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pilepsy Research (2007) 75, 112—121

journa l homepage: www.e lsev ier .com/ locate /ep i lepsyres

nfluence of levetiracetam on the anticonvulsantfficacy of conventional antiepileptic drugs againstudiogenic seizures in DBA/2 mice

ugenio Donato Di Paolaa, Pietro Gareri a, Alessandro Davoli a,anto Gratteri a, Francesca Scicchitanoa, Clara Naccarib,iovambattista De Sarroa,∗

Section of Pharmacology, Department of Experimental and Clinical Medicine, Faculty of Medicine and Surgery,niversity of Catanzaro, Catanzaro, ItalyDepartment Farmaco-Biologico, School of Pharmacy, University of Messina, Villaggio SS Annunziata, Contr. Annunziata,8168 Messina, Italy

eceived 8 February 2007; received in revised form 22 April 2007; accepted 24 April 2007vailable online 5 June 2007

KEYWORDSEpilepsy;Levetiracetam;Antiepileptic drug;Carbamazepine;Audiogenic seizure;DBA/2 mouse

Summary Levetiracetam (LEV, [S]-alpha-ethyl-2-oxo-1-pyrrolidine acetamide) is a newantiepileptic that has been used as adjunctive therapy to treat patients with intractableepilepsy. Systemic administration of levetiracetam (2.5—30 mg/kg, intraperitoneally (i.p.)) wasable to produce a dose-dependent decrease in DBA/2 audiogenic seizure severity score. In com-bination with conventional antiepileptic drugs, levetiracetam, 5 mg/kg, i.p., which per se didnot significantly affect the occurrence of audiogenic seizures in DBA/2 mice, potentiated theanticonvulsant activity of some antiepileptic drugs studied against sound-induced seizures inDBA/2 mice. The degree of potentiation induced by levetiracetam was greater, approximatelytwice, for carbamazepine, diazepam, felbamate, topiramate, gabapentin, and valproate, lessfor lamotrigine, phenobarbital and phenytoin. This increase was associated with a compara-ble impairment in motor activity; however, the therapeutic index of combined treatment ofantiepileptic drugs with levetiracetam was more favourable than the combination with salinewith the exception of lamotrigine, phenytoin and phenobarbital. Since levetiracetam did notsignificantly influence the total and free plasma and the brain levels of antiepileptics studied.

In addition, levetiracetam did not significantly affect the hypothermic effects of the anticon-vulsants tested. In conclusion,administered in combination witdiazepam, felbamate, gabapentrelevance of such drug combina© 2007 Elsevier B.V. All rights re

∗ Corresponding author at: Department of Experimental and Clinical Momini, Via T. Campanella 115, 88100 Catanzaro, Italy. Tel.: +39 0961 71

E-mail address: desarro@unicz.it (G. De Sarro).

920-1211/$ — see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.eplepsyres.2007.04.008

levetiracetam showed an additive anticonvulsant effect when

h some classical anticonvulsants, most notably carbamazepine,in, topiramate and valproate, implicating a possible therapeutictions.served.

edicine, School of Medicine at Catanzaro, Policlinico Mater2323; fax: +39 0961 774424.

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Levetiracetam enhances anticonvulsant effects of certain an

Introduction

Monotherapy is actually considered as the optimal treat-ment of epilepsy. However, around 30% of patients mayexperience seizures resistant to the currently availableanticonvulsant drugs (Kwan and Brodie, 2000a,b). Actually,polytherapy with two or more antiepileptic drugs remainsthe treatment of choice in patients who do not respondadequately to antiepileptic monotherapy (Perucca, 2006).Furthermore, these patients experience numerous prob-lems, including Central Nervous System (CNS) side effectsand idiosyncratic reactions, which are exacerbated by phar-macokinetic and/or pharmacodynamic interactions betweendrugs (Patsalos and Perucca, 2003a,b). With the recentintroduction of 10 new antiepileptics, the number of possi-ble antiepileptic combinations has exponentially increased.Actually, there are no rational two-drug combinations tochoose from, although through clinical experience severaldrug antiepileptic combinations have been identified asbeing efficacious against specific seizure types (Stephenand Brodie, 2002). Since the direct clinical evaluation ofthe anticonvulsant efficacy of drug combinations in epilep-tic patients is ethically and methodologically very difficult,potential combinations may be readily pre-selected in pre-clinical studies on animals, and only those, displayingsynergistic interactions in terms of seizure suppression couldbe considered for further clinical evaluation.

Levetiracetam ([S]-alpha-ethyl-2-oxo-1-pyrrolidine ace-tamide) is a new antiepileptic that has been licensedfor clinical use as adjunctive therapy to treat patientswith intractable partial-onset seizures with or without sec-ondary generalization (Betts et al., 2000; Cereghino etal., 2000; Shorvon et al., 2000; Siniscalchi et al., 2005).In the clinical setting, levetiracetam has demonstrated abroad spectrum of anticonvulsant activity, showing effi-cacy in suppressing juvenile myoclonic epilepsy (Kumar andSmith, 2004); tonic—clonic, absence and myoclonic epilepsy(Genton and Gelisse, 2000; Frucht et al., 2001; Krauss etal., 2003); atypical absence or atonic seizures and photo-sensitive epilepsy (Kasteleijn-Nolst Trenite et al., 1996).Additionally, levetiracetam is efficacious in children withpartial-onset seizures and in those with myoclonic seizures(Tan and Appleton, 2004). Levetiracetam has been suc-cessfully used in patients with refractory partial epilepsyconverted to monotherapy (Ben-Menachem and Falter, 2000;Alsaadi et al., 2002, 2004; Alsaadi and Thieman, 2003).

