0090-9556/03/3110-1251–1254$7.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
DMD 31:1251–1254, 2003
Vol. 31, No. 10
1124/1098232
Printed in U.S.A.
EFFECT OF P-GLYCOPROTEIN-MEDIATED EFFLUX ON CEREBROSPINAL
FLUID/PLASMA CONCENTRATION RATIO
TOMOYUKI OHE, MASAHIKO SATO, SACHIKO TANAKA, NAOKO FUJINO, MIKIKO HATA, YOSHIHIRO SHIBATA,
AKIO KANATANI, TAKEHIRO FUKAMI, MASAYO YAMAZAKI, MASATO CHIBA, AND YASUYUKI ISHII
Banyu Tsukuba Research Institute, Ibaraki , Japan (T.O., M.S., S.T., N.F., M.H., Y.S., A.K., T.F., M.C., Y.I.); and Merck Research Laboratories,
Merck & Co., Inc., West Point, Pennsylvania (M.Y.)
(Received March 18, 2003; accepted July 7, 2003)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
transport activity of the 20 compounds with P-gp (mdr1a)-transfected LLC-PK1 cells and calculated P-gp efflux index (PEI), indicating the extent of P-gp-mediated transport. A plot of the CSF/
plasma versus fp/PEI showed a strong correlation (r ⴝ 0.93), and
the absolute values were almost identical [CSF/plasma ⴝ fp/PEI].
These results suggest that P-gp quantitatively shifts the equilibrium of unbound drugs across the BBB. Although we cannot rule
out the possibility that endogenous transporters other than P-gp
on BBB and/or blood-CSF barrier may affect CSF levels of compounds, the present study indicated that fp and PEI measurements
may be useful in predicting in vivo CSF/plasma fractions for central
nervous system-targeting drugs.
Since cerebrospinal fluid (CSF1) is a very low protein fluid, and drug
in CSF is considered to be almost unbound, the ratio of drug concentration in CSF to plasma (CSF/plasma) in an equilibrium state has been
viewed as in vivo free fraction in plasma (fp) (CSF/plasma ⫽ fp), if no
active transport is involved in brain penetration (Lin and Lu, 1997).
Consistent with this notion, it has been reported that the in vitro free
fraction of phenytoin in serum (0.155) is almost equal to the CSF/serum
drug ratio (0.185) (Chou and Levy, 1981), and the in vitro fp of demethylchlorimipramine (0.035) is similar to the CSF/plasma ratio (0.026)
(Bertilsson et al., 1979). Furthermore, equilibrium CSF/plasma ratios of
eight benzodiazepines are known to be highly correlated with fp (r ⫽
0.93, regression line slope ⫽ 0.98) (Arendt et al., 1983). These results
suggest that in vitro fp may accurately reflect CSF/plasma. However,
CSF/plasma can be lower than fp, if certain active transporters such as
P-glycoprotein (P-gp) are involved in brain penetration.
P-gp is an ATP-dependent efflux pump that transports a variety of
amphiphilic and hydrophobic drugs and plays a major role in multidrug resistance in cancer cells. P-gp is also expressed in normal
tissues, including the apical membranes of intestinal and renal epithelia and the endothelial cells of the blood-brain barrier. The putative
function of P-gp in normal tissues is to act as a functional barrier for
endogenous and exogenous products that may be toxins or carcinogens. A more interesting function of this protein is the expression in
the brain, especially on the endothelium of capillary blood vessels,
which makes up so-called “blood-brain barrier” (BBB) that limits the
entry of many compounds into the brain.
In this way, P-gp seems to be one of the most important transporters
responsible for lower CSF/plasma ratio than expected from fp. In fact,
the HIV-1 protease inhibitor, indinavir, which is a well known P-gp
substrate (Kim et al., 1998), showed much lower CSF/plasma (Stahle
et al., 1997; Letendre et al., 2000; Zhou et al., 2000) than in vitro fp
(Vacca et al., 1994). In addition, CSF concentrations of quinidine, a
typical P-gp substrate (Kusuhara et al., 1997), in human subjects were
lower than unbound levels in serum and plasma (Ochs et al., 1980;
Sindrup et al., 1996).
