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Hindawi Publishing CorporationBioMed Research
InternationalVolume 2013, Article ID 140327, 5
pageshttp://dx.doi.org/10.1155/2013/140327
Research ArticleImpact of Pregnancy on
ZonisamidePharmacokinetics in Rabbits
Kamal M. Matar
Department of Pharmacology andTherapeutics, Faculty of Pharmacy,
Kuwait University, P.O. Box 24923, 13110 Safat, Kuwait
Correspondence should be addressed to Kamal M. Matar;
[email protected]
Received 6 October 2013; Accepted 22 November 2013
Academic Editor: Allegaert Karel
Copyright 2013 Kamal M. Matar. This is an open access article
distributed under the Creative Commons Attribution License,which
permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Pregnancy is associated with various physiological changes which
may lead to significant alterations in the pharmacokinetics ofmany
drugs. The present study was aimed to investigate the potential
effects of pregnancy on the pharmacokinetic profile ofzonisamide
(ZNM) in the rabbit. Seven female rabbits were used in this study.
The pregnant and nonpregnant rabbits receivedZNM orally at a dose
of 10mg/kg and blood samples were collected from the animals just
before receiving the drug and thenserially for up to 24 h. The
plasma samples were analyzed using tandem mass spectrometric
method. Following a single oraldose of ZNM to the rabbits, the mean
values of ZNM plasma concentrations at different times were
consistently low in pregnantcompared to nonpregnant rabbits. The
mean values of ZNMs max and AUC0 were significantly ( < 0.05)
decreased, whereasthe CL/F exhibited substantial increase ( <
0.05) in pregnant compared to nonpregnant rabbits. max, 1/2abs,
1/2el, MRT, and Vd/Fshowed no significant differences between the
two groups.The present study demonstrates that pregnancy decreased
ZNM plasmaconcentrations in rabbits and that the decrease could be
due to decreased extent of gastrointestinal absorption, induced
hepaticmetabolism, or enhanced renal elimination of the drug.
1. Introduction
Zonisamide (ZNM) is a 1,2-benzisoxazole-3-methanesulfon-amide,
structurally unrelated to other antiepileptic drugs(AEDs) in
clinical practice. The precise mechanism of ZNMby which it exerts
its antiepileptic effects is not yet known.However, it has been
proposed that ZNM stabilizes neuronalmembranes by blocking
voltage-sensitive Na+ channels andalso inhibiting low-threshold
T-type Ca2+ channels [1]. Itis used as an adjunctive therapy in the
treatment of par-tial seizures (with or without generalization) in
adults [1].Following oral administration, ZNM is almost
completelyabsorbed from the gastrointestinal tract, irrespective of
foodintake reaching maximal plasma concentrations within 25 h under
fasting conditions and 46 h with food [2, 3]. Itis predominantly
metabolized in the liver with subsequentelimination by the kidney.
About 50% of ZNM ismetabolizedby cytochrome P450 (CYP), CYP3A4
isoenzyme. However,CYP2C19 and CYP3A5 may also contribute [4]. In
addi-tion, acetylation accounts for 20% of ZNM metabolism
[3].Approximately 30% of the parent drug is excreted unchanged
in the urine [3]. Although ZNM is only 4060% plasma pro-tein
bound, it is extensively bound to erythrocytes [2, 3].
Thetherapeutic level of ZNM is usually in the range of 1040 g/mL
[5].
Epilepsy is the most common neurologic condition thatrequires
continuous management during pregnancy andAEDs are associated with
increased risk of teratogenicity inthis population
[6].Themanagement of epilepsy during preg-nancy requires a balance
between minimizing fetal exposureto AEDs and maintaining seizure
control [7]. Although ithas been reported that ZNM is teratogenic
in animals, verylimited data are available on the extent of
teratogenicity inhumans and further clinical investigations are
warranted toconfirm the preliminary findings [8, 9].
Pregnancy is associated with various physiologicalchanges which
may lead to considerable variations in the dis-position of many
drugs including AEDs [10]. During preg-nancy, the plasma levels of
AEDs tend to decrease as preg-nancy progresses, with potential
consequences of increas-ingseizure frequency [10, 11].
