Neuroscience Letters 453 (2009) 54–57

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Differential hippocampal pharmacokinetics of phenobarbital and carbamazepine

in repetitive seizures induced by 3-mercaptopropionic acid
Christian Höcht a,b,∗ , Alberto Lazarowski b,c,d , Nélida N. Gonzalez c , Marcos A. Mayer a,b ,
Javier A.W. Opezzo a,b , Carlos A. Taira a,b , Elena Girardi c
a
Cátedra de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (C1113AAD) Buenos Aires, Argentina
b
Instituto de Fisiopatología y Bioquímica Clínica (INFIBIOC), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, (C1113AAD) Buenos Aires, Argentina
c
Instituto de Biología Celular y Neurociencia “Prof. Eduardo De Robertis”, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
d
Departamento de Bioquímica Clínica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: Previous evidence has shown that chronic 3-mercaptopropionic acid (MP) administration induced brain P-
Received 12 December 2008 glycoprotein (P-gp) overexpression altering target site accumulation of phenytoin. The aim of the present
Received in revised form 26 January 2009 work was to assess the involvement of P-glycoprotein in carbamazepine and phenobarbital hippocam-
Accepted 30 January 2009
pal pharmacokinetics in an experimental model of epilepsy, induced by repetitive MP administration.
Seizures were induced in Wistar rats by injection of MP (45 mg kg−1 , i.p.) during 10 days. Control rats
Keywords:
(C) were injected with saline solution. In order to monitor extracellular brain antiepileptic levels, a
3-Mercaptopropionic acid
concentric probe was inserted into the hippocampus. Animals were administered with carbamazepine
Chronic epilepsy
Phenobarbital
(10 mg kg−1 , i.v.) or phenobarbital (20 mg kg−1 , i.v.) 30 min after intraperitoneal administration of vehicle
Carbamazepine or nimodipine (2 mg kg−1 ), a well known P-glycoprotein inhibitor. No differences were found in hippocam-
Microdialysis pal concentrations of carbamazepine comparing all groups. In vehicle pre-treated rats, hippocampal
Hippocampus phenobarbital concentrations were lower in MP (maximal concentration, Cmax : 6.0 ± 0.6 ␮g ml−1 , p < 0.05)
P-glycoprotein than in C animals (Cmax : 9.4 ± 0.9 ␮g ml−1 ). Control rats pre-treated with nimodipine showed similar
results (Cmax : 10.7 ± 0.6 ␮g ml−1 ) than those pre-treated with vehicle. Nimodipine pre-treatment in MP
rats enhanced hippocampal phenobarbital concentrations (Cmax : 10.2 ± 1.0 ␮g ml−1 , p < 0.05) as compared
with vehicle pre-treatment.
Results of our work suggest that P-glycoprotein (P-gp) overexpression by repetitive seizures induced
by MP administration does not modify brain bioavailability of carbamazepine. Conversely, hippocampal
levels of phenobarbital are reduced in MP rats with regard to non-epileptic rats, suggesting a potential
role of P-gp overexpression in pharmacoresistance to phenobarbital.
© 2009 Elsevier Ireland Ltd. All rights reserved.

Epilepsy is one of the most common neurological disorders [4].
Despite the existence of a large variety of antiepileptic drugs (AED),
almost 30% of epileptic patients are resistant to treatment [27]. Two
hypotheses have been put forward to explain pharmacoresistance
to AED [27]. The target hypothesis explains the pharmacoresistance
because of an altered sensitivity of drug targets [28]. However, the
fact that patients resistant to one AED are often resistant to other
drugs with different mechanism of action supports the transporter
hypothesis [13,16,28]. This hypothesis contends that expression or
function of multidrug transporters is augmented in the epileptic
brain [13,16,28].
P-glycoprotein, normally located on luminal cell membrane of
endothelial cells of the blood-brain barrier (BBB), reduces brain
accumulation of xenobiotics [6] and is thought to limit drug distri-
∗ Corresponding author. Tel.: +54 11 4964 8265; fax: +54 11 4508 3645.