It is considered that there are many cases other than indinavir and
quinidine where CSF/plasma is lower than fp, because quite a few
drugs are known to be P-gp substrates. In the present study, we
hypothesized that the lower CSF/plasma ratio can be quantitatively
explained by P-gp-mediated transport. To prove the hypothesis, we
chose 20 compounds that were synthesized in our institute and have
similar physicochemical properties, and examined their P-gp-mediated transport with P-gp (mdr1a)-transfected LLC-PK1 cells as well
as their CSF/plasma ratio and plasma protein binding in rats.
1
Abbreviations used are: CSF, cerebrospinal fluid; fp, free fraction in plasma;
P-gp, P-glycoprotein; BBB, blood-brain barrier; mdr1a, multidrug resistance 1a
P-gp; B, basolateral; A, apical; PEI, P-gp efflux index.
Address correspondence to: Tomoyuki Ohe, Banyu Tsukuba Research Institute, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan. E-mail: [email protected]
Materials and Methods
Chemicals. Vincristine was purchased from Sigma-Aldrich (St. Louis,
MO). Compounds A to T were synthesized at Banyu Tsukuba Research
Institute. (We cannot show the chemical names of compounds A to T in this
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The ratio of drug levels in cerebrospinal fluid (CSF) to plasma
(CSF/plasma) at equilibrium has been viewed as in vivo free fraction (fp) in plasma [CSF/plasma ⴝ fp], if no active transport is
involved in brain penetration. We determined the CSF/plasma level
following oral administration in rats and in vitro rat plasma protein
binding for 20 compounds that were synthesized in our institute
and have similar physicochemical properties. However, results
indicated that the CSF/plasma was not only poorly correlated with
fp but remarkably lower than fp in most of the compounds tested,
suggesting that certain transporters such as P-glycoprotein (P-gp)
located in blood-brain barrier (BBB) may decrease the unbound
drug concentration in the brain. We evaluated P-gp-mediated
1252
OHE ET AL.
TABLE 1
Calculated properties of compounds tested in the present study
Name
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
cLogP
Number
of HBD
Number
of HBA
vdw
Volume
366.5
456.5
350.3
373.4
400.4
400.4
418.4
388.4
424.4
388.4
388.4
390.4
388.4
388.4
406.4
406.4
433.6
449.6
366.4
396.4
3.4
3.1
0.9
2.6
2.2
2.0
2.2
2.3
2.2
1.7
1.7
3.3
1.7
1.7
2.0
2.0
3.9
2.7
2.6
3.7
0
1
1
1
1
1
1
2
1
1
1
2
1
1
1
1
1
2
2
1
4
8
5
4
7
7
7
7
7
7
7
5
7
7
7
7
5
6
5
6
511
609
433
477
531
531
534
510
519
512
512
500
512
512
515
515
633
641
465
532
cLogP, calculated logP; HBD, hydrogen-bond donor; HBA, hydrogen-bond acceptor; vdw
volume, van der Waals volume.
article due to the restriction of the disclosure of their chemical structures.) All
other reagents were of analytical grade.
