Antiepileptic Drugs, 5th Edition

Zonisamide

91

Mechanisms of Action

Robert L. Macdonald MD, PhD

Professor and Chairman, Department of Neurology, Vanderbilt University, Nashville, Tennessee

CLINICAL ANTIEPILEPTIC ACTIONS OF ZONISAMIDE

Zonisamide (Zonegran; Elan Pharmaceuticals, Gainesville, GA) is a 1,2-benzisoxazole compound with a sulfonamide side chain (1,2-benzisoxazole-3-methanesulfonamide) that is structurally different from other marketed antiepileptic drugs (AEDs). Zonisamide was shown to be an effective AED in patients with refractory partial seizures (1, 2, 3, 4, 5, 6) and generalized seizures (4,7, 8, 9, 10, 11, 12, 13). Based on these clinical trials, therapeutic plasma levels were determined to be 15 to 25 µg/mL (approximately 70 to 120 µmol/L) (14) or 20 to 30 µg/mL (85 to 140 µmol/L) (15), and zonisamide is 50% to 60% bound to plasma proteins (14).

ANTICONVULSANT ACTIONS OF ZONISAMIDE IN EXPERIMENTAL ANIMALS

Zonisamide is an effective anticonvulsant drug in experimental animal models of seizures and has an anticonvulsant profile that is similar but not identical to that of phenytoin and carbamazepine. Zonisamide was effective against maximal electroshock (MES) seizures at nontoxic doses in mice [median effective dose (ED₅₀), 19.6 mg/kg orally (p.o.)] and rats (ED₅₀, 7.9 mg/kg p.o.) and was more potent that phenytoin or carbamazepine. Similar to phenytoin and carbamazepine, zonisamide was not active against subcutaneous pentylenetetrazol (PTZ)-induced seizures (16,17). With MES seizures, effective nontoxic plasma levels ranged from 10 to 70 µg/mL (47 to 330 µmol/L) in rats (18). Zonisamide was effective against partial seizures in experimental animals, reducing hippocampus-kindled seizures in rat (19) and amygdala-kindled seizures in rat (20) and cat (21). Zonisamide also reduced kindled generalized seizures to partial seizures in the cat (22). In addition, zonisamide was effective against tonic-clonic and myoclonic seizures in the genetic animal model of reflex epilepsy in the Mongolian gerbil (23) and suppressed tonic, but not absence-like, seizures in spontaneously epileptic rats (SER) and sound-induced seizures in DBA/2 mice (24). However, zonisamide did not completely suppress spontaneous seizures in the EL mouse (25). Although not always predictive of efficacy against seizures in patients with epilepsy, this anticonvulsant profile suggests that zonisamide would be effective against partial seizures and secondarily generalized seizures and may have some efficacy against tonic and myoclonic seizures. The lack of effect against PTZ seizures and seizures in SER suggests that zonisamide would not be effective in the treatment of generalized absence seizures.

ZONISAMIDE ACTIONS ON EPILEPTIFORM DISCHARGES IN VIVO AND IN VITRO

Zonisamide prevented spread of epileptiform activity in the cortex of experimental animals. Zonisamide suppressed focal seizure activity induced by direct electrical stimulation of the cat visual cortex and increased afterdischarge threshold (22), and after unilateral injection of kainic acid into the amygdala of rat, zonisamide reduced spread of seizures to the contralateral side (26). Zonisamide also suppressed epileptogenic focal activity induced in the cortex of experimental animals. Zonisamide suppressed spiking activity induced by cortical freezing in cat cortex and interictal spikes induced by tungstic acid gel in rat cortex (27,28).

ZONISAMIDE MECHANISMS OF ACTION

Multiple mechanisms of action for zonisamide have been proposed. However, these can be divided into three basic mechanisms of drug action: an action on neuronal sodium channels to reduce sustained, high-frequency repetitive firing of action potentials, on T-type voltage-dependent calcium channels, and on synaptic transmission. Although evidence has been reported supporting all of these mechanisms, the weight of current experimental evidence

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suggests that the major mechanisms of action of zonisamide are to modify the ability of neurons to fire at high frequency by producing an enhancement of sodium channel inactivation and to reduce T-type calcium current. These mechanisms and the others are discussed in the following sections.

