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TRANSPORT OF THE
b
-LACTAM ANTIBIOTIC BENZYLPENICILLIN AND THE DIPEPTIDE
GLYCYLSARCOSINE BY BRAIN CAPILLARY ENDOTHELIAL CELLS IN VITRO
MICHAEL T ¨
OR ¨
OK, J ¨
ORG HUWYLER, J ¨
URGEN DREWE, HEIKE GUTMANN, AND GERT FRICKER
Departments of Research and Clinical Pharmacology, University Hospital (Kantonsspital and Childrens Hospital) (M.T., J.H., J.D., H.G.), Basel,
Switzerland, and Institute for Pharmaceutics and Biopharmacy (G.F.), Heidelberg, Germany
(Received March 9, 1998; accepted June 24, 1998)
This paper is available online at http://www.dmd.org
ABSTRACT:
Peripherally administered
b
-lactam antibiotics, which are struc-
tural analogs of tripeptides, may cause neurotoxic reactions or
induce seizures. Previous in vivo studies provided evidence for
brain uptake of these antibiotics. In the present work, we studied
the extent and mechanism of the uptake of benzylpenicillin and
glycylsarcosine by brain microvessel endothelial cells in vitro, us-
ing freshly isolated and cultured porcine brain capillary endothelial
cells. Characterization of the cell culture model demonstrated the
functional expression of the system transporting the neutral amino
acids leucine and phenylalanine. The initial rate of uptake of ben-
zylpenicillin was >3-fold greater than the rate of uptake of the
extracellular marker sucrose (ratio, 3.29 60.37), whereas uptake of
glycylsarcosine did not differ from that of sucrose. The differences
in cellular uptake correlated with the octanol/buffer partition co-
efficients for glycylsarcosine and benzylpenicillin (1.16 310
23
for
glycylsarcosine and 6.83 310
22
for benzylpenicillin). The concen-
tration-dependent uptake of benzylpenicillin (1–2000
m
M) was not
saturable and was not sensitive to shifts in pH or temperature. The
permeability-surface area product for the uptake of benzylpenicil-
lin at pH 7.4 was determined from these experiments and was
found to be 8.1 310
25
ml/sec/g of brain. This value was very close
to the value determined in in vivo studies. Uptake of benzylpeni-
cillin and glycylsarcosine was not reduced in the presence of 1 mM
ceftibuten or 100
m
M probenecid. The findings with cultured cell
monolayers were confirmed using freshly isolated endothelial
cells. These in vitro data are compatible with benzylpenicillin, but
not glycylsarcosine, being able to penetrate endothelial cells. Up-
take of benzylpenicillin by brain capillary endothelial cells occurs
by a slow nonsaturable process, with no evidence for carrier-
mediated transport.
Knowledge of drug disposition is an essential prerequisite for
estimating drug effectiveness. It is of particular importance when
drugs must cross epithelial or endothelial barriers to exert their actions
at the desired target sites. Antibiotics must cross the blood-brain
barrier to be suitable for use in the treatment of severe cerebral
infections such as bacterial meningitis. However, reports on the
blood-brain barrier permeation of
b
-lactam antibiotics and/or small
peptides are controversial. On one hand, small peptides seem to cross
the blood-brain barrier in only negligible amounts, which do not differ
from those of extracellular markers (Himmelseher et al., 1996;
Vasquez et al., 1992). On the other hand,
b
-lactam antibiotics (which
are structural analogs of tripeptides) (Suzuki and Sugiyama, 1994)
seem to penetrate the blood-brain barrier, because central neurotoxic
reactions or the induction of seizures can be caused by peripherally
administered
b
-lactam antibiotics (Grondahl and Langmoen, 1993;
Schliamser et al., 1991; Sunagawa and Nouda, 1996). Using an in situ
brain perfusion technique, a probenecid-sensitive mechanism was
suggested for the facilitated diffusion of some
b
-lactam antibiotics
across the blood-brain barrier (Spector, 1987; Suzuki et al., 1989).
Other reports, using the carotid artery injection technique in rats,
imply carrier-mediated uptake of antibiotics via the monocarboxylate
carrier (Kang et al., 1990).
Using in vivo techniques, such as those described above, the actual
contribution of a certain cell type within the brain microvasculature to
brain penetration of a given drug cannot be determined. The situation
is complicated by the presence of two distinct barriers, i.e. the choroid
plexus and the blood-brain barrier. For instance, it has been shown
that a transport system for benzylpenicillin exists in the rat choroid
plexus (Suzuki et al., 1987b).