Cell Lines and Cell Cultures. Human MDR1 transfectants L-MDR1,
mouse mdr1a transfectants L-mdr1a, and their parental cell line LLC-PK1
porcine kidney epithelial cells were kindly provided by Dr. Alfred H. Schinkel
(The Netherlands Cancer Institute, Amsterdam, The Netherlands) and used
under license agreement. Cells were cultured in Medium 199 supplemented
with 1 mM L-glutamine, penicillin (50 units/ml), streptomycin (50 ␮g/ml), and
10% (v/v) fetal calf serum. For L-MDR1 and L-mdr1a, cells were maintained
in the continuous presence of vincristine (640 nM). Confluent monolayers
were subcultured every 3 to 4 days by treatment with 0.25% trypsin/1 mM
EDTA in Ca2⫹- and Mg2⫹-free Hanks’ balanced salt solution. All cultures
TABLE 2
In vivo and in vitro results for compounds tested in the present study
In Vivo
In Vitro
Concentrationa
Name
Plasma Protein Bindingb
PEI (B to A/A to B)c
CSF/Plasma
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
a
b
c
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
Plasma
Brain
CSF
␮M
nmol/mg
␮M
4.1
4.8
3.8
2.0
3.0
3.9
4.0
6.1
3.5
6.5
2.9
1.1
6.7
1.5
6.4
1.1
0.9
0.6
0.7
2.8
2.7
0.6
12.3
4.4
2.9
0.3
0.4
0.5
0.9
2.8
3.1
2.3
0.5
0.4
4.0
1.4
4.4
0.2
0.1
1.1
0.390
0.029
0.228
0.012
0.095
0.126
0.065
0.059
0.058
0.416
0.276
0.057
0.091
0.045
0.310
0.077
0.011
0.003
0.021
0.033
0.096
0.006
0.060
0.006
0.032
0.033
0.016
0.010
0.017
0.064
0.094
0.052
0.014
0.030
0.048
0.071
0.013
0.005
0.030
0.012
% Binding
fp
LLC-PK1
L-MDR1
L-mdr1a
83.0%
93.5%
92.5%
98.8%
96.0%
86.4%
93.6%
85.7%
86.5%
74.7%
87.2%
82.7%
71.7%
83.9%
88.9%
92.1%
88.6%
82.5%
73.7%
97.6%
0.170
0.065
0.075
0.012
0.040
0.136
0.064
0.143
0.135
0.253
0.128
0.173
0.283
0.161
0.111
0.079
0.114
0.175
0.263
0.024
0.9
1.2
1.0
1.0
1.3
1.3
1.1
1.8
1.2
1.3
1.1
1.7
2.3
1.9
1.2
1.1
1.2
n.d.
1.1
1.4
1.2
2.6
2.0
1.2
1.1
1.8
1.7
4.4
2.1
2.0
1.1
1.4
6.1
2.4
1.7
1.2
5.4
21.6
2.7
0.8
1.7
10.6
1.2
2.0
1.1
4.9
5.4
24.5
7.8
6.1
1.7
2.7
21.1
7.1
4.9
1.9
16.2
20.0
15.1
2.8
fp/PEI in
L-MDR1
fp/PEI in
L-mdr1a
0.148
0.025
0.038
0.010
0.037
0.075
0.037
0.033
0.064
0.128
0.122
0.126
0.046
0.069
0.067
0.064
0.021
0.008
0.099
0.030
0.099
0.006
0.063
0.006
0.037
0.028
0.012
0.006
0.017
0.042
0.074
0.063
0.013
0.023
0.023
0.042
0.007
0.009
0.017
0.009
Plasma, brain, and CSF levels were determined at 2 h after oral administration of each compound.
Plasma protein binding was obtained by an ultrafiltration method.
P-gp efflux index (PEI) was calculated as basolateral-to-apical flux/apical-to-basolateral flux (B to A/A to B) in the transcellular transport study with P-gp-transfected cells.
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Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
Compound
mol. wt.
were incubated at 37°C in a humidified atmosphere of 5% CO2/95% air
(Schinkel et al., 1995; Yamazaki et al., 2001).