In preclinical studies, it has been found that levetirac-etam is virtually ineffective in acute models of epilepsy(i.e. the maximal electroshock (MES)- and pentylenete-trazole (PTZ)-induced seizures), which are routinely usedto screen for potential new antiepileptics (Loscher andSchmidt, 1988). In contrast, levetiracetam increased thethreshold for electroconvulsions and suppressed seizures inkindled and genetically epileptic animals (Gower et al.,1992, 1995; Loscher and Honack, 1993; Klitgaard et al.,1998; Loscher et al., 1998). The precise mechanism of actionof levetiracetam has not been fully understood. It reducesvoltage-operated K+ current and inhibits the delayed rec-

tifier K+ current in neurons (Madeja et al., 2003), reducesN-type and partially P/Q type high-voltage activated (HVA)Ca2+ currents (Niespodziany et al., 2001; Lukyanetz et al.,2002; Pisani et al., 2004) but not low-voltage-activated

natu

ileptics in DBA/2 mice 113

a2+ currents (Zona et al., 2001). Indeed, levetiracetamuppresses the inhibitory action of zinc and �-carbolinesn GABAA- and glycine-gated currents (Rigo et al., 2002).ecent molecular studies involving transgenic mice suggesthat levetiracetam binds to a synaptic vesicle protein 2ASV2A), which is involved in vesicle neurotransmitter exocy-osis, and that the affinity of binding to SV2A significantlyorrelates with anticonvulsant potency by a series of leve-iracetam derivatives (Lynch et al., 2004).

There is increasing evidence that levetiracetam is asso-iated with favourable pharmacodynamic interaction withumerous antiepileptics in various animal models includ-ng: diazepam (Mazarati et al., 2004), topiramate (Sills etl., 2004; Luszczki et al., 2005, 2006) oxcarbazepine andarbamazepine (Luszczki et al., 2007).

Consequently, the aim of the present study was tonvestigate the efficacy of conventional antiepileptic drugsuch as carbamazepine, diazepam, felbamate, phenytoin,abapentin, lamotrigine, phenobarbital, topiramate andalproate administered together with levetiracetam againstudiogenic seizures in DBA/2 mice. Additionally, we inves-igated the antiepileptic combinations in relation to motormpairment by the use of the rotarod test. Finally, brainevetiracetam and other antiepileptics concentrations wereeasured in order to ascertain whether any observed effectsere consequent to a pharmacodynamic and/or a pharma-okinetic interaction.

aterials and methods

nimals

he experiments were carried out on DBA/2 mice, weighing 6—12 g22—26 days old) or 20—28 g (48—56 days old). Animals were pur-hased from Harlan Italy (Correzzana, Milan, Italy) and housed inroups of 8—10 in colony cages at room temperature, under a 12-light/12-h dark cycle (lights on at 7:00 a.m.). The animals were

oused with free access to food pellets and tap water with foodnd water available ad libitum. Experimental groups, consistingf 10 animals, were assigned according to a randomised schedule,nd each mouse was used only once. Control animals were alwaysested on the same day as the respective experimental groups. Addi-ionally, all efforts were made to minimize animal suffering ando use only the number of animals necessary to produce reliablecientific data. Procedures involving animals and their care wereonducted in conformity with international and national law andolicies (European Communities Council Directive of 24th November986, 86/609EEC).

xperimental protocols

BA/2 mice were exposed to auditory stimulation, 30, 60, 90 or20 min following intraperitoneal (i.p.) administration of levetirac-tam (2.5—30 mg/kg) or saline and 30, 45, 60 or 120 min following.p. injection of some antiepileptics. Each mouse was placed under aemispheric perspex dome (diameter 58 cm) and 1 min was allowedor habituation and assessment of locomotor activity. Auditory stim-lation (12—16 kHz, 109 dB) was applied for 1 min or until tonicxtension occurred. The seizure response, as previously reported

o response, 1: wild running, 2: clonus, 3: tonus, 4: respiratoryrrest. The maximum response was recorded for each animal. Rec-al temperature was recorded immediately prior to auditory testingsing an Elektrolaboratoriet thermometer type T.E.3. Behavioural

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hanges were observed during the period between drug administra-ion and auditory testing.

etermination of the plasma and brain levels of thentiepileptic compounds

BA/2 mice (20—28 g) were given i.p. either saline and onentiepileptic drug or levetiracetam and one antiepileptic drug.he same protocol was used for behavioural and pharmacokinetictudies. Older DBA/2 mice were used for pharmacokinetic studiesecause it is very difficult to collect blood from younger DBA/2ice. No differences in pharmacokinetics were reported between

