198 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006)

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1 198 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) Schwindt, P.C., Crill, W.E., Amplification of synaptic current by persistent sodium conductance in apical dendrite of neocortical neurons. J. Neurophysiol. 74, Shimoyama, M., Shimoyama, N., Hori, Y., Gabapentin affects glutamatergic excitatory neurotransmission in the rat dorsal horn. Pain 85, Sills, G.J., Butler, E., Forrest, G., Ratnaraj, N., Patsalos, P.N., Brodie, M.J., Vigabatrin, but not gabapentin or topiramate, produces concentration-related effects on enzymes and intermediates of the GABA shunt in rat brain and retina. Epilepsia 44, Sills, G.J., The mechanisms of action of gabapentin and pregabalin. Curr. Opin. Pharmacol (Epub ahead of print). Silverman, R.B., Andruszkiewicz, R., Nanavati, S.M., Taylor, C.P., Vartanian, M.G., Alkyl-4-aminobutyric acids: the first class of anticonvulsant agents that activates l-glutamic acid decarboxylase. J. Med. Chem. 34, Stefani, A., Spadoni, F., Bernardi, G., Gabapentin inhibits calcium currents in isolated rat brain neurons. Neuropharmacology 37, Su, T.-Z., Feng, M.R., Weber, M.L., Mediation of highly concentrative uptake of pregabalin by l-type amino acid transport in Chinese hamster ovary and Caco-2 cells. J. Pharmacol. Exp. Ther. 313, Suman-Chauhan, N., Webdale, L., Hill, D.R., Woodruff, G.N., Characterisation of [ 3 H]gabapentin binding to a novel site in rat brain: homogenate binding studies. Eur. J. Pharmacol. 244, Taylor, C.P., Gabapentin mechanisms of action. In: Levy, R.H., Mattson, R.H., Meldrum, B.S., Perucca, E. (Eds.), Antiepileptic Drugs, fifth ed., Lippincott Williams & Wilkins, Philadelphia, pp Taylor, C.P., Meeting report. The biology and pharmacology of calcium channel 2 - proteins. Pfizer Satellite Symposium to the 2003 Society for Neuroscience Meeting, Sheraton New Orleans Hotel, New Orleans, LA November 10, CNS Drug Rev. 10, Taylor, C.P., Vartanian, M.G., Yuen, P.W., Bigge, C., Suman- Chauhan, N., Hill, D.R., Potent and stereospecific anticonvulsant activity of 3-isobutyl GABA relates to in vitro binding at a novel site labeled by tritiated gabapentin. Epilepsy Res. 14, Thurlow, R.J., Brown, J.P., Gee, N.S., Hill, D.R., Woodruff, G.N., [ 3 H]gabapentin may label a system-l-like neutral amino acid carrier in brain. Eur. J. Pharmacol. 247, van Hooft, J.A., Dougherty, J.J., Endeman, D., Nichols, R.A., Wadman, W.J., Gabapentin inhibits presynaptic Ca 2+ influx and synaptic transmission in rat hippocampus and neocortex. Eur. J. Pharmacol. 449, Vartanian, M.G., Radulovic, L.L., Kinsora, J., Serpa, K.A., Vergnes, M., Bertram, E., Taylor, C.P., Activity profile of pregabalin in rodent models of epilepsy and ataxia. Epil. Res. 68, Wang, M., Offord, J., Oxender, D. L., Su, T.-Z., Structural requirement of the calcium channel subunit 2 for gabapentin binding. Biochem. J. 342, Westphalen, R.I., Hemmings, H.C. Jr., Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals. J. Pharmacol. Exp. Ther. 304, Glutamate receptors Wojciech Danysz *, Chris G. Parsons Merz Pharmaceuticals, Eckenheimer Landstrasse 100, D Frankfurt, Germany Introduction The mechanism of action of many antiepileptic drugs involves enhancement of inhibitory GABAergic transmission. Therefore, shortly after the role of glutamate as a major excitatory transmission was accepted (Meldrum, 1985), similar efficacy was expected for the potential use of agents inhibiting glutamatergic transmission. Indeed, nearly 25 years ago Czuczwar and Meldrum (Czuczwar and Meldrum, 1982) showed that AP7, an antagonist of the NMDA subtype of glutamate receptor produced anticonvulsive effects in experimental animals. This was followed by a number of publications confirming this initial observation (Meldrum, 1991), however convincing palliative activity in animal models relevant for temporal epilepsy such as kindling seizures have