New horizons in the development of antiepileptic drugs

Epilepsy Research 50 (2002) 3 – 16 www.elsevier.com/locate/epilepsyres

New horizons in the development of antiepileptic drugs Wolfgang Lo¨scher a,*, Dieter Schmidt b a

Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Buenteweg 17, D-30551 Hanno6er, Germany b Epilepsy Research Group, Berlin, Germany

Abstract Significant advances have been made in the treatment of epilepsy over the past decades. However, despite the development of various novel antiepileptic drugs, about one third of patients with epilepsy is resistant to current pharmacotherapies. Even in patients in whom pharmacotherapy is efficacious, current antiepileptic drugs do not seem to affect the progression or underlying natural history of epilepsy. Furthermore, there is currently no drug available which prevents the development of epilepsy, e.g. after head trauma. Thus, there are at least three important goals for the future. (1) Better understanding of processes leading to epilepsy, thus allowing to create therapies aimed at the prevention of epilepsy in patients at risk; (2) improved understanding of biological mechanisms of pharmacoresistance, allowing to develop drugs for reversal or prevention of resistance; and (3) development of disease-modifying therapies, inhibiting the progression of epilepsy. The ultimate goal would be a drug combining these three properties, thus resulting in a complete cure for epilepsy. In this review, the current status of antiepileptic therapies is critically assessed, and innovative approaches for future therapies are highlighted. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Antiepileptic drugs; Seizures; Epileptogenesis; Pharmacoresistance; Neurotransplantation; Neurostimulation; Gene therapy

1. Introduction Epilepsy, one of the most common neurologic disorders, is a major public health issue, affecting about 4% of individuals over their lifetime (Browne and Holmes, 2001). Despite progress in understanding the pathogenesis of seizures and epilepsy (McNamara, 1999), the cellular basis of * Corresponding author. Tel.: + 49-511-953-8721; fax: + 49-511-953-8581 E-mail address: [email protected] (W. Lo¨scher).

human epilepsy remains a mystery. In the absence of a specific etiological understanding, approaches to drug therapy of epilepsy must necessarily be directed at the control of symptoms, i.e. the suppression of seizures by chronic administration of antiepileptic (anticonvulsant) drugs (AEDs). However, seizures remain uncontrolled in at least 30% of all epilepsies despite adequate AED therapy. During recent years, a large number of new AEDs have been marketed worldwide, but the proportion of patients failing to respond to drug treatment has not been changed to any significant extent. Furthermore, none of the old or new

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AEDs appears to represent a ‘cure’ for epilepsy or an efficacious means for preventing epilepsy or its progression. Thus, new concepts and original ideas for developing AEDs are urgently needed. In this review, the current status of antiepileptic therapies is critically assessed, and innovative approaches for future therapies are highlighted.

2. Pharmacotherapy of epilepsy Over the last decade, there has been considerable progress in the pharmacotherapy of epilepsy, including the introduction of several new AEDs and improved formulations of older drugs (Bazil and Pedley, 1998; McCabe, 2000). The neurologist can now choose from over 20 different medications, including older (‘first generation’) drugs such as phenytoin, carbamazepine, phenobarbital, and valproate, and new (‘second generation’) drugs such as lamotrigine, vigabatrin, tiagabine, topiramate, gabapentin, and levetiracetam. However, the various newly introduced AEDs also create a dilemma for the clinician because their individual places and their optimal use in the treatment of various forms of epilepsy are yet to be determined. The goal of therapy with an AED is to keep the patient free of seizures without interfering with normal brain function. In at least 60% of patients with epilepsy, the prognosis for seizure control is good. However, up to 40% of individuals with epilepsy suffer from intractable, i.e. pharmacoresistant epilepsy despite early treatment and an optimum daily dosage of an adequate AED (Regesta and Tanganelli, 1999; Kwan and Brodie, 2000). The problem of intractable or difficult-to-control seizures has not been changed to any significant extent by the introduction of new AEDs, although drug treatment has become better tolerable for a number of patients. In addition to the problem of pharmacoresistance, several AEDs, including second generation drugs, suffer from substantial problems with toxicity, particularly neurotoxic side effects and idiosyncratic reactions such as skin rash (Brodie, 2001). Thus, new AEDs with better safety, less toxicity, and higher efficacy in difficult-to-control patients are urgently needed.