1—26 and 48—56 days old mice (De Sarro et al., 1998, 2002; Garerit al., 2004). The animals were lightly anaesthetized with ethylther and killed by decapitation at appropriate times and bloodamples of approximately 1 ml were collected into Eppendorf tubes.he felbamate and lamotrigine assay was carried out using high-erformance liquid chromatography (HPLC) analysis (Rizzo et al.,997). The gabapentin assay was carried out using a HPLC methodreviously described by Tang et al. (1999). Levetiracetam con-entrations were determined by the HPLC method described byatnaraj et al. (1996). Blood samples were centrifuged at 2000 rpmor 15 min for carbamazepine, diazepam, phenytoin and pheno-arbital determination. The plasma was put into a MPS-1 systemAmicon, Danvers, MA, USA) for the separation of free from protein-ound microsolutes. Plasma samples, 60 �l, were transferred topecial sample cups and inserted into an Automatic Clinical AnalyserACA II, du Pont, Wilmington, DE, USA) which uses a method based onhe homogenous enzyme immunoassay technique. For the valproatessay a serum sample, 50 �l, was diluted twice with Tris buffer andnalysed using the same method. For brain antiepileptic determi-ation brains were removed from skulls, weighed and homogenizedy using bidistilled water (2:1, w/v). The homogenates were cen-rifuged at 10,000 rpm for 10 min. The supernatant samples (60 or0 �l for valproate only) were analysed as previously described forlasma samples. Control drug solutions were put before and afterhe respective antiepileptic experimental samples.

ffects on motor movements

ehavioural changes and their onset and duration were recordedfter drug injection until the time of the rotarod test. In particu-ar, two independent observers followed gross behavioural changesonsisting of locomotor activity, ataxia, squatting posture and pos-ible piloerection. These behavioural changes were noted but nottatistically analysed. Groups of 10 DBA/2 mice, 8—12 g and 22—26ays old, were trained to do coordinated motor movements continu-usly for 2 min on a 3-cm diameter rotarod turning at 8 rev min−1 (U.asile, Comerio, Varese, Italy). Impairment of coordinated motorovements was defined as inability of the mice to remain on the

otarod for a 2-min test period (Dunham and Miya, 1957). The abil-ty of the mice to remain on the rotarod was tested 45 min after the.p. administration of saline and one of the conventional antiepilep-ics, after the combined treatment with levetiracetam and one ofhe antiepileptic drugs.

tatistical analysis

tatistical comparisons of groups of control and drug treated ani-als were made using Fisher’s exact probability test (incidence of

he seizure phases and influence on motor performance) or anal-sis of variance (ANOVA) and Dunnett’s test (rectal temperature).

he percent incidence of each phase per dose of the administeredompound and the dose—response curves were fitted using linearegression analysis. ED50 values (±95% confidence limits) for eachompound and each phase of seizure response were estimated usingcomputer program (SPSS for windows, SPSS, Chicago, USA) for the

Tmawe

E. Donato Di Paola et al.

ethod of Litchfield and Wilcoxon (1949); the relative anticonvul-ant activities were determined by comparison of respective ED50alues. The lines of best fit for conventional antiepileptic drug plusaline or in association with levetiracetam were compared using a2-test, with results expressed for position, parallelism and hetero-eneity. TD50 values (±95% confidence limits) for each compoundere estimated using the method of Litchfield and Wilcoxon (1949).he plasma levels of the drugs are expressed as means ± S.E.M.f at least eight determinations and Student’s t-test was used fortatistical comparisons. Group differences of P < 0.05 consideredtatistically significant.

rugs

he drugs and their sources were: carbamazepine (Novartis, Basel,witzerland), diazepam (Hoffman La Roche, Basel, Switzerland),elbamate (Schering Plough, Milano, Italy), which were suspendedn a 1% solution of Tween 80. Valproate (Mg2+ salt) (Sigma Tau,omezia, Italy), gabapentin (Parke Davies, Milano, Italy), pheno-arbital (Na+ salt, Bracco, Milano, Italy), phenytoin (Na+ salt,ecordati, Milano, Italy), lamotrigine (Glaxo-Wellcome, Verona,taly), topiramate was a gift from Dr. R.P. Shank (Johnson & Johnsonharmaceutical Research & Development LCC, USA) and levetirac-tam (UCB Pharma, Braine-l’Alleud, Belgium) were dissolved in 0.9sterile saline. All drugs were administered intraperitoneally (i.p.),

he injection volume was always 0.1 ml/10 g of body weight. Theoses for the drugs and optimal treatment window are based onrevious studies (De Sarro et al., 1998, 2000, 2002; Gareri et al.,004). Control animals received equivalent volumes of the solventt the respective times before the test.

esults

nticonvulsant properties of levetiracetam inBA/2 mice

o allow better evaluation of anticonvulsant activity, wexposed the animals to auditory testing at different timesfter levetiracetam administration. Levetiracetam (10, 15nd 20 mg/kg, i.p.), produced dose dependent significantrotection (P < 0.01) against the clonic or tonic phase ofhe audiogenic seizure response in DBA/2 mice 30, 60,0 and 120 min after administration (Fig. 1). Significantrotection against the wild running phase was observed0, 60 and 90 min after levetiracetam 20 mg/kg, i.p.. Aarger dose of levetiracetam (30 mg/kg, i.p.) was necessaryo significantly antagonize the wild running phase whenevetiracetam was administered 120 min before auditoryesting. After levetiracetam 2.5 and 5 mg/kg, i.p., no signif-cant anticonvulsant activity or behavioural changes werebserved. ED50 values (±95% confidence limits) of levetirac-tam against the different phases of audiogenic seizures areeported in Table 1. The doses of levetiracetam used in theresent study carried neither ataxia nor a fall in rectal tem-eratures or a reduction in locomotor activity was evident.ince levetiracetam appeared to exert its maximal anticon-ulsant activity at 60 min (Table 1 and Fig. 1), we decidedo use this pretreatment time for the subsequent studies.

he dose of 5 mg/kg of levetiracetam was chosen, being theaximum ineffective dose in our animal model. In addition,

s in previous studies, all the conventional anticonvulsantsere administered 45 min before auditory testing (De Sarrot al., 1998, 2000, 2002; Gareri et al., 2004).