not yet been provided. The present commentary focuses on an analysis of the current status of modulators of glutamatergic system 23 years later. Glutamate receptors subtypes Glutamate interacts with two major classes of receptors: glutamate-gated ion channels (ionotropic glutamate receptors, iglurs) such as NMDA (N-methyl-daspartate), AMPA (alpha-amino-3-hydroxy-5-methylisoxalazole-4-propionate) and kainate receptors and G protein-coupled metabotropic receptors (mglurs) present in at least eight subtypes (Parsons et al., 1998; Schoepp et al., 1999a,b) which have been classified into group I mglurs (mglur1 and mglur5), group II mglurs (mglur2 and mglur3) and group III (mglur4 and mglur6-8) (Schoepp et al., 1999a). NMDA receptors are permeable to Ca 2+,Na + and K +, and consist of tetrameric, heteromeric subunit assemblies of two obligatory NR1 subunits and two further subunits from the group of NR2 subunits (A, B, C or D; the NR3 subunit is not discussed

2 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) here) (Parsons et al., 1998). NMDA receptor function can be influenced by multiple endogenous modulatory mechanisms such as: voltage dependent blockade by Mg 2+ ions; positive modulation (or co-agonism) by glycine (binding to the NR1 subunit); positive modulation by polyamines like spermine and spermidine (binding to the NR2B subunit); redox and ph. Exogenous agonists/antagonists for NMDA receptors can competitively interact with the glutamate recognition site or act at one of these modulatory sites, e.g. there are high affinity (+)-5-methyl-10,11- dihydro-5h-dibenzocyclohepten-5,10-imine maleate ((+)MK-801) and moderate affinity 1-amino-3,5- dimethyladamantane (memantine) channel blockers which act at a similar site to Mg 2+ (see Parsons et al., 1998; Hirai, 2001, for review). AMPA ( -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors are involved in mediating most forms of fast glutamatergic neurotransmission. There are four known subunits GluR1 to GluR4 (also named GluRA to GluRD), which are widely, but differentially, distributed throughout the CNS (Hollmann and Heinemann, 1994). Further non-nmda ionotropic receptor subunits, forming kainate receptors are GluR5, GluR6, KA1 and KA2 (Lerma et al., 1997). Both AMPA and kainite receptors also have allosteric modulatory sites allowing both positive and negative influence of receptor function. For example benzodiazepines such as 1-(4-aminophenyl)-4- methyl-7,8-methylenedioxy-5h-2,3-bendodiazepine hydrochloride (GYKI-52466) is a non-competitive AMPA receptor antagonist (Bleakman et al., 1996). In contrast to ionotropic receptors, activation of metabotropic (mglu receptors) (Pin and Acher, 2002; Spooren et al., 2003b; Schoepp et al., 1999a) produces slower modulatory response through second messenger systems. So far, eight subtypes have been described which are divided into three families based on type of second messenger coupling, pharmacology and amino acids homology. Group I mglurs are positively coupled to PLC through Gq protein and their activation leads to an increase in PI hydrolysis, i.e. production of DAG and IP 3 which evokes secondary effects such as release of Ca 2+ from ER stores in the case of IP 3. There are two subtypes of mglurs belonging to this group, i.e. mglur 1 and 5. Group II mglurs are negatively coupled to adenylate cyclase through Gi protein, i.e. their activation inhibits camp production. The two subtypes belonging to this group, i.e. mglur 2 and 3 are hardly distinguishable by presently available pharmacological tools but show different function, e.g. the majority of mglur3 is located on glial cells while mglur2 are mainly located presynaptically on neurons regulating release of transmitters. The least explored group III of mglurs contains four subtypes, i.e. mglur4, 6 8. All of them are, as with group II, coupled negatively to adenylate cyclase and show diverse distribution and function. For example, mglur6 are