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; kamal@hsc.edu.kw

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

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

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

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

BioMed Research International 5

care of the experimental animals. The technical assistance ofMss. B. Baby and A. George is highly appreciated.

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