Comparison of the effects of anticonvulsant drugs with diverse mechanisms of action in the formalin test in rats

Neuropharmacology 48 (2005) 1012e1020 www.elsevier.com/locate/neuropharm

Comparison of the effects of anticonvulsant drugs with diverse mechanisms of action in the formalin test in rats Harlan E. Shannon*, Elizabeth Lutz Eberle, Steven C. Peters Neuroscience Division, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA Received 1 October 2004; received in revised form 29 November 2004; accepted 20 January 2005

Abstract The purpose of the present studies was to compare anticonvulsant drugs with diverse mechanisms of action in a persistent pain model, the formalin test. In addition, the anticonvulsant effects of the compounds were determined in the threshold electroshock tonic seizure test and the 6-Hz limbic seizure test. The effects of the compounds were also determined on locomotor activity. Carbamazepine, oxcarbazepine, lamotrigine, gabapentin and ethosuximide all produced statistically significant analgesic effects in the formalin test whereas phenytoin, topiramate, zonisamide, phenobarbital, tiagabine, valproate and levetiracetam did not. All compounds were anticonvulsant. In addition, morphine and phenobarbital increased locomotor activity while ethosuximide had no effect and all other compounds decreased locomotor activity. For those compounds that were analgesic, the doses required to produce analgesia were larger in magnitude than the anticonvulsant ED50 values in the threshold electroshock and 6-Hz tests, as well as larger than doses that altered locomotor activity. The present results demonstrate that the anticonvulsant and analgesic effects of clinically used antiepileptic drugs do not necessarily correlate and therefore suggest that the anticonvulsant and analgesic efficacy of these drugs may be due to different pharmacologic mechanisms. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Antiepileptic drugs; Formalin test; Locomotor activity; Threshold electroshock test; 6-Hz test; Rats; Mice

1. Introduction Persistent pain states may arise from a number of etiologies, including nerve damage, trauma, disease states, chronic inflammation, amputation, or metabolic disturbances, which alter the processing or transmission of nociceptive and non-nociceptive stimuli in pain pathways. Pharmacological treatments for persistent pain include opioids, non-steroidal antiinflammatory drugs and tricyclic antidepressants (e.g., Collins et al., 2000; Pappagallo and Haldey, 2003). Persistent pain

* Corresponding author. Tel.: C1 317 276 4360; fax: C1 317 276 5546. E-mail address: [email protected] (H.E. Shannon). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.01.013

syndromes are considered by some to be poorly responsive to analgesics such as opioids (Arner and Meyerson, 1988; but see also review by McCleane, 2003) and non-steroidal antiinflammatory drugs (Max et al., 1988). Moreover, both opioids and non-steroidal antiinflammatory drugs produce problematic side-effects (e.g., Reisine and Pasternak, 1995; Insel, 1995), particularly with prolonged use as may be required in the treatment of persistent pain, which markedly limit the clinical utility of these two classes of analgesics. Tricyclic antidepressants are more amenable to longer term use, but also produce side-effects, ranging from dry mouth to the potential for cardiotoxicity, which can limit their clinical utility (e.g., Monks, 1994). Alternative pharmacological treatments for persistent pain are therefore needed.