Reduction of Sustained, High-Frequency Repetitive Firing by Zonisamide

In an early study, zonisamide was shown to reduce the excitability of Myxicola giant axons by reducing sodium current (29). This effect was specific for sodium currents because voltage-dependent potassium currents were unaffected. Zonisamide had no effect on sodium channel activation, but produced a shift in the steady-state fast inactivation curve to more negative voltages and slowed recovery from both fast and slow inactivation. The effect of zonisamide was produced only with intracellular zonisamide; extracellular zonisamide did not affect sodium channel inactivation. The zonisamide effect on fast inactivation occurred at relatively low zonisamide concentrations (1 to 100 µmol/L), with a half-maximal concentration of 12 µmol/L producing a 20-mV shift in the steady-state inactivation curve. The effect of zonisamide to slow recovery from slow inactivation occurred at even lower concentrations (0.1 to 10 µmol/L). Slow inactivation was slowed from 4.4 seconds to 11.5 and 16 seconds by 1 and 12 µmol/L zonisamide, respectively. Thus, this early study demonstrated that zonisamide directly affected sodium channels at clinically relevant concentrations.

The effect ofzonisamide on repetitive firing was not limited to axonal preparations. Zonisamide reduced high-frequency repetitive firing of action potentials recorded from fetal mouse spinal cord neurons grown in primary dissociated cell culture (30). When depolarized, spinal cord neurons fire high-frequency repetitive discharges (31). In the presence of zonisamide at or below therapeutic free serum concentrations (35 to 60 µmol/L), there was a concentration-dependent reduction in sustained repetitive firing [above 2 µmol/L; median therapeutic serum concentration (IC₅₀) of 17 µmol/L]. Thus, zonisamide limited sustained, high-frequency repetitive firing of action potentials at therapeutic free serum concentrations.

These results suggested that zonisamide was affecting sodium channels, and that the effect was likely on the inactivation process of sodium channels. It was proposed that zonisamide produced limitation of high frequency repetitive firing by binding to sodium channels in the inactive state and by slowing the rate of recovery of these channels from inactivation. The effect appeared to be selective for the inactive form of the closed channel. Thus, it is likely that zonisamide binds preferentially to the inactive form of the sodium channel to produce use- and voltage-dependent block of sodium channels, an action consistent with the modulated receptor hypothesis of local anesthetic drug action proposed by Hille (32). Zonisamide therefore would be more effective in reducing high-frequency repetitive firing when neurons were depolarized because more channels would be in the inactive state. Under normal physiologic conditions, it is likely that vertebrate myelinated and unmyelinated axons have a large negative membrane potential, and therefore propagated action potentials would be relatively resistant to the action of zonisamide. In contrast, the cell body of neurons is subject to synaptic depolarization and inward currents that produce burst firing. This is particularly true in neurons undergoing epileptic discharge. Zonisamide would be effective, therefore, in limiting high-frequency action potentials generated in bursting neurons.

In addition to altering neuronal excitability, zonisamide may alter the process of synaptic transmission by affecting presynaptic sodium channels. It has been demonstrated that [³H]BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones with a heterogeneous distribution in the nervous system (33). In the hippocampal slice, stimulation of stratum radiatum elicited extracellular field potentials recorded from the CA1 pyramidal cell layer. The field potentials consisted of a fiber spike, which reflects axonal propagation, and a population spike, which reflects effective synaptic transmission. Veratridine, which displaces [³H] BTX-B binding, produced a specific reduction in the synaptically evoked population spike without affecting the fiber spike. This effect of veratridine was antagonized by carbamazepine. It is likely, therefore, that zonisamide would block presynaptic sodium channels and the firing of action potentials; this would secondarily reduce voltage-dependent calcium entry and synaptic transmission.