mainly located in the retina, mglur4 are concentrated in the striatum and mglur7 in limbic structures. AMPA/kainate receptor antagonists as antiepileptic therapy Selected AMPA/kainate receptor antagonists which had either been or are still under development are presented in Table In principle, competitive AMPA antagonists show several obstacles such as low solubility in neutral ph, short half life and weak anticonvulsive effects, therefore in the presented table there are no pure competitive AMPA antagonists. Substances listed are either mixed antagonists of AMPA and other receptors such as kainate (NS-1209) or glycine (LU ) or act through noncompetitive, modulatory sites, e.g. talampanel. Three agents are in phase II, and it remains to be demonstrated whether the side-effect benefit ratio justifies further development and marketing of these agents. Surprisingly, LU acting at three receptors (AMPA, kainite and NMDA) which seemed to have good profile and activity in kindled animals has not been pushed for further development, and this must be assumed to be due to developmental problems not related to preclinical efficacy (Potschka et al., 1998). NMDA receptor antagonists as antiepileptic therapy NMDA antagonists that have been or are presently being tested for epilepsy belong to one of the following categories: channel blockers, competitive antagonists, glycine site antagonist, and NR2B selective agents. In general, NMDA channel blockers show good brain penetration due to high lipophylicity and have a relatively long half life. In the kindling model of epilepsy they inhibit the induction of seizures but not their expression once established. The first NMDA channel blocker to be tested in epileptic patients was

3 200 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) Table Selected AMPA/kainite receptor antagonists which are or have been under development for the indication epilepsy Agents Company Profile Preclinical data Stage of development Example reference (+) MES, (+) PTZ (+) kindling Phase II, (+) refractory partial seizures Novartis, Annual Report 2005 from Potschka et al. (1998) Becampanel Novartis AMPA competitive + kainate antagonist (+) kindling (+) NMDA (+) AMPA Preclinical, no development activity reported LU BASF (Knoll) AMPA (compet.) + GlycineB + kainate (5,7) antagonist (+) MES, (+) PTZ, (+) kindling Phase II, status epilepticus Nielsen et al. (1999) NS-1209, (SDP-502) NeuroSearch AMPA antagonist, GluR5 antagonist Czuczwar et al. (1998) (+) PTZ, (+) ECS, (+) kindling Phase II (+) N = 250, complex partial, (+) 25 % median reduction in seizure frequency (SE: dizziness, headache, semnolescence) IVAX, Lilly AMPA noncompetitive antagonist Talampanel, (LY , GYKI 53773) Gibbs et al., (2000), Reis et al. (2002) (+) MES, (+) PTZ (weak) Launched ( ) partial onset seizures, add on to primary generalised, intractable epilepsy J&J GABA-A enhancer( 2/ 3), Na + antagonist (glu. release), non-nmda antagonist Topiramate (Topimax, KW-6485) (+), positive effect. (+)MK-801 (dizocilpine) which showed no efficacy at doses producing appearance of side-effect (Leppik et al., 1988). Another compound, remacemide (which also effects sodium channels and in turn decreases glutamate release) has been shown to be active in animal models such as MES, audiogenic seizures and chemically induced seizures by NMDA, AMPA, but not by PTZ, bicuculline, strychnine (Palmer and Hutchison, 1997; Schachter and Tarsy, 2000). Subsequently, a phase II refractory epilepsy study showed some efficacy but it was inferior to carbamazepine (Jones et al., 2002). Development of remacemide has been discontinued as a clinical neuroprotection study in Huntington s disease failed to show any efficacy. It should be stressed that NMDA channel blockers in kindled rats may even have a pro-convulsive effect (Löscher and Hönack, 1990). ADCI is a low affinity uncompetitive NMDA receptor antagonist (developed by NIH) showing anticonvulsive activity