The Role of Probenecid-Sensitive Organic Acid Transport in

the Pharmacokinetics of N-Methyl-D-Aspartate Receptor

Antagonists Acting at the Glycine
B
-Site: Microdialysis and
Maximum Electroshock Seizures Studies
M. B. HESSELINK,
1,2,3
H. SMOLDERS,
1,2
B. EILBACHER, A. G. DE BOER,
1,4
D. D. BREIMER,
1,4
and W. DANYSZ
Department of Pharmacological Research (M.B.H., H.S., B.E., W.D.), Merz Co., Frankfurt/Main, Germany
Accepted for publication April 23, 1999 This paper is available online at http://www.jpet.org
ABSTRACT
The purpose of the present study was to determine whether the
probenecid-sensitive organic acid transporter is responsible for
the short duration of action of a new group of N-methyl-D-
aspartate receptor glycine
B
-site antagonists, MRZ 2/570,
2/571, and 2/576. A prolongation of their anticonvulsant activity
from 60 to 180 to 240 min, was found in mice after pretreatment
with probenecid (200 mg/kg i.p.). Microdialysis studies in rats
showed that this is likely due to a change in central nervous
system concentrations of these drugs because cotreatment
with probenecid caused an increase in the brain extracellular
fluid half-life (0.5- to 4-fold) and the brain area under the curve
(1.8- to 3.6-fold). In serum the half-life of MRZ 2/576 (30 mg/kg)
was also increased by coadministration of probenecid from
15.6 1.3 to 40.6 6.0 min. At steady state (MRZ 2/576, 20
mg/kg/h i.v.), brain extracellular fluid concentration was ele-
vated 2.5-fold by concomitant administration of probenecid.
These results clearly show that these glycine
B
-site antagonists
are rapidly cleared from the systemic circulation and the central
nervous systemby the probenecid-sensitive organic acid trans-
port system. Moreover, the present data show that MRZ 2/570,
2/571, and 2/576 reach the brain in concentrations (1.342.32
M) above the range of their in vitro potencies at the glycine
site of the N-methyl-D-aspartate receptor (0.11.0 M).
The glutamatergic N-methyl-D-aspartate (NMDA) receptor
complex consists of several distinct binding domains that
allow modulation of NMDA receptor-mediated responses.
One of them that has attracted considerable attention in
recent years is the strychnine-insensitive glycine coagonist
site (glycine
B
-site) (Johnson and Ascher, 1987; Danysz et al.,
1987; Danysz and Parsons, 1998). The term coagonist
stresses that both glutamate and glycine must be bound to
their respective binding sites simultaneously to allow the
NMDA receptor activation. Thus complete inhibition can be
achieved through an antagonistic action at either site (Kleck-
ner and Dingledine, 1988; Danysz and Parsons, 1998).
Antagonists of the NMDA receptor have a potentially wide
variety of therapeutic applications, ranging from neuropro-
tection to the treatment of epilepsy, drug dependence, and
many others (for review see Scatton 1994; Parsons et al.,
1998). Several uncompetitive NMDA receptor antagonists
are clinically used or are in late phases of clinical develop-
ment (Parsons et al., 1998). However, with competitive and
uncompetitive NMDA antagonists some side effects such as
PCP-like psychotomimetic effects and vacuolization of the
cingulate/retrosplenial cortex, are known to occur at high
doses (see Parsons et al., 1998). Thus recently more attention
has focused on glycine
B
-site antagonists, which have been
suggested to be apparently devoid of these side effects and
seem to have a more favorable therapeutic profile (Bristow et
al., 1996; Kretschmer et al., 1997; Danysz and Parsons, 1998;
Parsons et al., 1998).
To date the major problem with the use of glycine
B
-site
antagonists has been their poor bioavailability. However,
recently some full antagonists of the glycine
B
-site with better
brain penetration have become available (see Parsons et al.,
1998; Danysz and Parsons, 1998). Efficacy after systemic
administration has been shown in models of epilepsy (MDL
104,653; Chapman et al., 1995), hyperalgesia (ACEA-1011;
Vaccarino et al., 1993), focal ischemia (7-chlorothiokynurenic
Received for publication October 7, 1998.
1
Current address: Department of Pharmacology, Leiden/Amsterdam Cen-
ter for Drug Research, Einsteinweg 55, 2300 RA Leiden, the Netherlands.
2
On leave from Department of Pharmacology, Leiden/Amsterdam Center
for Drug Research, Einsteinweg 55, 2300 RA Leiden, the Netherlands.
3
Present address: Department of Neuropharmacology, Cerebrus Ltd., Oak-
dene Court, 613 Reading Road, Winnersh, Wokingham, RG41 5UA, UK.