Understanding the pharmacoki-netic alterations of AEDs during
pregnancy is clinically
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2 BioMed Research International
important as the decrease in the plasma concentrations ofthese
drugs has been associated with deterioration of seizurescontrol and
this could adversely affect the pregnancy out-comes. Therefore,
therapeutic drug monitoring for thesedrugs might be of value during
pregnancy [10]. There havebeen no systematic investigations on the
potential influenceof pregnancy on ZNM pharmacokinetics and the
data arelimited. There exists only one case report on ZNM
plasmalevels during pregnancy [5]. However, the report did
notexplore the underlying mechanisms responsible for thedecrease in
ZNM plasma levels.
The objective of the present study was to investigate
thepotential effects of pregnancy on the pharmacokinetic profileof
ZNM in the rabbit.
2. Materials and Methods
2.1. Chemicals and Reagents. Zonisamide pure standard
waspurchased from Sigma-Aldrich (St. Louis, MO, USA) andthe
internal standard (IS), [2H
4,15N]-zonisamide, was pur-
chased from Alsachim Co. (Strasbourg, France). Zonegran(100mg
capsule), the commercial formulation of ZNM (EisaiLtd,
Hertfordshire, UK), was purchased from a drug store.Water was
purified using a Milli-Q water device (Millipore,Bedford, MA, USA).
All other chemicals and reagents wereof analytical grade and
solvents were of HPLC grade.
2.2. Ethical Approval. Ethical approval for conducting thisstudy
was provided by the local Experimental AnimalResource Center Ethics
Committee, Health Sciences Center,Kuwait University. The study was
conducted in compliancewith theHelsinkiDeclaration for ethical
principles ofmedicalresearch.
2.3. Blood Sampling. Seven femaleNewZealandwhite rabbitsweighing
between 3.5 and 5 kg were used in this study.The animals were
acclimatized in a separate room, undercontrolled lighting and
heating conditions, in ExperimentalAnimal Resources Center for 1
week before inclusion in thestudy. The rabbits were maintained on
food and water adlibitum. Each animal was physically examined and
was con-sidered healthy before inclusion in the study. The study
wasconducted in a crossover design with a washout period of
sixweeks. The same animal was used as a control (nonpregnant)for
itself. Vulvas of the rabbits were thoroughly examined forany
potential infection before mating. The receptive female(vulva was
red and dilated) was cohabited with the malebreeder and they were
kept together in the cage for 24 hbefore they were separated. The
mating day was consideredas the day one of pregnancy. Two weeks
later, pregnancy wasverified by palpation of the abdomen, changes
in the bodyweight, and pulling off the animals fur near term.The
lengthof gestational period in rabbits normally varies between 3035
days. In the pregnant rabbit, the study was performed onday 28 of
gestation by administering a single dose of ZNMorally (by gavage)
at a dose of 10mg/kg in a freshly preparedsuspension (20mg/mL ZNM
in 0.5% CMC). Blood samples(0.5mL) were serially collected into
preheparinized (10 L)
plastic centrifuge tubes (1.5mL). The blood samples
werecollected through a cannula inserted into the marginal earvein
just before dosing and at 0.5, 1.0, 1.5, 2, 4, 6, 8, 12, 20,and 24
h following ZNM administration. The blood sampleswere immediately
centrifuged at 9,000g for 10min and thenplasma samples were
separated and stored at 80C pendinganalysis.
2.4. Analytical Procedure
2.4.1. Instrumentation. A liquid chromatographic system,Alliance
2695, consisted of a solvent delivery system, and anautosampler
(Waters Assoc., Milford, MA, USA) was used.Chromatographic
separation of the analytes was achieved onSymmetry C
18column (5m, 3.9 50mm) equipped with
a precolumn filter of the same packing material. The mobilephase
consisted of acetonitrile-0.1% triethylamine (80 : 20,v/v; pH =
9.9) and delivered at a flow rate of 0.2mL/min toa negative
electrospray ionization interface (ESI) of triplequadrupole mass
spectrometer (Quattro LC, Micromass,Manchester, UK). Tuning
parameters of MS were optimizedby direct infusion of solutions of
ZNM and IS in the mobilephase into the ionization probe at a flow
rate of 10 L/minusing Hamilton syringe. The ion source and
desolvationtemperatures were set at 150C and 350C, respectively.