It was, therefore, the aim of the present study to determine the
contribution of the brain capillary endothelial cells, which make up
the blood-brain barrier, to the penetration of a
b
-lactam antibiotic (the
tripeptide analog penicillin) and the metabolically stable dipeptide
glycylsarcosine. The present study was carried out in an in vitro
system, using freshly isolated or cultured porcine brain microvessel
endothelial cells (Huwyler et al., 1996). Because benzylpenicillin and
glycylsarcosine are substrates of the dipeptide (oligopeptide) carrier
expressed in small intestine (Dantzig and Bergin, 1988; Kramer et al.,
1992) and kidney (Boll and Daniel, 1995), special attention was paid
to the possible contribution of this carrier system.
Materials and Methods
Chemicals and Reagents. [2-
14
C]Glycyl[1-
14
C]sarcosine (110 mCi/mmol)
was from Movarek Biochemicals (Brea, CA). [phenyl-4-
3
H]Benzylpenicillin
(13.6 Ci/mmol), L-[4,5-
3
H]leucine (67 Ci/mmol), L-[4-
3
H]phenylalanine
This work was supported by the Swiss National Science Foundation (Grant
32–42179.94), a scholarship to M.T. from the Association of Chemical Industries
(Basel, Switzerland), and the ASTRA Research Fund of the Department of Internal
Medicine of the University Hospital (Basel, Switzerland).
Send reprint requests to: Dr. Ju¨ rgen Drewe, University Hospital, Divisions of
Gastroenterology and Clinical Pharmacology, Petersgraben 4, CH-4031 Basel,
Switzerland. E-mail: drewe@ubaclu.unibas.ch
0090-9556/98/2611-1144–1148$02.00/0
DRUG METABOLISM AND DISPOSITION Vol. 26, No. 11
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
1144
at Univ of Heidelberg on October 11, 2013dmd.aspetjournals.orgDownloaded from
(15 Ci/mmol), [6,69(N)-
3
H]sucrose (16.6 Ci/mmol), and [U-
14
C]sucrose (626
mCi/mmol) were from Amersham (Buckinghamshire, UK). Ceftibuten was a
gift from Essex Chemie (Luzern, Switzerland). Probenecid was from Sigma
Chemical Co. (St. Louis, MO). All other chemicals used were of the highest
quality available.
Determination of PC
1
Values. Radioactively labeled benzylpenicillin and
glycylsarcosine (0.3
m
Ci) were dissolved in 300
m
l of 100 mM phosphate
buffer, pH 7.4. The solution was added to the same volume of 1-octanol
(Merck, Darmstadt, Germany), and this mixture was equilibrated overnight at
room temperature before centrifugation for 30 min. The concentration of the
tracer in each phase was determined by liquid scintillation counting.
Microvessel Endothelial Cell Isolation. Primary cultures of porcine brain
capillary endothelial cells were prepared as described previously (Huwyler et
al., 1996). Briefly, cortical gray matter from six fresh porcine brains was
minced and digested enzymatically using 0.5% dispase. Cerebral microvessels
were obtained after centrifugation in 13% dextran and were subsequently
incubated in buffer containing 1 mg/ml collagenase/dispase. The resulting cell
suspension was supplemented with 10% horse serum and filtered through
150-
m
m nylon mesh, and brain capillary endothelial cells were separated on a
continuous 50% Percoll gradient (Pharmacia, Uppsala, Sweden). Isolated
endothelial cells were filtered through 35-
m
m nylon mesh before being seeded,
at a density of 150,000 cells/cm
2
, onto collagen/fibronectin (Boehringer-
Mannheim)-coated, 24-well, cell culture plates. Cells were cultured under
standard cell culture conditions [cell culture medium consisting of 45% min-
imal essential Eagle medium, 45% Ham’s F-12 nutrient mixture, 100
m
g/ml
streptomycin, 100
m
g/ml penicillin G, 100
m
g/ml heparin, 13 mM NaHCO
3
,
and 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (all from Sig-
ma), with 10% heat-inactivated horse serum (Gibco BRL, Basel, Switzer-
land)].