4
Present address: Department of Pharmacology, Leiden/Amsterdam Cen-
ter for Drug Research, Einsteinweg 55, 2300 RA Leiden, the Netherlands.
ABBREVIATIONS: NMDA, N-methyl-D-aspartate; CNS, central nervous system; ECF, extracellular fluid; AUC, area under the curve; glycine
B
-site,
strychnine-insensitive glycine coagonist site; BBB, blood-brain barrier; MES, maximal electroshock; aCSF, artificial cerebrospinal fluid; PNQX,
pyrido[3,4-f]quinoxaline-2,3-dione,1,4,7,8,9,10-hexahydro-9-methyl-6-nitromethanesulfonate.
0022-3565/99/2902-0543$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 2
Copyright 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 290:543550, 1999
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acid; Chen et al., 1993; GV 150526A; Bordi et al., 1997),
anxiety (MDL 100,458 and MDL 102,288; Kehne et al., 1995),
and psychosis (L-701,324; Bristow et al., 1996). Even though
these compounds show an improved brain accessibility, for
many the duration of action in the central nervous system
(CNS) is rather short. It has been suggested that these com-
pounds (strong acids) are actively transported out of the CNS
and the systemic circulation (P.D. Leeson, personal commu-
nication). The most likely carrier system responsible for this
phenomenon is the organic acid transport system located in
the epithelium of both the choroid plexus and kidney and
possibly at the endothelium of the blood-brain barrier (BBB;
Adkinson et al., 1994; Deguchi et al., 1995). It transports
unselectively xenobiotics with an organic acid structure at
physiological pH out of the cerebrospinal fluid into the blood-
stream and out of the systemic circulation into the urine.
This carrier can be competitively inhibited by probenecid,
which was developed in the 1950s to prolong the duration of
action of antibiotics by inhibition of renal clearance. Lately
attention has been focused again on the effect of probenecid
on the pharmacokinetics of a number of different classes of
drug with short durations of action, such as AZT (Dykstra et
al., 1993; Wang and Sawchuck, 1995), valproic acid (Golden
et al., 1993; Naora et al., 1996), and both -lactam and
quinolone antibiotics (Suzuki et al., 1997). Also the CNS
actions of NMDA receptor antagonists such as kynurenic
acid and quinoxaline derivatives have been shown to be pro-
longed by coadministration of probenecid (Taylor and
Vartanian, 1992; Santa-Maria et al., 1996; Danysz and Par-
sons, 1998).
We have recently developed a series of tricyclic pyrido-
pthalazine-diones, which are active in a number of animal
models after systemic administration, indicating sufficient
brain penetration, but their duration of action is rather short
(Parsons et al., 1997; Popik et al., 1998; Wenk et al., 1998;
Karcz-Kubicha et al., 1999). Hence, this study focused on the
CNS pharmacokinetic profile of these agents and the role of
the probenecid-sensitive organic acid transport in their clear-
ance. CNS presence was determined directly using brain
microdialysis in rats and indirectly by assessing the anticon-
vulsant action in maximal electroshock (MES)-induced con-
vulsions in mice. Some of these results were presented al-
ready in abstract form (Hesselink et al., 1997).
Materials and Methods
Subjects
Female Naval Medical Research Institute mice (1828 g) were
used for MES-induced convulsions and male Sprague-Dawley rats
(235270 g) were used for microdialysis experiments. All animals
were obtained from Charles River (Sulzfeld, Germany). The rats
were housed 5 per cage and the mice 15 per cage. During recovery
from microdialysis surgery the rats were housed separately. All
animals were kept under standard laboratory conditions: 12-h light/
dark cycle, 20C, and free access to food and water.
MES-Induced Convulsions
MRZ 2/570 (30 mg/kg), MRZ 2/571 (30 mg/kg), MRZ 2/576 (20 and
30 mg/kg), L-701,324 (6 mg/kg), or memantine (20 mg/kg) were
injected i.p. 30 min after probenecid (200 mg/kg) or vehicle (also i.p.).
Afterwards, MES (100 Hz, 0.5-s shock duration, 50 mA shock inten-
sity, 0.9-ms impulse duration, Ugo Basile, Basel, Switzerland) was
applied through corneal electrodes. The presence of tonic convulsions
was scored (tonic extension of hind paws with minimum angle to the
body of 90).