E-mail address: chocht@ffyb.uba.ar (C. Höcht).
bution within brain parenchyma. P-gp reduces brain distribution
of certain AEDs in experimental models [24,25,30,32]. This trans-
porter 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
[13,16], leading to enhanced extrusion of drugs from brain to blood-
stream. In this regard, Cucullo et al. [5] by using a humanized
dynamic in-vitro BBB model have established that BBB permeabil-
ity to phenytoin was 10-fold less in drug-resistant BBB models
than in controls. However, in-vivo clinical study of this hypoth-
esis is restricted by the limitation in obtaining suitable control
tissue to compare with epileptogenic brain of patients [26]. High
interindividual variability in AEDs extracellular levels was found
in cortical regions of patients with intractable epilepsy [26]. Even-
though lower extracellular concentrations of several antiepileptic
drugs were found in cerebrospinal fluid of patients with intractable
epilepsy, in the absence of data from non-epileptic tissues; it
was not possible to determine if their findings were related to

0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2009.01.079
 C. Höcht et al. / Neuroscience Letters 453 (2009) 54–57 55

overexpression of brain multidrug transporters [26]. Therefore,

animal models of epilepsy may be useful to test the transporter
hypothesis.
In our laboratory, we have developed an animal model of
refractory seizure to phenytoin by daily administration of 3-
mercaptopropionic acid (MP). In this model we have found P-gp
overexpression in BBB, astrocytes and neurons. Repetitive admin-
istration of MP produced a progressive refractoriness to phenytoin
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 [13]. More recently, we have
found that central pharmacokinetics of phenytoin is altered in
Fig. 1. Carbamazepine concentrations in corrected hippocampal dialysate of C non-
MP rats and pretreatment with NIMO normalizes phenytoin brain epileptic rats (circles) and MP animals (squares) after pre-treatment with vehicle
bioavailability, suggesting a role of P-gp in pharmacoresistance to (open symbols) or NIMO (black symbols). Mean ± S.E.M., n = 6 rats per group. No
phenytoin [9]. differences were found comparing all groups.
The objective of the present work is to evaluate the influence
of P-gp overexpression in hippocampal pharmacokinetics of car- wavelength was 210 nm. Mobile phase composed of 0.05 M phos-
bamazepine (CBZ) and phenobarbital (PhB) in rats with repetitive phate buffer/acetonitrile (60:40; pH 5.6) was pumped at a flow rate
seizures induced by MP administration. CBZ and PhB have been pre- of 1.4 ml min−1 . Limit of quantification was 20 and 10 ng ml−1 for
viously found to be effective in prevention of seizure induced by MP CBZ and PhB, respectively.
in a single dose administration [34,35]. Concentrations of CBZ and PhB in hippocampal and blood
Experiments were performed in 12-week-old male Wistar rats dialysate were corrected by in-vitro and in-vivo recovery of the
(250–280 g) according to Principles of Laboratory Animal Care [19]. concentric and shunt microdialysis probe, respectively. Mean in-
Rats were administered with MP solution (45 mg kg−1 ) during 10 vitro recovery of the concentric microdialysis probe in CBZ and PhB
days. MP administration resulted in onset of seizures episodes experiments was 0.12 ± 0.01 and 0.15 ± 0.03, respectively. In-vivo
occurring 5–10 min after convulsant drug injection and character- recovery of the shunt microdialysis probe, estimated by the retro-
ized by excitation with sudden running fits and seizures [8]. Rats dialysis method, was 0.32 ± 0.05 and 0.41 ± 0.04 for CBZ and PhB
injected with saline solution (SS) were used as control (C) group. experiments, respectively. No differences were found in the in-vivo
Blood and hippocampal concentrations of PhB or CBZ were sep- recovery of shunt microdialysis probe comparing all experimental
arately studied in rats anaesthetized with chloralose (50 mg kg−1 , groups (data not shown).
i.p.) and urethane (500 mg kg−1 , i.p.). Studies were undertaken 1–2 CBZ and PhB concentration-time profiles obtained from
days after last MP or SS administration. corrected microdialysis samples following bolus dosing were
A femoral vein was cannulated for intravenous administration described by a one-compartment, first-order elimination model.
of PhB (20 mg kg−1 ) or CBZ (10 mg kg−1 ) isotonic solution. In order Non-linear least-squares regression analysis was performed using
to determine hippocampal concentrations of CBZ or PhB, a micro- TOPFIT software. Elimination rate constant (Ke ) was determined
dialysis probe was inserted in the hippocampus (A/P −5.2 mm, L/M from terminal part of the curves. Area under the curve (AUC) of CBZ
4.8 mm, V/D 7.5 mm, from bregma) [22]. Concentric microdialysis and PhB levels was calculated using trapezoidal rule.