Levetiracetam enhances anticonvulsant effects of certain antiepileptics in DBA/2 mice 115

Table 1 ED50 values (+95% confidence limits) for levetiracetam on audiogenic seizures in DBA/2 mice 30, 60, 90 and 120 minafter drug administration

Treatment time (min) Seizure phase

Wild running Clonus Tonus

30 18.66 (14.35—24.25) 11.36 (8.50—15.18) 9.04 (6.15—13.27)60 15.38 (12.17—19.45) 9.77 (7.22—13.22) 7.89 (5.89—10.57)90 17.72 (13.44—23.37) 9.86 (6.83—14.25) 8.60 (5.95—12.44)

120 22.65 (17.07—30.04) 14.91 (11.47—19.39) 11.50 (8.32—15.580)

ding

response varied according to the different classes. Asshown in Table 2, diazepam, carbamazepine, felbamate,

All data above are expressed in mg/kg and were calculated accor

Influence of levetiracetam on the anticonvulsantactivity of conventional antiepileptic drugs against

audiogenic seizures

The influence of levetiracetam on the activity of the con-ventional antiepileptic drugs on the audiogenic seizure

Figure 1 Dose—response curves of anticonvulsant activity oflevetiracetam, 1—30 mg/kg, administered i.p. 30, 60, 90 and120 min before auditory test. Abscissae show the doses, ordinateshows the percentage of clonic (A) and tonic (B) seizures.

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to the method of Litchfield and Wilcoxon (1949).

abapentin, lamotrigine, phenobarbital, phenytoin, topi-amate and valproate exhibited anticonvulsant activity in

Table 2 ED50 values (+95% confidence limits) of saline +some antiepileptic drugs or in association with levetiracetam(5 mg/kg, i.p.) against audiogenic seizures in DBA/2 mice

Seizure phase Drug + saline Drug + levetiracetam

Wild runningCarbamazepine 10.6 (8.1—13.8) 5.3 (4.0—7.02)a

Diazepam 0.49 (0.34—0.71) 0.23 (0.15—0.35)a

Felbamate 114.6 (92—142.7) 54.8 (36.2—82.96)a

Gabapentin 38 (16—51) 23.2 (17.1—31.5)a

Lamotrigine 6.1 (4.6—8.1) 5.3 (3.8—7.4)Phenobarbital 7.1 (5.6—9) 5.4 (4.5—8.6)Phenytoin 4.3 (3.1—6) 3.7 (2.7—5.1)Topiramate 22.9 (15.8—33.9) 10.8 (8.7—13.4)a

Valproate 84 (63—114) 44 (29.1—61.7)a

ClonusCarbamazepine 4.4 (3.6—5.4) 2.2 (1.6—3.02)a

Diazepam 0.28 (0.2—0.39) 0.13 (0.10—0.17)a

Felbamate 48.8 (35.4—67.2) 25.6 (18.9—34.7)a

Gabapentin 20.3 (13.7—30.2) 9.9 (7.8—12.6)a

Lamotrigine 3.5 (2.4—5.1) 2.8 (1.9—4.1)Phenobarbital 3.4 (2.3—5) 2.5 (1.9—3.3)Phenytoin 2.5 (1.8—3.5) 1.9 (1.4—2.6)Topiramate 12.12 (6.94—21.15) 5.91 (4.22—8.28)a

Valproate 43 (33—56) 21.5 (16.1—28.7)a

TonusCarbamazepine 3.0 (2.6—3.8) 1.5 (1.1—2.7)a

Diazepam 0.24 (0.15—0.39) 0.11 (0.07—0.17)a

Felbamate 23.1 (12.1—44) 12.2 (9.9—15.0)a

Gabapentin 13.9 (8.7—22.3) 6.5 (4.9—8.6)a

Lamotrigine 1.1 (0.7—1.8) 0.9 (0.7—1.16)Phenobarbital 2.4 (1.7—3.4) 1.6 (1.2—2.13)Phenytoin 2.0 (1.6—2.5) 1.5 (1.1—2.05)Topiramate 6.12 (3.48—20.88) 3.19 (2.12-4.80)a

Valproate 31 (22—43) 15.4 (10.5—22.6)a

All data above are expressed in mg/kg and were calculatedaccording to the method of Litchfield and Wilcoxon (1949).Significant differences in the ED50 values between concurrentgroups of saline plus antiepileptic drug or levetiracetam plusantiepileptic drug-treated groups are denoted by aP < 0.01.

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he audiogenic seizure model of DBA/2 mice. Pretreatment60 min before anticonvulsant administration) with leve-iracetam (5 mg/kg, i.p.) was able to produce a consistenthift to the left of the dose—response curves and a signifi-ant reduction of ED50 values of antiepileptics, suggestingn increase in anticonvulsant activity. All dose—responseurves were parallel. The degree of potentiation by lev-tiracetam varied among the anticonvulsant drugs, beingreatest for carbamazepine, diazepam, felbamate, topira-ate, gabapentin and valproate and less for lamotrigine,henobarbital and phenytoin (Table 2).