In summary, zonisamide is likely to act both presynaptically to block release of neurotransmitter by blocking firing of action potentials and postsynaptically by blocking the development of high-frequency repetitive discharge initiated at cell bodies. This combined presynaptic and postsynaptic effect is likely to form the basis for the anticonvulsant actions of zonisamide. It appears that zonisamide blocks sustained, high-frequency repetitive firing of action potentials and spontaneous burst discharges by enhancing voltage-dependent sodium channel inactivation in both mature and immature neuronal preparations, which suggests that the mechanism of action of zonisamide is the same in both mature and immature animals. In addition to zonisamide, several other AEDs that are effective against generalized tonic-clonic and partial seizures have a similar mechanism of action and also block high-frequency repetitive firing (34, 35, 36, 37, 38), including phenytoin (31), carbamazepine (39,40), and valproic acid (41), possibly by a similar mechanism. Thus, zonisamide may share this mechanism of action with several currently used AEDs.

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Reduction of T-Type Calcium Current by Zonisamide

Zonisamide did not alter calcium-dependent action potentials in mouse spinal cord neurons that are primarily composed of high-threshold calcium currents (30). However, low-threshold, T-type calcium currents in cultured fetal rat cortical neurons were reduced by zonisamide in a concentration-dependent manner (42). The reduction in T current was maximal at 500 µmol/L (60% reduction), and reductions of 10% to 25% were produced by therapeutic concentrations of zonisamide (10 to 50 µmol/L). In contrast, the high-threshold L-type calcium current was unaffected by zonisamide at concentrations up to 500 µmol/L. In these experiments, zonisamide was dissolved in 50% dimethylsulfoxide (DMSO), which produced an 18% reduction in T current but did not affect L current. Thus, the reductions in T current were reported as reductions above 18%. The 7-methylated analog of zonisamide reduced T current only by 13.5%, similar to that of the DMSO control, suggesting that this effect was specific for zonisamide. Zonisamide also reduced the T-type calcium current recorded from cultured neuroblastoma cells (43). Zonisamide reduced T current by 38% at 50 mmol/L and shifted the inactivation curve to more negative potentials by 20 mV.

In addition to zonisamide, several other AEDs that are effective against generalized absence seizures also reduce T-type calcium currents, including ethosuximide (44,45), the active metabolite of trimethadione, dimethadione (44,45), and valproic acid (46). Generalized absence epilepsy is characterized clinically by brief periods of loss of consciousness and electrically by a generalized 3-Hz spike-and-wave discharge recorded on the electroencephalogram. Thalamic relay neurons play a critical role in the generation of the abnormal thalamocortical rhythmicity that underlies the 3-Hz spike-and-wave discharge. Low-threshold T-type and high-threshold calcium currents are present in rat thalamic neurons (47), and T-current activation was necessary and sufficient to cause the generation of low-threshold calcium spikes in thalamic relay neurons. Ethosuximide and dimethadione, the active metabolite of trimethadione, both reduced the T-type current of thalamic neurons isolated from guinea pigs and rats at clinically relevant concentrations (44,45). Valproic acid also decreased T-type current in rat nodose ganglion neurons (46). Thus, zonisamide may share this mechanism of action with several currently used AEDs.

Actions of Zonisamide on Neurotransmitter Systems

Zonisamide has been reported to alter neurotransmitter metabolism and levels and to modify neurotransmitter receptor function. The primary neurotransmitter systems studied include monoamine, γ-aminobutyric acid (GABA)ergic, glutamatergic, and cholinergic neurotransmitter systems.