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Several anticonvulsant drugs have emerged as being efficacious in treating several persistent pain syndromes and therefore as alternatives to opioids, non-steroidal antiinflammatory and tricyclic antidepressant drugs in the treatment of persistent pain. In particular, the sodium channel blocker carbamazepine is efficacious in the treatment of trigeminal neuralgia (e.g., Campbell et al., 1966) and diabetic neuropathy (e.g., Gomez-Perez et al., 1996). Similarly, gabapentin, the mechanism of action of which has not been fully delineated, has been reported to be efficacious in the treatment of painful diabetic neuropathy and postherpetic neuralgia (Backonja et al., 1998; Rowbotham et al., 1998). These clinical findings have engendered studies with newer anticonvulsant drugs such as the sodium channel blockers lamotrigine (e.g., Simpson et al., 2003) and zonisamide (see review by Backonja, 2002), but with mixed results (e.g., see review by Tremont-Lukats et al., 2000). The results of these clinical studies thus raise the question of whether all anticonvulsant drugs, or only particular mechanistic classes, may be efficacious in the treatment of persistent pain syndromes. Alternatively, the anticonvulsant and analgesic efficacy of drugs may be due to separate, unrelated, mechanisms. The major purpose of the present studies was to compare a broad range of clinically used anticonvulsant drugs in a persistent pain model, the formalin test, in order to determine if all anticonvulsants, only particular mechanistic classes of anticonvulsants, or unrelated anticonvulsants, are analgesic. The subcutaneous injection of formalin produces a biphasic electrophysiological response: an immediate and intense increase in the spontaneous activity of C-fiber primary afferents followed by a quiescent phase and then a more prolonged increase in cell firing of both primary afferents (Heapy et al., 1987; Puig and Sorkin, 1995) as well as dorsal horn neurons (Dickenson and Sullivan, 1987, 1988). Behaviorally, the injection of formalin elicits a temporally biphasic increase in licking, biting, flinching and other nocifensive behaviors (e.g., Dubuisson and Dennis, 1977). The initial phase is short lived and may reflect an acute nociceptive state, while the second phase is more extended and reflects a state of central sensitization (Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al., 1993). All drugs currently effective clinically in the treatment of persistent pain are efficacious in the formalin test (see e.g., Wheeler-Aceto et al., 1990). Accordingly, doseeresponse curves were determined in the formalin test for 13 clinically used anticonvulsants. For purposes of comparison, a doseeresponse curve was also determined for the opioid analgesic morphine. In addition, the anticonvulsant effects of all of the compounds tested, including morphine, were determined in both the threshold (10 mA) electroshock test, a model of tonic-clonic seizures (White et al., 1995), and the 6-Hz test, a model of limbic partial seizures (Barton et al., 2001,

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2003) in mice. As a measure of non-specific side-effects, doseeresponse curves were also determined for each of the compounds on locomotor activity in mice.

2. Methods 2.1. Subjects Male SpragueeDawley rats weighing 180e220 g and male CD1 mice weighing 20e25 g (Harlan Sprague Dawley, Indianapolis, IN) were used. Rats were housed up to 8 per cage and mice up to 12 per cage in a large colony room, and provided with food and water ad libitum using a 12-h light/dark cycle (lights on 6:00 am). Each animal was used only once. All experiments were conducted in accordance with the NIH regulations of animal care covered in Principles of Laboratory Animal Care, NIH publication 85-23 and were approved by the Institutional Animal Care and Use Committee. 2.2. Formalin test The methods used have been described in detail previously (Shannon and Lutz, 2000; 2002). All testing took place in commercially available startle behavior chambers (Model SR-Lab, San Diego Instruments, San Diego, CA) that detected movements of the rats by means of an accelerometer. At the beginning of an experiment, the rats were injected with vehicle or a dose of drug and individually placed in the restraint cylinders (i.d. 8.5 cm; length 16 cm). Thirty minutes later, the rats were removed from the cylinders, administered formalin (50 ml of a 5% solution in saline) subcutaneously into the plantar surface of the right hindpaw, and immediately placed back into the restraining cylinders. The magnitude of movements was monitored continuously for 60 min in 1 s bins. The number of ‘‘agitation’’ events, defined as the number of 1 s bins with a change in dynamic force that exceeded an empirically determined threshold value (20 arbitrary load units, which was previously determined in pilot experiments to be greater than that produced by animals quietly sniffing and breathing) were totaled in 5-min intervals. The formalininduced movements detected by the system included licking and flinching the affected paw as well as hopping and turning. 2.3. Locomotor activity Locomotor activity was measured with a 20 station Photobeam Activity System (San Diego Instruments, San Diego, CA) with seven photocells per station. Animals were weighed, injected, and placed individually in a polypropylene cage (40.6!20.3!15.2 cm). After a 30-min habituation and pretreatment period, recording