Morphological and Biochemical Characterization of Freshly Isolated
Cells and 10-Day-Old Cell Cultures. At day 10 of growth, all cell monolay-
ers used for transport experiments exhibited spindle-shaped cells (typical for
primary cultures of capillary endothelial cells) (Audus and Borchardt, 1986).
Contamination by cells of other shapes was rare, but cultures containing them
were rejected. Cultured endothelial cells expressed enzymatic markers such as
angiotensin-converting enzyme (EC 3.4.15.1.) (29.6 64.4 nmol/mg/min) and
alkaline phosphatase (EC 3.1.3.1) (549.8 634.5 nmol/mg/min). In addition,
the expression of the tight junctional marker protein ZO-1 by the cell cultures
could be shown by immunostaining (Huwyler et al., 1996). Tight junctions
play an important role in maintaining the integrity of the blood-brain barrier;
therefore, the expression of the zona occludens protein ZO-1 at confluency is
evidence of the formation of a structurally intact and polarized monolayer. This
has been demonstrated for several endothelial and epithelial cell layers in vitro
and in vivo,e.g. Madin-Darby canine kidney and endothelial cells (Smith and
Shine, 1992; Staddon et al., 1995).
Uptake Assays. Uptake assays were performed at 20°C, using 10-day-old
confluent monolayers of porcine brain capillary endothelial cells. Cells were
grown in 24-well cell culture plates. The surface area was 2 cm
2
/well. Cells
were washed using transport buffer [142 mM NaCl, 3 mM KCl, 1.4 mM CaCl
2
,
1.2 mM MgCl
2
, 1.5 mM K
2
HPO
4
, 4 mM D-glucose, 10 mM 4-(2-hydroxy-
ethyl)piperazine-1-ethanesulfonic acid, pH 6.5 or, where indicated, pH 7.4].
The reaction was initiated by addition of 250
m
l of transport buffer containing
0.3
m
Ci of
3
H- or
14
C-labeled tracer for the respective substrate, sufficient
unlabeled substrate or inhibitor to bring the medium to the desired final
concentration, and 0.3
m
Ci of the labeled extracellular marker sucrose. Incu-
bations were terminated after 2 min ([
3
H]benzylpenicillin, [
3
H]leucine, or
[
3
H]phenylalanine) or 5 min ([
14
C]glycylsarcosine) by rapid removal of the
incubation medium by aspiration, followed by washing of the cells using
ice-cold transport buffer. Incubations in the presence of inhibitors were per-
formed with 1 mM ceftibuten or 0.1 mM probenecid. Cells were then detached
from the wells by incubation with trypsin (0.25%) for 10 min and were
transferred to scintillation vials. The amount of radiolabeled substrate taken up
by the cells was determined by scintillation counting.
Uptake in freshly isolated brain capillary endothelial cells was assayed at
20°C. The viability of the cells was determined by trypan blue exclusion. Only
cell populations with a viability of .95% were used for additional experi-
ments. Total cell numbers were measured with a hemocytometer, and 2.0 3
10
6
cells were used in each incubation. The reaction was initiated by mixing
100
m
l of cell suspension in transport buffer with 50
m
l of transport buffer
containing substrate (0.3
m
Ci of [
3
H]benzylpenicillin or [
14
C]glycylsarcosine),
an extracellular marker (0.3
m
Ci of
14
C- or
3
H-labeled sucrose), and, when
indicated, the inhibitor ceftibuten at a final concentration of 0.66 mM. The
cells were incubated for 2 min ([
3
H]benzylpenicillin) or 5 min ([
14
C]glycyl-
sarcosine) on a rotary shaker. One hundred microliters of the cell suspension
were then transferred to microcentrifuge tubes containing 50
m
lof3MKOH
and 150
m
l of silicone oil (1:1, v/v, mixture of silicone oil types Ar20 and
Ar200; Wacker Chemie, Mu¨nchen, Germany). The cells were immediately
centrifuged in a tabletop microfuge (Hettich, Tuttlingen, Germany) capable of
rapid acceleration. The centrifugation tubes were then transferred to liquid
nitrogen. The amount of tracer taken up was quantified by cutting the frozen
centrifugation tube just above the KOH/oil interface and placing the tip of the
tube (with the cell pellet layer) in a scintillation vial containing 500
m
lof
Solutron tissue solubilizer (Kontron, Zu¨rich, Switzerland). Samples were in-
cubated overnight, sonicated in a water bath sonicator, and neutralized using
70
m
l of glacial acetic acid. The amount of radiolabeled substrate in the pellet
layer was determined by scintillation counting.