in various animal models and a favourable side effect profile (as compared to classical antiepileptic drugs) which was discontinued in Phase I of clinical trials (Rogawski et al., 1995). An obstacle for development of NMDA channel blockers for epilepsy is the fact that, similarly to competitive NMDA receptor antagonists (see below), side effects of NMDA channel blockers also seem to be more pronounced in kindled animals (Dziki et al., 1992). Competitive NMDA receptor antagonists showed initially promising activity in animal models where tonic convulsions were assessed (e.g. MES) (Meldrum, 1991), however in animals with established kindled seizures no activity was observed and in fact strong enhancement of side effects was documented (Löscher, 1998; Löscher and Honack, 1991). Indeed, these findings predicted the results of a clinical study performed with CPPene (Midafotel, Sandoz, Novartis) in patients with temporal lobe epilepsy, i.e. no satisfactory antiepileptic activity and enhancement of side effects (Sveinbjornsdottir et al., 1993). Glycine site antagonists could have some advantages over NMDA channel blockers such as lower potential for inducing psychotomimetic side effects. However, they have not yet been tested in clinical trials for epilepsy most likely due to numerous pharmacokinetic problems such as low solubility potentially resulting in kidney damage due to crystallization (e.g. ACEA 1021), low penetration to the CNS, short half life, and last but not least by doubtful efficacy in kindled

4 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) seizure models (Ebert et al., 1997; Wlaz and Löscher, 1998). Another hope in the development of NMDA antagonists was connected with NR2B selective agents. Although most of them were profiled not for epilepsy but rather for acute neuroprotection (stroke, trauma) or pain, they are potentially interesting for this indication as well (Wang and Bausch, 2004). Generally speaking, CNS side effects profile of these agents has been suggested to be very favourable with a lower risk of psychotomimetic effects (Chenard and Menniti, 1999) (however see Chaperon et al., 2003; De Vry and Jentzsch, 2003 for contrasting conclusions). One of the agents that has been under development for epilepsy was PD which was however discontinued (Table ). Although seldom officially admitted, though major obstacle of this group of substances seems to be strong blockade of cardiac herg (potassium) channels resulting in potential prolongation of QT duration in the ECG. Another controversial aspect is the role of NR2B subunits in learning and memory (Massey et al., 2004). On one hand it has been claimed that NR2B antagonists do not impair, but under some conditions may even improve learning (Higgins et al., 2005), but on the other hand over-expression of the NR2B subunit in transgenic mice has been shown to improve learning and age related impairment has been connected with a decline in NR2B expression (Clayton and Browning, 2001; Tang et al., 1999). Another, probably preferential, but not very selective NR2 antagonist is felbamate (Harty and Rogawski, 2000; Pellock, 1999) which was in fact launched for the indication epilepsy (Lennox Gastaut syndrome and partial complex seizures) and has favourable profile in animal models including kindling (Wlaz and Löscher, 1997). However, felbamate had to be subsequently withdrawn due to the occurrence of aplastic anaemia cases connected with the drug use (Pennell et al., 1995). Conantokin G which also preferentially interacts with NR2B subunit is still under development for epilepsy, and is presently in phase I (Donevan and McCabe, 2000; White et al., 1997, 2000). Metabotropic glutamate receptor ligands as antiepileptic therapy It has been reported that the mglur1 antagonist LY and mglur5 antagonist MPEP are effective in mice in the 6 Hz model (Barton et al., 2003; Shannon et al., 2005). However, these findings could not be replicated using