Interactions between zonisamide and conventional antiepileptic drugs in the mouse maximal electroshock test model

European Neuropsychopharmacology (2007) 17, 265 — 272

www.elsevier.com/locate/euroneuro

Interactions between zonisamide and conventional antiepileptic drugs in the mouse maximal electroshock test model Kinga K. Borowicz a,*, Jarogniew J. Luszczki a, Grzegorz Sobieszek a, Neville Ratnaraj b, Philip N. Patsalos b, Stanislaw J. Czuczwar a,c a

Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland Pharmacology and Therapeutics Unit, Department of Clinical and Experimental Epilepsy, Institute of Neurology, Queen Square, London, UK c Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland b

Received 16 March 2006; received in revised form 9 May 2006; accepted 20 June 2006

KEYWORDS Zonisamide; Drug interactions; Antiepileptic drugs; Isobolographic analysis; Maximal electroshock; Epilepsy

Abstract Despite the major advances in antiepileptic drug (AED) therapeutics, about one third of patients with epilepsy still do not have adequate seizure control with currently available AEDs when prescribed as monotherapy. Typically, in this setting polytherapy with two or more AEDs is used. Zonisamide (ZNS) is a new AED effective in the treatment of refractory epilepsy and since it is only prescribed in polytherapy regimens, its interactions with other AEDs is of particular importance. The aim of this study was to isobolographically determine interactions between ZNS and four conventional AEDs: carbamazepine (CBZ), phenytoin (PHT), phenobarbital (PB), and valproate (VPA), in the mouse maximal electroshock (MES)induced seizure model. The total brain concentrations of conventional AEDs and ZNS were measured with immunofluorescence and high-pressure liquid chromatography (HPLC), respectively, in order to determine any pharmacokinetic contribution in any observed interactions. With isobolography, synergistic interactions were observed for the combination of ZNS plus VPA and ZNS plus PHT at the fixed-ratio of 1:1, while additivity was observed for their combinations at the remaining dose ratios of 1:3 and 3:1. In contrast, the interactions between ZNS and PB and between ZNS and CBZ, applied at the fixed-ratios of 1:3, 1:1 and 3:1 proved to be additive. None of these AED combinations were associated with motor and long-term memory impairment. Furthermore, whilst brain AED concentrations were unaffected by ZNS, PHT significantly increased and PB reduced brain ZNS concentrations. Thus, the resultant interactions between ZNS and PHT and between ZNZ and PB were consequent to both pharmacodynamic and pharmacokinetic components. Finally, one can conclude that because of

* Corresponding author. Tel.: +48 81 7425837; fax: +48 81 7415828. E-mail address: [email protected] (K.K. Borowicz). 0924-977X/$ - see front matter D 2006 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2006.06.008

266

K.K. Borowicz et al. the synergistic pharmacodynamic interaction between ZNS and VPA, this combination might be useful in clinical practice.

D 2006 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction Epilepsy, a chronic neurological disorder, affects about 1—2% of the population (Browne and Holmes, 2001). Although significant progress in the pharmacotherapy of epilepsy has been achieved during last decade, about one third of patients are resistant to the current first-line conventional antiepileptic drugs (AEDs; Lo ¨scher, 2002). Furthermore, although since 1989 ten new antiepileptic drugs (AEDs) have been introduced into clinical practice, and with many patients having been rendered seizure-free, overall these new drugs have not had a significant impact on the prognosis of patients with intractable epilepsy (Walker and Sander, 1996). Consequently, in these patients, when monotherapy AED treatment fails, two or more AEDs are usually prescribed (Deckers et al., 2000) and commonly problematic pharmacokinetic and pharmacodynamic AED interactions can occur (Patsalos et al., 2002; Patsalos and Perucca, 2003; Patsalos, 2005). These interactions can best be anticipated, and advantageous drug combinations that are associated with potent anticonvulsant activity with minimal adverse effects should be identified, using animal models of epilepsy. Zonisamide (ZNS; 1,2-benzisoxazole-3-methanesulfonamide) is a new AED that is licensed for clinical use as adjunctive treatment of refractory epilepsy (Bialer et al., 1999; Sobieszek et al., 2003). Its mechanism of action relates to preventing repetitive neuronal firing by blocking voltage-dependent Na+ channels. ZNS also inhibits T-type Ca2+ channels without affecting L-type currents (Rock et al., 1989; Suzuki et al., 1992). Other postulated mechanisms of action include: blockade of K+-evoked glutamate-mediated synaptic excitation, an effect on monoamine neurotransmission, inhibition of carbonic anhydrase activity and an inhibitory effect on the excessive nitric oxide production and free radical scavenging activity (Bialer et al., 1999; Mori et al., 1998 (Okada et al., 1995, 1999). In animal models of epilepsy, ZNS protected rodents against maximal electroshock (MES)-induced seizures (Uno et al., 1979), and flurothyl-induced seizures (Hashimoto et al., 2003). The drug is also effective in the seizure susceptible EL mouse mutant strain (Nagatomo et al., 1996), but is ineffective against clonic seizures induced by pentylenetetrazole (PTZ) (Lo ¨scher, 2002). Moreover, ZNS raised the seizure threshold in amygdala kindled rats, suppressed convulsions produced by cortical application of tungstic acid gel in rats, and reduced the duration of cortical focal seizures induced by electrical stimulation of visual cortex in cats (Ito et al., 1980, 1986). Several multicenter double-blind placebo-controlled long-term clinical trials have documented efficacy of ZNS in the adjunctive treatment of refractory simple and complex partial seizures (Faught, 2004). It is also effective against atonic—clonic seizures, absence attacks and myoclonic seizures (Sackellares et al., 1985; Henry et al., 1988). Findings from one long-term study indicate that ZNS is also effective in monotherapy (Faught, 2004). The drug

