basicity, independently of the vasopressin receptors.
We also examined the conversion of C-AVP4-9 to AVP4-9 in
the cerebrum in vitro. The conversions of C-AVP4-9 by the
authentic PPCE and the mouse cerebral homogenate were
inhibited by the PPCE-specific inhibitor ZPP (Fig. 8), and the
CLint of the C-AVP4-9 conversion by the mouse cerebral homogenate agreed well with the value by the authentic PPCE
(Fig. 9), suggesting that C-AVP4-9 is converted to AVP4-9 by
PPCE in the cerebrum.
In conclusion, the results of our studies suggest that
C-AVP4-9 is transported through the BBB via AME to the
cerebral parenchyma more effectively than its parent peptide, AVP4-9. The results also suggest that C-AVP4-9 is converted to AVP4-9 in the cerebrum, consistent with our molecular design concept of C-AVP4-9. Chemical modification to
obtain a peptide with basicity and convertibility to the active
form in the CNS is a promising strategy for the conversion of
native physiologically active peptides into neuropharmaceuticals.
Vol. 290