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began. Data were collected for 60 min and expressed as total ambulations. 2.4. Threshold electroshock test Threshold electroshock (TES) seizures were induced via corneal stimulation (60 Hz, 0.2 s, 10 mA; White et al., 1995) using an apparatus similar to that originally described by Woodbury and Davenport (1952). At the time of drug administration and again prior to stimulation, a drop of 0.5% tetracaine solution was applied to the eyes of all animals. For all studies, the effect of each compound was quantitated by varying the dose between 0% protection and 100% protection. Protection was defined as the absence of the hindlimb tonic extensor component.

and the total number of events during the third to ninth 5-min block (15e45 min) were considered to be phase II. Locomotor activity was recorded as the number of ambulations, where ambulation was defined as the sequential breaking of adjacent photobeams. For both the formalin test and locomotor activity, treatment groups were compared to appropriate control groups using ANOVA and Dunnett’s t-test. Data are expressed as meansGSEM. A p value of !0.05 was considered as significant. The median effective doses (ED50) in the seizure tests were determined by probit analysis (Finney, 1971). All computations were done using JMP v4.04 (SAS Institute Inc., Cary, NC).

3. Results 2.5. 6-Hz seizure test 3.1. Formalin test in rats Limbic partial seizures were induced via corneal stimulation (6 Hz, 0.2 ms rectangular pulse width, 3 s duration) using a Grass S48 stimulator and a 300-ohm resistor in series with the animal. The 6-Hz-induced (limbic) seizure was characterized by immobility, forelimb clonus, twitching of the vibrissae, and Straub-tail (Barton et al., 2001, 2003). All of the compounds were tested at a current intensity of 32 mA. Protection in the 6-Hz model was defined as the complete absence of a seizure. 2.6. Drugs Morphine SO4, carbamazepine, ethosuximide, chlordiazepoxide, phenobarbital, phenytoin, sodium valproate (Sigma Chemical Co., St. Louis, MO), gabapentin, lamotrigine, oxcarbazepine, levetiracetam, tiagabine, topiramate and zonisamide (Lilly Research Laboratories, Indianapolis, IN) were used. Morphine, ethosuximide, chlordiazepoxide, phenobarbital, and valproate were dissolved in deionized water; phenytoin was dissolved in deionized water with a minimal amount of 1 N NaOH. All other drugs were dissolved in 25% 2-hydroxypropylb-cyclodextrin (RBI, Natick, MA). Doses refer to the form of the drug listed. Drugs were administered intraperitoneally, except morphine, which was administered subcutaneously, in a volume of 1.0 ml/kg in rats and 10 ml/kg in mice. 2.7. Data analysis In the formalin experiments, the time course of the number of ‘‘agitation’’ events was totaled in 5-min intervals and data were analyzed by repeated measures ANOVA. For constructing doseeresponse curves, the total number of events during the first 5-min block after formalin administration were considered to be phase I,

In animals administered vehicle SC 30 min before formalin, there was an initial peak in the number of events during the first 5-min block after formalin, followed by a decrease in the number of events in the second 5-min block, and subsequently an increase again in blocks 3e9 (Fig. 1, open circles, all panels). When morphine was administered SC 30 min before formalin, it produced a dose- and time-related analgesic effect in the formalin test in rats (Fig. 1, upper left panel). Morphine (1.0e10 mg/kg SC) was efficacious in reducing formalin-induced behaviors in both phase I (the first 5-min block), and phase II (5-min blocks 3e9); however, only the effects of the 10 mg/kg dose were statistically different compared to vehicle. A dosee response curve was constructed by summing all of the events in phase II and is presented in Fig. 2 (upper panel). Several antiepileptic drugs statistically significantly reduced formalin-induced behaviors. Representative time courses are presented in Fig. 1, doseeresponse curves are presented in Fig. 2, and minimal effective dose values are presented in Table 1. Like morphine, lamotrigine produced dose-related decreases in formalin-induced behaviors in both phases I and II of the formalin test (Fig. 1, upper right panel and Fig. 2, upper panel), and the effects of 30 mg/kg were significantly different from vehicle in phase II. Gabapentin also reduced the frequency of formalin-induced behaviors in both phase I and II (Fig. 1, lower left panel and Fig. 2, upper panel). However, the effects of gabapentin were similar across the time and dose range tested in the present study (30e300 mg/kg), although the effects of the 30 and 300 mg/kg doses were statistically different from vehicle. Lower doses of gabapentin were without effect (data not presented). Oxcarbazepine also produced dose-related decreases in formalin-induced

