Nimodipine restores the altered hippocampal phenytoin pharmacokinetics in a refractory epileptic model.
ABSTRACT The present work was undertaken to examine the central pharmacokinetics of phenytoin (PHT) in an experimental model of epilepsy, induced by administration of 3-mercaptopropionic acid (MP), and possible participation of P-glycoprotein in this model of epilepsy. Repeated seizures were induced in male Wistar rats by injection of 3-MP (45 mg kg(-1), i.p.) during 10 days. Control rats (C) were injected with saline solution. In order to monitor extracellular PHT levels, either a shunt microdialysis probe or a concentric probe was inserted into carotid artery or hippocampus, respectively. All animals were administered with PHT (30 mg kg(-1), i.v.) 30 min after intraperitoneal administration of vehicle (V) or nimodipine (NIMO, 2 mg kg(-1)). No differences were found in PHT plasma levels comparing all experimental groups. In pre-treated rats with V, hippocampal PHT concentrations were lower in MP (maximal concentration, C(max): 2.7+/-0.3 microg ml(-1), p<0.05 versus C rats) than in C animals (C(max): 5.3+/-0.9 microg ml(-1)). Control rats pre-treated with NIMO showed similar results (C(max): 4.5+/-0.8 microg ml(-1)) than those pre-treated with V. NIMO pre-treatment of MP rats showed higher PHT concentrations (C(max): 6.8+/-1.0 microg ml(-1), p<0.05) when compared with V pre-treated MP group. Our results indicate that central pharmacokinetics of PHT is altered in MP epileptic rats. The effect of NIMO on hippocampal concentrations of PHT suggests that P-glycoprotein has a role in reduced central bioavailability of PHT in our epileptic refractory model.
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ABSTRACT: A sensitive quantitative fluorescence method was used to explore the time course and regional pattern of blood-brain barrier (BBB) opening after transient middle cerebral artery occlusion (MCAo). Male Sprague-Dawley rats were anesthetized with halothane and subjected to 2 h of temporary MCAo by retrograde insertion of an intraluminal nylon suture, coated with poly-l-lysine, through the external carotid artery into the internal carotid artery and MCA. Damage to the BBB was judged by extravasation of Evans Blue (EB) dye, which was administered either 2, 3, 24 or 48 h after onset of MCAo. Fluorometric quantitation of EB was performed 1 or 2 h later in six brain regions. Cerebral infarction volumes were quantitated from histopathological material at 72 h. EB extravasation first became grossly visible in the ipsilateral caudoputamen and neocortex following 3 h of MCAo, was grossly unapparent at 24–26 h, and was maximal at 48–50 h. Fluorescence quantitation confirmed that BBB opening was absent at 2–3 h but present at all later times. In the hemisphere ipsilateral to MCAo, a 179% mean increase in extravasation of EB (compared to sham rats) was measured at 4 h, 407% at 5 h, 311% at 26 h and 264% at 50 h. (in each case, P < 0.05 vs. sham). The volume of infarcted tissue at 72 h in this model was 163.6 ± 7.7 mm3. Our results indicate that an initial, acute disruption of the BBB occurs between 3 and 5 h following MCAo, and that a later, more widespread increase in regional BBB permeability is present at 48 h. Regional measurement of Evans Blue extravasation offers a precise means of quantitating BBB disruption in focal cerebral ischemia; this method will be of considerable utility in assessing the BBB-protective properties of pharmacological agents.Brain Research 11/1996; · 2.88 Impact Factor