Metabolic Stability. The radiochemical purity of tracers, as determined by
the supplier using HPLC analysis, was 98.9% for [
14
C]glycylsarcosine and
97.5% for [
3
H]benzylpenicillin. The metabolic stability of [
3
H]benzylpenicil-
lin was examined by TLC (Spector, 1986; Suzuki et al., 1987a). Samples were
spotted on a silica gel plate (precoated silica gel 60; Merck, Darmstadt,
Germany) and then developed in a solvent system of methanol/isopropanol
(7:3, v/v). The locations of unlabeled benzylpenicillin and its possible metab-
olites were determined under UV light. An autoradiograph of the silica gel
plate was used to locate isotope-labeled benzylpenicillin and possible radio-
labeled metabolites. During a 2-min incubation of the substrate with cells,
there was negligible formation of metabolites.
Data Analysis. To represent data and to obtain estimates of kinetic param-
eters, a nonlinear regression program was used (Microcal Origin, version 3.5;
Origin, Microcal Software, Inc., Northampton, MA). For statistical compari-
son, data of groups were compared by analysis of variance. The level of
significance was p50.05. If this analysis revealed significant differences,
pairwise comparisons within groups were performed with two-sided unpaired
ttests. The pvalues were adjusted by Bonferroni’s correction for multiple
comparisons.
Results
Structural and Functional Characterization of the In Vitro Cell
Culture System. It was our goal to study the contribution of brain
microvessel endothelial cells to brain uptake of
b
-lactam antibiotics,
with benzylpenicillin and glycylsarcosine as model compounds, and
to correlate the in vitro data with previous in vivo findings. Primary
cultures of porcine brain capillary endothelial cells were used as an in
vitro system. This system was first characterized to establish its
suitability.
Microscopic inspection of the cultured cells clearly demonstrated a
homogeneous cell population. Biochemical and immunochemical
characterization demonstrated the preservation of important charac-
teristics in isolated and cultured brain endothelial cells, such as
endothelial marker enzymes and the zona occludens-associated pro-
tein ZO-1 (see Materials and Methods), thus confirming our own
recent data (Huwyler et al., 1996, 1997). In this report, validation of
the model was extended to functional assays, to demonstrate the
intactness of a carrier system (the L-system of neutral amino acid
transport). Initial rates of uptake of leucine and phenylalanine as
functions of their concentrations in the incubation mixture were
determined. The overall uptake of leucine consisted of two compo-
nents, i.e. a linear term representing passive diffusion and a second
term representing a specific transport system. The latter was saturable
and showed Michaelis-Menten-type kinetics, with a K
M
of 19
m
M and
1
Abbreviations used are: PC, octanol/buffer partition coefficient; PS, perme-
ability-surface area.
1145ENDOTHELIAL UPTAKE OF PEPTIDES
aV
max
of 6.9 pmol/min/cm
2
. The parameters for uptake of phenylal-
anine were determined in a separate set of experiments; the K
M
was 40
m
M and the V
max
was 12 pmol/min/cm
2
. When the uptake of leucine
was measured in the presence of increasing concentrations of phenyl-
alanine, which shares the leucine carrier in other cell types (Olden-
dorf, 1971b), transport was apparently competitively inhibited, with a
K
i
of 13
m
M (fig. 1B). Here again, only specific transport could be
inhibited, resulting in a reduction of leucine uptake by a maximum of
70%; the remainder represents passive diffusion. In these experi-
ments, sucrose was used as an extracellular marker. Cell-associated
sucrose was found to be minimal, and ,0.07% of the applied dose of
sucrose was typically recovered. This value did not change with
incubation time (determined for incubation times ranging from 1 to 30
min).