Thecapillary voltage was adjusted at 3.18 kV, cone voltage at20V,
collision energy at 12 eV, and collision gas pressure at 118.8
and216 > 122.8 were selected for quantification of ZNM and
IS,respectively. Data acquisition, handling, and system controlwere
performed by MassLynx Software (Version 4.1, Micro-mass,
Manchester, UK).
2.4.2. Preparation of Calibration Standards and Quality Con-trol
Samples. Stock solutions of ZNM and the internal stan-dard ([2H
4,15N]-zonisamide) were prepared by dissolving
the compounds in methanol to yield 1.0mg/mL solutions.Aliquots
of ZNM and the IS stock solutions were furtherdilutedwithmethanol
to yield theworking standard solutionsof 500g/mL and 100 g/mL,
respectively. The calibrationstandards of ZNM at concentrations of
0.5, 1.0, 5, 10, 15, 20,30, and 50 g/mL were prepared by spiking
drug-free rabbitplasma with ZNM standard solution. Similarly,
quality con-trol (QC) samples at concentrations of 2, 12, 25, and
40 g/mLwere prepared by spiking drug-free rabbit plasma with
ZNMsolution.The spiked plasma samples were aliquoted (200L)into
Eppendorf polypropylene tubes and kept frozen at80Cpending
analysis.
2.4.3. Sample Preparation. Prior to assay, frozen rabbitplasma
samples, including calibrators or QC samples, werethawed at ambient
temperature. A 100 L aliquot of eachplasma sample was transferred
to a 1.5mL Eppendorf tubeand then 20L of IS (100 g/mL) was added
and vortex-mixed for 30 sec. To each tube, 20L of ammonium
acetate(1.0mM) and 1.0mL of diethylether were added and
vortex-mixed for 30 sec. The tube was centrifuged at 9000g
for10min, the organic layer was separated and evaporated under
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BioMed Research International 3
stream of purified N2gas and then reconstituted with 150 L
of mobile phase. A 10 L of this sample was then injected intothe
LC-MS system for analysis. The assay method was fullyvalidated for
linearity, accuracy, precision, selectivity, stabil-ity, and matrix
effect according to the standard guidelines[12].
2.5. Pharmacokinetics and Statistical Analyses. ZNM
phar-macokinetic parameters were estimated by standard
non-compartmental methods using Kinetica software, version
5.1(Thermo Fisher Scientific, USA).Themaximum plasma con-centration
(max) and time needed to attain this concentra-tion (max)were
directly obtained from the drug plasma pro-files; the drug plasma
elimination half-life (
1/2el) values werecalculated as ln 2/el, where el is the
elimination rate con-stant. The absorption half-life (
1/2abs) was determined fromthe plasma concentration-time
profiles employing method ofresiduals (feathering technique). The
area under the plasmaconcentration-time curves (AUC
0) was calculated from themeasured data points from time zero to
time of last quantifi-able concentration by the linear trapezoidal
rule and the areaunder the plasma concentration-time curves
extrapolated totime infinity (AUC
0) was calculated using the equationAUC0 = AUC0 +
/el, where
is the last quantifiabledrug plasma concentration.The mean
residence time (MRT)was calculated as AUMC
0/AUC0, where AUMC0 isthe area under the first moment of plasma
concentration-time curve from time zero to time infinity. Oral body
clear-ance (CL/F) was calculated as CL/F = Dose/AUC
0 andthe volume of distribution (Vd/F) was calculated as Vd/F
=(CL/F)/el. The pharmacokinetic parameters were presentedas mean
SD. Differences between the pharmacokineticparameters of ZNM among
the groups were consideredstatistically significant if < 0.05
using Wilcoxon matched-pair signed-rank test (two-tailed). The
statistical analysis wasperformed using the statistical package for
social sciences(SPSS) software, version 20 (SPSS Inc., Chicago, IL,
USA).
3. Results
The tandem mass spectrometric (LC-MS/MS) assay methodwas
developed, fully validated, and employed for quantifica-tion of ZNM
plasma samples.The linear range of the methodwas 0.550g/mL (2 >
0.99) and the lower limit of quan-tification was 0.5 g/mL. The
intra- and interrun precisions,as measured by relative standard
deviations (RSD,%), of themethod were less than 7%.