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Fig. 1. Dose- and time-related effects of morphine, lamotrigine, gabapentin and oxcarbazepine in reducing nocifensive behaviors produced by the intraplantar injection of formalin in rats. Each point represents the mean of one observation in each of 8 rats. Vertical lines representGSEM and are absent when less than the size of the point. Abscissa: consecutive 5-min blocks; ordinate, number of events.

behaviors when tested over the dose range of 3.0e 30 mg/kg (Fig. 1, lower right panel and Fig. 2, upper panel), with the maximal effect (30 mg/kg) being similar in magnitude to that produced by gabapentin. The 30 mg/kg dose of oxcarbazepine produced effects that were significantly different from vehicle. In addition, carbamazepine, ethosuximide and chlordiazepoxide also reduced formalin-induced behaviors (Fig. 2, upper panel). A dose of 30 mg/kg of carbamazepine produced effects that were significantly different from vehicle, as did a dose of 300 mg/kg of ethosuximide (Fig. 2, upper panel). Doses of 3.0, 10 and 30 mg/kg of chlordiazepoxide significantly reduced formalin-induced behaviors (Fig. 2, upper panel). In contrast, the antiepileptic drugs levetiracetam (10e 300 mg/kg), phenobarbital (3e30 mg/kg), phenytoin (3e30 mg/kg), tiagabine (0.3e10 mg/kg), topiramate (3e30 mg/kg), valproate (10e300 mg/kg) and zonisamide (3e30 mg/kg) were without significant effect on formalin-induced behaviors over the dose ranges tested in the present study (Fig. 3, upper panel, and Table 1). 3.2. Locomotor activity in mice Morphine produced modest but non-significant decreases in locomotor activity at doses of 1.0 and 3.0 mg/kg, but significantly increased locomotor activity at a dose of 10 mg/kg, an effect which is well

documented in mice (e.g., Shannon et al., 1976). Among the antiepileptic drugs that were effective in reducing formalin-induced behaviors, gabapentin (100e1000 mg/ kg) produced a dose-related decrease in locomotor activity, with doses of 100e1000 mg/kg producing effects that were significantly different from vehicle (Fig. 2, lower panel). Lamotrigine (3e30 mg/kg), carbamazepine (3e30 mg/kg) and oxcarbazepine (3e 30 mg/kg) also significantly reduced locomotor activity at a dose of 30 mg/kg for each drug. Chlordiazepoxide (1e30 mg/kg) did not significantly alter locomotor activity over the dose range tested. Ethosuximide (30e 560 mg/kg) also did not significantly affect locomotor activity, although doses of 300 and 560 mg/kg nonsignificantly increased locomotor activity. Among antiepileptic drugs that were ineffective in reducing formalin-induced behaviors, levetiracetam (0.3e300 mg/kg) significantly reduced locomotor activity at a dose of 300 mg/kg (Fig. 3, lower panel and Table 1). Similarly, tiagabine (0.3e10 mg/kg) significantly reduced locomotor activity at doses of 3 and 10 mg/kg, and zonisamide (10e300 mg/kg) significantly reduced locomotor activity at a dose of 300 mg/kg (Fig. 3, lower panel and Table 1). In contrast, phenobarbital (1e30 mg/ kg) increased locomotor activity after a dose of 30 mg/kg (Fig. 3 and Table 1). Valproate (10e300 mg/kg) tended to decrease locomotor activity, but these effects were not statistically significant. Further, phenytoin (1e30 mg/kg)

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1.4 mg/kg, to valproate with an ED50 value of 90 mg/ kg. In the 6-Hz electroshock test, all of the antiepileptic drugs, with the exception of topiramate, were effective in blocking the limbic seizures elicited by the 32 mA, low frequency stimulation. The potencies of the compounds in the 6-Hz test ranged from tiagabine with an ED50 value of 0.32 mg/kg, to gabapentin with an ED50 value of 327 mg/kg. However, several compounds (i.e., carbamazepine, gabapentin, lamotrigine, and phenytoin) did not produce 100% protection in the 6-Hz model, although they produced at least 50% protection, allowing for estimates of the ED50 value.