Uptake of Benzylpenicillin and Glycylsarcosine by Cultured
Brain Microvessel Endothelial Cells. The initial rates of uptake of
the peptide analog benzylpenicillin and the dipeptide glycylsarcosine
were measured with confluent monolayers of cultured porcine brain
capillary endothelial cells. The cells were incubated with the respec-
tive drug on the luminal side, which corresponds to the blood-directed
cell surface in vivo. The initial rates of uptake were directly compared
with the uptake of the extracellular marker sucrose. The benzylpeni-
cillin/sucrose uptake ratio was 3.29 60.37 (N510, mean 6SE, pH
6.5). The glycylsarcosine/sucrose uptake ratio was 0.74 60.03 (N5
10, pH 6.5). Thus, cellular uptake of benzylpenicillin was .3-fold
greater than that of sucrose (statistically significant difference by
Student’s ttest, p,0.001), whereas that of glycylsarcosine did not
differ from the corresponding sucrose value (p50.39). These dif-
ferences in cellular uptake correlated with differences in the lipophi-
licity of these compounds. The PC of benzylpenicillin was .1-log
unit greater than that of sucrose (log PC of [
14
C]sucrose 522.971 6
0.092, N53; log PC of [
3
H]benzylpenicillin 521.165 60.004, N5
5; p,0.001). The PC of glycylsarcosine (log PC of [
14
C]glycylsar-
cosine 522.935 60.008, N55) equals that of sucrose (p50.62).
To determine whether there was carrier-mediated transport, initial
rates of cellular uptake of benzylpenicillin were determined at pH 7.4
and pH 6.5, over a wide range of concentrations (1–2000
m
M) (fig. 2).
Uptake was linear at pH 7.4, as well as at pH 6.5, over the entire range
of concentrations and could be approximated by linear regression
(coefficients of correlation under the two pH conditions were
.0.999), thus giving no indication for carrier-mediated transport. The
PS product for the uptake of benzylpenicillin at pH 7.4 could be
calculated from the data shown in fig. 2. The slope of the line
represents the PS product and equals 48.9 (pmol/min/cm
2
)/mM. This
corresponds to a PS product of 8.1 310
25
ml/sec/g of brain (assum-
ing a value of 100 cm
2
/g of brain) for the surface area of the
blood-brain barrier in vivo (Pardridge et al., 1990).
Uptake experiments with benzylpenicillin and glycylsarcosine were
also performed at different temperatures and in presence of inhibitors
of the dipeptide carrier, to clarify the potential involvement of this
carrier system (table 1). The cephalosporin antibiotic ceftibuten is a
substrate of the dipeptide carrier (Matsumoto et al., 1995; Sugawara
et al., 1994) and was therefore used as a potential competitive inhib-
itor of benzylpenicillin and glycylsarcosine uptake. Probenecid has
been reported to interact with in vivo brain uptake of benzylpenicillin
(Suzuki et al., 1989). In addition, it was suggested that a probenecid-
sensitive drug efflux system might exist at the blood-brain barrier
(Dykstra et al., 1993; Wong et al., 1992). In the inhibition experi-
ments, the concentrations of ceftibuten and probenecid were 1 and 0.1
mM, respectively. These concentrations correspond to those of pre-
viously performed studies with ceftibuten (Matsumoto et al., 1995) or
probenecid (Suzuki et al., 1987a). Our results indicate that uptake of
benzylpenicillin and uptake of glycylsarcosine at low temperatures
and in the presence of inhibitors of the dipeptide carrier were not
statistically significantly different from controls determined at pH 6.5.
Uptake of Benzylpenicillin and Glycylsarcosine by Freshly Iso-
FIG.1.Functional characterization of cultured brain microvessel endothelial
cells.
A, Carrier-mediated, and therefore saturable, uptake of leucine by endothelial
cells (E), shown as a function of increasing substrate concentration. This curve
represents the difference between the overall uptake of leucine (f) and the calcu-
lated simple diffusion term (dashed line). B, Competitive inhibition of leucine
uptake by cultured brain endothelial cells in the presence of different concentrations
of phenylalanine. Data points are means 6SE (N58).
FIG.2.Concentration-dependent uptake of benzylpenicillin by brain microvessel
endothelial cells at pH 6.5 and pH 7.4.
Inset, magnification of the first section of the graph. Data points represent
means 6SE (N54).
1146 TO¨RO¨KET AL.
lated Brain Capillary Endothelial Cells. The uptake of benzylpen-
icillin and glycylsarcosine was measured in freshly isolated brain
capillary endothelial cells (table 2) to exclude the possibility of a
regulatory effect of the culture conditions on the expression of carrier
proteins by brain capillary endothelial cells. These experiments con-
firmed the results obtained with confluent monolayers, in that the ratio
between the uptake of benzylpenicillin and the uptake of sucrose was
4.41 60.47 (N55, pH 6.5, p,0.001). The ratio between the uptake
of glycylsarcosine and the uptake of sucrose was 0.89 60.01 (N55,
p50.78). Furthermore, the uptake of benzylpenicillin and glycylsar-
cosine in the presence of ceftibuten was not statistically significantly
different from control values (p.0.1).