is well tolerated, probably because of its favorable pharmacokinetic profile with excellent oral bioavailability (Bialer et al., 2001; 2002). Recently, clinical and experimental studies have suggested some additional indications for ZNS including mania, neuropathic pain, Parkinson’s disease and migraine prophylaxis (Bialer et al., 1999; Murata et al., 2001; McCumber et al., 2002; Hashimoto et al., 2003). Also, there is substantial evidence to suggest that ZNS has neuroprotective properties (Hayakawa et al., 1994; Minato et al., 1997; Owen et al., 1997; Mori et al., 1998). Since ZNS is primarily prescribed in polytherapy regimens, its interaction with other AEDs is of particular importance. Therefore, in the present study we assessed the effect of ZNS on the anticonvulsant effects of some conventional AEDs: carbamazepine [CBZ], phenytoin [PHT], phenobarbital [PB], and valproate [VPA] in the mouse MESinduced seizure model using isobolographic analysis. Isobolography is considered as a bgold standardQ in determining the precise characteristic of interactions between two drugs, and thus allowing classification of such interactions as synergistic, additive, antagonistic or indifferent (Berenbaum, 1989; Gessner, 1995; Deckers et al., 2000). The adverse effects of the various drug combinations were assessed in the chimney test, a measure of motor coordination, and the step-through passive avoidance task, a measure of long-term memory. Furthermore, the effect of ZNS on AED brain concentrations and, inversely, the effect of these AEDs on brain ZNS concentrations were evaluated in order to ascertain any pharmacokinetic interactions that may contribute to any observed pharmacodynamic interactions (anticonvulsant or adverse effects).

2. Materials and methods 2.1. Animals The experiments were carried out on male Swiss mice weighing 25— 30 g. The animals were housed in colony cages with free access to food and tap water, under standard laboratory conditions with constant temperature 22 F 1 8C and natural light—dark cycle. After 7 days of acclimatization to laboratory conditions the animals were challenged with experimental tests. The tested groups consisting of 8—10 mice were chosen by means of a randomized schedule. All experiments were performed between 10:00 a.m. and 2:00 p.m. The Local Ethical Committee at the Medical University of Lublin approved all experimental procedures undertaken in this study.

2.2. Drugs The following AEDs were used in the study: zonisamide (ZNS; Sigma, St. Louis, U.S.A.), valproate (VPA; ICN Polfa, Rzeszow, Poland), carbamazepine (CBZ; Polfa, Starogard Gdanski, Poland), phenobarbital (PB; Polfa, Krakow, Poland), and phenytoin (PHT; Sigma, St. Louis, U.S.A.). CBZ, PHT, PB and ZNS were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, U.S.A.) in distilled water, whereas VPA was dissolved in sterile water. All the AEDs were

Interactions between zonisamide and conventional antiepileptic drugs in the mouse maximal electroshock test model administered intraperitoneally (i.p.), in a volume of 10 ml/kg, PHT — 120 min., PB — 60 min, VPA, CBZ, and ZNS — 30 min before the tests.

2.3. Electroconvulsions Electroconvulsions were produced by a Hugo Sachs generator (Type 221, Freiburg, Germany) equipped with internal system stabilizing the current intensity (25 mA, 500 V). An alternating current (50 Hz) was delivered with the use of auricular electrodes, the stimulus duration being 0.2 s. The end point was the tonic extension of the hindlimbs (Borowicz et al., 2002b; Luszczki et al., 2003). To evaluate a median effective dose (ED50), corresponding to a dose of the drugs protecting 50% of animals against MES-induced seizures, mice pre-treated with different doses of AED were challenged with an electroshock of 25 mA. At least four groups of mice were used to estimate each ED50 value. A dose—response curve was subsequently calculated on the basis of the percentage of mice protected against tonic convulsions according to Litchfield and Wilcoxon (1949).

2.4. Chimney test The effects of ZNS and conventional AEDs administered alone or in combination on motor coordination impairment were determined in the chimney test in which the animals had to climb backward up a plastic tube (3-cm inner diameter, 25-cm length). Motor impairment was indicated by the inability of the animals to perform the test within 60 s. Results were expressed as percentage of mice failing to perform this test. The doses of AEDs tested in the chimney test corresponded to the ED50s previously determined in the MES test.

2.5. Passive avoidance task The effects of ZNS and conventional AEDs administered alone or in combination, at doses corresponding to their ED50s from the MEStest, on long-term memory were assessed by the use of step-through passive avoidance task according to Venault et al. (1986). In this test, drug pre-treated animals were placed separately in an illuminated box (10  13  15 cm) connected to a large dark box (25  20  15 cm), which was equipped with an electric grid floor. As it is a natural characteristic of rodents to avoid illuminated places and to prefer dark ones, entrance of the animals to the dark box was punished by an electric footshock (0.6 mA for 2 s; facilitation of acquisition). The animals which did not enter the dark box within 60 s were excluded from the experiment. On the subsequent day (24 h later) the same animals were placed into the illuminated box and observed for 180 s. The time it took the animals to enter the dark box was measured and expressed as a retention time in seconds (s) and the results presented as medians with 25th and 75th percentiles.