nfluence of levetiracetam on the motormpairment induced by antiepileptic drugs

o gross behavioural changes and neurological deficits wereetected at any doses of levetiracetam. Levetiracetamdministered at dose levels up to 50 mg/kg, i.p., did notignificantly affect locomotor performance. When appliedt doses equal to their ED50 values against the clonic phasef the audiogenic seizures, carbamazepine (4.4 mg/kg),iazepam (0.28 mg/kg), gabapentin (20.3 mg/kg), fel-amate (48.8 mg/kg), lamotrigine (3.5 mg/kg), pheny-oin (2.5 mg/kg), phenobarbital (3.4 mg/kg), topiramate12.12 mg/kg) and valproate (43 mg/kg) did not influencehe motor performance of DBA/2 mice. Higher doses wereecessary to produce motor impairment (Table 3). Concomi-ant treatment with one antiepileptic and levetiracetam

5 mg/kg, i.p.) resulted usually in a weak increase of motormpairment (Table 3). In fact, the therapeutic index ofombined treatment with carbamazepine + levetiracetam,iazepam + levetiracetam, felbamate + levetiracetam, topi-amate + levetiracetam, gabapentin + levetiracetam or valp-

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Table 3 TD50 values (±95% confidence limits) of saline + various athe rotarod

Treatment TD50

Saline + carbamazepine 46.5 (37.9Levetiracetam + carbamazepine 34.1 (24.6Saline + diazepam 3.8 (3.0—Levetiracetam + diazepam 2.9 (1.8—Saline + felbamate 816 (590—1Levetiracetam + felbamate 764 (636—9Saline + gabapentin 290.3 (218.Levetiracetam + gabapentin 187.2 (140.Saline + phenytoin 48.3 (50.9Levetiracetam + phenytoin 28.9 (20.2Saline + lamotrigine 81 (55—11Levetiracetam + lamotrigine 51.2 (43.1Saline + phenobarbital 139 (115—1Levetiracetam + phenobarbital 87 (63—12Saline + topiramate 162 (110—2Levetiracetam + topiramate 102.4 (83.1Saline + valproate 290 (240—2Levetiracetam + valproate 235 (178—3

All data are expressed as mg/kg and were calculated according to the mindex which represents the ratio between TD50 and ED50 from the clowere observed between concurrent groups.

E. Donato Di Paola et al.

oate + levetiracetam was more favourable than thator carbamazepine + saline, diazepam + saline, felbamate +aline, topiramate + saline, gabapentin + saline or valproatesaline. On the contrary, the therapeutic index of combined

reatment with phenobarbital + levetiracetam, lamotrig-ne + levetiracetam or phenytoin + levetiracetam was lessavourable than that for saline, phenobarbital + saline, lam-trigine + saline or phenytoin + saline or (Table 3).

ffects of combined treatment with levetiracetamith antiepileptic compounds on body temperature

he body temperature was recorded in animals given salineanticonvulsant drugs or levetiracetam + anticonvulsantrugs. Hypothermic effects were observed only after admin-stration of saline + the highest doses of carbamazepine (20,0 and 50 mg/kg, i.p.), diazepam (3 and 5 mg/kg, i.p.)nd valproate (100, 200 and 300 mg/kg, i.p.). In particular,e observed a dose dependent reduction in body tem-erature of 2—4 ◦C only after these drugs. No significantifferences among groups treated with saline + felbamate,abapentin, lamotrigine, phenytoin, phenobarbital, topira-ate or low doses of carbamazepine, diazepam or valproateere evident (data not shown). A no-significant increasef hypothermic effects was observed in mice receiv-ng carbamazepine, diazepam or valproate + levetiracetamhen compared to their concurrent saline + carbamazepine,

iazepam or valproate group. On the contrary, mice treatedith levetiracetam (5 mg/kg, i.p.) + the other antiepilepticrugs showed no significant changes of hypothermic effectshen compared with concurrent saline + antiepileptic drugs-

reated animals.

ntiepileptics and levetiracetam + antiepileptics obtained with

Locomotor deficit TD50/ED50

—57) 10.6—44.5) 15.54.8) 13.64.67) 22.3

024) 16.717.8) 29.83—386) 14.37—249.1) 18.9—68.4) 19.3—41.3) 15.28) 23.1—60.8) 18.368) 40.90.1) 34.838.6) 13.4—126.18) 17.351) 6.710.2) 10.9

ethod of Litchfield and Wilcoxon (1949). TD50/ED50 = therapeuticnic phase of the audiogenic seizures. No significant differences

Levetiracetam enhances anticonvulsant effects of certain antiepileptics in DBA/2 mice 117

Table 4 Influence of levetiracetam on total and free plasma levels of some antiepileptic compounds (carbamazepine,diazepam, felbamate, gabapentin, lamotrigine, phenytoin, phenobarbital, topiramate and valproate) in DBA/2 mice

Treatment (time) (dose mg/kg) Saline + compound Levetiracetam + compound

Total Free Total Free

Carbamazepine (60 min) (15 mg/kg) 5.2 ± 0.7 0.62 ± 0.2 5.1 ± 0.5 0.66 ± 0.2Diazepam (60 min) (5 mg/kg) 2.1 ± 0.2 0.15 ± 0.05 2.0 ± 0.3 0.21 ± 0.05Felbamate (60 min) (100 mg/kg) 4.2 ± 0.3 3.1 ± 0.3 4.1 ± 0.3 3.0 ± 0.3Gabapentin (45 min) (70 mg/kg) 10.2 ± 1.5 10.3 ± 1.4Lamotrigine (45 min) (10 mg/kg) 1.8 ± 0.2 0.67 ± 0.07 1.9 ± 0.2 0.69 ± 0.1Phenytoin (120 min) (10 mg/kg) 8.8 ± 1.8 0.9 ± 0.1 8.6 ± 1.9 1.2 ± 0.1Phenobarbital (60 min) (20 mg/kg) 35.3 ± 3.1 4.4 ± 0.3 35.2 ± 3.2 4.5 ± 0.3Phenobarbital (120 min) (20 mg/kg) 22.4 ± 2.5 3.1 ± 0.3 21.3 ± 2.4 3.0 ± 0.3Topiramate (90 min) (30 mg/kg) 7.43 ± 0.58 4.51 ± 0.39 7.38 ± 0.62 4.56 ± 0.41Valproate (30 min) (200 mg/kg) 251 ± 22 40.2 ± 3.9 245 ± 23 43 ± 3.9Valproate (60 min) (200 mg/kg) 309 ± 29 49.4 ± 4.1 298 ± 31 60 ± 4.2