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ABSTRACT: The anticonvulsant activities of some 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)/kainate receptor antagonists, noncompetitive (2,3-benzodiazepines) and a competitive 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX), were compared in different experimental seizure models. In particular, compounds were evaluated against audiogenic seizure in DBA/2 mice, maximal electroshock seizure (MES) test and various chemoconvulsant models; both groups showed a protective action against audiogenic seizure, MES- and pentylenetetrazole (PTZ)-induced seizures. All 2,3-benzodiazepines were also protective against clonic and tonic seizures and lethality induced by 4-aminopyridine, kainate, AMPA and 3-mercaptopropionic acid but were ineffective against NMDA-induced seizures. NBQX was unable to affect 4-aminopyridine-, mercaptopropionic acid- and NMDA-induced seizures. The duration of anticonvulsant action of 33 micromol/kg of some 2,3-benzodiazepine in DBA/2 mice, genetically susceptible to audiogenic seizures, was also investigated. The derivatives possessing a thiocarbonyl group at the C-4 position of heptatomic ring showed higher anticonvulsant activities and longer lasting protective effects. We conclude that all 2,3-benzodiazepines studied are effective against various models of experimental epilepsy and the presence of thiocarbonyl groups at the C-4 position of heptatomic ring is able to increase the anticonvulsant effect of these compounds.Pharmacology Biochemistry and Behavior 03/2003; 74(3):595-602. · 2.61 Impact Factor
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ABSTRACT: Transport by ATP-dependent efflux pumps such as P-glycoprotein is an increasingly recognized determinant of drug disposition. P-glycoprotein does not only contribute to multidrug resistance (MDR) in tumor cells, it is also expressed in normal tissues with excretory function such as liver, kidney and intestine. Apical expression of P-glycoprotein in such tissues results in reduced drug absorption from the gastrointestinal tract and enhanced drug elimination into bile and urine. Moreover, expression of P-glycoprotein in the endothelial cells of the blood-brain barrier prevents entry of certain drugs into the central nervous system. Human P-glycoprotein has been shown to transport a wide range of structurally unrelated drugs such as digoxin, quinidine, cyclosporine and HIV-1 protease inhibitors. Drug administration to P-glycoprotein knock-out and control mice provided data on the importance of P-glycoprotein for absorption after oral administration and penetration through the blood-brain barrier. Moreover, P-glycoprotein knock-out mice were used to identify inhibition of P-glycoprotein-mediated transport as a mechanism for drug interactions such as the digoxin-quinidine interaction. Studies in humans indicate a particular importance of intestinal P-glycoprotein for bioavailability of the immunosuppressant cyclosporine. Moreover, induction of intestinal P-glycoprotein by rifampin has now been identified as the major underlying mechanism of reduced digoxin plasma concentrations during concomitant rifampin therapy. In summary, P-glycoprotein functions as a defense mechanism, which determines bioavailability and CNS concentrations of drugs. Modification of P-glycoprotein function is an important underlying mechanism of drug interactions in humans. However, disposition of a drug and its metabolites frequently is not only determined by P-glycoprotein, but also by drug-metabolizing enzymes and possibly by drug transporters other than P-glycoprotein [e.g. members of the MRP family (MRP = multidrug resistance-associated proteins)].International journal of clinical pharmacology and therapeutics 03/2000; 38(2):69-74. · 1.20 Impact Factor
Neuroscience Letters 413 (2007) 168–172
Nimodipine restores the altered hippocampal phenytoin
pharmacokinetics in a refractory epileptic model
Christian H¨ ochta,∗, Alberto Lazarowskib,c, N´ elida N. Gonzalezb, Jer´ onimo Auzmendib,
Javier A.W. Opezzoa, Guillermo F. Bramugliaa, Carlos A. Tairaa, Elena Girardib
aC´ atedra de Farmacolog´ ıa, Facultad de Farmacia y Bioqu´ ımica, Universidad de Buenos Aires, Jun´ ın 956, (C1113AAD) Buenos Aires, Argentina
bInstituto de Biolog´ ıa Celular y Neurociencia “Prof. Eduardo De Robertis”, Facultad de Medicina, Universidad de Buenos Aires, Argentina
cDepartamento de Bioqu´ ımica Cl´ ınica, Facultad de Farmacia y Bioqu´ ımica, Universidad de Buenos Aires, Argentina
Received 21 August 2006; received in revised form 16 October 2006; accepted 24 November 2006
The present work was undertaken to examine the central pharmacokinetics of phenytoin (PHT) in an experimental model of epilepsy, induced