2.6. Brain AED concentrations Brain AED concentrations were measured in animals that were administered an AED alone + vehicle or a combination of an AED with ZNS at the fixed drug dose ratio of 1:1. Mice were killed by decapitation, at times scheduled for the MES test, and brains removed from skulls, weighed and homogenized with Abbott buffer (2:1 vol/wt) using an Ultra-Turrax T8 homogenizer (IKA, Staufen, Germany). The homogenates were centrifuged at 10,000 g for 15 min and supernatant samples (75 Al) were analysed by immunofluorescence using an Abbott TDx analyzer (Irvine, TX, USA) for PHT, PB, CBZ and VPA content. Results were expressed in `l/ml ı (means F S.D. of at least eight determinations). Brain ZNS concentrations were determined by high-performance liquid chromatography (HPLC) using an automated Gilson (Anachem) HPLC system. The system comprised of a Gilson 234 autosampler,

267

Gilson 306 pumps and a Gilson UV 155 variable wave length detector set at 215 nm. The mobile phase comprised of phosphate (50 mmol) and acetonitrile in a ratio of 70: 30. Chromatographic separation was achieved using a LiChrospher 60 RP-select B 5 Am column (VWR International). Brain homogenate samples were prepared for analysis as follows: 50 Al brain homogenate were pipetted into a 1.5-ml plastic tube to which was added 100 Al acetonitrile and the sample was vortex-mixed for 1 min. Samples were centrifuged for 5 min. Subsequently 90 Al of the supernatant were transferred into an autosampler vial, from which 10 Al were injected automatically into the column. Quantization was achieved by use of chromatographic peak areas and these were linearly related over the range 0.4—10 Ag/ml ZNS. The within-batch and between-batch precision was b5% and b6% respectively.

2.7. Isobolographic analysis Isobolographic analysis was used to characterize pharmacological interactions between drugs co-administered in various fixed-ratio combinations. The experimental ED50 mix and theoretical ED50 add values were calculated from the dose—response curves of combined drugs according to Litchfield and Wilcoxon (1949) or Tallarida (2001). The 95% confidence limits of ED50 values were transformed in standard errors of the mean (SEM) according to Tallarida (1992a,b). The ED50 add represents a total additive dose of two drugs in the mixture calculated from the line of additivity that theoretically protected 50% of animals against MES-induced seizures. Analogously, the ED50 mix represents a total effective dose of a two-drug mixture determined experimentally that protected 50% of animals against electroconvulsions. Statistical comparison of experimentally determined ED50 mixs with theoretically calculated ED50 adds was performed using the unpaired Student’s t-test according to Porecca et al. (1990) and Tallarida (2001). If the experimental ED50 mix is not statistically different from the respective theoretical ED50 add then the characteristic of interaction is considered to be additive. If the ED50 mix is statistically lower than the theoretically additive ED50 add, the interaction is considered to be synergistic. Otherwise, when the ED50 mix value is significantly greater than the respective ED50 add, the interaction is considered to be antagonistic. In our study we examined the following fixed drug dose ratio combinations: 1:1, 1:3, 3:1.

2.8. Statistics ED50 values for ZNS and conventional AEDs were calculated based on a computer log-probit analysis according to Litchfield and Wilcoxon (1949). Statistical analysis of drug interactions was performed according to Porecca et al. (1990) and Tallarida (2001). The experimental ED50 mix values were compared with the respective theoretical ED50 add values by the use of unpaired Student’s t-test. Data from the chimney test was analyzed with the Fisher’s exact probability test. The results from the passive-avoidance task were compared by using Kruskal—Wallis nonparametric ANOVA test followed by the post-hoc Dunn’s test for multiple comparisons. The brain concentrations of AEDs were statistically analyzed by use of the Student’s t-test.

3. Results 3.1. Isobolographic analysis of interactions between ZNS and conventional AEDs in maximal electroshock-induced seizures The ED50 mix values F SEM, obtained experimentally in the MES test for all the studied AEDs are presented in Table 1.

268

K.K. Borowicz et al.

Table 1 Effect of zonisamide and conventional antiepileptic drugs against maximal electroshock-induced seizures Drug

ED50 (mg/kg)

ZNS VPA CBZ PHT PB

46.9 249.0 9.5 10.4 16.5

(38.2—57.3) (229.0—270.8) (7.3—12.0) (8.9—12.2) (13.5—19.8)

Data are presented as median effective doses (ED50 values with 95% confidence limits). Drugs were administered i.p. at times corresponding to their peak of maximal activity: ZNS, VPA and CBZ — 30 min, PHT — 120 min, and PB — 60 min before the MES test. ZNS — zonisamide; VPA — valproate; CBZ — carbamazepine; PHT — phenytoin; PB — phenobarbital.

Isobolographic analysis demonstrates a synergistic interaction between ZNS and VPA at the fixed-ratio of 1:1, whilst their interaction at the fixed-ratios of 1:3 and 3:1 was additive and for illustrative purposes these interactions are shown graphically in Fig. 1. A similar profile was seen with ZNS and PHT in combination with synergy occurring at the fixed-ratio of 1:1 and additivity at the fixed-ratio combinations of 1:3 and 3:1. All examined combinations of ZNS with PB or CBZ in the MES test were additive (Table 2).

that PHT significantly increased brain ZNS concentrations whilst PB significantly decreased brain ZNS concentrations. The interactions of the remaining AED combinations appear to be purely pharmacodynamic in nature, since neither VPA nor CBZ affected brain ZNS concentrations. Moreover, none of the examined combinations were associated with any motor coordination or long-term memory impairment in mice. The most interesting interaction, which may prove to be of clinical interest, is the synergism between ZNS and VPA. The main mechanism of ZNS action relates to its blocking of voltage-dependent Na+ channels. ZNS also inhibits T-type Ca2+ channels, carbonic anhydrase activity and the excessive nitric oxide production, blocks K+evoked glutamate-mediated synaptic excitation, influences monoamine neurotransmission and exerts free radicals scavenging properties (Rock et al., 1989; Suzuki et al., 1992; Okada et al., 1995; Mori et al., 1998; Bialer et al., 1999). It is noteworthy that VPA primarily acts by inhibiting Na+ channel function, enhancing GABA-mediated inhibition and affecting K+ channels (Czuczwar and Patsalos, 2001; White, 1997). Although some authors (Mimaki et al., 1989) have hypothesized that central benzodiazepine receptors are specific for ZNS binding, ZNS does not intensify GABAA receptor-related events (Rock et al., 1989; Bialer et al., 1999). In contrast, at very high concentrations, VPA can act on GABA aminotransferase and glutamate decarboxylase

3.2. Chimney test and step-through passive avoidance task ZNS co-administered with conventional AEDs at the fixedratio of 1:1 did not affect motor performance of animals as assessed in the chimney test (Table 3). Similarly, these combinations had no effect on long-term memory in mice as assessed by the step-through passive avoidance task (Table 3).