It has been suggested that alteration of monoamine neurotransmission may be involved in the actions of zonisamide. The threshold for inducing electroshock seizures was shown to be reduced after administration of drugs that deplete brain monoamines (48, 49, 50, 51, 52). In contrast, the threshold for inducing electroshock seizures was elevated by administration of monoamine precursors or inhibitors of monoamine catabolism (50,53,54). The anticonvulsant effect of zonisamide was reduced but not abolished by reserpine, and zonisamide had increased potency in reserpinized rats (16), suggesting that its anticonvulsant effect may be mediated, at least in part, by altered monoaminergic function. Zonisamide has been shown to increase both total and extracellular levels of striatal and hippocampal dopamine (DA) (55, 56, 57) and serotonin [(5-hydroxytryptamine (5-HT)] (55,56,58,59).

Administration of therapeutic doses of zonisamide (20 and 50 mg/kg) altered DA metabolism in rat striatum and hippocampus (55,57). Acute administration of zonisamide increased striatal extracellular dihydroxyphenylalanine (DOPA) levels and intracellular striatal and hippocampal 3,4-DOPA levels, and stimulated DOPA accumulation in both striatum and hippocampus; zonisamide also increased striatal and hippocampal intracellular and extracellular DA and homovanillic acid (HVA) levels and decreased 3,4dihydroxyphenylacetic acid (DOPAC) levels (57). Acute administration of zonisamide had no effect on calcium-dependent dopamine release in rat striatum (55) or DA reuptake in rat striatum or hippocampus (57), but did weakly inhibit monoamine oxidase B (MAOB) activity more than MAOA activity (57). Chronic (3 weeks) administration of zonisamide increased intracellular and extracellular DA, DOPA, DOPAC, and HVA levels in striatum and hippocampus (57). Acute and chronic high-dose (100 mg/kg) administration of zonisamide decreased intracellular levels of all compounds and inhibited DOPA accumulation (57). These data suggest that acute administration of therapeutic doses of zonisamide enhances DA function by enhancing DA synthesis and inhibiting DA degradation, resulting in an increase in intracellular DA. Chronic administration of therapeutic doses of zonisamide also enhances DA function by increasing DA synthesis without altering DA degradation, leading to increased intracellular DA. Administration of supratherapeutic doses of zonisamide inhibits DA function by reducing intracellular DA, suggesting that zonisamide inhibits DA synthesis and degradation, leading to decreased DA turnover. Thus, zonisamide has biphasic effects on DA function, with therapeutic doses enhancing and supratherapeutic doses decreasing DA function. Acute and chronic therapeutic and supratherapeutic administration of carbamazepine had effects on DA metabolism that were similar to those of zonisamide (60).

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Zonisamide also altered 5-HT turnover (55,56,58,59). Both acute and chronic administration of therapeutic doses of zonisamide increased striatal and hippocampal levels of 5-HT, its precursor 5-hydroxytryptophan (5-HTP), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), thus increasing 5-HT turnover (56,59). In contrast, supratherapeutic doses of zonisamide had the opposite effect of either decreasing or not changing 5-HT, 5-HTP, and 5-HIAA levels. Thus, zonisamide has biphasic effects on 5-HT function, with therapeutic doses enhancing and supratherapeutic doses decreasing 5-HT function.

Administration of therapeutic doses of zonisamide (20 mg/kg) altered acetylcholine (ACh) metabolism in rat striaturn (61). Acute or chronic administration of zonisamide increased ACh release and metabolism, thus increasing ACh turnover, without affecting acetylcholinesterase or butyrylcholinesterase activities. Supratherapeutic doses of zonisamide decreased ACh turnover and extracellular ACh levels without affecting cholinesterase activity. Similar experiments with carbamazepine demonstrated similar acute and chronic effects. However, the relationship of this effect of zonisamide on ACh metabolism in rat striatum to its antiepileptic effects is unclear.