Surgical treatment of epilepsy with resection of epileptogenic tissue or neurostimulation, e.g. vagus nerve stimulation, may be an alternative if AEDs fail (see below), but resective surgery is restricted to patients with focal epilepsy in which the epileptogenic zone can be adequately identified (Foldvary et al., 2001). Although epilepsy surgery is often considered the only causal treatment (or cure) of epilepsy, in most patients AED treatment has to be continued after surgery to achieve seizure control (see below). This suggests that in many patients undergoing epilepsy surgery, the focal tissue contributing to intractability is removed rather than the complex epileptogenic network underlying the epileptic process. Traditionally, pharmacological strategies for treatment of epilepsy have been aimed at suppressing initiation or propagation of seizures rather than the processes leading to epilepsy (Lo¨ scher, 1998). As a result, none of the currently available AEDs is capable of preventing epilepsy, e.g. after brain injury (Hernandez, 1997; Temkin et al., 2001). Furthermore, there is increasing evidence that— despite early onset of treatment and suppression of seizures— AEDs do not affect the progression or underlying natural history of epilepsy (Lo¨ scher, 1998). Thus, in addition to new drugs needed for pharmacoresistant patients, another important goal for the future will be to develop anti-epileptogenic and disease-modifying drugs, i.e. drugs which prevent the development or progression of epilepsy and not just treat the symptoms. Such therapies would be referred to as truly antiepileptic.

3. Strategies in the development of AEDs In epilepsy research, a major goal in the past has been the development of new AEDs with higher anticonvulsant efficacy and less toxicity than existing AEDs (Lo¨ scher, 1998; Lo¨ scher and Schmidt, 1994; Upton, 1994). Most clinically effective AEDs have been found by screening (i.e. serendipity) or structural variation of known drugs and not by rational strategies based on knowledge of pathophysiologic processes involved

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in seizures or epilepsy. The only exceptions are the GABA-mimetic drugs vigabatrin and tiagabine, which have been developed by a rational strategy, the ‘GABA hypothesis’ of epilepsy, viz. the idea that impaired GABAergic inhibitory neurotransmission is critically involved in the pathogenesis of several types of epilepsy. Several other rational strategies failed to produce efficacious AEDs, most notably a strategy which was based on the ‘glutamate hypothesis’ of epilepsy. Whereas there is ample evidence that exaggerated activity of glutamatergic neurotransmission may contribute to various types of epilepsy (Dalby and Mody, 2001), drugs which counteract this exaggerated activity by antagonism of the N-methylD-aspartate (NMDA) subtype of glutamate receptors showed unimpressive antiepileptic activity and a disturbing incidence of adverse effects in clinical trials, so that further development of such drugs was terminated (Lo¨ scher, 1998). Apart from the bromides and phenobarbital, the anticonvulsant effect of all first and second generation AEDs was first determined in animal models, such as the maximal electroshock seizure (MES) or the pentylenetetrazole (PTZ) seizure tests in mice or rats, demonstrating that clinical activity can be predicted by such simple laboratory models (Lo¨ scher and Schmidt, 1994). Therefore, seizure models in laboratory animals are still the most important prerequisite in the preclinical search for new AEDs. However, the fact that preclinical models used for identification and development of novel AEDs have been originally validated by ‘old’ AEDs may explain why none of the new AEDs possesses significant advantages in efficacy and toxicity compared with the old drugs (Lo¨ scher, 1998). Drugs identified by the MES test commonly resemble phenytoin, which acts by modulating voltage-dependent sodium channels, while drugs identified by the PTZ test have often a benzodiazepine-like mode of action, potentiating the inhibitory effect of GABA (Meldrum, 1997). Because of logistical problems when testing large numbers of compounds, more laborious models of epilepsy such as kindling, which resembles the most common type of epilepsy in adult humans (temporal lobe epilepsy), are only used at later stages of drug development, although drug