3.3. Influence of ZNS on brain AED concentrations ZNS applied at doses corresponding to the drug in the mixture at the fixed-ratio of 1:1 did not affect total brain concentrations of VPA, CBZ, PHT and PB (Table 4).

3.4. Influence of AEDs on brain ZNS concentrations Mice were administered with ZNS alone or combinations of ZNS and the respective AED (VPA, CBZ, PHT, and PB) at the fixed-ratio of 1:1. Whilst CBZ and VPA did not affect ZNS brain concentrations, PHT significantly increased ( P b 0.01) and PB significantly decreased (P b 0.001) ZNS brain concentrations (Table 5).

4. Discussion This study demonstrates that ZNS interacts synergistically with VPA and PHT at the fixed-ratio combination of 1:1 and additively at the fixed- ratio combinations of 1:3 and 3:1. Additivity was also shown for the combination of ZNS with CBZ and ZNS and PB. However, pharmacokinetic interactions contributed to the pharmacodynamic interaction observed between ZNS and PHT as well as ZNS and PB in

Figure 1 Isobologram illustrating interactions between zonisamide (ZNS) and valproate (VPA) against MES-induced seizures. The median effective doses (ED50) for ZNS and VPA are shown plotted graphically on X- and Y-axes, respectively. The solid line on the axes represents the 95% confidence limits (CLs) for the AEDs administered alone. The straight line connecting these two ED50 values on each graph represents the theoretical line of additivity for a continuum of different fixed dose ratios. The open points (o) depict the experimentally-derived ED50 mixs (with 95% CLs as the error bars) for total dose expressed as the proportion of ZNS and a conventional AED that produced a 50% anticonvulsant effect. The dashed lines represent on each isobologram the theoretical additive 95% CLs of ED50 adds. The dotted line reflects the experimentally-derived characteristic of interactions. The experimental ED50 mix of the mixture of ZNS + VPA at the fixed-ratio of 1:1 was significantly below the line of additivity and thus indicates supra-additivity ( P b 0.05). The remaining ED50 mixs for the mixtures of ZNS + VPA at the fixed-ratios of 1:3 and 3:1 are near to the theoretical isobole of additivity, indicating additive interactions.

Interactions between zonisamide and conventional antiepileptic drugs in the mouse maximal electroshock test model

269

Table 2 Isobolographic analysis of interactions of zonisamide with conventional antiepileptic drugs against maximal electroshock-induced seizures (mg/kg)

Drug combination

FR

ED50

ZNS + VPA ZNS + VPA ZNS + VPA ZNS + PB ZNS + PB ZNS + PB ZNS + CBZ ZNS + CBZ ZNS + CBZ ZNS + PHT ZNS + PHT ZNS + PHT

1:3 1:1 3:1 1:3 1:1 3:1 1:3 1:1 3:1 1:3 1:1 3:1

198.5 F 9.2 147.9 F 7.7 97.4 F 6.3 24.1 F 2.5 31.7 F 3.3 39.2 F 4.1 18.8 F 2.0 28.2 F 2.9 37.5 F 3.9 19.5 F 1.8 28.6 F 2.8 37.7 F 3.8

add

N add

ED50

36 36 36 28 28 28 28 28 28 36 36 36

204.8 F 11.3 120.7 F 9.1* 94.0 F 8.2 29.4 F 2.6 39.5 F 3.6 46.7 F 3.4 18.5 F 1.6 23.1 F 2.4 37.0 F 3.2 15.2 F 1.7 20.2 F 2.5* 28.7 F 3.1

mix

(mg/kg)

N mix 16 16 24 24 16 16 8 16 16 16 24 16

Data are presented as ED50 mix and ED50 add values (FSEM) and were calculated by computerized log-probit analysis according to Litchfield and Wilcoxon (1949) followed by the method elaborated by Tallarida et al. (1997) and Porecca et al. (1990). Each ED50 mix was obtained from at least four groups of mice injected with different amounts of drugs at a fixed-ratio combination. Statistical evaluation of data was performed with Student’s t-test. ZNS — zonisamide; VPA — valproate; CBZ — carbamazepine; PHT — phenytoin; PB — phenobarbital. N add and N mix — total number of animals at those doses whose anticonvulsant effects were between 4 and 6 probits. *p b 0.05 vs. respective ED50 add.

and elevate GABA concentration in the CNS (Lo ¨scher, 1999; Johannessen, 2000) whilst at therapeutic doses, VPA can enhance the activity of neuronal and glial GABA transporTable 3 Effect of different combinations of zonisamide with conventional AEDs on motor performance and long-term memory Treatment (mg/kg)

FR

Animals showing motor deficit (%)

Retention time (s)

Control (vehicle) ZNS (12.1) + VPA (191.1) ZNS (19.1) + VPA (101.6) ZNS (33.9) + VPA (60.1) ZNS (11.5) + CBZ (7.0) ZNS (18.4) + CBZ (3.7) ZNS (34.7) + CBZ (2.4) ZNS (9.1) + PHT (6.1) ZNS (18.3) + PHT (4.1) ZNS (26.7) + PHT (2.0) ZNS (14.3) + PB (15.1) ZNS (28.2) + PB (9.9) ZNS (41.8) + PB (4.9)

