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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:1018 –1025, 1997
Vol. 283, No. 3
Printed in U.S.A.
Selective Brain to Blood Efflux Transport of paraAminohippuric Acid across the Blood-Brain Barrier: In Vivo
Evidence by Use of the Brain Efflux Index Method1
ATSUYUKI KAKEE, TETSUYA TERASAKI and YUICHI SUGIYAMA
Central Pharmaceutical Research Institute, Japan Tobacco Inc., Murasaki-cho, Takatsuki, Osaka, 569 –11, Japan (A.K.), Department of
Pharmaceutics, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramakiaza, Aoba-ku, Sendai, 980 –77, Japan (T.T.) and
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113, Japan (Y.S.)
Accepted for publication July 29, 1997
Numerous drugs, including nonsteroidal anti-inflammatory drugs, b-lactam antibiotics, quinolone antibiotics and
the anti-AIDS agent azidothymidine, have exhibited limited
distribution in the brain (Terasaki and Pardridge, 1988; Suzuki et al., 1989a). Based on pharmacokinetic analysis, it has
been suggested that this low distribution is caused by a
significant efflux compared with the influx at the BBB (Adkison et al., 1994; Wang and Sawchuk, 1995). However, with
the exception of P-glycoprotein, only limited information is
available regarding selective efflux transport at the BBB
(Suzuki et al., 1996).
Recently we developed a novel method, termed the “brain
efflux index method,” to estimate directly the efflux rate of
various substances at the BBB (Kakee et al., 1996). The
validity of the BEI method has been demonstrated with wa-
Received for publication December 13, 1996.
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 Japan
Health Foundation Drug Innovation Science Project.
change significantly, which suggested that blood-cerebrospinal
fluid barrier was not responsible for the elimination of PAH from
the brain after microinjection. No significant metabolism of PAH
was demonstrated in the brain for at least 20 min after microinjection, and most of the radioactivity in the ipsilateral and
contralateral carotid veins was as the intact form. With the
distribution volume of PAH, 0.800 ml/g brain, obtained from the
brain slice uptake experiment, the apparent efflux clearance
was calculated as 46.9 ml/min/g brain. In addition, the influx
clearance of PAH across the BBB determined by the in vivo
brain uptake index method was much smaller than the efflux
clearance, which demonstrates that BBB transports PAH selectively from the brain to the circulating blood.
ter and 3-O-methyl-D-glucose, the former used as a substrate
representing blood flow limited elimination and the latter a
substrate representing symmetrical elimination at the BBB.
The BEI method may enable us to detect the selective efflux
transport systems.
PAH, an organic anion, is known to be excreted in the urine
via the active organic anion transport systems located in the
kidney (Pritchard, 1987, 1988; Hori et al., 1993; Takano et al.,
1994). It has also been reported as a substrate for the organic
anion efflux transport systems at the BCSFB (Holloway and
Cassin, 1972; Bass and Lundborg, 1973; Domer, 1973).
Azidothymidine or valproic acid is also expected to be transported by putative probenecid-sensitive organic anion efflux
systems at the BBB (Wong et al., 1993; Adkison et al., 1994),
leading to extremely limited cerebral distribution. It would
be very useful to know if the BBB has a role in pumping out
organic anions from brain to blood.
The present study investigated the hypothesis that the
BBB transports PAH, an organic anion, selectively from the
brain to the circulating blood by the BEI method.
ABBREVIATIONS: BBB, blood-brain barrier; CSF, cerebrospinal fluid; BCSFB, blood-cerebrospinal fluid barrier; BEI, brain efflux index; BUI, brain
uptake index; Par2, Parietal Cortex Area2; i.c.v., intracerebroventricular; HEPES, 4-(2-hydroxyethyl)-piperazineethanesulfonic acid; PAH, paraaminohippuric acid; AIDS, acquired immune deficiency syndrome; MDR, multi-drug resistance;
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ABSTRACT
Efflux transport of para-aminohippuric acid (PAH) across the
blood-brain barrier (BBB) has been demonstrated by use of the
brain efflux index (BEI) method. PAH was eliminated from the
ipsilateral cerebrum extensively with an apparent efflux rate
constant of 0.0587 (min21) after microinjection into a cerebral
cortex region termed Par2. This efflux transport showed a
saturation with the Michaelis constant of approximately 400
mM. No more than 3% dose of PAH and carboxyl-inulin, used
as a reference compound showing limited permeability at the
BBB, were found in the contralateral cerebrum, cerebellum or
cerebrospinal fluid up to 20 min after administration. Under
saturated conditions for carrier-mediated efflux of PAH via the
blood-cerebrospinal fluid barrier, the BEI value of PAH did not
1997
BBB Efflux Transport of PAH
clearance, CLefflux, across the BBB was obtained as follows.