The mean (SD) plasma concentration-time profile fol-lowing a
single oral dose of ZNM (10mg/kg) administeredto pregnant and
nonpregnant (control) rabbits is depicted inFigure 1 and the mean
(SD) pharmacokinetic parametersof ZNM in the two groups are
presented in Table 1. Figure 1and Table 1 demonstrated substantial
interanimal variabilityamong the two groups. Statistical comparison
of the meanvalues of ZNMpharmacokinetic parameters resulted in a
sig-nificant ( < 0.05) decrease in max, AUC0, and AUC0in
pregnant compared to nonpregnant rabbits. In addition,the oral
clearance (CL/F) was significantly ( < 0.05)
Time (h)0 5 10 15 20 25 30
Plas
ma c
once
ntra
tion
(mg/
L)
0
2
4
6
8
10
12
14
NonpregnantPregnant
Figure 1: Mean (SD) plasma concentration-time profile
followingan oral dose of 10mg/kg zonisamide to pregnant and
nonpregnantrabbits; = 7.
Table 1: Mean (SD) pharmacokinetic parameters of zonisamide
inpregnant and nonpregnant rabbits.
Parameter Nonpregnant Pregnant valuea
max (h) 4.33 1.97 3.33 1.03 0.344max (g/mL) 10.24 2.90 7.21 1.84
0.0361/2abs (h) 1.86 1.62 0.94 0.36 0.3131/2el (h) 13.72 5.11 10.84
3.43 0.313MRT (h) 20.92 6.44 16.38 5.17 0.156AUC
0 (gh/mL) 150.35 36.70 97.88 24.87 0.031AUC
0 (gh/mL) 213.86 45.59 124.10 31.60 0.031Vd/F (L/kg) 0.98 0.51
1.32 0.59 0.313CL/F (mL/min/kg) 0.82 0.21 1.40 0.27 0.031max: time
needed to reach maximum plasma concentration; max: max-imum plasma
concentration; 1/2abs: absorption half-life; 1/2el:
eliminationhalf-life; MRT: mean residence time; AUC0: area under
the plasmaconcentration-time curve from time 0 to time of last
quantifiable concentra-tion; AUC0: area under the plasma
concentration-time curve from time 0to infinity; Vd/F: volume of
distribution; CL/F: oral clearance.aStatistically significant if
< 0.05, using Wilcoxon matched-pair signed-rank test; = 7.
increased in pregnant rabbits in comparison with nonpreg-nant
group. The other pharmacokinetic parameters, involv-ing max,
1/2abs, 1/2el, MRT, and the volume of distribution(Vd/F), were not
significantly different in both pregnant andnonpregnant rabbits (
> 0.05). However, the mean value ofVd/F was increased by about
35% in pregnant compared tononpregnant rabbits.
4. Discussion
The present study was aimed at investigating the influence
ofpregnancy on the pharmacokinetic profile of ZNM in rabbitsbecause
well-studied, detailed pharmacokinetic investiga-tions involving
alterations and time course of alterations arelacking and knowledge
of pharmacokinetic profile of ZNM
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4 BioMed Research International
during pregnancy is important in order to optimize drugtherapy.
The present study was performed at late pregnancystage (28-29 days
gestation) because at this period, thephysiological alterations as
a consequence of pregnancy areexpected to be pronouncedwhichwould
potentially affect thepharmacokinetics of ZNM.
To the best of my knowledge, this is the first attempt
toinvestigate the potential effects of pregnancy on the
phar-macokinetics of ZNM. The present data demonstrated
thatpregnancy significantly ( < 0.05) modified the
pharma-cokinetic parameters (max, AUC0, AUC0, and CL/F) ofZNM in
rabbits. However, the other pharmacokinetic param-eters (max,
1/2abs, 1/2el, MRT, and Vd) demonstrated no sig-nificant
alterations by pregnancy ( > 0.05), Table 1.Although the mean
Vd/F was increased by about 35% inpregnant compared to nonpregnant
rabbits, it did not reachthe level of significance.
There has been only one published case report on
ZNMconcentrations during pregnancy [5]. In that report, ZNMplasma
levels were followed regularly in a pregnant patientstarting from
the fifth gestational week until the last week ofpregnancy. The
patient was on ZNM monotherapy at a dailydose of 200mg until week
29 and the plasma levels of thedrug were in the range of 7.5 to
10.1 g/mL (from fifth weekto week 22) and 4.4 g/mL in week 27.