— 1:3 1:1 3:1 1:3 1:1 3:1 1:3 1:1 3:1 1:3 1:1 3:1

0 0 0 12.5 0 0 0 0 0 12.5 12.5 12.5 12.5

180 180 180 180 180 180 180 180 180 180 180 180 180

(180; 180) (180; 180) (180; 180) (141; 180) (180; 180) (180; 180) (180; 180) (180; 180) (180; 180) (180; 180) (180; 180) (80.2; 180) (150; 180)

Results are shown as percentage of animals showing motor deficits in the chimney test and as median retention times (with 25th and 75th percentiles in parentheses), determined form the step-through passive avoidance task. Statistical evaluation of the data from the chimney test was performed with Fisher’s exact probability test, whereas the data from the step-through passive avoidance task were analyzed by use of the Kruskal— Wallis nonparametric ANOVA test followed by the post-hoc Dunn’s test. ZNS — zonisamide; VPA — valproate; CBZ — carbamazepine; PHT — phenytoin; PB — phenobarbital; FR — fixed-ratio combination.

ters by up to 10% (Whitlow et al., 2003). Moreover, whilst both AEDs inhibit T-type Ca2+ channels (Hashimoto et al., 2003) they differently modulate Na+ conductance across cell membranes. Finally, whilst ZNS enhances slow Na+ current inactivation, VPA has no effect on the recovery of Na+ channels from the inactivated state (Kuo, 1998). Thus it can be postulated that the synergism of ZNS and VPA is probably the consequence of their different mechanisms of action. Indeed the synergism between VPA and ZNS has also been confirmed by Nagamoto et al. (2000) who have observed that VPA increased the influence of ZNS on monoamine neurotransmission. However, this effect was accompanied by a VPA-induced increase in brain and free plasma ZNS concentrations (Nagamoto et al., 2000). In contrast, Seino et al. (1991) have reported that VPA coadministration did not affect ZNS concentrations and are in agreement with the data reported in the present study. It is worth highlighting, however, that the synergistic interaction between ZNS and VPA was observed only at the

Table 4 Effect of zonisamide on total brain antiepileptic drug concentrations Treatment (mg/kg)

Brain concentration (Ag/ml)

VPA (101.6) + saline VPA (101.6) + ZNS (19.1) CBZ (3.7) + saline CBZ (3.7) + ZNS (18.4) PHT (4.0) + saline PHT (4.0) + ZNS (18.3) PB (9.9) + saline PB (9.9) + ZNS (28.2)

60.13 F 6.57 65.72 F 7.81 0.47 F 0.10 0.52 F 0.12 0.66 F 0.11 0.72 F 0.18 5.33 F 0.39 5.68 F 0.48

Values are the means F SD of eight determinations. Brain tissue samples were taken at times scheduled for the MES test. Student’s t-test was used for statistical evaluation of the data. ZNS — zonisamide; VPA — valproate; CBZ — carbamazepine; PHT — phenytoin; PB — phenobarbital.

270

K.K. Borowicz et al.

Table 5 Effect of conventional antiepileptic drugs on brain concentrations of zonisamide Treatment (mg/kg)

Brain concentration (Ag/ml)

ZNS ZNS ZNS ZNS ZNS ZNS ZNS ZNS

2.95 F 0.27 2.90 F 0.21 2.76 F 0.30 2.91 F 0.31 3.78 F 0.29 4.27 F 0.30** 6.00 F 0.61 3.72 F 0.53***

(19.1) + saline (19.1) + VPA (101.6) (18.4) + saline (18.4) + CBZ (3.7) (18.3) + saline (18.3) + PHT (4.1) (28.2) + saline (28.2) + PB (9.9)

Values are the means F SD of eight determinations. Brain tissue samples were taken at times scheduled for the electroconvulsive test. Student’s t-test was used for statistical evaluation of the data. ZNS — zonisamide; VPA — valproate; CBZ — carbamazepine; PHT — phenytoin; PB — phenobarbital. **p b 0.01 and ***p b 0.001 vs. the respective control (ZNS alone-treated) group.

fixed-ratio of 1:1, whereas the remaining combinations of these drugs (1:3, 3:1) had additive characteristics. Thus, the dose ratio seems to be an important factor in influencing the final outcome of interactions between AEDs. Indeed, in several preclinical studies it has been observed that the pharmacological profile of AED combinations depends on the proportion of drugs used in the mixture and this phenomenon may be the consequence of complex mechanisms of action of AEDs involving different neurotransmitter systems in the brain (Borowicz et al., 2002a; Luszczki et al., 2003). In contrast to VPA, PHT and CBZ have similar pharmacological profiles to that of ZNS. The main mechanism of action of these drugs is a blockade of voltage-dependent Na+ channels via their binding to the A-subunit of the Na+ channel which serves to stabilize its inactive form in a voltage-, frequency-, and time-dependent fashion (Kuo, 1998). However, there are some differences between PHT and CBZ both experimentally (Czuczwar et al., 1999; Borowicz et al., 2002b) and clinically (Ragsdale and Avoli, 1998). CBZ compared to PHT has a 3-fold lower affinity for depolarized Na+ channels and binds to the channels at five times the rate, so the drug is much more effective against seizures with relatively brief depolarization shifts (Kuo et al., 1997; Ragsdale and Avoli, 1998). Furthermore, it is considered that CBZ and PHT bind to different receptors within A-subunit of Na+ channel, although these different receptors do not co-exist in one channel conformation (Kuo, 1998; Deckers et al., 2000). The differences between these drugs may also depend on other mechanisms of their antiepileptic activity. In the present study, ZNS and PHT in combination were associated with synergy whilst ZNS and CBZ were associated with additivity. These data can be explained by the pharmacokinetic interaction that occurred between ZNS and PHT and relates to the 13% increase in total brain ZNS concentrations. In the present study, whereby both drugs were administered as a single injection, brain CBZ, PHT, PB and VPA concentrations were unaffected by ZNS, whereas PHT (4.1 mg/kg) significantly increased and PB (9.9 mg/kg) decreased brain ZNS concentrations. That PHT increased