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testing in the kindling model was found to be predictive for the negative outcome from clinical testing of NMDA antagonists (Lo¨ scher and Ho¨ nack, 1991). In kindling, repeated electrical stimulation of limbic areas leads to progressive intensification of stimulus-induced seizure activity, culminating in complex partial seizures with secondary generalisation (Lo¨ scher, 1999). Once established, the increased seizure susceptibility in kindled rats is permanent. As indicated by drug testing in kindled rats and subsequently confirmed in patients with epilepsy, the structural and functional brain alterations associated with epilepsy can dramatically change the pharmacology of AEDs, so that preclinical drug development should involve chronic models such as kindling to avoid false positive predictions (Wlaz and Lo¨ scher, 1998). One lesson to be learned from the failure of several rational strategies aimed towards more and more selective drugs is that an absolute selectivity for one target may not be desirable for a multifactorial disease such as epilepsy (Lo¨ scher and Schmidt, 1994). Thus, most clinically efficacious AEDs act by a combination of several mechanisms (e.g. blockade of voltage-dependent sodium channels, potentiation of GABA, limitation of glutamatergic excitation), so that a ‘rational’ combination of mechanisms in a single drug, e.g. by combinatory chemistry, may be a more successful strategy for creating novel broadly-acting AEDs than development of highly selective compounds. Furthermore, ‘reverse target development’, in which only anticonvulsant drugs not acting by known mechanisms of AEDs are further characterised, may be an interesting strategy for the future. With respect to adverse effects, the emerging evidence for the role of polymorphisms (Pirmohamed and Park, 2001) will certainly have an impact on new AED developments, resulting in personalised medicines, whereby administration of the drug and dosage is tailored to an individual genotype. One major risk of the limited advances in epilepsy therapy which have been achieved by novel AEDs is that no further drugs are likely to be developed by the pharmaceutical industry for this indication. However, in this respect it is im-

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portant to consider that several AEDs have indications other than epilepsy, including bipolar disorders, neuropathic pain, and migraine, and are very successful in these indications (MacPherson, 2000; Ross, 2000; Bowden, 2001). The fact that the beneficial effects of AEDs in other indications have been identified only subsequent to their development as epilepsy therapies suggests that the mechanisms involved in anticonvulsant effects are also operative in other conditions. In line with this suggestion, a number of experimental and clinical observations suggest that kindling-like mechanisms are involved in epilepsy, pain states and affective disorders (Weiss and Post, 1998; Post, 2002), so that development of new AEDs is very likely to result also in drugs with beneficial effects in a number of common neurological and psychiatric disorders, thereby substantially increasing the returns from such drugs.

4. Strategies for developing drugs for pharmacoresistant epilepsy Only a small minority (less than 5%) of patients refractory to old AEDs has been reported to become seizure-free with new AEDs (Regesta and Tanganelli, 1999). Thus, previous and current strategies in AED development are not suited to change the situation in pharmacoresistant epilepsies. A major problem in developing new strategies is that mechanisms of pharmacoresistance are only poorly understood (Devinsky, 1999; Kwan and Brodie, 2000). Several factors have been associated with intractability, including early onset of seizures (within the first year of life), length of time from first seizure and number of seizures before onset of therapy, high seizure frequency, a history of febrile seizures or febrile status epilepticus, the type of seizures (about 60% of patients with intractable epilepsy suffer from partial seizures), the presence of multiple seizure types, the persistence of seizures on treatment, the type of epilepsy syndrome, structural brain lesions (particularly hippocampal sclerosis), brain tumours, developmental brain abnormalities, associated neurological and cognitive deficits, and gross EEG abnormalities (Regesta and Tanganelli,

1999). However, none of these factors constitutes a possible target for new treatment strategies. There are many possible causes of refractory epilepsy: it is likely to be a multifactorial process (Lo¨ scher and Potschka, 2002). Genetic factors, e.g. polymorphisms, may be important and explain why two patients with the same type of epilepsy or seizures may differ in their response to AEDs. Disease-related factors are certainly important, including the etiology of the seizures, progression of epilepsy under treatment with AEDs, alterations in drugs targets, seizure-induced synaptic reorganisation, and alterations in drug uptake into the brain. Furthermore, drug-related factors are most likely involved in insufficient seizure control, including loss of anticonvulsant efficacy during treatment (i.e. development of tolerance), or ineffective mechanisms of action of currently available AEDs in difficult-to-control types of epilepsy. With respect to tolerance, which is commonly ignored by most neurologists in epilepsy therapy, a recent analysis of add-on studies in patients with refractory partial epilepsy showed that in almost 30% of the patients the added AEDs (including carbamazepine, phenytoin, lamotrigine, and gabapentin) were associated only with a temporary response, indicating development of tolerance (Boggs et al., 2000). Tolerance can be predicted from long-term studies in rodents and should be considered during preclinical drug development (Lo¨ scher, 1999). An important characteristic of pharmacoresistant epilepsy is that most patients with refractory epilepsy are resistant to most, and often all, AEDs (Regesta and Tanganelli, 1999). As a consequence, patients not controlled on monotherapy with the first AED have a chance of only about 10% to be controlled by other AEDs, even when using AEDs, which act by diverse mechanisms. This argues against epilepsy-induced alterations in specific drug targets as a major cause of pharmacoresistant epilepsy, but rather points to nonspecific and possibly adaptive mechanisms, for instance decreased drug uptake into the brain by seizure-induced overexpression of multidrug transporters in the blood– brain barrier (see below).