probes were home-made using fibers of cuproammonium rayon Data were expressed as mean ± S.E.M. Statistical analysis was
(2 mm long, o.d. 200 ␮m and 10,000 molecular weight cutoff, Asahi performed by two-way ANOVA followed by Bonferroni post-hoc
Medical Co., Japan), stainless steel tubing (25 G) and silica tubing test or unpaired Student’s t-test using GraphPad Prism v.5.01
(o.d. 145 ␮m). Microdialysis probe was perfused with Ringer solu- (GraphPad Software, San Diego, CA, USA). Statistical significance
tion consisting in 147 mM NaCl, 2.4 mM CaCl2 , 4.0 mM KCl, pH 7.3, at was defined as p < 0.05. PhB, CBZ, NIMO and MP were obtained from
a rate of 2 ␮l min−1 . Samples were collected every 15 min after CBZ Sigma–Aldrich.
or PhB administration during 3 h period. An equilibration period CBZ concentration-time profile obtained from hippocampal
of 2 h preceded drug administration in order to minimize BBB dis- microdialysis from C and MP animals is shown in Fig. 1. In V pre-
ruption [3]. The in-vitro recovery of the microdialysis probe was treated rats, no differences were found between CBZ hippocampal
determined after each experiment. concentrations of C and MP treated rats. NIMO preadministration
In a separately experiment, a “shunt” microdialysis probe [10] did not modify pharmacokinetic profile of CBZ in both C and MP
was used for examining the time course of PhB or CBZ plasma treated rats. Pharmacokinetic analysis of CBZ hippocampal lev-
concentrations. Both the inlet and vascular outlet of the hep- els did not show any difference between experimental groups
arinized probe (50 U ml−1 ) were inserted in a carotid artery, while for different estimated parameters (Table 1). Repetitive seizures
the remainder vascular outlet was used for heparin (25 U ml−1 ) induced by MP administration did not affect central disposition of
administration as necessary. In-vivo recovery of CBZ or PhB was CBZ, considering that both Cmax and AUC were not different com-
determined by retrodialysis before drug administration by perfus- paring non-epileptic and MP treated rats. P-gp seems not to be
ing the microdialysis probe with a solution of CBZ (1 ␮g ml−1 ) or
PhB (1 ␮g ml−1 ) and by taking the proportion of lost across dialysis
membrane as an estimate of recovery [9]. Table 1
Pharmacokinetic parameters of carbamazepine in rats obtained from hippocam-
Experimental groups were pre-treated 30 min before CBZ or PhB
pal dialysate from control and MP animals after pre-treatment with vehicle (V) or
administration with vehicle (V, i.p.) or NIMO (2 mg kg−1 , i.p.), a nimodipine (NIMO).
calcium channel blocker with inhibitory actions on P-gp activity
Control rats MP rats
[11,21].
CBZ and PhB dialysate levels were determined by liquid V (n = 6) NIMO (n = 6) V (n = 6) NIMO (n = 6)
chromatography with ultraviolet detection. Dialysate samples Tmax (h) 0.33 ± 0.06 0.28 ± 0.06 0.33 ± 0.05 0.49 ± 0.13
were injected into the chromatographic system equipped with a Cmax (␮g ml−1 ) 2.16 ± 0.32 2.53 ± 0.21 3.27 ± 1.00 3.26 ± 0.46
Phenomenex Luna 5 ␮m, C18, 250mm × 4.60 mm column and ultra- t1/2 (h) 1.95 ± 0.65 1.53 ± 0.54 1.03 ± 0.19 1.19 ± 0.27
AUC (␮g ml−1 h−1 ) 5.88 ± 1.30 6.88 ± 2.78 5.11 ± 1.14 7.34 ± 1.40
violet detector (UVIS 204, Linear Instruments, Reno, USA). Lecture
56 C. Höcht et al. / Neuroscience Letters 453 (2009) 54–57

Involvement of P-gp in pharmacoresistance to AEDs was inves-

tigated in an experimental model of MP-induced seizures. MP
is a classic seizure-inductor in animal epilepsy models [7,8,33],
showing P-gp overexpression in brain capillary endothelial cells,
astrocytes, and neurons after repetitive seizures [12]. A refrac-
tory response to phenytoin was observed after several days of MP
administration and additional treatment with NIMO was able to
restore the protective effect of phenytoin [13]. In addition, involve-
ment of P-gp in phenytoin resistance in MP treated rats was
confirmed by the fact that P-gp inhibition enhances phenytoin brain
Fig. 2. Phenobarbital concentrations in corrected hippocampal dialysate of C non- disposition in this model of repetitive seizures [9].