Materials and Method
1
BEI (%) 5 1 2
2
Amount of reference in the brain
Amount of test drug injected
Amount of reference injected
3 100
(1)
the apparent elimination rate constant of PAH from the brain, kel,
can be obtained by the nonlinear regression analysis of a semilogarithmic plot of (1002BEI) values versus time. A least squares regression analysis program, MULTI (Yamaoka et al., 1981) was used for
the calculation. Multiplying the apparent efflux rate constant by the
distribution volume of PAH in the brain, Vbrain, the apparent efflux
BBB
CL
5 kel Vbrain
efflux
(2)
The Vbrain was determined by the in vitro uptake into brain slices as
described below.
Kinetic parameters for the efflux transport of PAH across the BBB
were estimated according to the following equation:
Vo 5
VmaxC
(3)
Km 1 C
where Km, Vmax and C are the Michaelis constant (mM), the maximum transport velocity across the BBB (nmol/min/g brain) and the
concentration of PAH in the brain (mM), respectively. The concentration of PAH in the brain was estimated from the concentration in
the injectate, assuming that the 0.5-ml injectate was diluted 30.3-fold
in the brain as reported previously (Kakee et al., 1996). Accordingly,
PAH concentration in the brain was calculated by dividing the drug
concentration in the injectate by 30.3. To obtain the kinetic parameters, Km and Vmax, Eadie-Hofstee plot analysis was performed by
the following equation derived from equation 3.
Vo
C
5 2
1
Km
Vo 1
Vmax
Km
(4)
Fitting equation 4 to the data sets was carried out by an iterative
nonlinear least-squares method using a program “MULTI” to obtain
the kinetic parameters (Yamaoka et al., 1981). The input data were
weighed as the reciprocal of the square of the observed values, and
the algorithm used for the fitting was the damping Gauss Newton
Method. The Km and Vmax for the efflux of PAH across the BBB
obtained were 396 6 73 (mM) and 23.4 6 2.7 (nmol/min/g brain),
respectively.
Intracerebroventricular injection of [3H]PAH and [14C]inulin. [3H]PAH (2.5 ml; 1 mCi/rat) and [14C]inulin (0.05 mCi/rat) were
administered to the lateral cerebral ventricle of the left cerebrum in
the absence or presence of 100 mM unlabeled PAH as reported
previously (Suzuki et al., 1985, 1988, 1989b). After injection of drug,
CSF was sampled from the cisterna magna at appropriate times as
described above. The elimination clearances of [3H] PAH and
[14C]inulin in the CSF were calculated from the following equation:
Injected dose in the CSF
CSF
CL
5
efflux
AUCCSF
(5)
Because the CSF volume has been reported to be 250 ml/rat (Cserr
and Berman, 1978), the PAH concentration in the CSF was estimated assuming that the injectate was immediately and homogeneously distributed in the CSF compartment.
To investigate the effect of a saturable efflux system at the BCSFB
on the apparent elimination of PAH from the brain after microinjection into Par2, 100 mM unlabeled PAH was administered to the rat
cerebral ventricle 30 sec before the BEI study with [3H]PAH. The
osmolarity of the injectate was, if required, adjusted by removal of
NaCl from the ECF buffer to give an isotonic solution.
Metabolism of [3H]PAH after cerebral microinjection. An
aliquot of 1 ml [3H]PAH (2 mCi/rat) was administered to the Par2
region of the left cerebrum. One milliliter of blood from the ipsilateral and contralateral carotid veins was withdrawn 5 min after
administration; then rats were decapitated and the left cerebrum
was removed. Plasma samples were obtained by centrifugation at
3,000 rpm for 10 min. Analytical samples were prepared following
deproteinization with methanol. In the case of brain samples, they
were suspended in 1.8 ml ECF buffer using a Teflon homogenizer
(Iuchi Seieido Co., Ltd., Tokyo, Japan), and then centrifuged at 3,000
rpm for 10 min. A 3 ml supernatant was added to 6 ml methanol to
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Reagents. [3H]PAH (181.3 Gbq/mmol), [3H]3-O-methyl-D-glucose
(2782.4 GBq/mmol), [3H]inulin (11.7 GBq/g), [14C]carboxyl-inulin
([14C]inulin; 0.093 GBq/g) and [14C]butanol (0.059 GBq/mmol) were
purchased from New England Nuclear (Boston, MA). HEPES, from
Dojin Chemicals (Kumamoto, Japan), and xylazine, from Sigma
Chemical Co. (St. Louis, MO), were both analytical grade. Ketaral 50
[ketamine hydrochloride (Sankyo Co., Tokyo, Japan)] was used as an
anesthetic. All other reagents were of reagent grade and used without further purification.