Drugs were administered i.p. Saline or levetiracetam (5 mg/kg, i.p.) + lamotrigine and gabapentin 45 min, carbamazepine, diazepam,and felbamate 60 min, phenobarbital 60 and 120 min, phenytoin 120 min, topiramate 90 min and valproate 30 and 60 min before drawingthe sample. Values are means (�g/ml) of at least eight determinations ± S.E.M. Student’s t-test was used for statistical analysis of thedata.

Table 5 Influence of levetiracetam on brain levels of some antiepileptic compounds (carbamazepine, diazepam, felbamate,gabapentin, lamotrigine, phenytoin, phenobarbital, topiramate and valproate) in DBA/2 mice

Treatment (time) (dose mg/kg) Saline + compound Levetiracetam + compound

Carbamazepine (60 min) (15 mg/kg) 3.65 ± 0.49 3.69 ± 0.44Diazepam (60 min) (5 mg/kg) 2.14 ± 0.18 2.16 ± 0.19Felbamate (60 min) (100 mg/kg) 3.57 ± 0.39 3.61 ± 0.35Gabapentin (45 min) (70 mg/kg) 9.8 ± 1.5 9.9 ± 1.5Lamotrigine (45 min) (10 mg/kg) 1.52 ± 0.18 1.58 ± 0.2Phenytoin (120 min) (10 mg/kg) 1.54 ± 0.22 1.61 ± 0.20Phenobarbital (60 min) (20 mg/kg) 9.04 ± 0.72 8.75 ± 0.74Phenobarbital (120 min) (20 mg/kg) 6.12 ± 0.56 6.3 ± 0.39Topiramate (90 min) (30 mg/kg) 4.41 ± 0.31 4.49 ± 0.32Valproate (30 min) (200 mg/kg) 66.14 ± 5.71 72.01 ± 5.92Valproate (60 min) (200 mg/kg) 91.29 ± 7.89 80.60 ± 5.22

Drugs were administered i.p. Saline or levetiracetam (5 mg/kg, i.p.) + lamotrigine and gabapentin 45 min, carbamazepine, diazepam,and felbamate 60 min, phenobarbital 60 and 120 min, phenytoin 120 min, topiramate 90 min and valproate 30 and 60 min before drawing

tions

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the sample. Values are means (�g/ml) of at least eight determinadata.

Influence of levetiracetam on the total and freeplasma levels of antiepileptic drugs

Blood concentrations of carbamazepine, diazepam, felba-mate, gabapentin, lamotrigine, phenytoin, phenobarbital,topiramate and valproate are presented in Table 4.The dose of levetiracetam studied did not signif-icantly modify the plasma levels of carbamazepine(15 mg/kg, i.p.), felbamate (100 mg/kg, i.p.), lamotrigine(10 mg/kg, i.p.), phenytoin (10 mg/kg, i.p), phenobarbital(20 mg/kg, i.p), gabapentin (70 mg/kg, i.p.), topiramate

(30 mg/kg, i.p.), valproate (200 mg/kg, i.p.) and diazepam(5 mg/kg, i.p.). A small increase of free plasma levelsof carbamazepine, diazepam, phenytoin and valproate,was observed following concomitant treatment withlevetiracetam.

D

Ttb

± S.E.M. Student’s t-test was used for statistical analysis of the

nfluence of levetiracetam on the brain levels ofntiepileptic drugs

rain concentrations of carbamazepine, diazepam, felba-ate, gabapentin, lamotrigine, phenytoin, phenobarbital,

opiramate and valproate were not significantly differenthen these anticonvulsants were administered in combina-

ion with saline or levetiracetam (Table 5). Similarly, thenticonvulsant drugs studied did not significantly affect therain level of levetiracetam (Table 6).

iscussion

he present data indicate that the protective action of cer-ain antiepileptics against audiogenic seizures is enhancedy co-administration of levetiracetam.

118

Table 6 Influence of some antiepileptic compounds(carbamazepine, diazepam, felbamate, gabapentin, lamot-rigine, phenytoin, phenobarbital, topiramate and valproate)on levetiracetam brain levels in DBA/2 mice

Treatment (time)(dose mg/kg)

Saline +levetiracetam

compound +levetiracetam

Carbamazepine (60 min; 15 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.46 ± 0.06Levetiracetam (10) 1.82 ± 0.18 1.90 ± 0.19Levetiracetam (20) 4.16 ± 0.35 4.32 ± 0.39

Diazepam (60 min; 5 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.44 ± 0.05Levetiracetam (10) 1.82 ± 0.18 1.86 ± 0.19Levetiracetam (20) 4.16 ± 0.35 4.30 ± 0.37

Felbamate (60 min; 100 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.43 ± 0.05Levetiracetam (10) 1.82 ± 0.18 1.89 ± 0.18Levetiracetam (20) 4.16 ± 0.35 4.22 ± 0.35