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Dose (mg/kg) Fig. 2. Upper panel: doseeresponse curves for antiepileptic drugs with analgesic activity in the formalin test in rats. Each point represents the total number of events during phase II (third to ninth 5-min block) of the 45-min time course after formalin was injected (see Fig. 1). Each point represents the mean of one observation in each of 8 rats. Lower panel: doseeresponse curves for the effects of antiepileptic drugs with analgesic activity on locomotor activity in mice. Each point represents the mean of one observation in each of 5e8 mice. Vertical lines representGSEM and are absent when less than the size of the point. Points above Veh represent the effects of vehicle. Abscissa, dose of drug in mg/kg; ordinate, upper panel, total number of events during the final 35 min (phase II) of the formalin test; ordinate, lower panel, total number of ambulations during 1 h. *p!0.05 vs. Vehicle, Dunnett’s t-test.

and topiramate (1e30 mg/kg) also failed to significantly alter locomotor activity over the dose ranges tested (Fig. 3 and Table 1). 3.3. Anticonvulsant effects The ED50 values for the anticonvulsant effects of the drugs evaluated in the present study are presented in Table 1. In the threshold electroshock test (TES), all the antiepileptic compounds, except ethosuximide and levetiracetam, were effective in blocking tonic extensor seizures elicited by the 10 mA, 60 Hz stimulation. The potencies of the compounds in the TES test ranged from tiagabine and topiramate with ED50 values of

There has been growing interest in the potential utility of anticonvulsant drugs in the treatment of persistent pain, but systematic studies comparing the various clinically used drugs have not been conducted. The present studies therefore sought to directly compare the analgesic, anticonvulsant and locomotor activityaltering effects of a number of clinically used anticonvulsant drugs with differing mechanisms of action. The present findings demonstrated that the anticonvulsant and analgesic effects of clinically used AEDs do not necessarily correlate and therefore suggest that the anticonvulsant and analgesic efficacy of these drugs may be due to different pharmacologic mechanisms. The sodium channel blocker carbamazepine has been shown to be efficacious in the treatment of trigeminal neuralgia (Campbell et al., 1966) and diabetic neuropathy (Gomez-Perez et al., 1996). The sodium channel blocker lamotrigine has been reported to be efficacious in the treatment of trigeminal neuralgia (Zakrzewska et al., 1997), central poststroke pain (Vestergaard et al., 2001), spinal cord injury pain (Finnerup et al., 2002), and diabetic neuropathy (Eisenberg et al., 2001; but see also McCleane, 1999). Oxcarbazepine has also been reported to be efficacious in trigeminal neuralgia (Zakrzewska and Patsalos, 2002). Other sodium channel blockers, such as phenytoin, have been reported to have efficacy in trigeminal neuralgia but not painful neuropathic disorders (e.g., Backonja, 2002). Thus, clinically, AEDs that act in whole or in part as sodium channel blockers appear to be efficacious in the treatment of trigeminal neuralgia, but, with the possible exception of carbamazepine and lamotrigine, there has been little evidence to suggest broader efficacy in neuropathic and other persistent pain disorders. In the present studies, the putative sodium channel blockers carbamazepine, lamotrigine, and oxcarbazepine all produced statistically significant analgesic effects in the formalin test, while phenytoin, topiramate and zonisamide were ineffective. The present findings are consistent with previous reports that carbamazepine and lamotrigine were efficacious in the