BBB efflux study of [3H]PAH. [3H]PAH and [14C]inulin, used as
a reference compound, were dissolved in physiological buffer containing 122 mM NaCl, 25 mM NaHCO3, 10 mM D-glucose, 3 mM KCl, 1.4
mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4 and 10 mM HEPES.
This buffer was adjusted to pH 7.4 by 2 N NaOH (ECF buffer).
Sprague-Dawley male rats (supplied by Charles River, Yokohama,
Japan) weighing 200 to 250 g were anesthetized with an intramuscular injection of ketamine-xylazine (1.22 mg xylazine and 125 mg
ketamine per kg b.wt.) and placed in a stereotaxic frame (Narishige
Co., Tokyo, Japan). The BEI experiments were carried out as reported previously (Kakee et al., 1996). After removing part of the
scalp, a midline incision was performed to expose the reference point
on the skull called the bregma. A small hole was drilled at the Par2
region (0.2 mm anterior and 5.5 mm lateral to the bregma and 4.5
mm deep) of the left cerebrum to allow entry of an injection needle,
and then 0.5 ml of a mixture of [3H]PAH (2.5 nCi or 0.2 mCi/rat) and
[14C]inulin (0.25 nCi or 1.5 nCi/rat) was injected by use of a 5-ml
microsyringe (Hamilton, Reno, NE) fitted with a needle (i.d. 100 mm,
o.d. 350 mm, Seiseido Medical Industry, Tokyo, Japan) in the absence
and presence of unlabeled PAH. The osmolarity of the injectate was
adjusted by removal of NaCl from the ECF buffer to give an isotonic
solution, if required. The craniometric data and the precise localization of the region to be injected were determined with a stereotaxic
atlas (Paxinos and Watson, 1986). After microinjection of drug into
the cerebrum, CSF was sampled from the cisterna magna at appropriate times. A small hole was opened at the sagittal midline through
the suture between the interparietal and supraoccipital bones with
an electrical drill (Natsume Seisakusho Co., Ltd., Tokyo, Japan). A
syringe was introduced into the hole to a depth of 6 mm. Gentle
suction was applied by means of this syringe to withdraw 50 to 150
ml CSF. Immediately after CSF sampling, rats were decapitated and
left and right cerebrum and cerebellum were removed. After measuring the wet weight of each excised cerebrum or cerebellum, they
were dissolved in 2.5 ml 2 N NaOH by incubating at 50°C for 3 hr.
Then, 14 ml liquid scintillation cocktail (Hionic-fluor; Packard Instruments Corp., Meriden, CT) was added to the sample at room
temperature. Radioactive counting was performed by a double-channel system for the 3H, 14C mixed samples using an LC-6000 liquid
scintillation counter (Beckmann Instruments Corp., Fullerton, CA).
Each sample was measured twice for 5 min and the average was used
for calculations. The BEI value was obtained by the following equation (Kakee et al., 1996):
Amount of test drug in the brain
1019
1020
Kakee et al.
Vol. 283
BUI (%) 5
Edrug
Ereference
3 100
Results
3
Efflux of [ H]PAH from the brain across the BBB.