by administration of 3-mercaptopropionic acid (MP), and possible participation of P-glycoprotein in this model of epilepsy. Repeated seizures
were induced in male Wistar rats by injection of 3-MP (45mgkg−1, i.p.) during 10 days. Control rats (C) were injected with saline solution. In
order to monitor extracellular PHT levels, either a shunt microdialysis probe or a concentric probe was inserted into carotid artery or hippocampus,
respectively. All animals were administered with PHT (30mgkg−1, i.v.) 30min after intraperitoneal administration of vehicle (V) or nimodipine
(NIMO, 2mgkg−1). No differences were found in PHT plasma levels comparing all experimental groups. In pre-treated rats with V, hippocam-
pal PHT concentrations were lower in MP (maximal concentration, Cmax: 2.7±0.3?gml−1, p<0.05 versus C rats) than in C animals (Cmax:
5.3±0.9?gml−1). Control rats pre-treated with NIMO showed similar results (Cmax: 4.5±0.8?gml−1) than those pre-treated with V. NIMO
pre-treatment of MP rats showed higher PHT concentrations (Cmax: 6.8±1.0?gml−1, p<0.05) when compared with V pre-treated MP group. Our
results indicate that central pharmacokinetics of PHT is altered in MP epileptic rats. The effect of NIMO on hippocampal concentrations of PHT
suggests that P-glycoprotein has a role in reduced central bioavailability of PHT in our epileptic refractory model.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Refractory epilepsy; P-glycoprotein; Nimodipine; Phenytoin; Microdialysis
Epilepsy is one of the most common neurological disorders
. Despite the existence of a large variety of antiepileptic
drugs (AED), almost 30% of epileptic patients are resistant
to treatment . Two hypotheses have been put forward to
explain pharmacoresistance to AED . The target hypothesis
explained the pharmacoresistance because of an altered sen-
sitivity of drug targets . However, the fact that a patient
resistant to one AED is often resistant to other drugs with dif-
ferent mechanism of action supports the transporter hypothesis
[15,17,28]. This hypothesis contends that expression or func-
P-glycoprotein (P-gp) is a defense mechanism located on
luminal cell membrane of endothelial cells of the blood–brain
∗Corresponding author. Tel.: +54 11 4964 8265; fax: +54 11 4508 3645.
E-mail address: firstname.lastname@example.org (C. H¨ ocht).
barrier (BBB), that reduces brain accumulation of naturally
occurring toxins and xenobiotics [5,32] and is thought to limit
drug distribution within brain parenchyma. P-gp reduces distri-
bution of certain AED into the brain of experimental models
[21,22,29,31]. Rizzi et al. using the Kainate model of epilepsy
showed that overexpression of P-gp in the hippocampus is asso-
ciated with reduced brain concentrations of phenytoin (PHT)
when compared with control animals . However, Potschka
and L¨ oscher did not find altered levels of PHT in the hippocam-
Recently, van Vliet et al.  reported that administration of
of PHT, suggesting that co-administration of P-gp inhibitors
with AED might be a promising therapeutic strategy to avoid
pharmacoresistance in epileptic patients .
In a previous report, we found an overexpression of P-gp
in the BBB, as well as immunoreative astrocytes and neurons,
in 3-mercaptopropionic acid (MP) induced epileptic rats .
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
C. H¨ ocht et al. / Neuroscience Letters 413 (2007) 168–172
Repetitive administration of MP produced a progressive refrac-
toriness to PHT treatment, until a total loss of protective effect
of PHT at days 7–13 was reached. Additional treatment with
nimodipine (NIMO) reversed this refractory phenotype [8,15].
Taking into account these findings and that calcium channel
blockers, such as nimodipine, have been shown to have anticon-
the mechanism of the beneficial effect of NIMO on antiepilep-
tic efficacy of PHT in MP induced epilepsy. We studied PHT
hippocampal pharmacokinetics and its potential modification
after NIMO treatment at the same dose as previously used for
reversion of PHT refractory phenotype in this model.