Brain Microdialysis
Microdialysis Surgery. The rats were anaesthetized with Hyp-
norm (1.0 ml/kg i.m.; Janssen-Cilag, Buckinghamshire, UK), placed
in a stereotaxic frame, and the skull was exposed. A small hole was
drilled to allow the implantation of a microdialysis guide cannula
(CMA/10, CMA Microdialysis, Solna, Sweden) in the anterior stria-
tum relative to bregma (anterior/posterior: 1.0; lateral: 2.5, vertical:
3.0). A second hole was drilled, a screw was secured into the skull,
and the screw and guide cannula were cemented together onto the
skull using dental cement (Paladur, Heraeus, Germany). A microdi-
alysis probe (CMA/10, membrane length of 3.0 mm) was inserted into
the guide cannula immediately after surgery and the animals were
allowed to recover for 22 to 26 h. In the animals that received MRZ
2/576 as an i.v. infusion, the femoral vein was cannulated immedi-
ately after guide cannula implantation.
Microdialysis Experiment. At the start of the experiment the
inflow tubing was connected to a syringe pump (CMA/100, syringe
pump) by means of a dual channel swivel (CMA) and the probe was
perfused with artificial cerebrospinal fluid (aCSF, composition in
mM: NaCl, 145; KCl, 0.6; MgCl
2
, 1.0; CaCl
2
, 1.2; ascorbic acid, 0.2, in
a 2 mM potassium phosphate buffer, pH 7.4) at a flow rate of 3
l/min. The outlet tubing was connected to a microfraction collector
(CMA/140) and 10-min fractions were collected. After 2 h of dialysis
MRZ 2/570, 2/571, 2/576 (30 mg/kg or 1040 mg/kg for MRZ 2/576),
or memantine (20 mg/kg) was injected i.p. Microdialysate samples
were collected for up to 180 min for the glycine
B
-antagonists and 240
min for memantine. Probenecid (200 mg/kg) was administered 30
min before the administration of the glycine antagonists or meman-
tine.
In Vitro Recovery. A microdialysis probe (CMA 10, membrane
length 3 mm) was placed in a stirred aCSF solution maintained at
37C and containing 1 M MRZ 2/570, 2/571, 2/576, or memantine.
The probe was perfused (CMA/100, syringe pump) with aCSF at a
speed of 3 l/min for 2 h to allow establishment of steady state before
three 20-min samples were collected.
In Vivo Zero-Net Flux Method. The animals were connected to
an infusion pump (WPI, SPI100i; World Precision Instruments,
Sarasota, FL) 2 h before perfusion of the microdialysis probe started
for steady-state plasma concentrations to develop. MRZ 2/576 was
infused at a dose of 20 mg/kg/h i.v. Subsequently, the microdialysis
probe was continuously perfused with aCSF containing different
concentrations MRZ 2/576 (four different concentrations ranging
from 05 M). Every inflow concentration was perfused for 2 h
before three 20-min samples were collected.
Serum Pharmacokinetics
Canulla Implantation. For serum sampling the femoral artery
was cannulated using silicone tubing (i.d. 0.508, o.d. 0.9398) under
Nembutal (Sanofi, Hannover, Germany) anesthesia. The cannula
was led subcutaneously to the neck of the animal, where a small
incision was made in the skin. The outlet of the cannula was secured
using a small rodent jacket (Lomir Biomedical, Malone, NY). The
animals were allowed to recover for 20 to 24 h before the experiment
started.
Serum Sample Collection. A 30-cm-long polyethylene tubing,
filled with a heparin solution (500 U/ml), was connected to the
cannula outlet. The animals were placed in a plastic rat bowl (CMA)
separately and were allowed to move freely during the experiment.
MRZ 2/570, 2/571, or 2/576 was injected i.p. at a dose of 30 mg/kg and
250-l samples were taken after 2, 5, 10, 20, 40, 60, and 90 min.
When probenecid (200 mg/kg i.p.) was given 30 min before adminis-
tration of the glycine
B
-antagonists, serum samples were also taken
after 120 and 150 min. Memantine (20 mg/kg) was given i.p. and
samples were taken after 15, 30, 60, 120, 180, 240, 300, 360, and 480
544 Hesselink et al.
Vol. 290
min. Aliquots (50 l) of serum were pipetted into 2-ml screw-capped
test tubes. After addition of 500 l trifluoroacetic acid solution (0.1%)
the samples were mixed for 1 min on a vortex mixer and 1 h on a BC1
mixer. After centrifugation for 10 min the sample was directly in-
jected on the HPLC system (column switching). The memantine
samples were analyzed using gas chromatography as described pre-
viously (Hesselink et al., 1999).