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Phase I Phase II Analgesic compounds Morphine 10 Carbamazepine 30 Ethosuximide >300 Gabapentin 300 Lamotrigine 10 Chlordiazepoxide 30 Oxcarbazepine >30 Ineffective compounds Levetiracetam Phenobarbital Phenytoin Tiagabine Topiramate Valproate Zonisamide a

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8.7a >30 5.5 12.5a >560 179 10.5 327a 1.7 30a 5.7 0.78 2.9 11.1

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formalin test, whereas phenytoin was not (BlackburnMunro et al., 2002). The efficacy of oxcarbazepine, and the lack of efficacy of topiramate and zonisamide in the formalin test have not been previously reported. Interestingly, carbamazepine was efficacious in an animal model of trigeminal neuralgia only at a dose that produced motor impairment (Ida¨npa¨a¨n-Heikkila¨ and Guilbaud, 1999). In addition, carbamazepine was reported not to be efficacious in the sciatic nerve ligation and chronic constriction injury models of neuropathic pain (Hunter et al., 1997; Gonzalez et al., 2000; Fox et al., 2003). On the other hand, lamotrigine was efficacious in the sciatic nerve ligation and chronic constriction injury models (Hunter et al., 1997; Fox et al., 2003). However, phenytoin (Hunter et al., 1997) and oxcarbazepine (Fox et al., 2003) were not efficacious in the sciatic nerve ligation model. Recently, zonisamide was reported to be efficacious in alleviating thermal, but not mechanical, allodynia in the chronic constriction injury model (Hord et al., 2003). Topiramate has been reported to be moderately efficacious in nerve injury models (Bischofs et al., 2004; Wieczorkiewicz-Plaza et al., 2004). In the present study, doses of the sodium channel blockers that produced analgesia in the formalin test were larger in magnitude than doses required to produce anticonvulsant effects or changes in locomotor activity, or both. Taken together, the data available to date, both clinically and preclinically, suggest that AEDs with sodium channel blocking activity are not uniformly efficacious in producing analgesia in models involving central

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Dose (mg/kg) Fig. 3. Upper panel: Doseeresponse curves for antiepileptic drugs that did not have analgesic activity in the formalin test in rats. Each point represents the total number of events during phase II (third to ninth 5-min block) of the 45-min time course after formalin was injected (see Fig. 1). Each point represents the mean one observation in each of 8 rats. Lower panel: Doseeresponse curves for the effects of antiepileptic drugs that did not have analgesic activity on locomotor activity in mice. Each point represents the mean of one observation in each of 5e 8 mice. Vertical lines representGSEM and are absent when less than the size of the point. Points above Veh represent the effects of vehicle. Abscissa, dose of drug in mg/kg; ordinate, upper panel, total number of events during the final 35 min (phase II) of the formalin test; ordinate, lower panel, total number of ambulations during 1 h. *p!0.05 vs. Vehicle, Dunnett’s t-test.

sensitization and/or neuropathy such as the formalin test and sciatic nerve ligation and chronic constriction injury models. Moreover, sodium channel blockade is not sufficient for analgesic efficacy, at least for AEDs in the formalin test. The present findings thus suggest that the mechanism of action of sodium channel blockers in blocking seizures and in producing analgesia are different. The GABA-uptake inhibitor tiagabine was without effect in the formalin test at anticonvulsant doses and even up to doses that produced marked decreases in locomotor activity. The present findings are in contrast to those of Laughlin et al. (2002) who found that tiagabine was efficacious in both acute and persistent