Discussion
b
-Lactam antibiotics are used for the treatment of central nervous
system infections, although their therapeutic use is often hampered by
the low blood-brain barrier permeability of many common antibiotics.
There is still controversy regarding how the transfer of
b
-lactam
antibiotics is mediated. Central nervous system side effects have been
clearly observed for some of these drugs (Schliamser et al., 1991), and
there is evidence from in vivo studies showing that
b
-lactam antibi-
otics may penetrate the brain (Matsushita et al., 1991; Spector, 1987).
However, in vitro data describing the mechanism of transport in more
detail are lacking. Therefore, the uptake of benzylpenicillin and the
dipeptide glycylsarcosine was characterized using an in vitro model of
the blood-brain barrier that consists of cultured or freshly isolated
porcine brain capillary endothelial cells. Furthermore, we correlated
our in vitro findings with previous in vivo studies on brain uptake of
b
-lactam antibiotics, to obtain information on the predictive ability of
our cell culture model.
The
b
-lactam antibiotic benzylpenicillin is a derivative and struc-
tural analog of a tripeptide and shares the uptake system for di- and
tripeptides in the intestine and in the kidney (Ganapathy et al., 1995).
Glycylsarcosine, an hydrolysis-resistant dipeptide, was used as a
second substrate. This compound was used previously to characterize
the peptide carriers PEPT 1 and PEPT 2 in intestine and kidney
(Ganapathy et al., 1995; Thwaites et al., 1993; Tomita et al., 1995).
Glycylsarcosine is hydrophilic and has a low PC. Its uptake was
marginal and even lower than the uptake of the extracellular marker
sucrose. In contrast, the uptake of benzylpenicillin, which has a
considerably higher PC than sucrose, was .3-fold greater than the
endothelial uptake of sucrose. This in vitro finding parallels in vivo
observations where, using a single-pass brain-uptake technique, pen-
icillin was shown to penetrate from the blood into rat brain, although
the rate of transport was low (Oldendorf, 1971a).
From the concentration-dependent uptake experiment with ben-
zylpenicillin, the PS product was determined to be 8.1 310
25
ml/sec/g of brain. This in vitro value is very close to the in vivo PS
product of 9 310
25
ml/sec/g of brain that was determined using a
brain perfusion technique, at a substrate concentration where transport
was most efficient (Suzuki et al., 1989). Thus, our results, which were
obtained using a cell culture system, are in very good agreement with
in vivo data.
Experiments were carried out to determine whether the uptake of
benzylpenicillin or glycylsarcosine might be mediated by a carrier
system. Special attention was thus given to a possible involvement of
the dipeptide carrier system. Concentration-dependent uptake of ben-
zylpenicillin by brain endothelial cells showed no saturation and was
not temperature sensitive, indicating that endothelial uptake of ben-
zylpenicillin represents simple diffusion. In addition, the uptake of
benzylpenicillin was not stimulated by acidification of the incubation
medium to pH 6.5, which is in contrast to the pH-sensitive stimulation
of transport by the intestinal and renal peptide carriers (Ganapathy et
al., 1995; Thwaites et al., 1993). Also, the uptake of glycylsarcosine
TABLE 2
Uptake of benzylpenicillin and glycylsarcosine by freshly isolated brain capillary endothelial cells
Tracer Condition Tracer/Sucrose Ratio NpValue
a
% of control
[
3
H]Benzylpenicillin pH 6.5 100
b
5
Ceftibuten (0.66
m
M), pH 6.5 139.6 65.4 5 NS
[
14
C]Glycylsarcosine pH 6.5 100
b
5
Ceftibuten (0.66
m
M), pH 6.5 97.9 61.6 5 NS
Values are means 6SE of Nexperiments.
a
By two-tailed Student’s ttest. NS, statistically not significant (p.0.05, adjusted by Bonferroni’s correction for multiple comparisons).
b
Control value, which was defined as 100%.