Substances
The following compounds were used: MRZ 2/570, MRZ 2/571, and
MRZ 2/576 (8-Br-, 8-F, and 8-Cl-4-hydroxy-1-oxo-1,2-dihydropyr-
idaliono(4,5-b)quinoline-5-oxide choline salt, Merz Co., Frankfurt/
Main, Germany), L-701,324 (7-chloro-4-hydroxy-3-(-phenoxy-)-phe-
nyl-2(H)quinolone; Tocris Cookson, Bristol, UK), memantine (HCl,
1-amino-3,5-dimethyladamantane, Merz Co), and probenecid
(Sigma, Deisenhofen, Germany). Probenecid was suspended in 0.5%
CMC (carboxymethylcellulose) with 0.1% Tween 80 in water. MRZ
2/570, 2/571, and 2/576 were dissolved in water and memantine was
dissolved in saline. L-701,324 was suspended in 25% polyethylene
glycol and adjusted with 1 N NaOH to pH 8.0. All compounds were
dissolved at a concentration to assure a injection volume of 1 ml/kg
for rats i.p. or i.v. and 10 ml/kg for mice i.p.
Results
MES-Induced Convulsions. To investigate a functional
interaction between the tested glycine
B
-site antagonists and
probenecid the duration of anticonvulsive activity of MRZ
2/570, 2/571, and 2/576 was measured with and without
preadministration of probenecid. Previously it has been
shown that this dose of probenecid (200 mg/kg i.p.) does not
have an effect on MES-induced convulsions on its own (Tay-
lor and Vartanian, 1992; Parsons et al., 1997). MRZ 2/576 at
doses of 20 and 30 mg/kg i.p. protected against MES-induced
convulsions (Fig. 1, A and B) with a short duration of action,
Fig. 1. Probenecid prolongs the anticonvulsant action of the glycine
B
-site NMDA antagonists: MRZ 2/576 (A and B), MRZ 2/570 (C), MRZ 2/571 (D),
and L-701,324 (E) but not of the uncompetitive NMDA antagonist memantine (F) in the MES model in mice. Probenecid (200 mg/kg i.p.) was given
30 min before administration of the test compounds. MRZ 2/576 was given at a dose of 20 (A) and 30 (B) mg/kg i.p. MRZ 2/570, MRZ 2/571, L701,324,
and memantine were given at doses of 30, 40, 6, and 20 mg/kg, respectively. Data are expressed as mean S.E.M. (n 46).
1999
Interaction between Probenecid and Glycine
B
-Site Antagonists 545
i.e., less than 60 min. When probenecid (200 mg/kg i.p.) was
administered 30 min before MRZ 2/576, a prolongation of the
anticonvulsive activity to 180 to 240 min was seen. A similar
effect of probenecid was observed with the other NMDA
receptor glycine
B
-site antagonists MRZ 2/570, MRZ 2/571,
and L-701,324 (Fig. 1, CE). In case of the channel blocker
memantine, only a minor difference between the probenecid-
pretreated animals and nontreated animals existed 6 h after
injection of memantine, i.e., 6.5 h after injection of probene-
cid (Fig. 1F). This was probably not directly related to pro-
benecid treatment because its effect starts to decrease al-
ready after 3 h as shown by the experiment with glycine
B
antagonist MRZ 2/576 (Fig. 6), hence after 6 h its action
should be marginal.
In Vitro and In Vivo Recovery. The three glycine
B
-site
antagonists, tested, i.e., MRZ 2/570, 2/571, and 2/576, showed
similar in vitro recovery (15.5 4.7, 17.9 2.4, and 12.9
3.2%, respectively). For memantine an in vitro recovery of
18.0 4.8% was obtained. Figure 2 shows the in vivo recov-
ery of MRZ 2/576 using the zero-net flux method for four
separate animals. Using this method, the in vivo recovery
can be estimated by taking the inverse of the slope of the
linear regression line when the difference between ingoing
and outgoing dialysate concentration (C
out
C
in
) is plotted
against the ingoing concentration (C
in
). For MRZ 2/576 an in
vivo recovery of 32 5% was found. All subsequent micro-
dialysate concentrations of MRZ 2/570, 2/571, and 2/576 were
corrected for this in vivo recovery. The in vivo recovery of
memantine (39 2%) was determined previously (Hesselink
et al., 1999).