Owing to low ZNMplasma levels, the daily dose was increased to
300mg at week29 and maintained at this level until delivery. The
case reportdid not investigate the pharmacokinetic profile that
couldelucidate the underlyingmechanisms responsible for reducedZNM
plasma concentrations during pregnancy because thestudy was based
on sparse concentration measurements ofonly one pregnant patient.
However, the investigators haverecommended utilization of
therapeutic monitoring of ZNMlevels throughout pregnancy.
Several mechanisms have been proposed to explain thedecrease in
the plasma concentrations of AEDs during preg-nancy. These include
reduced gastrointestinal drug absorp-tion, increased volume of
distribution, altered drug proteinbinding, enhanced metabolism, and
increased drug renalclearance [10, 13]. The findings of the present
study demon-strated that ZNM plasma concentrations were
consistentlylow in pregnant compared to nonpregnant rabbits and
thedecline in the plasma levels demonstrates that the
presentinvestigation supports and concurs with the observation
ofthe previous case report [5], Figure 1. However, the advantageof
the present investigation over the previous case report is
anendeavor to explain the underlying mechanisms causing thedecline
in ZNM plasma levels during pregnancy. The declinein ZNM plasma
levels during pregnancy in the present studyismost likely caused by
a combination of severalmechanisms.One of these mechanisms could be
a reduced gastrointestinalabsorption of ZNM. As shown in Table 1,
both max andAUC0
were significantly ( < 0.05) decreased in pregnantrabbits in
comparison with nonpregnant group leading toa reduction in the
extent of ZNM absorption, whereas therate of ZNM absorption was not
significantly altered, asdemonstrated by the unaltered max, 1/2abs,
and MRT, valuesin both groups ( > 0.05) [10]. Other mechanisms
thatcould be attributed to the reduced ZNM plasma levels during
pregnancy could be enhanced ZNM elimination as reflectedby a
marked increase ( < 0.05) in ZNM oral clearancein pregnant
compared to nonpregnant rabbits. Pregnancyis known to induce
changes in the hepatic metabolism ofmany drugs by affecting the
enzymes responsible for thedrug metabolism such as CYP450 and UGT.
Activity ofenzymes involving CYP3A4, CYP2D6, CYP2C9, UGT1A4,and
UGT2B7 is increased during pregnancy, whereas theactivity of CYP1A2
and CYP2C19 is decreased [1315]. ZNMis primarilymetabolized
byCYP3A4 isoenzymes, where theiractivities are known to be induced
during pregnancy, whichin turn may contribute to the low plasma
concentrations ofZNM during pregnancy in rabbits. In addition, a
significant( < 0.05) increase in ZNM oral clearance could
explainthe reduced plasma levels of the drug owing to enhancedrenal
blood flow and glomerular filtration rate (GFR) duringpregnancy
[16]. In pregnancy, however, the renal plasma flowincreases leading
to an increase in the GFR (up to 5080%).This results in an increase
of 2065% in the renal clearanceof many drugs [13, 16]. It has been
reported that 30% ofZNM is eliminated as unchanged in the urine
[3]. So, duringpregnancy ZNM renal clearance is expected to be
enhanced,and this may contribute to low ZNM plasma concentrationsin
the present study. Moreover, pregnancy is associated withan
increase in the volume of distribution of many drugs dueto an
increase in the plasma volume and a decrease in plasmaprotein
binding. Although the mean value of Vd/F of ZNMhas been increased
by about 35% in pregnant compared tononpregnant rabbits, this
effect is unlikely to influence ZNMplasma levels because the drug
is not significantly bound toplasma proteins (4060%) [3, 10].