Figure 1 shows the time course of the remaining percentage
of [3H]PAH in the ipsilateral cerebrum after microinjection
into Par2 of rat brain. Approximately 60% of the administered dose of [3H]PAH was eliminated from the ipsilateral
cerebrum within 20 min, which indicates a significant elimination of [3H]PAH from the cerebrum (fig. 1). The apparent
elimination rate constant, kel, was found to be 5.87 3 1022 6
0.65 3 1022 min21 by nonlinear least squares regression
analysis of the observed values up to 20 min after microinjection. As shown in table 1, no significant amount of administered [3H]PAH was found in the contralateral cerebrum,
cerebellum or CSF compartment at 20 min, even after an
intracerebral microinjection of a high dose of radioactivity. In
addition, no significant difference in the remaining percentage of [14C]inulin, an internal reference compound, was observed at 20 min after microinjection (table 1). The compartmental model analysis based on the drug elimination
obtained by the BEI study and i.c.v. injection study as described below indicated that only 0.52% of the apparent elim-
(6)
where Edrug and Ereference represent the extraction of the test and
reference compound in the brain, respectively. In these experiments,
[14C]butanol was used as a reference compound, with an Ereference
value of 64% (Pardridge and Fierer, 1985). The BUI value was
obtained as follows:
Amount of test drug in the brain
BUI (%) 5
Amount of reference in the brain
Amount of test drug in the injectate
3 100
(7)
Amount of reference in the injectate
CLinf was calculated from the following equation:
CLinf 5 QEdrug
(8)
where Q is the cerebral blood flow rate reported previously as 0.93
ml/min/g brain (Pardridge and Fierer, 1985). PSinf was determined
as follows:
PSinf 5 2 Q ln (1 2
CLinf
Q
)
(9)
Fig. 1. Time courses of [3H]PAH in the ipsilateral cerebrum after intracerebral microinjection in the presence of [14C]inulin as an internal
reference. A mixture of [3H]PAH (2.5 nCi) and [14C]inulin (0.25 nCi)
dissolved in 0.5 ml ECF buffer was injected into Par2 of rat cerebrum in
the absence or presence of 100 mM unlabeled PAH. Rats were decapitated at 2, 5, 10, 15 and 20 min after microinjection. Closed and open
symbols represent the time courses of the percentage of [3H]PAH
remaining in the brain after administration in the absence and presence
of 100 mM PAH (the mean 6 S.E.; n 5 3–7). The slope of the solid line
represents the elimination rate constant of tracer amount of [3H]PAH,
i.e. 5.87 3 1022 6 0.65 3 1022 min21, obtained by the nonlinear least
squares regression analysis.
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denature proteins. After centrifugation at 3,000 rpm for 10 min, 8 ml
supernatant was evaporated under N2 and then the samples were
analyzed by the imaging analyzer system (BAS-3000; Fuji Photo Co.,
Ltd., Tokyo, Japan) following 10 3 10 cm thin-layer chromatography
(Kieselgel 60F254, Merck Co., Ltd., Darmstadt, Germany). The solvent system for the thin-layer chromatography was n-butanol/acetic
acid/water (25:4:10).
Measurement of distribution volume of [3H]PAH in the
brain. The distribution volume of PAH in the brain was determined
by the in vitro brain slice uptake technique. Brain slices were prepared as reported previously with minor modification (Newman et
al., 1991). After decapitating rats, brains were immediately removed
and dissected in ice-cold oxygenated ECF buffer. A hypothalamic
slice, 300 mm thick, was cut using a brain microslicer (DTK-2000,
Dosaka EM Co., Ltd., Kyoto, Japan), and kept in oxygenated ECF
buffer equilibrated with 95% O2-5% CO2. After preincubation for 5
min at 37°C, the brain slice (40–50 mg) was transferred to 50 ml
oxygenated incubation medium containing 0.05 mCi/ml [3H]PAH and
0.01 mCi/ml [14C]inulin at 37°C. At appropriate times, brain slices
and part of the incubation medium were stored at 220°C for the
determination of drug concentrations. The apparent zero-time intercept of the [14C]inulin uptake time profile, i.e., 0.136 6 0.006 ml/g
slice (mean 6 S.E., n 5 6), obtained from the same study with the
brain slices, was used to correct for the adsorbed water volume.
BUI of [3H]PAH, [3H]3-O-methyl-D-glucose or [3H]inulin.
The influx clearance of [3H]PAH across the BBB was determined by
the BUI method reported previously (Oldendorf, 1970; Terasaki et
al., 1986). An aliquot of 250 ml Ringer’s/HEPES buffer (pH 7.4, 5 mM
HEPES) was injected rapidly into the left common carotid artery.
The injection solution contained [3H]PAH (100 mCi/ml) and [14C]butanol (0.05 mCi/ml). In the BUI studies with [3H]3-O-methyl-D-glucose or [3H]inulin, [3H]3-O-methyl-D-glucose (10 mCi/ml) and
[14C]butanol (0.5 mCi/ml) or [3H]inulin (100 mCi/ml) and [14C]butanol
(0.05 mCi/ml) were dissolved in the injectate, respectively. Fifteen
seconds after the carotid artery injection, the rats were decapitated.
The radioactivity in the injection solution and the hemisphere ipsilateral to the injection were determined. The BUI value, extraction
ratio, apparent influx clearance (CLinf) and intrinsic BBB permeability surface area product (PSinf) were calculated as follows (Oldendorf,
1970):
1997
1021
BBB Efflux Transport of PAH
TABLE 1
Recovered percentage of [3H]PAH and [14C]inulin in the ipsilateral, contralateral cerebrum, cerebellum or CSF after intracerebral
microinjection
[3H]PAH (0.2 mCi) and [14C]inulin (1.5 nCi) dissolved in 0.5 ml ECF buffer were injected intracerebrally for 1 sec into normal male rats. Rats were decapitated 2, 5 and
20 min after administration. In this experiment, the detection limits were 0.050% ([3H]PAH) and 2.9% ([14C]inulin) of the administered dose.