Microdialysis Studies after Acute Injection of MRZ
2/570, 2/571, 2/576, or MemantineInteraction with Pro-
benecid. Microdialysis studies were performed to determine
the brain pharmacokinetics of the selected glycine
B
-site an-
tagonists. After administration of 30 mg/kg i.p. the peak
concentration of the glycine
B
-site antagonists in striatal ex-
tracellular fluid (ECF) ranged from 1.34 to 2.32 M. (MRZ
2/576 and 2/571, respectively; Fig. 3, AC.). The short dura-
tion of their anticonvulsive activity in the MES-induced con-
vulsion model in mice corresponds to short brain ECF half-
life ranging from 14.7 min for MRZ 2/571 to 20.2 min for MRZ
2/576 (Table 1). To evaluate whether the interaction between
probenecid and glycine
B
-site antagonists in the MES test was
of a pharmacokinetic nature, microdialysis studies were per-
formed with probenecid (200 mg/kg). Pretreatment with pro-
benecid increased the brain ECF half-life and area under the
curve (AUC; 1.8- to 3.6-fold) for all three glycine
B
-site antag-
onists (Fig. 3, AC; Table 1). Some variation was observed
Fig. 2. In vivo recovery of MRZ 2/576 using the zero-net flux method for
four individual rats. MRZ 2/576 was continuously infused i.v. (20 mg/
kg/h) starting 2 h before the start of the experiment. Four different
ingoing concentrations (C
in
) of MRZ 2/576 (05 M) were included in the
perfusate and the outgoing dialysate concentrations were measured
(C
out
). The intercept with the x-axis is the estimate of the brain ECF
concentration. The slope is the inverse in vivo recovery.
Fig. 3. Coadministration of probenecid increases both the peak concen-
trations and the brain ECF half-lives of the glycine
B
-site NMDA receptor
antagonists MRZ 2/570 (A), MRZ 2/571 (B), and MRZ 2/576 (C) (each 30
mg/kg). Probenecid (200 mg/kg i.p.) was given 30 min before the admin-
istration of the glycine
B
-site antagonists. Data are expressed as mean
S.E.M. (n 5).
546 Hesselink et al.
Vol. 290
between the tested agents in the effect of probenecid on their
brain ECF half-life (Table 1). For MRZ 2/576, only a 50%
increase was apparent, from 20.2 to 31.2 min, whereas in the
case of MRZ 2/570 its half-life increased 4-fold (from 14.7 to
57.7 min). A difference between the three compounds was
also apparent in a delay and magnitude of peak concentra-
tion (Fig. 3, AC). Administration of probenecid did not affect
the peak concentration of either MRZ 2/570 or 2/571. In the
case of MRZ 2/576 however, pretreatment with probenecid
caused a 2-fold increase the peak concentration reached in
the striatal ECF (Fig. 3C).
The effect of probenecid on the pharmacokinetics of the
NMDA receptor-channel blocker memantine served as a neg-
ative control to ascertain that the interaction seen for the
glycine
B
-site antagonists is a specific one for this class of
compounds. Memantine is an uncompetitive antagonist of
the NMDA receptor, which binds to a different site on the
NMDA receptor than the glycine
B
-site antagonists (Parsons
et al., 1999). Memantine is structurally unrelated to MRZ
2/570, MRZ 2/571, and MRZ 2/576 and shows high lipophi-
licity and very good penetration to the brain (Hesselink et al.,
1999). It does, however, have a comparable effect on MES-
induced convulsions in mice. Hence, it was considered an
appropriate control to determine whether the interaction
with probenecid is specific for the glycine
B
-site antagonists or
whether it occurred with all antagonists of the NMDA recep-
tor. Pretreatment with probenecid did not affect the concen-
tration of memantine in striatal ECF (Fig. 4A).
Serum Pharmacokinetics of MRZ 2/576 and Meman-
tineInteraction with Probenecid. Serum concentrations
of MRZ 2/576 were considerably higher than brain ECF con-
centrations. The peak concentration in serum was 94.4 M
(Fig. 5A) whereas in the brain ECF only 1.34 M was
reached. Thus by comparing the AUCs in serum and ECF a
brain penetration of 2% can be calculated. Coadministration
of probenecid increased the AUC of MRZ 2/576 in serum
1.6-fold and in ECF 3.6-fold (Table 1), enhancing brain ECF
versus serum ratio to 4%. The effect of probenecid on the
serum elimination half-life was more pronounced, i.e., it in-
creased from 15.6 to 40.6 min by pretreatment with probe-
necid (Table 1).
Serum concentrations of the NMDA receptor channel
blocker memantine were not affected by pretreatment with
probenecid (Fig. 4B)
Microdialysis Studies after Continuous i.v. Infusion
of MRZ 2/576Interaction with Probenecid. The concen-
tration of MRZ 2/576 in the striatal ECF was measured
under steady-state conditions achieved through continuous
i.v. infusion (20 mg/kg/h). Probenecid was subsequently ad-
ministered systemically and changes in brain ECF concen-
trations were determined (Fig. 6). Before administration of
TABLE 1
Pharmacokinetic parameters (AUC and brain half-life, T
1/2
) determined
using brain microdialysis after acute administration of MRZ 2/570,
MRZ 2/571, or MRZ 2/576 (30 mg/kg i.p.) in combination with
probenecid (200 mg/kg i.p., 30 min before administration of the
glycine
B
-antagonists). Additionally for MRZ 2/576 these parameters for
serum (serial sampling) are shown. Data are expressed as mean
S.E.M.