epileptic rats (circles) and MP animals (squares) after pre-treatment with vehicle Role of P-gp in reducing central disposition of CBZ is controver-
(open symbols) or NIMO (black symbols). Mean ± S.E.M., n = 6 rats per group. sial. Several lines of evidence reported conflicting results regarding
*p < 0.05 vs. C rats pretreated with V (two-way ANOVA with Bonferroni post hoc affinity of CBZ to drug transporters. Potschka et al. [24] found
test); # p < 0.05 vs. MP rats pretreated with V (two-way ANOVA with Bonferroni post
that both P-gp and multidrug resistance associated protein (MRP)
hoc test).
participate in regulation of CBZ brain concentrations. Moreover,
enhanced anticonvulsant activity of CBZ was found in ABCC2 defi-
playing a role in brain bioavailability of CBZ, considering that phar- cient rats [23]. Nevertheless, these in-vivo results are not supported
macokinetic parameters did not differed comparing V and NIMO by in-vitro findings, which suggest a lack of affinity of CBZ to P-gp.
pretreatments in both control and MP rats. In addition, no differ- By using different approaches, several authors have found that CBZ
ences were found in plasma levels of CBZ comparing C and MP is not a substrate for P-gp [1,18,20,36]. In the present work, we did
treated rats, both after V and NIMO pretreatment (data not shown). not find changes in CBZ brain accumulation after i.v. administration
Fig. 2 shows PhB concentration/time profile obtained from when comparing MP treated rats with control animals, suggesting
hippocampal microdialysis of C and MP animals. After V pretreat- that brain P-gp overexpression induced by MP administration does
ment, PhB hippocampal concentrations were significantly lower in not modify CBZ hippocampal disposition. Moreover, P-gp inhibition
MP rats when compared to C animals. Pharmacokinetic analysis by systemic NIMO administration did not modify CBZ hippocampal
showed a lower Cmax and AUC in MP rats versus C group (Table 2). concentrations in both control and MP animals. Our results are in
No differences were found in t1/2 comparing both groups (Table 2). accordance with the fact that CBZ is not a good substrate of P-gp.
In control animals, pre-treatment with NIMO did not modify hip- Although, our results disagree with findings from Potschka et al.
pocampal concentrations of PhB compared to C rats pre-treated [24], it is important to mention that our experimental design dif-
with V (Fig. 2) and therefore no differences were found in phar- fers from that reported by these authors. Meanwhile in the present
macokinetic parameters comparing both groups. In MP rats, PhB work P-gp inhibitor was administered i.p. previous to CBZ, Potschka
hippocampal concentrations were significantly higher in rats pre- et al. [24] infused P-gp blocker continuously in the hippocampus
treated with NIMO compared to V pre-treated animals (Fig. 2). by means of reverse microdialysis. Although reverse microdialysis
Moreover, NIMO pre-administration showed slightly higher PhB allows maintenance of constant levels of the inhibitor in hippocam-
hippocampal levels in MP rats than in control animals (Table 2). pal extracellular space, the existence of non-specific effects of the
No differences were found in plasma levels of PhB comparing C P-gp inhibitor is possible due to achievement of high levels as a
animals and MP treated rats, both after V and NIMO pretreatment consequence of local administration.