Time after
administration
(min)
[3H]PAH
2
5
20
14
[ C]Inulin
2
5
20
a
Remaining Percentage Relative to the Injected Amounta
n
Ipsilateral
Cerebrum
Contralateral
Cerebrum
Cerebellum
CSF
3
3
3
82.9 6 6.1
85.1 6 9.1
60.5 6 7.6
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
3
3
3
119.0 6 7.8
127.0 6 11.1
109.4 6 16.7
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Each value represents the mean 6 S.E. N.D., not detected.
3
Fig. 2. (a) Concentration dependence of the efflux of [ H]PAH at the
BBB. A mixture of [3H]PAH (2.5 nCi) and [14C]inulin (0.25 nCi) dissolved
in 0.5 ml ECF buffer was injected into Par2 of rat cerebrum in the
presence of 1, 10, 25, 50 or 100 mM unlabeled PAH. Rats were
decapitated at 2 and 20 min after microinjection. Closed symbols
represent the efflux rate constant of [3H]PAH at the BBB (the mean 6
S.E.; n 5 6). (b) Eadie-Hofstee plot of the efflux transport of [3H]PAH at
the BBB. The reciprocal of the slope of solid line, obtained by the
nonlinear least squares regression analysis, represents the Km value for
the transport of PAH at the BBB, as described under “Materials and
Method.”
CSF, the elimination of [3H]PAH from the CSF compartment
was delayed significantly (fig. 3).
After administering unlabeled PAH in the cerebral ventricle, the BEI study of [3H]PAH was also performed to examine
Fig. 3. Time courses of the percentage of the dose of [3H]PAH and
[14C]inulin remaining in the CSF after i.c.v. injection. A mixture of
[3H]PAH (1 mCi) and [14C]inulin (0.05 mCi) dissolved in 2.5 ml ECF buffer
was injected into the cerebroventricle in the absence and presence of
100 mM unlabeled PAH in the injectate. Rats were decapitated at 5, 15
and 30 min after administration. Closed and open circles represent the
time courses of the percentage dose of [3H]PAH remaining in the CSF
in the absence and presence of 100 mM unlabeled PAH in the injectate,
respectively (n 5 4). Closed squares represent the time courses of the
percentage dose of [14C]inulin remaining in the CSF (n 5 6). The
elimination clearance of tracer amounts of [3H]PAH and [14C]inulin was
calculated to be 26.7 and 4.0 ml/min/rat, respectively.
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ination rate constant in the BEI study contributed to efflux
from the CSF.
The BEI study of [3H]PAH was also performed at 2 and 20
min with 100 mM PAH in the injectate. Twenty minutes
after injections, the remaining percentage of [3H]PAH after
coadministration of unlabeled PAH was found to be 79.6 6
1.9%, which was significantly greater that that in the absence of unlabeled PAH, i.e., 31.8 6 3.4% (fig. 1).
The apparent elimination rate constant was decreased in a
dose-dependent manner (fig. 2a). The Michaelis constant,
Km, and the maximum velocity, Vmax, for the efflux of PAH
across the BBB obtained were 396 6 73 mM and 23.4 6 2.7
nmol/min/g brain by Eadie-Hofstee plot analysis, respectively (fig. 2b).
Efflux of [3H]PAH from the CSF after i.c.v. injection.
Figure 3 shows the time course of the percentage dose of
[3H]PAH and [14C]inulin in the CSF after cerebroventricular
administration. Based on equation 5, the apparent elimination clearance of [3H]PAH from the cerebral ventricle,
CLefflux, CSF, was estimated to be 26.7 and 4.0 ml/min/rat for
PAH and inulin, respectively. In the presence of 1 mM PAH
in the
1022
Kakee et al.
Vol. 283
Discussion
Several acidic drugs have been reported to show significantly limited cerebral distribution compared with neutral or
basic drugs, by BUI, brain perfusion, brain microdialysis or
in vivo intravenous injection techniques (Nau and Loscher,
1982; Cornford et al., 1985; Suzuki et al., 1989a). Also, the Kp
TABLE 2
BEI of [3H]PAH after intracerebroventricular injection of excess
unlabeled PAH
PAH Concentration in the CSF (mM)a
n
BEI at 20 minb (%)
0
1
4
4
54.6 6 3.3
51.3 6 1.5c
a
Aliquots (2.5 ml) in the presence or absence of 100 mM PAH were administered to the cerebral ventricle, 30 sec before the microinjection of [3H]PAH into
Par2 of rat hemisphere. PAH concentration in CSF was estimated by the CSF
volume of 250 ml assuming immediate mixing of the injectate with CSF.
b
Each value represents the mean 6 S.E.
c
Not significantly different at P , .05.