Treatment AUC T
1/2
g h/ml min
MRZ 2/570 0.217 0.040 14.7 2.1
MRZ 2/570 probenecid 0.666 0.107 57.7 16.2
MRZ 2/571 0.496 0.074 19.7 1.1
MRZ 2/571 probenecid 0.869 0.110 39.8 4.1
MRZ 2/576 0.188 0.076 20.2 0.2
MRZ 2/576 probenecid 0.671 0.075 31.2 2.5
Serum: MRZ 2/576 9.7 0.6 15.6 1.3
Serum: MRZ 2/576 probenecid 16.6 1.7 40.6 6.0
Fig. 4. Coadministration of probenecid does not effect the brain ECF (A)
and plasma pharmacokinetics (B) of the uncompetitive NMDA antagonist
memantine. Probenecid (200 mg/kg i.p.) was given 30 min before the
administration of 20 mg/kg of memantine. Data are expressed as mean
S.E.M. (n 5).
Fig. 5. The effect of coadministration of probenecid on the serum phar-
macokinetics of the glycine
B
-site NMDA antagonist MRZ 2/576. Probe-
necid (200 mg/kg i.p.) was given 30 min before the administration of 30
mg/kg of the glycine
B
-site antagonists. Data are expressed as mean
S.E.M. (n 4).
1999
Interaction between Probenecid and Glycine
B
-Site Antagonists 547
probenecid a steady-state concentration of MRZ 2/576 of
0.174 0.015 M was found, which was not significantly
different between four control measurements. Administra-
tion of probenecid (200 mg/kg i.p.) caused a 2.5-fold increase
in brain ECF concentration (Fig. 6). A clear effect of probe-
necid could be seen 40 to 200 min after its administration.
Dose Dependence of Brain Pharmacokinetics of
MRZ 2/576. If MRZ 2/576 is transported out of the CNS by an
active transport system, this transport system could puta-
tively be saturated by higher concentrations of MRZ 2/576
itself. To determine whether this is the case, increasing doses
of MRZ 2/576 (1040 mg/kg i.p.) were administered and the
brain ECF concentration was measured. A nonlinear rela-
tionship exists between the dose administered and the brain
ECF concentration of MRZ 2/576 (Fig. 7). The AUC found
after administration of 10 to 30 mg/kg MRZ 2/576 ranged
from 0.071 0.019 to 0.188 0.076 g h/ml for 10 and 30
mg/kg i.p., respectively, whereas after 40 mg/kg of MRZ 2/576
considerably higher levels were found i.e., an AUC of 0.484
0.060 g h/ml.
Discussion
The present results clearly show a functional potentiation
of duration of anticonvulsive action of glycine
B
-site antago-
nists (MRZ 2/570, MRZ 2571, MRZ 2/576, and L-701,324)
against MES seizures when probenecid was preadminis-
tered. Such an interaction was not observed with the NMDA
channel blocker memantine. Based on the microdialysis data
it can be concluded that this interaction is most likely phar-
macokinetic in nature because the brain ECF half-lives of the
glycine
B
-site antagonists were increased by probenecid from
0.5- to 4-fold.
The data presented are the first direct proof that after
systemic administration of 30 mg/kg the tested glycine
B
an-
tagonists reached concentrations in the brain ECF (corrected
for the in vivo recovery) high enough to block NMDA recep-
tor-mediated neurotransmission (see Parsons et al., 1997). It
was reported previously that in patch-clamp experiments,
steady-state inward current responses of cultured hippocam-
pal neurons to NMDA (200 M, glycine 1 M) were antago-
nized by MRZ 2/570, 2/571, and 2/576 with IC
50
values of
0.14, 1.02, and 0.54 M, respectively (Parsons et al., 1997).
These concentration are exceeded in the brain ECF for a
duration of ca. 60 min for all three compounds (see Fig. 4,
AC). This is in line with present MES-induced convulsion
studies because mice were protected from MES-induced con-
vulsions when the concentrations of MRZ 2/570, 2/571, and
2/576 in the brain ECF were high enough to block NMDA
receptor-mediated transmission. Of course differences be-
tween species, rats versus mice, should also be taken into
account. Previous studies demonstrated the efficacy of these
compounds in animal tests related to opioid dependence
(Popik et al., 1998), parkinsonism (Karcz-Kubicha et al.,
1999), and neuroprotection (Wenk et al., 1998). However in
the case of MRZ 2/576, when brain ECF concentrations are
compared to serum levels (AUCs) it becomes clear that only
2% brain penetration is achieved.