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pain models, including the formalin test, in mice, whereas lamotrigine and gabapentin were efficacious only in persistent pain models in mice. Ipponi et al. (1999) also reported analgesic efficacy with tiagabine in acute pain models at doses of the uptake inhibitor that increased brain levels of GABA; moreover, the analgesic effects were blocked by a GABAB receptor antagonist. The reasons for the apparent discrepancies between the present and previous reports in pain models are not readily apparent, but may be due to differences in species (rat vs. mouse) or doses (10 vs. 30 mg/kg) used. In contrast to the lack of efficacy with the GABA uptake inhibitor tiagabine in the formalin test, the benzodiazepine GABAA receptor positive modulator chlordiazepoxide was efficacious in the formalin test. Chlordiazepoxide does not appear to have been previously evaluated in the formalin or neuropathic pain models. However, there is a growing body of evidence that GABA receptors, particularly of the GABAB subtype, are involved in antinociception, although the efficacy may be limited due to rapid desensitization of GABAB receptors (e.g., see review by Bowery et al., 2002). The mechanism of action of gabapentin is unknown, but may involve modulation of calcium channels (Gee et al., 1996) or enhancement of GABA neurotransmission (Lo¨scher et al., 1991). Gabapentin is a cyclized analog of GABA, but it does not interact with GABA receptors, nor does it inhibit GABA uptake, or prevent the degradation of GABA (Taylor et al., 1998). However, in vivo, gabapentin increases GABA accumulation in rat brain induced by amino-oxyacetic acid (Lo¨scher et al., 1991). The present findings that gabapentin is efficacious in the formalin test is consistent with numerous previous reports of efficacy not only in the formalin test (e.g., Field et al., 1997; Yoon and Yaksh, 1999) but also in neuropathic (Laughlin et al., 2002; Field et al., 2002) and inflammatory pain models (e.g., Field et al., 1997). Similarly, valproate does not directly interact with GABA receptors, but it has been reported to increase brain levels of GABA, possibly by enhancing glutamate decarboxylation or inhibiting GABA transaminase (Lo¨scher, 2002). There is no clear consensus on the analgesic properties of valproate. Preclinical and clinical data have been inconsistent (see e.g., Tremont-Lukats et al., 2000 and references therein), showing efficacy in some studies but not in others. In the present studies, valproate was ineffective in the formalin test at doses larger than those required to produce anticonvulsant effects. It would be of interest to determine the efficacy of valproate in neuropathic pain models in rodents for purposes of comparison. Ethosuximide is thought to produce its anticonvulsant effects by blocking T-type calcium channels (Coulter et al., 1989). There do not appear to be any

reports of the use of ethosuximide to treat neuropathic or persistent pain in humans. However, recent reports have supported a role for T-type calcium channels in nociception. Matthews and Dickenson (2001) demonstrated that ethosuximide inhibited the electrophysiological response of dorsal horn neurons to mechanical and thermal evoked responses in the spinal nerve ligation model. In addition, Todorovic and co-workers have reported that ethosuximide and mibefradil, another T-type calcium channel blocker, were antinociceptive in thermal and mechanical nociceptive tests (Todorovic et al., 2002, 2003). Consistent with these findings, ethosuximide was efficacious in the formalin test in the present studies, providing further support for a role for T-type calcium channels in nociception. Little information is available on the potential antinociceptive effects of the AED levetiracetam, which has recently been reported to bind to the synaptic vesicle protein SV2A (Lynch et al., 2004). There have been no formal studies on the effects of levetiracetam on neuropathic or persistent pain disorders in humans. However, Ardid et al. (2003) have recently reported that levetiracetam produces antihyperalgesic effects in neuropathic pain models in rats, although the doses required were well above those required to produce anticonvulsant effects. In the present studies, levetiracetam was ineffective in the formalin test at doses approximately 50-fold higher than the anticonvulsant dose. In summary, carbamazepine, oxcarbazepine, lamotrigine, gabapentin, and ethosuximide all produced statistically significant analgesic effects in the formalin test and were anticonvulsant in the TES tonic-clonic seizure test or the 6-Hz limbic seizure test. For each drug, the minimally effective dose that produced a significant analgesic effect in the formalin test was larger in magnitude than the anticonvulsant ED50 dose in either the TES or the 6-Hz test. At analgesic doses, all of the compounds, except ethosuximide, also decreased locomotor activity. In addition, the anticonvulsants phenytoin, topiramate, zonisamide, phenobarbital, tiagabine, valproate and levetiracetam did not produce statistically significant analgesic effects in the formalin test at doses greater than or equal to their respective anticonvulsant ED50 dose. The present findings suggest that the anticonvulsant and analgesic effects of AEDs may be mediated through different pharmacologic mechanisms.

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