Fig. 4. Time courses of [3H]PAH uptake by rat brain slices. Rat brain
slices were incubated with 0.05 mCi/ml [3H]PAH and 0.01 mCi/ml
[14C]inulin at 37°C. At appropriate times, the radioactivity in the brain
slices and incubation medium were measured, and the slice-to-medium concentration ratio was estimated.
TABLE 3
BUI, brain extraction and BBB influx clearance of [3H]PAH,
[3H]inulin or [3H]3-O-methyl-D-glucosea
An aliquot of 250 ml RHB containing [3H]PAH, [3H]inulin or [3H]3-O-methyl-Dglucose in the presence of [14C]butanol was injected rapidly into the left common
carotid artery as indicated under “Materials and Method.” Fifteen seconds after
injection, the rats were decapitated, and the radioactivity of the injection solution
and the ipsilateral hemisphere were determined.
[3H]PAH
[3H]Inulin
[3H]3-O-Methyl-Dglucose
a
n
BUI (%)
Brain Extraction
(%)
PSinf
(ml/min/g brain)
4
4
3
2.0 6 0.1
2.8 6 0.2
15.8 6 1.3
1.3 6 0.1
1.8 6 0.1
10.1 6 0.8
12.1 6 0.9
16.6 6 1.2
99.1 6 7.8
Each value represents the mean 6 S.E.
value, the concentration ratio of drug in brain to that in
plasma, has been found to be well below unity at steady state.
The following four possibilities have been proposed to explain
the limited cerebral distribution of acidic drugs: 1) protein
binding in plasma was very high compared with that in
brain, 2) the efflux clearance was greater than the influx
clearance across the BBB, 3) the efflux via BCSFB was significant and 4), all these possibilities combined. Recently,
pharmacokinetic model analysis after i.c.v. injection, perfusion or brain microdialysis experiments have led us to consider the second possibility to explain the limited distribution
of the acidic drugs such as valproic acid (Adkison et al., 1994).
As reported previously, the BEI method has been shown to
characterize the BBB efflux transport process (Kakee et al.,
1996). By this newly developed BEI method, we have determined the efflux rate of PAH, selected as a model substrate of
an acidic drug.
PAH is well known to be actively excreted at the kidney or
choroid plexus via organic anion transporters (Holloway and
Cassin, 1972; Bass and Lundborg, 1973; Domer, 1973). This
compound also has a low distribution in the brain similar to
several other substances. Because the protein binding of
PAH in the plasma has been reported to be only 2.4% (Elbourne et al., 1990), the first possibility can be rejected as an
explanation of the limited distribution of PAH in the brain.
PAH molecules microinjected into Par2 of the rat hemisphere
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the contribution of the saturable elimination pathway of
PAH via the BCSFB to the apparent elimination from the
ipsilateral cerebrum. As shown in table 2, no significant
difference was observed in the BEI values of [3H]PAH at 20
min compared in the absence and presence of 1 mM unlabeled PAH in the CSF, which suggests the minimal contribution of the elimination pathway via the BCSFB.
Metabolism of [3H]PAH after cerebral microinjection. Metabolism of [3H]PAH was investigated by the autoradiogram of the thin-layer chromatography of [3H]PAH in
the ipsilateral cerebrum and carotid vein after microinjection
into Par2. All the major spots of the ipsilateral cerebrum, and
ipsilateral and contralateral carotid veins had the same Rf
value (Rf 5 0.58) as that of authentic [3H]PAH used as a
standard (data not shown). Moreover, no significant metabolites were found for the brain and blood samples examined.
Comparison of efflux and influx clearance of
[3H]PAH at the BBB. The distribution volume of PAH in
the brain, Vbrain, was determined in the in vitro brain slice
uptake study. Figure 4 shows the time course of the brain
slice-to-medium concentration ratio of [3H]PAH. No significant difference in the slice-to-medium concentration ratio
between 60 and 120 min was observed after incubation, giving a steady-state slice-to-medium ratio of 0.800 6 0.051 ml/g
brain (n 5 6, mean 6 S.E.). Incorporating the apparent
elimination rate constant (5.87 3 1022 6 0.65 3 1022 min21,
fig. 1), and the distribution volume in the brain (0.800 6
0.051 ml/g brain) into equation 2, the apparent BBB efflux
clearance of PAH was calculated to be 46.9 6 3.8 ml/min/g
brain.