The findings of the present study indicate that
thepharmacokinetic profile of ZNM in rabbits is altered in
lategestational period. Although limited by a relatively
smallsample size and a large interanimal variability in ZNM
phar-macokinetics observed within the same group, the presentstudy
suggests that the decline in ZNM plasma levels duringpregnancy is
most likely caused by decreased extent ofgastrointestinal
absorption, induced enzyme induction, andenhanced renal elimination
of the drug. Further studies ona large number of appropriate
subjects using single dose aswell as multiple doses at various
stages of pregnancy are war-ranted. Extrapolation of the findings
of the present study tohumans suggests consideration of therapeutic
drug moni-toring for ZNM during pregnancy and understanding
thepharmacokinetic alterations of ZNM during pregnancy isclinically
important in an attempt to optimize drug therapysince these
alterations may potentially affect the seizurecontrol.
Conflict of Interests
The author declared that there is no conflict of interests.
Acknowledgments
The author would like to acknowledge the Animal ResourcesCenter,
HSC, Kuwait University, for providing and taking full
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BioMed Research International 5
care of the experimental animals. The technical assistance
ofMss. B. Baby and A. George is highly appreciated.
References
[1] S. M. Hoy, Zonisamide: a review of its use in the
managementof adults with partial seizures, Drugs, vol. 73, pp.
13211338,2013.
[2] A. Schulze-Bonhage, Zonisamide in the treatment of
epilepsySchulze-Bonhage zonisamide, Expert Opinion on
Pharma-cotherapy, vol. 11, no. 1, pp. 115126, 2010.
[3] G. J. Sills and M. J. Brodie, Pharmacokinetics and drug
inter-actions with zonisamide, Epilepsia, vol. 48, no. 3, pp.
435441,2007.
[4] S. V. Kothare and J. Kaleyias, Zonisamide: review of
pharma-cology, clinical efficacy, tolerability, and safety, Expert
Opinionon Drug Metabolism and Toxicology, vol. 4, no. 4, pp.
493506,2008.
[5] K. S. Oles and W. L. Bell, Zonisamide cncentrations
duringpregnancy, Annals of Pharmacotherapy, vol. 42, no. 7-8,
pp.11391141, 2008.
[6] L. B. Holmes, The teratogenicity of anticonvulsant drugs:
aprogress report, Journal of Medical Genetics, vol. 39, no. 4,
pp.245247, 2002.
[7] D. Battino and T. Tomson, Management of epilepsy
duringpregnancy, Drugs, vol. 67, no. 18, pp. 27272746, 2007.
[8] T. Kondo, S. Kaneko, Y. Amano, and I. Egawa,
Preliminaryreport on teratogenic effects of zonisamide in the
offspring oftreated womenwith epilepsy, Epilepsia, vol. 37, no. 12,
pp. 12421244, 1996.
[9] Y. Terada, S. Fukagawa, K. Shigematsu et al.,
Reproductionstudies of zonisamide. Part 4. Teratogenicity study
inmice, dogsandmonkeys, Japanese Pharmacology andTherapeutics, vol.
15,pp. 44354451, 1987.
[10] T. Tomson, C. Landmark, and D. Battino, Antiepileptic
drugtreatment in pregnancy: changes in drug disposition and
theirclinical implications, Epilepsia, vol. 54, pp. 405414,
2013.
[11] A. Sabers and V. Petrenaite, Pharmacokinetics of
antiepilepticdrugs in pregnancy, Expert Review of Clinical
Pharmacology,vol. 1, no. 1, pp. 129136, 2008.
[12] V. P. Shah, K. K. Midha, J. W. A. Findlay et al.,
Bioanalyticalmethod validationa revisit with a decade of progress,
Phar-maceutical Research, vol. 17, no. 12, pp. 15511557, 2000.
[13] G. D. Anderson, Pregnancy-induced changes in
pharmacoki-netics: a mechanistic-based approach, Clinical
Pharmacokinet-ics, vol. 44, no. 10, pp. 9891008, 2005.
[14] C. Johannessen Landmark and P. Patsalos, Methodologies
usedto identify and characterize interactions among
antiepilepticdrugs, Expert Review of Clinical Pharmacology, vol. 5,
pp. 281292, 2012.
[15] C. Johannessen Landmark and P. N. Patsalos, Drug
inter-actions involving the new second- and
third-generationantiepileptic drugs, Expert Review of
Neurotherapeutics, vol. 10,no. 1, pp. 119140, 2010.
[16] P. B. Pennell, Antiepileptic drug pharmacokinetics
duringpregnancy and lactation, Neurology, vol. 61, no. 6, pp.
S35S42,2003.
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