A number of anticonvulsant drugs have been demonstrated to enhance GABA_A receptor function (62). Zonisamide inhibited [³H]flunitrazepam and [³H]muscimol binding in rat brain (63). Zonisamide reduced [³H]flunitrazepam to 65% (1 mmol/L) and 92% (100 µmol/L) of control by increasing K_d but not altering B_max. Zonisamide also reduced [³H]muscimol binding to 28% (1 mmol/L) and 68% (100 µmol/L) of control. In addition, [³H]zonisamide was found to bind to a crude synaptosomal fraction of whole rat brain with a K_d of 90 nmol/L (64,65). Clonazepam reduced and GABA increased [³H] zonisamide binding, suggesting that the zonisamide binding site was coupled to benzodiazepine receptors. However, on spinal cord neurons in cell culture, no effect of zonisamide was found on postsynaptic responses to iontophoretically applied GABA (30). Thus, there is no direct evidence that zonisamide modifies GABA_A receptor currents.

Zonisamide has been reported to modify excitatory amino acid receptor function (66). The effects of zonisamide and carbamazepine administration on extracellular levels of glutamate in the hippocampus were studied using in vivo microdialysis. Perfusion of the microdialysis probe with zonisamide (1 mmol/L) or carbamazepine (100 µmol/L) reduced glutamate release evoked by KCI and the enhanced glutamate release evoked by KCI and Ca²⁺. No affect of zonisamide was observed, however, on glutamate responses evoked on spinal cord neurons in cell culture (30). These data suggest that zonisamide may inhibit excitatory glutamatergic synaptic transmission by reducing the presynaptic release of glutamate. However, whether this is a direct action of zonisamide on the release or an indirect action on voltage-gated sodium or calcium channels is unclear. Therefore, there is no compelling evidence that zonisamide acts directly on glutamatergic synaptic transmission to produce an antiepileptic effect.

Thus, zonisamide appears to alter DA, 5-HT, and ACh metabolism but not directly to affect GABA_A receptor or glutamate receptor function. These actions of zonisamide are shared by carbamazepine. It is unclear, however, if these effects on DA, 5-HT, and ACh metabolism have any relationship to the antiepileptic actions of zonisamide or carbamazepine.

Carbonic Anhydrase Inhibitory Activity of Zonisamide

Zonisamide has a sulfonamide side chain that is common to the carbonic anhydrase inhibitor acetazolamide, and zonisamide has been shown to have activity as a carbonic anhydrase inhibitor (67). Acetazolamide inhibited rat red blood cell carbonic anhydrase with an IC₅₀ of 15 µmol/L, and although zonisamide and 7-methylated zonisamide also inhibited carbonic anhydrase, they were much less potent carbonic anhydrase inhibitors, with IC₅₀s 200 times higher than that of acetazolamide (68). Furthermore, although both zonisamide and 7-methyated zonisamide inhibited carbonic anhydrase, only zonisamide had anticonvulsant activity in the MES test in mice, and the anti-MES activity of zonisamide was dose dependent and correlated with increasing brain levels of zonisamide (68). These data suggest that the anticonvulsant action of zonisamide against MES seizures or against seizures in humans is unrelated to inhibition of carbonic anhydrase.

CONCLUSION

Zonisamide has been shown to limit sustained, high-frequency repetitive firing of sodium-dependent action potentials and reduce T-type calcium currents at clinically effective free serum concentrations. In addition, zonisamide appears to alter DA, 5-HT, and ACh metabolism but not directly to affect GABA_A receptor or glutamate receptor function. These data are consistent with the human and animal data suggesting that zonisamide has multiple mechanisms of action. Carbamazepine produces similar effects, except that it does not alter T-type calcium currents. Phenytoin also alters sustained, high-frequency repetitive firing of sodium-dependent action potentials, but does not have the reported effects on T-type calcium currents or neurotransmitter metabolism. Although it is unclear if the effects on DA, 5-HT, and ACh metabolism have any relationship to the anticonvulsant actions of zonisamide, it has been well established that AEDs act on sodium and T-type calcium channels to block simple partial, generalized tonic-clonic, and absence seizures. Zonisamide appears to have “broad-spectrum” antiepileptic activity using similar mechanisms

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of action, and thus would be expected to have actions on simple partial, generalized tonic-clonic, and absence seizures.

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