Probenecid effect in the MES model seems to be specific
because the anticonvulsant activity as well as pharmacoki-
netics of the channel-blocker memantine (negative control)
were not changed significantly. Moreover, the pharmacoki-
netic data support the hypothesis that the interaction be-
tween probenecid and the glycine
B
-site antagonists is in fact
a pharmacokinetic one because pretreatment with probene-
cid increased the brain ECF half-life and the brain AUCs of
the glycine
B
-site antagonists. Taken together, this indicates
that the elimination of MRZ 2/570, 2/571, and 2/576 from the
CNS and the systemic circulation involves the probenecid-
sensitive organic acid transporter. Because this transporter
is also located at the epithelium of the kidney, the effect of
probenecid on the serum pharmacokinetics of these com-
pounds was determined to ascertain whether an effect of
probenecid in the kidney could solely explain the increased
brain concentrations. The AUC of MRZ 2/576 in serum was
Fig. 6. Systemic administration of probenecid causes a 2.5-fold increase
in the steady-state ECF concentration of MRZ 2/576 as determined using
intrastriatal microdialysis. MRZ 2/576 was infused i.v. (20 mg/kg/h) for
1 h before probenecid (200 mg/kg) was administered i.p. Data are ex-
pressed as mean S.E.M. (n 5).
Fig. 7. Brain ECF concentrations of MRZ 2/576 after systemic adminis-
tration of 10, 20, 30, or 40 mg/kg i.p. Data are expressed as mean
S.E.M. (n 35).
548 Hesselink et al.
Vol. 290
increased 1.7-fold, whereas for the brain ECF the AUC was
increased 3.6-fold, which would indicate that there is an
additive effect of the transporters located in the choroid
plexus, BBB, and kidney. However, the effect on elimination
out of the brain seems to predominate. Moreover, a possible
interaction of probenecid and the glycine
B
-site antagonists
could arise at the level of plasma protein binding. Probenecid
binds strongly to plasma protein and might then through
reduction of the protein binding of the glycine
B
-site antago-
nists increase the free fraction. This might be particularly
true for MRZ 2/576 because peak levels in the brain were
increased by probenecid. However, it has previously been
determined that warfarin, which is highly plasma protein-
bound, has only a minor effect on MRZ 2/576 anticonvulsant
effects in MES-induced convulsions in mice (Parsons et al.,
1997). Moreover, preliminary experiments indicated that
warfarin did not change brain pharmacokinetics of MRZ
2/570 (M.B.H. and W.D., not published). Moreover, displac-
ing tested glycine antagonists from plasma proteins should
rather result in shortening than prolonging the half-lives.
Because the probenecid-sensitive organic acid transporter
is saturable it is likely that these compounds could saturate
this carrier system on their own at higher doses. If this holds
true their brain ECF concentration would be disproportion-
ally increased at high doses. This can in fact be observed with
MRZ 2/576 at doses that exceed 30 mg/kg. On the basis of
these results this cannot be exclusively attributed to reduced
transport out of the brain and might be explained by a
decrease in clearance from the blood by a saturation of the
transporter in the kidney or a combination of both.
First indications that compounds acting on glutamate re-
ceptors are transported out of the CNS by the probenecid-
sensitive organic acid transporter came from studies with
kynurenic acid (Moroni et al., 1988). Systemic administration
of probenecid increases endogenous levels of either endoge-
nous kynurenic acid or that provided by its systemically
available precursor L-kynurenine (Miller et al., 1992). This
approach provided protection against NMDA, quinolinic acid,
and hypoxia/ischemia-induced neurotoxicity (Santa-Maria et
al., 1996; Miranda et al., 1997).
Similarly, for quinoxaline-based AMPA receptor antago-
nists 2,3,-dihydroxy-6-nitro-7-sulfamoylbenzo(F)-quinoxa-
line or pyrido[3,4-f]quinoxaline-2,3-dione,1,4,7,8,9,10-
hexahydro-9-methyl-6-nitromethanesulfate (PNQX) it has
been shown that their anticonvulsant and/or antidystonic
effects are prolonged by coadministration of probenecid (Tay-
lor and Vartanian, 1992; Richter et al., 1993; Borosky et al.,
1996). For PNQX also the neuroprotective dose in focal isch-
emia in rats could be reduced by 50% when probenecid was
coadministered (Kupina et al., 1997). Microdialysis and se-
rial blood sampling showed that the concentration of PNQX
in brain ECF and blood increased 4-fold after probenecid
administration (Jordan et al., 1997). This indicates that not
only the clearance from the brain contributes to PNQXs
short brain half-life, but also clearance from the systemic
circulation probably by the probenecid-sensitive organic acid
transporter located on the kidney epithelium.