Gabapentin (45 min; 70 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.42 ± 0.05Levetiracetam (10) 1.82 ± 0.18 1.81 ± 0.19Levetiracetam (20) 4.16 ± 0.35 4.19 ± 0.34

Lamotrigine (45 min; 10 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.38 ± 0.05Levetiracetam (10) 1.82 ± 0.18 1.76 ± 0.17Levetiracetam (20) 4.16 ± 0.35 4.08 ± 0.31

Phenytoin (120 min; 10 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.44 ± 0.08Levetiracetam (10) 1.82 ± 0.18 1.85 ± 0.21Levetiracetam (20) 4.16 ± 0.35 4.26 ± 0.41

Phenobarbital (60 min; 20 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.40 ± 0.04Levetiracetam (10) 1.82 ± 0.18 1.78 ± 0.16Levetiracetam (20) 4.16 ± 0.35 4.12 ± 0.32

Topiramate (90 min; 30 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.41 ± 0.05Levetiracetam (10) 1.82 ± 0.18 1.79 ± 0.17Levetiracetam (20) 4.16 ± 0.35 4.1 ± 0.36

Valproate (60 min; 200 mg/kg)Levetiracetam (5) 0.42 ± 0.05 0.47 ± 0.06Levetiracetam (10) 1.82 ± 0.18 1.91 ± 0.19Levetiracetam (20) 4.16 ± 0.35 4.34 ± 0.39

Drugs were administered i.p. Saline or levetiracetam (5 mg/kg,i.p.) + lamotrigine and gabapentin 45 min, carbamazepine,diazepam, and felbamate 60 min, phenobarbital 60 and120 min, phenytoin 120 min, topiramate 90 min and valproate

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30 and 60 min before drawing the sample. Values are means(�g/ml) of at least eight determinations ± S.E.M. Student’st-test was used for statistical analysis of the data.

In particular, the potency of carbamazepine, diazepam,elbamate, gabapentin, valproate and topiramate was much

ore enhanced than that of other antiepileptic studied.

n contrast, the anticonvulsant potency of lamotrigine,henytoin and phenobarbital was not significantly modified.he mechanisms causing the observed augmentation of the

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E. Donato Di Paola et al.

ffectiveness of the conventional antiepileptics mentionedre most probably not related to a pharmacokineticsnteraction, as the plasma and the brain levels of allompounds studied remained unchanged in the presence ofevetiracetam.

Our data in agreement with previous experiments show-ng that levetiracetam exerts anticonvulsant activity inudiogenic sensitive DBA/2 mice (Noyer et al., 1995; Lyncht al., 2004).

In addition, the observed synergistic interactionsetween levetiracetam and carbamazepine are in agree-ent with recent reports (Luszczki et al., 2005, 2006, 2007)

nd may probably be due to the fact that levetiracetam actsith a different mechanism of action than other antiepilep-

ics (i.e. on SV2A proteins which are involved in neuro-ransmitter exocytosis). Conversely, additional mechanismsould include an effect of levetiracetam on N- and P/Q-ypes HVA Ca2+ channels and an effect of carbamazepinen Na+-channel blockade or activation of the adenosiner-ic inhibitory neurotransmitter system (Schmidt and Elger,004). In contrast to carbamazepine, lamotrigine and pheny-oin (two drugs whose main mechanism of action is alsoelated with Na+ channel blockade) did not interact syner-istically with levetiracetam in the audiogenic seizure test.

The fixed dose of 5 mg/kg tested in the present studyas chosen on the bases that it they would moderatelyffect the audiogenic seizures in DBA/2 mice.

Furthermore, the present data showing the synergisticnteraction between levetiracetam and topiramate are gen-rally in agreement with those reported in a previous studySills et al., 2004). The latter authors, demonstrated thatevetiracetam (50 and 250 mg/kg) in combination with top-ramate (25 and 125 mg/kg) was associated with a potentnticonvulsant effect, suppressing tonic—clonic seizures inhe MES test, and that levetiracetam (50 and 250 mg/kg)n combination with topiramate (125 mg/kg) protected thenimals against PTZ-induced seizures, despite the inac-ivity of the two drugs in PTZ test when administeredlone (Sills et al., 2004). Topiramate possesses multipleechanisms of action (Russo et al., 2004), then it maye possible that the synergistic interactions observed areue to one or more mechanisms. For example, topira-ate inhibits excitatory neurotransmission at AMPA/kainite

eceptors and levetiracetam block neurotransmitter exo-ytosis via SV2A proteins. Another synergistic interactionhat might be envisaged entails the modulation of neu-onal L-type HVA Ca2+ channels mediated by topiramateith the inhibition of N- and P/Q-type HVA Ca2+ channelsue to levetiracetam. A synergistic interaction betweeniazepam (1 mg/kg) and levetiracetam has also been pre-iously observed with regard to control of status epilepticusnduced by perforant-path stimulation in rats (Mazarati etl., 2004). Interestingly, the synergistic interaction of leve-iracetam with all antiepileptics studied occurred at lowerose of levetiracetam (5 mg/kg; Table 2) than those usedn the previous reports (Luszczki et al., 2005, 2006, 2007;azarati et al., 2004; Sills et al., 2004). This observation is

ction of levetiracetam with carbamazepine, diazepam andopiramate appears to be more evident in this experimentalodel than that observed in the epileptic models previously

eported.