Male Wistar rats (280–330g) were used. Experiments were
performed according to Principles of Laboratory Animal Care
. Rats were administered with a 3-mercaptopropionic acid
(45mgkg−1), a convulsant drug, solution during 10 days. MP
administration resulted in onset of seizures episodes ocurring
5–10min after convulsant drug injection and characterized by
excitation with sudden running fits and seizures [8,9]. Rats
Blood and hippocampal concentrations of PHT were sepa-
rately studied in rats anesthetized with chloralose (50mgkg−1,
i.p.) and urethane (500mgkg−1, i.p.). Pharmacokinetic studies
A femoral vein was cannulated for intravenous administration
of PHT isotonic solution (30mgkg−1).
In order to determine hippocampal concentrations of PHT,
a concentric microdialysis probe was inserted in the hippocam-
pus (A/P −5.2mm, L/M 4.8mm, V/D 7.5mm, from bregma)
. Dialysis probes of concentric design were home-made
using fibers of cuproammonium rayon (3mm long, o.d. 200?m
microdialysis probe was perfused with a solution consisting in
147mM NaCl, 2.4mM CaCl2, 4.0mM KCl, pH 7.3, pumped
at a rate of 2?lmin−1. Samples were collected every 15min
after PHT administration (30mgkg−1, i.v.) during 3h period.
An equilibration period of 1h preceded drug administration in
order to minimize BBB disruption . The in vitro recovery of
the microdialysis probe was determined after each experiment.
vascular outlets  was used for examining the time course of
PHT plasma concentrations. Both the inlet and vascular outlet
of the heparinized probe (50Uml−1) were inserted in a carotid
artery, while the remainder vascular outlet was used for heparin
(25Uml−1) administration as necessary. In vivo recovery of
PHT was determined by retrodialysis before intravenous injec-
of PHT (1?gml−1) and by taking the proportion of lost across
the dialysis membrane as an estimate of the recovery .
administration with vehicle (V, i.p.) or nimodipine, a calcium
PHT dialysate levels were determined by high-performance
liquid chromatography (HPLC) with ultraviolet detection.
Dialysate samples were injected without pre-treatment into the
chromatographic system equipped with a Phenomenex Luna
5?m, C18, 250mm×4.60mm column and a ultraviolet detec-
tor (UVIS 204, Linear Instruments, Reno, USA). The lecture
phosphate buffer/acetonitrile (60:40; pH 5.6) was pumped at a
flow rate of 1.4mlmin−1. Limit of quantification of our HPLC
method was 20ngml−1.
Concentrations of PHT in hippocampal and blood dialysate
were corrected by the in vitro and in vivo recovery of the con-
centric and shunt microdialysis probe, respectively.
PHT concentration–time profiles obtained from corrected
microdialysis samples following bolus dosing were described
least-squares regression analysis was performed using the TOP-
FIT program. Elimination rate constant (Ke) was determined
from the terminal linear part of the curves. Area under the curve
(AUC0–180) of PHT levels was calculated using the trapezoidal
rule between drug administration and the end of the experiment.
Clearance (Cl) and volume of distribution (Vd) were calculated
by standard methods , where Cl=dose/AUC and Vd=Cl/Ke.
Data were expressed as mean±S.E.M. Statistical analy-
sis was performed by Student’s t-test. Statistical tests were
performed using GraphPad Prism Version 3.02 for Windows
(GraphPad Software, San Diego, CA, USA). Statistical signifi-
cance was defined as p<0.05.
PHT, NIMO and MP were obtained from Sigma–Aldrich.
The in vivo recovery of the “shunt” microdialysis probe in
all experiments was 0.30±0.05. Fig. 1 shows the PHT con-
centration/time profile obtained from blood microdialysis from
C and MP animals after PHT administration. No differences
were found in the unbound plasma levels of PHT of C and
MP animals pre-treated with V. NIMO pre-treatment did not
alter PHT dialysate levels in both experimental groups. Con-
sequently, no differences were found in the pharmacokinetic
parameters calculated from blood dialysate between C and MP
animals (Table 1).