Transport Studies. Transepithelial transport study was carried out as
described previously (Kim et al., 1998; Yamazaki et al., 2001), with minor
modifications. L-MDR1, L-mdr1a, and LLC-PK1 cells were plated at a density
of 4 ⫻ 105 cells/12-mm well on porous (3.0-␮m) polycarbonate membrane
filters (Transwell; Costar, Cambridge, MA). Cells were supplemented with
fresh media every 2 days and used in the transport studies on the fifth to sixth
day after plating. Transepithelial resistance was measured in each well using a
Millicell ohmmeter (model ERS; Millipore Corporation, Bedford, MA); wells
registering a resistance of 300 ⍀ or greater were used in the transport experiments. About 1 to 2 h before the start of the transport experiments, the
medium in each compartment was replaced with serum-free Hanks’ balanced
salt solution with 10 mM Hepes (pH 7.4). The transport experiment was then
initiated (t ⫽ 0) by replacing the medium in each compartment with 700 ␮l of
transport medium with and without each test compound (1 ␮M). After 3 h,
100-␮l aliquots were taken from the opposite compartment into a 96-well
plate. An equivalent volume of ethanol containing the internal standard was
added to each well and mixed well. Samples were stored at ⫺20°C until use.
Plasma Protein Binding. Binding of compounds to rat plasma was determined by an ultrafiltration method (n ⫽ 3). The compounds were added to
plasma to yield a final concentration of 1 ␮M. After incubation of plasma
samples for 15 min, 0.4 ml of plasma was immediately transferred to a
Centri-free tube (Millipore Corporation). The tube was then centrifuged at
1,500g for 10 min. The unbound fraction of the compound was estimated
directly from the ratio of drug concentration in the ultrafiltrate to the total drug
concentration in the original plasma samples before centrifugation.
Animal Study. Each compound at 10 mg/kg was given by gavage as a
suspension in 0.5% methylcellulose (5.0 ml/kg) to fed male Sprague-Dawley
rats (n ⫽ 3). The rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg), and blood samples were obtained from the abdominal
aorta at 2 h postdose. Plasma was prepared by immediate centrifugation at
2,000g for 15 min at 4°C. CSF was obtained by cisternal puncture of the
atlanto-occipital membrane with a 30-gauge needle that had a 40-cm length of
polyethylene tubing attached to a syringe. From 50 ␮l to 100 ␮l of CSF was
then collected through the tubing into a micro test tube. Collection was
terminated as soon as blood appeared in the tubing and the blood-tainted
P-gp-MEDIATED EFFLUX AND CSF/PLASMA CONCENTRATION RATIO
1253
portion of CSF was prevented from entering the collection. Immediately after
CSF collection, the brain was removed. Plasma, brain, and CSF were stored at
⫺80°C until analysis. All animal care and treatment procedures were approved
by the Banyu Tsukuba Research Institutional Animal Care and Use Committee
prior to initiating these studies.
Sample Preparation. Plasma and CSF samples were extracted with 3
volumes of ethanol containing an internal standard and then centrifuged at
10,000g for 10 min to obtain supernatant. Brain samples were homogenized
with equivalent or 4 volumes of water. An aliquot of homogenates was
deproteinized with 3 volumes of ethanol and then centrifuged at 10,000g for 10
min to obtain supernatant.
Quantification. Quantification was achieved by liquid chromatography/
tandem mass spectrometry with a PerkinElmerSciex (Boston, MA) API 300/
365/3000 triple quadrupole mass spectrometer and a Waters 2690/2790 HPLC
(Waters, Milford, MA) equipped with an Inertsil ODS-3 column (2.1 ⫻ 150
mm, 5 ␮m; GL Sciences Inc., Tokyo, Japan). Isocratic elution of the mobile
phase was carried out with 50 to 80% acetonitrile containing 10 mM ammonium acetate, at a flow rate of 0.2 ml/min. TurboIonSpray was used as an ion
source. The turbo probe was set at 425°C, with the nebulizing gas pressure and
turbo gas flow set at 50 p.s.i. and 7.0 l/min, respectively. Other mass spectrometric conditions such as orifice potential were optimized for each compound.
Detection of all compounds was carried out by multiple reaction monitoring,
whereby the first quadrupole (Q1) transmitted the [M ⫹ H]⫹ ions of the
compound. After collision-induced dissociation of the [M ⫹ H]⫹ ion in the
second quadrupole (Q2), an appropriate product ion was selected in the third
quadrupole (Q3).