The present results obtained using i.v. infusion of MRZ
2/576 are comparable with the previously mentioned data
with PNQX (Jordan et al., 1997). In the case of MRZ 2/576 the
concentration in the brain ECF was increased 2.5-fold after
administration of probenecid, whereas the PNQX concentra-
tion increased 4-fold. However, in the present study probe-
necid was administered as a single i.p. injection, whereas in
combination with PNQX probenecid was continuously in-
fused, apparently leading to more efficient transporter inhi-
bition due to a higher probenecid concentration in the brain
(Jordan et al., 1997). Moreover, these differences might also
be explained by different transport affinities of the probene-
cid-sensitive organic acid transporter for PNQX, MRZ 2/576,
and probenecid.
One of the major challenges to tackle when using microdi-
alysis in pharmacokinetic studies is the issue of recovery, i.e.,
relating dialysate to brain ECF concentrations. The recovery,
also called extraction fraction, is defined as the ratio between
medium (here brain ECF) and dialysate concentrations. Sev-
eral methods for determining the recovery have been devel-
oped, ranging from the simplest in vitro method to the more
complicated in vivo zero-net flux and mass-transfer methods
(Benveniste and Hu ttemeier, 1990; Bungay et al., 1990). For
the glycine
B
-site antagonists comparable in vitro recoveries
were found for all three compounds. They all have a pyrido-
phtalazinedione structure but are substituted by different
halogens at position 8: bromo-, fluoro-, and chloro- for MRZ
2/570, 2/571, and 2/576, respectively (Parsons et al., 1997).
With the homology in chemical structure one could assume
the same or comparable physicochemical properties and dif-
fusion kinetics.
Surprising was the observation that MRZ 2/576 had a
higher in vivo than in vitro recovery. The recovery of a
compound is determined by its diffusion kinetics, the perfu-
sion flow rate, the microdialysis membrane characteristics,
and the surrounding medium; in the case of in vitro recovery,
an artificial CSF solution (Benveniste and Hu ttemeier,
1990). In general it has been found that for hydrophilic com-
pounds the in vivo recovery is lower than the in vitro recov-
ery. This is due to the complex pattern of diffusion in the
brain, the glutinous character of the extracellular matrix,
and the fact that diffusion is limited to the ECF (Benveniste
et al., 1989). However, exceptions to this have been noted for
compounds that are actively transported. Menacherry and
coworkers (1992), explained the fact that a higher in vivo
than in vitro recovery was found for cocaine as an effect of
microvasculature transport, i.e., BBB transport. The concen-
tration gradient of cocaine in close vicinity of the probe is
dramatically changed when BBB transport is taken into ac-
count (Morrison et al., 1991). This phenomenon of distur-
bance in concentration gradient of a solute in close vicinity of
the probe as a result of removal of solute from the tissue by
transport or metabolism, was first described by Bungay and
colleagues (1990) for the reference compounds [
3
H]H
2
O, su-
crose, and 3,4-dihydroxyphenylacetic acid. If MRZ 2/576 is in
the fact transported by the organic acid transporter, this
might cause a change in the concentration gradient in close
vicinity of the microdialysis probe. When the depletion zone
in close vicinity of the probe is replenished by a gradient of
MRZ 2/576 created by active transport, a steeper concentra-
tion gradient is created, which in turn leads to a higher probe
recovery. The presented data once more point out the impor-
tance of calibrating the microdialysis probe using in vivo as
well as in vitro recovery, because comparing the two offers a
better understanding of the diffusion kinetics in close vicinity
of the microdialysis probe.
The present study provides the first direct proof that MRZ
1999
Interaction between Probenecid and Glycine
B
-Site Antagonists 549
glycine
B
antagonist at the doses effective in a number of
animal models reach the brain ECF at concentrations suffi-
cient to influence NMDA receptor-mediated transmission.
Moreover, it also clearly shows that the glycine
B
-site antag-
onists are rapidly cleared from the CNS and the systemic
circulation by the probenecid-sensitive organic acid transport
system, i.e., their short duration of action can be prolonged by
pretreatment with probenecid and this cotreatment approach
could be a valid therapeutic strategy in the future.
Acknowledgments
We thank Bianka Lorenz for her assistance with the MES exper-
iments.
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Send reprint requests to: Dr. Wojciech Danysz, Merz Co., Dept. of Phar-
macological Research, Eckenheimer Landstrasse 100104; 60318 Frankfurt/
Main, Germany. E-mail: wojciech.danysz@merz.de
550 Hesselink et al.
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