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Synergistic interactions between levetiracetam andgabapentin have not been described until now, the presentresults suggest being of interest because they are associatedwith a favourable therapeutic index (Table 3).

In the clinical setting, adverse pharmacodynamic inter-actions have been reported between levetiracetam andcarbamazepine (Sisodiya et al., 2002) and between leve-tiracetam and topiramate (Glauser et al., 2002), and morerecently, Kelly et al. (2004) reported that in their clinicalexperience, the most frequently discontinued combinationsdue to undesirable adverse effects were those of levetirac-etam with carbamazepine and lamotrigine. In the presentstudy, is noteworthy that levetiracetam had no signifi-cant effect on the adverse-effect profiles of the variousantiepileptics investigated with regard to motor perfor-mance (rotarod). However, it should be highlighted thatthe evaluation of acute neurotoxic effects in this studywas performed only at doses of levetiracetam and the var-ious antiepileptics that corresponded to the active dosesof ED50 in this genetic animal model of epilepsy. More-over, it is notable that the median toxic dose (TD50) oflevetiracetam with respect to the impairment of motorcoordination of animals in the rotarod test, denoted ina previous study, was 1601 (1324—1935) mg/kg (Luszczkiet al., 2005) and such a dose is several time largerthan that used in the present study. Furthermore, thesame authors have reported that levetiracetam (adminis-tered at a constant dose of 150 mg/kg) potentiated theacute neurotoxic effects of topiramate and carbamazepinein the rotarod test in mice, reducing considerably theirTD50 values from 423 to 246 mg/kg and from 53.6 to37.3 mg/kg, respectively (Luszczki et al., 2005). Since thedoses used in the present study are lower than thosereported by previous authors a comparison with these stud-ies is very difficult. The other antiepileptics tested incombination with levetiracetam in the rotarod (i.e. felba-mate, gabapentin, lamotrigine, phenytoin, phenobarbitaland valproate) produced no significant impairment of motorcoordination in animals. The latter data are also in agree-ment with recent reports (Luszczki et al., 2005, 2006).In addition, because neither antiepileptic’s levels brainwere affected by levetiracetam nor were levetiracetambrain affected by antiepileptic administration, the observedenhanced neurotoxicity was considered to be the conse-quence of pharmacodynamic interactions. Similarly, in thepresent study, brain antiepileptic concentration measure-ments (Tables 5 and 6) reveal that levetiracetam not onlydid not significantly affect brain levels of carbamazepine,diazepam, felbamate, gabapentin, lamotrigine, phenytoin,phenobarbital, topiramate or valproate, but that theseantiepileptics did not significantly affect brain levetirac-etam concentrations when administered in combination.Therefore the more evident synergistic anticonvulsant effi-cacy between levetiracetam and carbamazepine, diazepam,felbamate, gabapentin, topiramate and valproate can beconsidered to be the consequence of a pharmacodynamic,and not a pharmacokinetic, interaction. Indeed the lack of

a pharmacokinetic interaction between these antiepilepticswith levetiracetam would concur with its clinical pro-file (Patsalos, 2000, 2004a,b, 2006; Patsalos and Perucca,2003a,b). It should be highlighted that although in this study,antiepileptic brain concentrations were measured at only

A

B

ileptics in DBA/2 mice 119

xed-dose combination, higher doses would not be expectedo interact pharmacokinetically, because levetiracetam isevoid of pharmacokinetic characteristics that are associ-ted with such interactions (May et al., 2003). Luszczki etl. (2005) have also recently observed that levetiracetamt much higher doses than those investigated in the presenttudy had no effect on brain carbamazepine (37.3 mg/kg) oropiramate (246.2 mg/kg) concentrations, and conversely,rain levetiracetam concentrations were unaffected by car-amazepine and topiramate administration.

What might account for such a selective action? Underur experimental conditions, the enhancing effect was lessvident with drugs possessing membrane-stabilizing effectsi.e. lamotrigine and phenytoin). An alternative explanationay also be suggested for the different mechanism of action

nderlying the anticonvulsant activity of some antiepilep-ic drug studied, diazepam, felbamate and topiramate actainly via other specific mechanisms (augmentation of �-

minobutyric acid transmission, selective action on NMDAnd AMPA/kainate receptors and on voltage Ca2+ channelsDe Sarro et al., 1994, 1996; White, 1999; Gibbs et al., 2000;usso et al., 2004; Skradski and White, 2000; Zhang et al.,000). Assuming that the potention of antiepileptic activ-ty observed here is probably related to the binding affinityo SV2A by levetiracetam, it is possible to hypothesize thathis compound should enhance the anticonvulsant action ofome antiepileptics.

In conclusion, our preclinical data would suggest that lev-tiracetam in combination with carbamazepine, diazepam,elbamate, gabapentin, valproate and topiramate is asso-iated with synergistic anticonvulsant pharmacodynamicnteractions and that these data are, in part, in line withther anticonvulsant pharmacodynamic interactions thatave been reported with levetiracetam in other animal mod-ls (Luszczki et al., 2005, 2006, 2007).

Whereas the action of lamotrigine, phenytoin andhenobarbital was less markedly changed by the co-dministrations of levetiracetam. Although experimentalata may be hard to extrapolate to clinical pratice, it cane hypothesized that the use of drugs binding SV2A mighte potentially useful in the human therapy of so me typesf pharmacoresistant epilepsies.

cknowledgments

inancial support from the Italian Ministry of Education,niversity and Research (MIUR, COFIN 2005, Rome) andhe National Research Council (CNR, Rome) is gratefullycknowledged.

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