Fig. 1. Phenytoin concentrations in corrected plasma dialysate of C non-
C. H¨ ocht et al. / Neuroscience Letters 413 (2007) 168–172
Pharmacokinetic parameters of phenytoin obtained from plasma dialysate from control and MP animals after pre-treatment with vehicle (V) or nimodipine (NIMO)
Pharmacokinetic parameterControl rats+VControl rats+NIMO MP rats+V MP rats+NIMO
9.0 ± 0.9
0.93 ± 0.14
3.5 ± 0.4
50.4 ± 3.0
8.6 ± 0.8
8.5 ± 1.2
0.93 ± 0.20
3.2 ± 0.5
41.4 ± 3.9
10.1 ± 1.2
9.4 ± 1.8
1.23 ± 0.25
3.6 ± 0.5
52.2 ± 4.1
7.4 ± 0.9
7.9 ± 1.1
0.77 ± 0.11
4.2 ± 0.6
49.4 ± 4.8
8.8 ± 0.7
Mean±S.E.M., n=6 rats per group (Cmaxmaximal concentration, Keconstant of elimination, Vdvolume of distribution, Cl clearance, AUC0–180area under the
Fig. 2. Phenytoin concentrations in corrected hippocampal dialysate of C non-
obtained from hippocampal microdialysis from C and MP ani-
rats, PHT dialysate concentrations were significantly lower in
MP rats when compared to C animals. The pharmacokinetic
analysis showed a lower Cmaxand AUC0–180in MP rats versus
C group (Table 2). No differences were found in Kecomparing
both groups (Table 2).
In control animals, pre-treatment with NIMO did not modify
hippocampal concentrations of PHT compared with C rats pre-
in pharmacokinetic parameters comparing C rats pre-treated
with V and NIMO.
showed lightly higher PHT hippocampal levels in MP rats than
in control animals (Table 2).
netics of PHT is altered in MP treated rats, and NIMO treatment
can enhanced PHT concentrations at the target site. In previ-
ous studies [8,16], we found that NIMO administration reverses
beneficial effect of NIMO on PHT antiepileptic effectiveness in
P-gp was found to be overexpressed in endothelial cells, and
present in neurons and glial cells from human drug-resistance
epileptic brain tissue, as well as in experimental refractory
epilepsy rat brain [15,17], leading to enhanced extrusion of
drugs from the brain to the bloodstream. P-gp overexpression
may lower AED concentrations in epileptogenic tissue ren-
dering epilepsy resistant to treatment [15,17]. Clinical study
of this hypothesis is restricted by the limitation in obtaining
suitable control tissue to compare with epileptogenic brain tis-
sue of patients . This limitation was reflected in a recent
study, in which authors found a high interindividual variabil-
ity in extracellular levels of AED of cortical regions of patients
with intractable epilepsy . Eventhough authors found lower
extracellular concentration of several antiepileptic drugs in the
cerebrospinal fluid of patients with intractable epilepsy, in the
tidrug transporters in the brain . Therefore, animal models
of epilepsy may be useful to test the transporter hypothesis.
The involvement of the P-gp in the pharmacoresistance of
antiepileptic drugs was investigated in an experimental model
of MP-induced seizures. MP is a classic seizure-inductor in ani-
mal epilepsy models [4,7,9,33]. P-gp overexpression was found
in brain capillary endothelial cells, astrocytes, and neurons dur-
ing repetitive MP-induced seizures . A refractory response
Pharmacokinetic parameter Control rats+VControl rats+NIMO MP rats+V MP rats+NIMO
5.3 ± 0.5
0.63 ± 0.10
8.3 ± 1.4
4.6 ± 0.2
0.39 ± 0.09
8.8 ± 0.7
2.7 ± 0.3*
0.40 ± 0.10
4.7 ± 0.5*
6.9 ± 1.0#
0.45 ± 0.11
12.9 ± 2.4#
Mean±S.E.M., n=6 rats per group (Cmaxmaximal concentration, Keconstant of elimination, AUC0–180area under the curve).
*p<0.05 vs. control rats.
#p<0.05 vs. MP rats+NIMO.