different, selective antagonists such as EMQMCM and MTEP (Löscher et al., submitted for publication). Similarly, in the kindling model of epilepsy both positive (Shannon et al., 2005) and negative findings have been reported with mglur1 antagonists (Löscher et al., submitted for publication). Another mglur1 antagonist, BAY has been shown to inhibit PTZ seizures but failed to provide protection against MES or kindled seizures (Chapman et al., 2000; Spooren et al., 2003a). It should be pointed out that the use of either mglur1 and mglur5 antagonists may be connected with possible learning impairing effects (Gravius et al., 2005; Steckler et al., 2005). Several reports indicate potential anticonvulsive utility of metabotropic group II agonists. The orthosteric agonist LY has been shown to attenuate clonic convulsions produced by PTZ or picrotoxin, but not by NMDA (Klodzinska et al., 1999, 2000). Moreover, the GluR2/3 agonists LY and LY inhibited sound-induced clonic seizures in DBA/2 mice and partially inhibited seizure score and afterdischarge duration in amygdala-kindled rats (Moldrich et al., 2001). However, these agonists did not inhibit soundinduced seizures in genetically epilepsy-prone rats, but were even pro-convulsant following the sound stimulus (Moldrich et al., 2001). Daily injections of mglur3 antisense oligonucleotides into the hippocampi of hippocampal kindled rats had no effect on seizure progression rate (Greenwood et al., 2000). It should be also noted that chronic treatment with LY revealed pro-convulsive activity in mice causing suspension of a phase II clinical trial in anxiety with this agent (Danysz, 2005). Another problem related to the use of group II agonists is learning impairment (Danysz, 2005). Possibly, some of the negative aspects could be avoided by use of agents selectively targeting either mglur2 or rather mglur3 receptors. Little is known about the role of group III metabotropic receptors due to the very limited number of selective ligands. In principle, anticonvulsive effects could be expected from either agonists or positive modulators. (R,S)-4-Phosphonophenylglycine (PPG), group III agonist but not AP4 (having a similar profile) inhibited MES (Barton et al., 2003). PPG failed to inhibit PTZ-induced convulsions in mglur7 KO mice indicating a primary role of this receptor subtype

5 Table Selected NMDA receptor antagonists and Na + channels inhibitors (suggested glutamate release inhibitors) which are or have been under development for the indication epilepsy Agents Company Profile Preclinical data Stage of development Example reference Conantokin-G (CGX-1007) Cognetic + Medtronic NMDA NR2B antagonist (+) kindling (more expression) Phase I, refractory epilepsy White et al., (1997), White et al. (2000) Felbamate Fosphenytoin (Cerebyx) Carter-Wallace (MedPointe) Pfizer Na + channel blocker, NMDA antagonist (NR2B?) Harty & Rogawski, 2000 Na + channel blocker (glutamate release inhibitor) Lamotrigine Glaxo Wellcome Na + channel blocker (glutamate release inhibitor) Midafotel (SDZ-EAA-494, CPPene, D-CPPene), (+) MES, (+) PTZ, (+) kindling 1993 Approved, Lennox Gestaut Syndrome (second line therapy aplastic anaemia discontinued) Ebert et al. (2000), Harty and Rogawski (2000), Pellock, (1999), Wlaz and Löscher (1997) Launched Bialer et al. (1999); Emilien and Maloteaux (1998) (+) kindling Launched Ebert et al. (2000) Novartis (Sandoz) Competitive NMDA antagonist (+) MES, (+) epilepsy prone rats Phase I., complex partial ( ) discontinued, psychotomimetic side-effects (III Trauma) Sveinbjornsdottir et al. (1993); Löscher and Honack (1991), Potschka et al. (1999) Remacemide AstraZeneca NMDA channel blocker (metabolite) Na + channel blocker (+) MES (+) kindling ( ) PTZ Discontinued Palmer and Hutchison, (1997), Schachter and Tarsy (2000) RPR Aventis GlycineB site (5 nm) (+) MES Preclinical, discontinued W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) (+), positive effect.