Results and Discussion
We chose 20 compounds for this study. The calculated properties of
the compounds are shown in Table 1. Molecular weight ranges from
350 to 450 and calculated logP ranges from 0.9 to 3.9, which indicates
that the compounds tested are relatively lipophilic and have suitable
molecular weight for good membrane permeability. We determined
CSF and plasma levels at 2 h after oral administration in rats and
calculated CSF/plasma concentration ratio for the 20 compounds
(Table 2). Rat plasma protein binding was also evaluated with an
ultrafiltration method to obtain in vitro fp (Table 2).
Results indicated that the CSF/plasma was not only poorly correlated with fp but remarkably lower than fp in most of the compounds
tested (Fig. 1), suggesting that certain transporters such as P-gp
FIG. 2. A plot of in vivo CSF/plasma concentration ratio versus in vitro fp/PEI.
PEI (P-gp efflux index) was calculated as the ratio of basolateral-to-apical
permeability versus apical-to-basolateral permeability in the monolayer efflux assay. a, PEI in L-mdr1a was used; b, PEI in L-MDR1 was used.
located in BBB may decrease the unbound drug concentration in the
brain extracellular cerebral fluid, which is in equilibrium with CSF.
We examined the extent of P-gp-mediated transport by using the
polarized pig kidney epithelial cell line LLC-PK1 and L-mdr1a, a
subclone stably transfected with mouse P-gp (mdr1a) cDNA (Table
2). For most of the compounds, the basolateral-to-apical (B-to-A) flux
in L-mdr1a exceeded that in the opposite direction, whereas no
B-to-A-directed transport was observed in the parental LLC-PK1
cells. From this experiment, P-gp efflux index (PEI) was calculated as
B-to-A flux/A-to-B flux. PEI is the relative rate of polarized transport
of each compound; therefore, the value might represent an equilibrium
constant across P-gp-expressing cell monolayers at equilibrium.
A plot of the CSF/plasma versus fp divided by PEI in L-mdr1a
showed an excellent correlation (r ⫽ 0.93), and the absolute values
were almost identical (regression line slope ⫽ 1.0) (Fig. 2a). These
results demonstrate that P-gp quantitatively shifts the equilibrium of
unbound drugs across the BBB. In contrast, the division by PEI in
L-MDR1, a subclone stably transfected with human P-gp (MDR1)
cDNA, did not lead to a good correlation (r ⫽ 0.80), and the slope was
0.55 (Fig. 2b), which implies that the species difference in P-gp
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FIG. 1. A plot of in vivo CSF/plasma concentration ratio versus in vitro fp.
1254
OHE ET AL.
Acknowledgments. We gratefully thank Dr. Alfred H. Schinkel
from The Netherlands Cancer Institute for permission to use MDR1/
mdr1a-transfected LLC-PK1 cell lines. We are grateful to Tomoko
Iguchi, Kazuo Marutsuka, and Yoshio Sawazaki in our institute for
their technical support.
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susceptibility between humans and rats might be greater than that
between mice and rats, at least in the compounds used in this study.
Thus, the greater 1:1 correlation between CSF/plasma and fp/PEI
clearly demonstrates that it is desirable to consider PEI as well as fp
to more precisely predict CSF/plasma.
In conclusion, although we cannot rule out the possibility that
endogenous transporters other than P-gp on BBB and/or blood-CSF
barrier may affect CSF levels of compounds, the present study demonstrated that in vitro measurements of both fp and PEI may be
necessary for predicting in vivo CSF/plasma fractions in an equilibrium state. As described above, the compounds used in this study have
similar lipophilicity and molecular weight. In fact, most of the compounds originate from a lead compound. Therefore, it cannot be
denied that our finding may be true only in a special case. However,
in some cases, our theory should be useful for explaining CSF levels
of various drugs which are known to be related to their pharmacological (side) effects on the central nervous system. Therefore, the
findings in the present study are of great importance for the discovery/
development of central nervous system-targeting drugs.