brain ZNS concentrations can be explained by the fact that PHT can inhibit the metabolism of ZNS (Patsalos, 2005). As for the observed decrease in brain ZNS concentrations by PB, the effect can be explained on the basis that PB is known to induce the metabolism of ZNS. These data are in partial agreement with that reported by Nagamoto et al. (2000) in EL mice. They reported an increase in brain (60%) and plasma (66%) ZNS concentrations following the administration of 50 mg/kg PHT whilst only plasma ZNS concentrations were increased (62%) following the administration of 10 mg/kg PHT. In another animal study it has been reported that PB and CBZ, but not PHT, decreased the T 1/2 of ZNS (Kimura et al., 1992). Since the protein binding and erythrocyte distribution of ZNS were unaffected by the presence of these AEDs, the decreased T 1/2 value was considered to be the consequence of hepatic enzyme induction by PB and CBZ (Kimura et al., 1992). However, PHT is also a potent hepatic enzyme inducer and it might be anticipated that it too would decrease ZNS T 1/2 values. In clinical studies of add-on ZNS, ZNS was not observed to affect steady-state plasma concentrations of PHT, CBZ, PB and VPA (Bialer et al., 1999; Patsalos and Perucca, 2003). In contrast, PHT, CBZ and PB, but not VPA, decreased plasma ZNS concentrations (Patsalos and Perucca, 2003). Furthermore, whilst the ZNS plasma concentration—ZNS dose ratio was significantly reduced by PHT (39%), CBZ (39%) and PB (30%) administration, the ratio was unaffected by VPA (Leppik, 2004). ZNS clearance values calculated after a single 100 mg ZNS dose administration were not changed in patients that underwent chronic treatment with CBZ or PHT (Ojemann et al., 1986). Finally, after chronic (4-week) ZNS administration the mean plasma T 1/2 of ZNS (60 h) was decreased to 52 h, 28 h and 36 h in VPA, PHT and CBZ co-administered patients respectively (Bialer et al., 2002). The isobolographic analysis of interaction revealed that the combination of ZNS and PB was additive. Considering the molecular mechanisms of action of PB it is known that barbiturates bind to a specific site within the GABAA receptor complex, which results in prolongation of chloride channel opening and neuronal hyperpolarization. PB may also antagonize the activation of AMPA receptors, glutamate-induced excitation (Steppuhn and Turski, 1993; Czuczwar and Patsalos, 2001), and block N and L types of Ca2+ channels (Gross and Macdonald, 1988). The influence of barbiturates on Na+ channels is rather weak (Meldrum, 1996). As highlighted earlier, the influence of ZNS on the GABA-ergic system is controversial. It has been suggested that ZNS may bind to GABAA receptor complex, since the neuropharmacology of specific binding sites for [3H] ZNS in rat brain are similar to that of the benzodiazepine receptor (Mimaki et al., 1989). Moreover, it has been observed that ZNS does not intensify GABAA receptorrelated events (Rock et al., 1989; Bialer et al., 1999). Consequently, because of the different mechanisms of action of ZNS and PB, the additive interaction observed between these two AEDs was rather unexpected. However, because PB significantly decreased brain ZNS concentrations, it can be hypothesized that this pharmacokinetic interaction masked a possible synergistic interaction between these two drugs. Thus if PB did not decrease brain ZNS concentrations the interaction might have been synergistic and this highlights the importance of ascertain-

Interactions between zonisamide and conventional antiepileptic drugs in the mouse maximal electroshock test model ing pharmacokinetic variables in pharmacodynamic studies of AED combinations. In conclusion, the present study suggests that ZNS and VPA in combination is associated with a beneficial pharmacodynamic (anticonvulsant) interaction and may be a useful combination clinically. Also ZNS in combination with CBZ may be useful. In contrast, any useful pharmacodynamic interaction between ZNS plus PHT and ZNS plus PB, is complicated by concurrent pharmacokinetic interactions.

Acknowledgements The study was supported by a grant from the Ministry of Education and Science, Poland (grant No. 2 P05A 045 28). The authors express thanks to ICN-Polfa (Rzeszow, Poland) and Polfa (Starogard, Poland) for the generous supply of valproate sodium and carbamazepine, respectively.