6 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) (Sansig et al., 2001). ACPT-I (preferential mglur4 agonist) inhibited sound convulsions in DBA/2 mice and GEP rats (Chapman et al., 2001). Supportive to this, is a finding indicating that the mglur4 gene falls within a susceptibility locus for juvenile myoclonic epilepsy suggesting a potential link to this form of epilepsy (Wong et al., 2001). This data indicate that also mglur4 may be a candidate for anticonvulsive therapy. However, data obtained in kindled animals are scarce precluding any extrapolation to temporal lobe epilepsy which has the higher medical need. The only report shows that in kindled rats L-AP4 responses are not changed (Friedl et al., 1999). Glutamate release inhibitors There are several Na + channel blockers (with claimed selectivity for the glutamatergic system, i.e. producing release decrease) that have been, or are under development for epilepsy (Table ). In most cases they have multiple actions, e.g. topiramate also affects GABA-A and NMDA receptors, remacemide acts at NMDA receptors, and felbamate affects NR2B containing NMDA receptors (Table ). More selective seem to be fosphenytoin and lamotrigine. This group of agents seems to be the most promising as topiramate, fosphenytoin, lamotrigine and felbamate have all been launched, but the last agent was withdrawn due to side effects. Conclusions The aim of the present commentary was to provide an opinion on glutamate receptors (or the glutamatergic system in general) as a potential target for anticonvulsive therapy, in particular complex partial seizures. In general, it can be summarized as follows: 1. It is rather unlikely that antagonists of glutamate receptors acting through a single target/receptor could offer substantial benefit as compared to existing therapies. Although there are some examples of such drugs under development, e.g. talampanel, their future is not certain. 2. There are several combinations that could turn out to be useful such as actions at two or more glutamate receptors (e.g. kainate + AMPA and or NMDA) (Czuczwar et al., 1995; Löscher and Honack, 1994; Potschka et al., 1998) 3. Combination of agents acting at glutamate receptors with classical antiepileptic drugs offers another possibility (Borowicz et al., 1995, 1996, 1999, 2003; Czuczwar, 1990; Czuczwar et al., 1996). If such combinations offer a better side effects profile and enhanced efficacy development of such combinations seems to be justified. 4. It is too early to judge the validity of metabotropic glutamate receptors as a target for antiepileptic treatment. More well-controlled studies with selective agents in models of kindled seizures are needed. 5. So far, the most promising results have been obtained with claimed glutamate release inhibitors being de facto sodium channel blockers with claimed selectivity towards the glutamatergic system. References Barton, M.E., Peters, S.C., Shannon, H.E., Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models. Epilepsy Res. 56, Bialer, M., Johannessen, S.I., Kupferberg, H.J., Levy, R.H., Loiseau, P., Perucca, E., 99. Progress report on new antiepileptic drugs: a summary of the fourth Eilat conference (EILAT IV). Epilepsy Res. 34, Bleakman, D., Ballyk, B.A., Schoepp, D.D., Palmer, A.J., Bath, C.P., Sharpe, E.F., Woolley, M.L., Bufton, H.R., Kamboj, R.K., Tarnawa, I., Lodge, D., Activity of 2,3-benzodiazepines at native rat and recombinant human glutamate receptors in vitro: stereospecificity and selectivity profiles. Neuropharmacology 35, Borowicz, K.K., Gasior, M., Kleinrok, Z., Czuczwar, S.J., The non-competitive AMPA/kainate receptor antagonist, GYKI 52466, potentiates the anticonvulsant activity of conventional antiepileptics. Eur. J. Pharmacol. 281, Borowicz, K.K., Gasior, M., Kleinrok, Z., Czuczwar, S.J., Competitive NMDA-receptor antagonists, LY and LY , enhance the protective efficacy of various antiepileptic drugs against maximal electroshock-induced seizures in mice. Epilepsja 37, Borowicz, K.K., Luszczki, J., Szadkowski, M., Kleinrok, Z., Czuczwar, S.J., Influence of LY , an antagonist of AMPA/kainate receptors, on the anticonvulsant activity of clonazepam. Eur. J. Pharmacol. 380, Borowicz, K.K., Piskorska, B., Luszczki, J., Czuczwar, S.J., Influence of SIB 1893, a selective mglur5 receptor antagonist, on the anticonvulsant activity of conventional antiepileptic drugs in two models of experimental epilepsy. Pol. J. Pharmacol. 55, Chaperon, F., Muller, W., Auberson, Y.P., Tricklebank, M.D., Neijt, H.C., Substitution for PCP, disruption of prepulse inhibition and hyperactivity induced by N-methyl-d-aspartate receptor