By means of the in vivo carotid artery injection technique,
the BBB influx clearances for [3H]PAH, [3H]inulin and [3H]3O-methyl-D-glucose were determined. As shown in table 3,
[3H]3-O-methyl-D-glucose was significantly taken up by the
brain and the brain extraction percentage, i.e., 10.1 6 0.8%,
was very similar to that reported by us and others (Oldendorf
et al., 1982; Kakee et al., 1996). In contrast to the significant
efflux of [3H]PAH from the brain after cerebral microinjection, very limited BBB influx clearance of [3H]PAH was obtained, i.e., 12.1 6 0.9 ml/min/g brain, which was not significantly different from that of [3H]inulin, used as a vascular
space marker (table 3).
1997
1023
Fig. 5. A schematic diagram of the three-compartmental pharmacokinetic model in the BEI study.
caused by elimination from the CSF. Moreover, to assess the
contribution of CSF elimination, we have performed a BEI
study of [3H]PAH under saturated conditions of active efflux
at the BCSFB. No significant difference between the two
conditions was observed for the BEI values at 20 min after
microinjection into Par2 (table 2), which suggests that elimination from the BCSFB is not responsible for the apparent
elimination of [3H]PAH from the ipsilateral cerebrum.
PAH has been known to exist in its intact form in plasma
after intravenous administration, but excreted into urine,
80% as the intact form and 20% as an acetylated metabolite,
which indicates that PAH is partly transformed to acetylated
PAH in the kidney (Elbourne et al., 1990). However, there is
no information on the metabolism of PAH in the brain. No
major metabolite was found in the ipsilateral cerebrum, ipsilateral and contralateral carotid veins up to at least 20 min
after administration, which shows that PAH was effluxed
from the brain at the BBB in its intact form.
Recently, several reports have shown asymmetrical transport at the BBB and these have used pharmacokinetic model
analysis after i.c.v. injection, cerebroventricular perfusion or
brain microdialysis with model compounds such as valproic
acid, an anti-AIDS agent azidothymidine or quinolone antibiotics, all well known to exhibit limited distribution in the
brain (Wong et al., 1993; Adkison et al., 1994; Wang and
Sawchuk, 1995). Moreover, it has been also found that Pglycoprotein, i.e. the MDR-1 gene product, functions at the
BBB, pumping out several antitumor agents including vincristine and immunosuppressants such as cyclosporin A; this
has been achieved by in vitro uptake experiments with primary brain capillary endothelial cells (Tatsuta et al., 1992;
Tsuji et al., 1992, 1993), an in vivo brain ischemia model
(Sakata et al, 1994; Ohnishi et al, 1995) and the in vivo MDR
1a gene knockout mouse (Schinkel et al., 1994). The influx
rate of PAH has been determined by the intracarotid artery
injection technique and has an extremely low BUI value
similar to that of inulin used as a vascular space marker
(table 3). The apparent PSinf value was calculated to be 12
ml/min/g brain (table 3). Suzuki et al. have already reported
that the relevance of carrier-mediated transport can be detected using the brain perfusion technique for the influx of
benzylpenicillin, an organic anion with an influx clearance of
5.48 ml/min/g brain (Suzuki et al., 1989a). This value is below
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
disappeared rapidly from the ipsilateral cerebrum with an
elimination rate constant of 5.87 3 1022 min21 (fig. 1). Also,
we could not detect the eliminated PAH in the contralateral
cerebrum, cerebellum or CSF (table 1), which indicated that
only a limited amount of [3H]PAH is transferred from the
injection site to the other cerebral regions, and the elimination of [3H]PAH is attributed to efflux from the brain to blood
across the BBB.
Several acidic drugs such as b-lactam antibiotics, including
benzylpenicillin, or valproic acid have been recognized as
substrates for probenecid-sensitive anion transport systems
at the BCSFB, by use of in vivo i.c.v. injection, perfusion or in
vitro uptake techniques involving the isolated choroid plexus
(Suzuki et al., 1987a,b, 1989b, 1996; Adkison et al., 1994;
Ogawa et al., 1994). The elimination clearance of these compounds in the CSF after i.c.v. injection was approximately
22.5 ml/min/rat (Suzuki et al., 1989b), which was 5.6-fold
greater than that of inulin used as a CSF bulk-flow marker.