References Bialer, M., Johannessen, S.I., Kupferberg, H.J., Levy, R.H., Loiseau, P., Perucca, E., 1999. Progress report on new antiepileptic drugs: a summary of the Fourth Eilat Conference (EILAT IV). Epilepsy Res. 34, 1 – 41. Bialer, M., Johannessen, S.I., Kupferberg, H.J., Levy, R.H., Loiseau, P., Perucca, E., 2001. Progress report on new antiepileptic drugs: a summary of the Fifth Eilat Conference (EILAT V). Epilepsy Res. 43, 11 – 58. Bialer, M., Johannessen, S.I., Kupferberg, H.J., Levy, R.H., Loiseau, P., Perucca, E., 2002. Progress report on new antiepileptic drugs: a summary of the Sixth Eilat Conference (EILAT VI). Epilepsy Res. 51, 31 – 71. Berenbaum, M.C., 1989. What is synergy? Pharmacol. Rev. 41, 93 – 141. Borowicz, K.K., Swiader, M., Luszczki, J., Czuczwar, S.J., 2002a. Effect of gabapentin on the anticonvulsant activity of antiepileptic drugs against electroconvulsions in mice: an isobolographic analysis. Epilepsia 43, 956 – 963. Borowicz, K.K., Zadroz˙niak, M., Czuczwar, S.J., 2002b. Low affinity kainate receptor mediated events reduce the protective activity of phenobarbital and diphenylhydantoin against maximal electroshock in mice. Neuropharmacology 43, 1082 – 1086. Browne, T.R, Holmes, G.L., 2001. Epilepsy. N. Engl. J. Med. 344, 1145 – 1151. Czuczwar, S.J., Patsalos, P.N., 2001. The new generation of GABA enhancers. CNS Drugs 15, 339 – 350. Czuczwar, S.J., Kaminski, R., Kleinrok, Z., 1999. Influence of gabapentin on the anticonvulsive activity of the conventional antiepileptic drugs in mice. Epilepsia 40, 125 (Suppl.). Deckers, C.L.P., Czuczwar, S.J., Hekster, Y.A., Keyser, A., Kubova, H., Meinardi, H., Patsalos, P.N., Renier, W.O., Van Rijn, C.M., 2000. Selection of antiepileptic drug polytherapy based on mechanisms of action: the evidence reviewed. Epilepsia 41, 1364 – 1374. Faught, E., 2004. Review of United States and European clinical trials of zonisamide in the treatment of refractory partial-onset seizures. Seizure 13, S59 – S65 (Suppl.). Gessner, P., 1995. Isobolographic analysis of interactions: an update on applications and utility. Toxicology 105, 161 – 179. Gross, R.A., Macdonald, R.L., 1988. Differential actions of pentobarbitone on calcium current components of mouse sensory neurones in culture. J. Physiol. 405, 187 – 203. Hashimoto, Y., Araki, H., Futagami, K., Kawasaki, H., Gomita, Y., 2003. Effects of valproate, phenytoin, and zonisamide on clonic

271

and tonic seizures induced by acute and repeated exposure of mice to flurothyl. Physiol. Behav. 78, 465 – 469. Hayakawa, T., Higuchi, H., Nigami, H., Hattori, H., 1994. Zonisamide reduces hypoxic—ischemic brain damage in neonatal rats irrespective of its anticonvulsive effect. Eur. J. Pharmacol. 257, 131 – 135. Henry, T.R., Leppik, I., Gumnit, R.J., Jacobs, M., 1988. Progressive myoclonic epilepsy treated with zonisamide. Neurology 38, 928 – 931. Ito, T., Hori, M., Masuda, Y., Yoshida, K., Shimizu, M., 1980. 3Sulfamoylmethyl-1,2-benzisoxazole, a new type of anticonvulsant drug. Electroencephalographic profile. Arzneimittelforschung 30, 603 – 609. Ito, T., Hori, M., Kadokawa, T., 1986. Effects of zonisamide (AD-810) on tungstic acid gel-induced thalamic generalized seizures and conjugated estrogen-induced cortical spike-wave discharges in cats. Epilepsia 27, 367 – 374. Johannessen, C.U., 2000. Mechanisms of action of valproate: a commentatory. Neurochem. Int. 37, 103 – 110. Kimura, M., Tanaka, N., Kimura, Y., Miyake, K., Kitara, T., Fukuchi, H., 1992. Pharmacokinetic interactions of zonisamide in rats. Effect of other antiepileptics on zonisamide. J. Pharmacobio-dyn. 15, 631 – 639. Kuo, C.C., 1998. A common anticonvulsant binding site for phenytoin, carbamazepine, and lamotrigine in neuronal Na+ channels. Mol. Pharmacol. 54, 712 – 721. Kuo, C.C., Chen, R.S., Lu, L., Chen, R.C., 1997. Carbamazepine inhibition of neuronal Na currents: quantitative distinction from phenytoin and possible therapeutic implications. Mol. Pharmacol. 51, 1077 – 1083. Leppik, I.E., 2004. Zonisamide: chemistry, mechanism of action, and pharmacokinetics. Seizure 135, 55 – 59. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose—effect experiments. J. Pharmacol. Exp. Ther. 96, 99 – 113. Lo ¨scher, W., 1999. Valproate: a reappraisal of its pharmacodynamic properties and mechanisms of actions. Prog. Neurobiol. 58, 31 – 59. Lo ¨scher, W., 2002. Current status and future directions in the pharmacotherapy of epilepsy. Trends Pharmacol. Sci. 23, 113 – 118. Luszczki, J.J., Borowicz, K.K., Swiader, M., Czuczwar, S.J., 2003. Interactions between oxcarbazepine and conventional antiepileptic drugs in the maximal electroshock test in mice: an isobolographic analysis. Epilepsia 44, 489 – 499. McCumber, D., Chen, K.S., Meyer, K.B., Yaksh, T.L., 2002. A study of zonisamide (Zonegran) in the formalin model of nociception. J. Pain 3, 29. Meldrum, B.S., 1996. Update on the mechanism of action of antiepileptic drugs. Epilepsia 37, 4 – 11. Mimaki, T., Suzuki, Y., Tagawa, T., Tanaka, J., Itoh, N., Yabuuchi, H., 1989. [3H] Zonisamide binding in rat brain. Jpn. J. Psychiatry Neurol. 42, 640 – 642. Minato, H., Kikuta, C., Fuijtani, B., Masuda, Y., 1997. Protective effect of zonisamide, an antiepileptic drug against transient focal cerebral ischemia with middle cerebral artery occlusion— preperfusion in rats. Epilepsia 38, 975 – 980. Mori, A., Noda, Y., Packer, L., 1998. The anticonvulsant zonisamide scavenges free radicals. Epilepsy Res. 30, 153 – 158. Murata, M., Horiuchi, E., Kanazawa, I., 2001. Zonisamide has beneficial effects on Parkinson’s disease patients. Neurosci. Res. 41, 397 – 399. Nagatomo, I., Akasaki, Y., Nagase, F., Nomaguchi, M., Takigawa, M., 1996. Relationships between convulsive seizures and serum and brain concentrations of phenobarbital and zonisamide in mutant inbred strain EL mouse. Brain Res. 731, 190 – 198. Nagamoto, I., Akasaki, Y., Uchida, M., Tominaga, M., Hashiguchi, W., Takigawa, M., 2000. Effects of combined administration