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9 206 W. Löscher, D. Schmidt / Epilepsy Research 69 (2006) Wlaz, P., Löscher, W., Weak anticonvulsant effects of two novel glycine(b) receptor antagonists in the amygdala-kindling model in rats. Eur. J. Pharmacol. 342, Wong, C.G., Scherer, S.W., Snead, O.C., III, Hampson, D.R., Localization of the human mglur4 gene within an epilepsy susceptibility locus(1). Brain Res. Mol. Brain. Res. 87, Developing new antiepileptics: targeting the presynaptic terminal? Berkley A. Lynch 1, Henrik Klitgaard 2, Bruno Fuks 2 1 Cell and Molecular Biology, UCB Pharma, Granta Park, Cambridge CB1 6GS, UK; 2 UCB CNS Research, Braine L Alleud, Belgium The discovery that the antiepileptic drug Levetiracetam (LEV; Keppra TM ) binds to the synaptic vesicle protein SV2A (Lynch et al., 2004) has important implications for understanding the pathophysiology of epilepsy and for developing new antiepileptics. Levetiracetam has a unique pharmacological profile in animal models of seizures and epilepsy, as demonstrated by its lack of activity in acute seizure models used for drug screening, in which most other antiepileptic drugs (AEDs) are active, and as contrasted by its potent seizure protection in animal models of chronic epilepsy including kindling and genetic models (Klitgaard et al., 1998). LEV s unique pharmacological profile has been presumed to relate to a novel mechanism of action (Margineanu and Klitgaard, 2002). This hypothesis is supported by the absence of effect on any of the three main mechanisms currently accepted for the anti-seizure action of established AEDs (GABAergic facilitation, inhibition of Na + currents, or inhibition of low-voltage activated Ca 2+ currents) (Margineanu and Klitgaard, 2002). Previous studies had revealed that LEV binds saturably, reversibly, and stereospecifically to an unidentified binding site in rat brain (Noyer et al., 1995). Testing a series of LEV analogs revealed a strong correlation between their affinities for the brain binding site and their anti-seizure potencies in the audiogenic mouse model of epilepsy (Noyer et al., 1995). This indicates a functional role for the brain binding site in the antiseizure actions of LEV. Identification of the brain binding site of Levetiracetam as the synaptic vesicle SV2A was reported in 2004 (Lynch et al., 2004). SV2A, an integral membrane protein present on all synaptic vesicles, is the most widely distributed SV2 isoform, being ubiquitous in the central nervous system as well as being present in endocrine cells (Buckley and Kelly, 1985; Bajjalieh et al., 1994). SV2A knockouts (KOs) exhibit a severe seizure phenotype, and die at a young age (Crowder et al., 1999; Janz et al., 1999). Studies of the SV2A KOs indicate that SV2A has a crucial role in the regulation of vesicle function, though not in vesicle biogenesis or synaptic morphology (Crowder et al., 1999; Janz et al., 1999). From studies of SV2A knockout mice, the most significant cellular effect caused by the absence of SV2A is a reduction in stimulated exocytosis in neurons and endocrine cells. Current evidence supports that this results from a smaller readily-releasable pool of docked and primed synaptic vesicles at the active zone of the presynaptic terminal, or at the endocrine cell membrane (Crowder et al., 1999; Xu and Bajjalieh, 2001; Custer et al., 2006). The molecular function of SV2A is currently unknown, as is the effect of LEV binding on modulation of SV2As function (Fig ). The SV2A is a 12 transmembrane integral membrane glycoprotein with significant homology to the major facilitator superfamily (MFS) of transporters found in both bacteria and eukaryotes (Bajjalieh, et al., 1992). Given their universal presence in synaptic vesicles, it has been proposed that the SV2s might transport a common component of the synaptic vesicle lumen, such as calcium or ATP (Bajjalieh et al., 1994). However, no transport function has yet been found to rely on a SV2 protein. SV2A interacts with the presynaptic protein synaptotagmin, considered the primary calcium trigger for initiating synaptic vesicle exocytosis, in a calciumdependent manner, and may modulate synaptotagmin s function (Pyle et al., 2000; Schivell et al., 1996). SV2 proteins appear to be the dominant glycoproteins of synaptic vesicles, and it has been proposed that their glycosylation sequences might form a neurotransmitter binding matrix within the synaptic vesicle (Reigada et al., 2003). The question of what presynaptic function of SV2A that LEV might modulate remains to be elucidated. The correlation between anti-seizure properties of LEV derivatives and their affinity for SV2A strongly supports a mechanistic link between the two events. There are reports of effects of LEV on neuronal function, including the partial inhibition of N-type voltage-gated Ca 2+ channels; the reduction of caffeine-induced intra-