C. H¨ ocht et al. / Neuroscience Letters 413 (2007) 168–172
to PHT was also observed after several days of MP adminis-
tration and additional treatment with NIMO was able to restore
the protective effect of PHT . To disclose the potential role
for P-gp overexpression as a clearance mechanism in the refrac-
tory epilepsy, we studied the pharmacokinetic profile of PHT
in both MP and control rats, pre-treated either with vehicle or
NIMO, by means of microdialysis sampling. Microdialysis has
been extensively used in pharmacokinetic studies in laboratory
animals because of its ability to monitor extracellular unbound
levels of drugs without fluid loss .
PHT is a P-gp substrate  and central administration of
P-gp inhibitors enhanced hippocampal levels of PHT in non-
epileptic rats [23,24]. However, only few studies evaluated the
els with conflicting results. While Rizzi et al.  using the
of PHT compared with control animals, Potschka and L¨ oscher
 did not find altered levels of phenytoin in hippocampus
and amygdala of kindled when compared to non-kindled rats.
Recently, van Vliet et al.  found a reduced brain/plasma
ratio of PHT in the entorhinal cortex but not in the ventral
hippocampus of chronic epileptic rats.
In the present study, we evaluated the hippocampal and
plasma pharmacokinetics of PHT using the microdialysis tech-
nique. Although it is methodologically possible to undergo
plasma and central microdialysis simultaneously, we evaluated
these pharmacokinetic studies in different rats of independent
experiments to prevent a potential disruption of BBB integrity
Gupta et al.  have demonstrated that concomitant admin-
istration of NIMO enhanced carbamazepine plasma levels in
rhesus monkeys. In the present study, pharmacokinetic analy-
sis of plasma dialysate showed that blood kinetics of PHT is
not altered in MP rats compared to control animals. In addition,
pre-treatment with NIMO did not modify blood concentration
of PHT in both experimental groups. These results are in accor-
dance with the fact that NIMO did not affect the activity of the
cytochrome P-450 2C  and therefore clearance of PHT .
dialysate of MP rats compared to non-epileptic control animals.
Consequently, a lower Cmaxand AUC0–180were determined in
the epileptic group. These results suggested a role of central
ings are also supported by the fact that plasma PHT levels are
similar between MP and C rats. In order to confirm these find-
ings we evaluated the effect of NIMO pre-administration on the
hippocampal profile of PHT in both experimental groups.
In C rats, pre-treatment with NIMO did not alter pharma-
cokinetic profile of PHT when compared to V pre-treated rats,
suggesting that, in our experimental conditions, P-gp does not
limit PHT distribution in the hippocampus of non-epileptic ani-
in the hippocampus of non-epileptic rats enhanced PHT levels
within this central nucleus [23,24]. Although our findings are
contradictory with these works, it must be taken into considera-
PHT levels in the control group could be explained by several
factors, including the use of a low dose of NIMO, low P-gp
expression observed in non-epileptic animals  and weak
affinity of PHT to P-gp .
NIMO pre-treatment in MP animals significantly enhanced
hippocampal concentrations of PHT compared to MP rats pre-
treated with V. Moreover, hippocampal PHT levels of MP
animals pre-treated with NIMO are similar or lightly higher to
those obtained from C animals in the setting of a decreased
possible differences in NIMO bioavailability between MP and
C rats or by alternative mechanism of action for the beneficial
also a potent L-type voltage sensitive calcium channel blocker
and this action could participate in the experimental outcome.
in MP epileptic animals reduces hippocampal bioavailability of
PHT affecting its antiepileptic efficacy.
In conclusion, our results suggest that nimodipine could
ifying altered hippocampal PHT pharmacokinetics. These data
of pharmacoresistance to PHT. Our findings also suggested that
tance to PHT antiepileptic treatment. However, it is important
to take into consideration that chronic treatment with PHT will
tion between both drugs due to induction of NIMO metabolism.
This work was supported by grants of UBACYT M033 from
Universidad de Buenos Aires, and CONICET (PIP No. 02267
and PIP 5798) from the Consejo Nacional de Investigaciones
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