(data not shown). We also assessed the role of P-gp overexpression in MP ani-
In the last years, several authors have doubt regarding the role mals in PhB hippocampal bioavailability. PhB has been found to
of P-gp overexpression in pharmacoresistance to AEDs [17,29,31]. be a weak substrate for P-gp in several works [36,37]. Interest-
Although there is consensus that chronic epilepsy induces higher ingly, Luna-Tortós et al. [17] evaluated in-vitro transport of different
expression of several ABC transporters, the fact that AEDs are sub- AED by human P-gp, showing that concentration equilibrium trans-
strate for drug transporters efflux is controversial, considering that port assay, but not the conventional assay, identifies phenytoin
most in-vitro studies do not demonstrate efflux of AEDs by means of and PhB as substrates of human P-gp. Compromise of P-gp in
P-gp activity. However, in-vitro testing has some limitations, includ- PhB brain disposition was also established by in-vivo studies.
ing uncertainty whether P-gp levels in the in-vitro model accurately Potschka et al. [25] have found that intrahippocampal perfusion
represent P-gp levels in brain capillaries of epileptic subjects [31]. with verapamil, a P-gp inhibitor, enhances brain access of PhB in
Robey et al. [31] also hypothesized that AEDs are P-gp substrates non-epileptic rats. A reduced central disposition of PhB was also
but are not so well transported that they can be detected by in-vitro demonstrated in different animal models with brain P-gp overex-
models. pression as compared to control rats. Increased P-gp expression and
Considering this controversy, we tested the role of P-gp decreased phenobarbital distribution was observed in the brain of
overexpression on hippocampal bioavailability of CBZ and PhB. pentylenetetrazole-kindled rats as compared to non-epileptic ani-
mals [15]. Relationship between multidrug transporters and brain
Table 2 accumulation of PhB was further confirmed by Liu et al., consider-
Pharmacokinetic parameters of phenobarbital in rats obtained from hippocampal
ing that streptozotocin-induced diabetic mice showed an increased
dialysate from control and MP animals after pre-treatment with vehicle (V) or
nimodipine (NIMO).
brain distribution of PhB associated with attenuated function of P-
gp at BBB [14]. In the present work, we found that MP treated rats
Control rats MP rats showed lower hippocampal extracellular levels of PhB with regards
V (n = 6) NIMO (n = 6) V (n = 6) NIMO (n = 6) to non-epileptic animals, suggesting a role of P-gp overexpression
Tmax (h) 0.46 ± 0.10 0.43 ± 0.11 0.49 ± 0.08 0.49 ± 0.08 in reduced brain accumulation of PhB in this model of repetitive
Cmax (␮g ml−1 ) 9.4 ± 0.9 10.7 ± 0.6 6.0 ± 0.6* 10.2 ± 1.0# seizures.
t1/2 (h) 1.87 ± 0.40 2.55 ± 0.36 2.69 ± 0.54 2.06 ± 0.40 P-gp inhibition by systemic administration of NIMO enhanced
AUC (␮g ml−1 h−1 ) 33.5 ± 3.0 45.2 ± 6.4 24.2 ± 2.8* 33.4 ± 2.7# central bioavailability of PhB in MP treated rats, but not in control
*
p < 0.05 vs. control rats (unpaired Student’s t-test). animals. The absence of P-gp involvement in central bioavailability
#
p < 0.05 vs. MP rats pretreated with MP (unpaired Student’s t-test). of PhB in non-epileptic rats is opposite to findings by Potshka et al.
 C. Höcht et al. / Neuroscience Letters 453 (2009) 54–57
[25]. Nevertheless, as comment above, a possible explanation for
these divergent results is the different design of experiments.
In conclusion, results suggest that P-gp overexpression in repet-
itive seizures induced by MP administration does not reduce brain
bioavailability of CBZ. Conversely, hippocampal levels of PhB are
reduced in MP rats with regard to non-epileptic rats, suggesting a
potential role of P-gp overexpression in brain distribution of PhB,
giving support for the “transporter hypothesis” to the pharmacore-
sistance to PhB. NIMO pretreatment enhances hippocampal PhB
disposition, suggesting that NIMO could be an effective strategy
to enhance PhB brain bioavailability during antiepileptic treat-
ment. At this point, it is important to mention that Basic et al.
[2] have recently found that C3435T polymorphism of ABCB1 gene
significantly influences cerebrospinal concentrations of PhB and
seizure frequency in patients with generalized epilepsy. Neverthe-
less, further studies are needed to demonstrate if reduced central
distribution of PhB results in PhB refractory epilepsy in MP treated
rats.
Acknowledgements
This work was supported by grants of UBACYT from Universidad
de Buenos Aires, and CONICET (PIP 5798) from the Consejo Nacional
de Investigaciones Científicas y Técnicas. C.A. Taira, and E. Girardi
are members of Research Career, CONICET, Argentina.
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