Similarly, the CSF elimination clearance of PAH was 26.7
ml/min/rat after i.c.v. injection, which was 6.7-fold greater
than the bulk-flow rate (fig. 3), and its elimination profile
was similar to that reported previously (Jakobson, 1987). The
elimination clearance of PAH in the CSF can be considered to
include the bulk-flow rate, active efflux at the BCSFB and
efflux at the BBB after diffusion in the brain interstitial
space. The active efflux clearance of PAH at the BCSFB could
be estimated as 26.7 ml/min/rat at the most. On the other
hand, in [3H]inulin, the apparent elimination clearance from
the CSF, 4.0 ml/min/rat, which was similar to the reported
value, 2.9 ml/min/rat estimated by use of blue dextran (Suzuki et al., 1985), indicated only the CSF bulk-flow rate,
because its diffusion was extremely limited by its high molecular weight, 5000. The elimination of [3H]PAH was significantly reduced by unlabeled 1 mM PAH in the CSF, which
shows that the active efflux system at the BCSFB was significantly inhibited under these conditions (fig. 3). In this
situation, it would be possible to predict the contribution of
BCSFB to the elimination from the brain by the BEI method.
The proposed BEI pharmacokinetic model contained three
compartments, brain, CSF and plasma, and the rate constants among each compartment were defined as follows: k12,
brain to CSF which means the diffusion process in the brain
interstitial space, k10; brain to plasma which means the
efflux process at the BBB; and k20, CSF to plasma which
means the efflux process at the BCSFB (fig. 5). The diffusion
process in the brain interstitial space from CSF to brain was
neglected in this model to give the maximum value to the
rate constant of k12 in the BEI study. The drug amount in the
CSF compartment (X2) after intracerebral administration
can be obtained by the equation as described in figure 5. In
this condition, the summation of k12 and k10 was already
obtained as 0.058 min21 by the BEI study. Similarly, the
maximum k20 value was also obtained as 0.520 min21 by the
initial elimination curve of [3H]PAH up to 5 min in the i.c.v.
injection study. According to the equation as described in
figure 5, the drug amount in the CSF (X2) showed the highest
level at 4.7 min after intracerebral microinjection to the Par2
region. Considering the injected dose (0.2 mCi/rat) and the
detection limit of radioactivity in the CSF, the ratio of k12 to
the summation of k12 and k10 was calculated as 5.2 3 1023.
Accordingly, only 0.52% of the apparent elimination constant, estimated by the BEI technique, was estimated to be
BBB Efflux Transport of PAH
1024
Kakee et al.
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the detection limit of the BUI experiment. Considering that
the apparent BBB efflux clearance of PAH obtained using
BEI and the in vitro uptake technique with brain slices (figs.
1 and 4) was 46.9 ml/min/g brain, the efflux clearance is at
least 3-fold greater than the influx clearance. These results
led us to conclude that there is asymmetrical transport of
PAH across the BBB, with selective transportation from the
brain to blood.
We have measured the partition coefficient (P) of PAH in
octanol/ECF buffer and obtained 8.69 3 1024 as the P value.
Levin (1980) has already reported a good relationship between the parameter given by the octanol/water partition
coefficient divided by the square root of the molecular weight
and rat brain capillary permeability. Based on this report,
the permeability of PAH was calculated to be 2.6 ml/min/g
brain, which was 1/18 the observed efflux clearance. Moreover, the apparent elimination constant obtained by the BEI
method decreased 88% in the presence of 100 mM PAH in the
injectate, which suggests that passive diffusion is not a significant factor in the elimination from the brain. In fact, this
efflux transport showed the saturation with the Km value of
396 mM (fig. 2). This value was very similar to the Km value
for the transport of PAH in the kidney (Hori et al., 1993). As
yet, only MDR1 has been reported to pump out several agents
from the brain to blood at the BBB (Tsuji et al., 1992;
Schinkel et al., 1994). However, because it is generally believed that the substrates for MDR 1 seem to be cationic and
hydrophobic in nature, one could assume that non-P-glycoprotein efflux transport systems are responsible for the efflux
of PAH from the brain to blood across the BBB.
In a previous report, we demonstrated the validity of the
BEI method, with [3H]water and [3H]3-O-methyl-D-glucose,
the former used as a substrate representing blood flow limited elimination and the latter a substrate representing symmetrical elimination at the BBB (Kakee et al., 1996). This
sensitive method allowed us to obtain an accurate efflux rate
for the test substances. With the BEI method, we have demonstrated the asymmetrical transport of PAH across the
BBB. It is important to identify the efflux transport mechanisms of PAH across the BBB and clarify the difference
between the putative efflux system at the BBB and the
organic anion transport systems in the kidney.
In conclusion, the present study provides direct in vivo
evidence that the BBB selectively transports PAH, an organic anion, from brain to circulating blood.
Vol. 283
1997
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BBB Efflux Transport of PAH
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Send reprint requests to: Yuichi Sugiyama, Ph.D., Professor, Department of
Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113, Japan.
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