TABLE 1
Uptake of benzylpenicillin and glycylsarcosine under different incubation conditions
Tracer Condition Tracer/Sucrose Ratio NpValue
a
% of control
[
3
H]Benzylpenicillin pH 6.5 100
b
4
pH 7.4 88.5 64.5 4 NS
Ceftibuten (1 mM), pH 6.5 166.7 627.8 10 NS
Probenecid (100
m
M), pH 6.5 88.3 67.6 4 NS
4°C, pH 6.5 88.1 62.7 4 NS
4°C, pH 7.4 94.6 65.2 4 NS
[
14
C]Glycylsarcosine pH 6.5 100
b
4
pH 7.4 95.9 62.8 4 NS
Ceftibuten (1 mM), pH 6.5 87.3 65.3 6 NS
Probenecid (100
m
M), pH 6.5 95.7 61.3 4 NS
4°C, pH 6.5 103.2 61.3 4 NS
4°C, pH 7.4 102.1 61.9 4 NS
Values are means 6SE of Nexperiments.
a
By two-tailed Student’s ttest. NS, statistically not significant (p.0.05). The pvalues were adjusted by Bonferroni’s correction for multiple comparisons.
b
Control value, which was defined as 100%.
1147ENDOTHELIAL UPTAKE OF PEPTIDES
at pH 7.4 showed no statistically significant difference from the
uptake at pH 6.5. To assess whether uptake of benzylpenicillin and
glycylsarcosine could be inhibited by another substrate of the dipep-
tide carrier, we used the cephalosporin antibiotic ceftibuten (Matsu-
moto et al., 1995; Saito et al., 1995). Ceftibuten caused no statistically
significant reduction of uptake. However, there may be differences
between swine and other species in the transport of benzylpenicillin
and ceftibuten.
Although in vitro systems have repeatedly been shown to accu-
rately predict the blood-brain barrier permeability found in vivo (Au-
dus and Borchardt, 1986; Dehouck et al., 1992; this study), a possible
underestimation of carrier-mediated transport using in vitro systems
may be attributed to the loss of expression of carrier proteins in
endothelial cell culture (Pardridge et al., 1990). Biochemical and
functional characterization of the cell culture model used suggested,
however, that typical attributes of capillary endothelial cells were
maintained. Carrier systems such as the amino acid transporter and
P-glycoprotein (Huwyler et al., 1996) were functionally active. To
exclude the possibility of the loss of carrier activity during the culture
period, uptake of benzylpenicillin and glycylsarcosine was also de-
termined using freshly isolated brain capillary endothelial cells. The
rates of uptake of benzylpenicillin and glycylsarcosine were very
similar to the values obtained using endothelial cells in culture. Here
again, incubations with and without the inhibitor ceftibuten were not
significantly different.
Endothelial uptake of benzylpenicillin and glycylsarcosine was not
affected by the presence of the organic anion probenecid. Therefore,
it is unlikely that carrier-dependent uptake was masked by an active
drug efflux system in brain endothelial cells, as was recently sug-
gested for zidovudine (39-azido-39-deoxythymidine) (Dykstra et al.,
1993; Wong et al., 1992). It is important to note that our in vitro
model offers the advantage of direct access to the brain capillary
endothelial cells, thus avoiding interference with other structures of
the brain. This could be important in the present case, because
penicillins and some cephalosporins are known to be accumulated in
the choroid plexus by a probenecid-sensitive active transport system
(Spector, 1987). Subsequent animal studies demonstrated probenecid
inhibition of penicillin efflux from the cerebrospinal fluid (Suzuki et
al., 1987a). Thus, the slow entry of penicillins and cephalosporins in
the brain is counteracted by a vigorous transport system in the choroid
plexus, which transfers these agents from the cerebrospinal fluid into
the blood. It is tempting to speculate that this mechanism could
impede brain penetration of other antibiotics, such as cephalexin
(Sakane et al., 1991).
In summary, the present study provides a very good correlation
between in vitro results obtained with cultured capillary endothelial
cells and in vivo data obtained previously. The cell culture model can
be used to study transport processes separate from other structures in
the brain. The data show that benzylpenicillin, but not glycylsar-
cosine, is able to penetrate endothelial cells by simple diffusion. There
was no indication for carrier-mediated transport of benzylpenicillin by
porcine brain capillary endothelial cells.
Acknowledgments. We thank U. Behrens for excellent technical
assistance and Dr. J. Baumann for critical revision of the manuscript.
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1148 TO¨RO¨KET AL.