272 of zonisamide and valproic amid or phenytoin to nitric oxide production, monoamines and zonisamide concentrations in the brain of seizure-susceptible EL mice. Brain Res. Bull. 53, 211 – 218. Okada, M., Kaneko, S., Hirano, T., Mizuno, K., Kondo, T., Otani, K., Fukushima, Y., 1995. Effects of zonisamide on dopaminergic system. Epilepsy Res. 22, 193 – 205. Okada, M., Hirano, T., Kawata, Y., Murakami, T., Wada, K., Mizuno, K., Kondo, T., Kaneko, S., 1999. Biphasic effects of zonisamide on serotoninergic system in rat hippocampus. Epilepsy Res. 34, 187 – 197. Ojemann, L.M., Shastri, R.A., Wilensky, A.J., Friel, P.N., Levy, R.H., McLean, J.R., Bauchanan, R.A., 1986. Comparative pharmacokinetic of zonisamide (CI-912) in epileptic patients on carbamazepine or phenytoin monotherapy. Ther. Drug Monit. 8, 293 – 296. Owen, A.J., Ijaz, S., Miyashita, H., Wishart, T., Howlett, W., Shuaib, A., 1997. Zonisamide as neuroprotective agent in an adult gerbil model of global forebrain ischemia: a histological, in vivo microdialysis and behavioral study. Brain Res. 770, 115 – 122. Patsalos, P.N., 2005. Anti-epileptic Drug Interactions. A Clinical Guide. Clarius Press, Guildford, UK. Patsalos, P.N., Perucca, E., 2003. Clinically important interactions in epilepsy: general features and interactions between antiepileptic drugs. Lancet Neurol. 6, 347 – 356. Patsalos, P.N., Froscher, W., Pisani, F., van Rijn, C.M., 2002. The importance of drug interactions in epilepsy therapy. Epilepsia 43, 365 – 385. Porecca, F., Jiang, Q., Tallarida, R.J., 1990. Modulation of morphine antinociception by peripheral [Leu5]enkephalin: a synergistic interaction. Eur. J. Pharmacol. 179, 463 – 468. Ragsdale, D.S., Avoli, M., 1998. Sodium channels as molecular targets for antiepileptic drugs. Brain Res. Rev. 26, 16 – 28. Rock, D.M., Macdonald, R.L., Taylor, C.P., 1989. Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD810, C1912), a novel anticonvulsant. Epilepsy Res. 3, 138 – 143. Sackellares, J.C., Donofrio, P.D., Wagner, J.G., Abou-Khalil, B., Berent, S., Aasved-Hoyt, K., 1985. Pilot study of zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in patients with refractory partial seizures. Epilepsia 26, 206 – 211.

K.K. Borowicz et al. Seino, M., Miyzaki, H., Ito, T., 1991. Zonisamide. In: Pisani, F., Perucca, E., Avanzini, G., Richens, A. (Eds.), New Antiepileptic Drugs, Amsterdam: Epilepsy Res. (Suppl. 3). Elsevier Science, pp. 169 – 174. Sobieszek, G., Borowicz, K.K., Kimber-Trojnar, Z., Malek, R., Piskorska, B., Czuczwar, S.J., 2003. Zonisamide: a new antiepileptic drug. Pol. J. Pharmacol. 55, 683 – 689. Steppuhn, K.G., Turski, L., 1993. Modulation of the seizure threshold for excitatory amino acids in mice by antiepileptic drugs and chemoconvulsants. J. Pharmacol. Exp. Ther. 265, 1063 – 1070. Suzuki, S., Kawakami, K., Nishimura, S., Watanabe, Y., Yagi, K., Seino, M., Miyamoto, K., 1992. Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex. Epilepsy Res. 12, 21 – 27. Tallarida, R.J., 1992a. A further comment on testing for drug synergism. Pain 51, 381 – 382. Tallarida, R.J., 1992b. Statistical analysis of drug combinations for synergism. Pain 49, 93 – 97. Tallarida, R.J., 2001. Drug synergism: its detection and applications. J. Pharmacol. Exp. Ther. 298, 865 – 872. Tallarida, R.J., Stone, D.J., Raffa, R.B., 1997. Efficient designs for studying synergistic drug combinations. Life Sci. 61, 417 – 425. Uno, H., Kurokawa, M., Masuda, Y., Nishimura, H., 1979. Studies on 3-substituted 1,2-benzisoxazole derivatives. Syntheses of 3(sulfamoylmethyl)-1,2-benzisoxazole derivatives and their anticonvulsant activities. J. Med. Chem. 22, 180 – 183. Venault, P., Chapouthier, G., de Carvalho, L.P., Simiand, J., Morre, M., Dodd, R.H., Rossier, J., 1986. Benzodiazepines impair and beta-carbolines enhance performance in learning and memory tasks. Nature 321, 864 – 866. Walker, M.C., Sander, J.W., 1996. The impact of new antiepileptic drugs on the prognosis of epilepsy: seizure freedom should be the ultimate goal. Neurology 46, 912 – 914. White, H.S., 1997. Clinical significance of animal seizure models and mechanism of action studies of potential antiepileptic drugs. Epilepsia 38, 9 – 17. Whitlow, R.D., Sacher, A., Loo, D.D., Nelson, N., Eskandari, S., 2003. The anticonvulsant valproate increases the turnover rate of gamma-aminobutyric acid transporters. J. Biol. Chem. 278, 17716 – 17726.