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In order to enhance our understanding about the mechanisms of pharmacoresistance in epilepsy, studies on brain tissue from drug-resistant patients and suitable experimental models of intractable epilepsy are mandatory. There is increasing evidence from studies on epileptic brain tissue that overexpression of multidrug transporters may be one important mechanism of pharmacoresistance (see below). Studies in an animal model (pharmacoresistant subgroups of kindled rats) have indicated that both genetic and disease-related factors may be involved in pharmacoresistance (Lo¨ scher, 1997). Intruigingly, 6% of patients with drug resistant temporal-lobe epilepsy who become seizure free after surgery develop drug-resistancy (again) when withdrawn from their medication (Schiller et al., 2000, see below). If confirmed, these patients can be examined as a clinical model for processes leading to drug-resistancy and further studies are warranted. Drug resistance is not unique to epilepsy or neurology, but occurs in other conditions such as arthritis or cancer, and may involve similar mechanisms in these different conditions. For instance, drug transport-based mechanisms are amongst the most intensively studied mechanisms of pharmacoresistance in oncology (Tan et al., 2000; Litman et al., 2001). As in oncology, study of the basis of drug resistance in epilepsy may allow prediction of poor response to AED treatment and should offer new rational approaches to treatment, for instance by design of AEDs that are not targets for brain-expressed resistance mechanisms.

5. Potential targets for intractability in the epileptic brain As outlined above, most epileptic patients resistant to drug treatment do not become seizure-free with any of a broad range of AEDs, although these drugs act by different mechanisms. This suggests the involvement of nonspecific mechanisms of resistance. Furthermore, there must be mechanisms that make some patients more resistant to treatment than others. Likely candidates in this respect are multidrug resistance systems, i.e. membrane transporters such as P-glycoprotein

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(PGP) or the family of multidrug-resistance associated proteins (MRPs), which were first found in pharmacoresistant cancer cells, but now are known to be located in various normal tissues, including capillary endothelial cells of the bloodbrain barrier (BBB) (Tan et al., 2000). Unlike endothelial cells in most tissues, brain capillary endothelial cells are joined by tight junctions and lack intercellular pores and pinocytotic vesicles. Processes from pericapillary astrocytes (‘glial endfeet’) terminate on the capillary and contribute to the barrier function. The BBB passively excludes strongly ionized (polar), hydrophilic drugs, but nonpolar, highly lipid-soluble drugs (like most AEDs) penetrate easily into the brain by simple diffusion. As an active defence mechanism of the BBB, ATP-dependent multidrug transporters, which are located in the apical (luminar) cell membrane of capillary endothelial cells of the BBB, act as outwardly directed active efflux pumps, transferring drugs back into blood after they have entered endothelial cells from blood, thus limiting penetration of many lipophilic drugs into brain parenchyma. Overexpression of these transporters, including PGP and members of the MRP family, as shown in epileptogenic brain tissue (Lo¨ scher and Potschka, 2002), is therefore likely to result in reduced drug penetration into brain parenchyma. This overexpression not only occurs in endothelial cells of the BBB, but also in glial foot processes extending onto capillaries, suggesting that glial overexpression of multidrug transporters represents a ‘second barrier’ that limits drug penetration (Sisodiya et al., 2002), resulting in the simultaneous expression of resistance to a variety of unrelated lipophilic AEDs. Tishler et al. (1995) were the first to report that brain expression of the multidrug resistance gene (MDR1 ), which encodes the multidrug transporter PGP, is markedly increased in patients with medically intractable epilepsy. In line with enhanced MDR1 expression, immunohistochemistry for PGP showed increased staining in capillary endothelium and astrocytes. Tishler et al. (1995) proposed that PGP may play a clinically significant role by limiting access of AEDs to the brain parenchyma, so that increased MDR1 ex-

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pression may contribute to the refractoriness of seizures in patients with pharmacoresistant epilepsy. Subsequently, it was shown that, in addition to PGP, MRP1 and MRP2 are overexpressed in brain tissue of pharmacoresistant patients (Sisodiya et al., 1999, 2001; Abbott et al., 2002; Sisodiya et al., 2002). While MRP2 is found in BBB endothelial cells, MRP1 is found in the choroid plexus epithelium and is thought to contribute to the blood –CSF barrier (Rao et al., 1999). Because of the overexpression of these drug transporters in pharmacoresistant epilepsy, it is of major clinical interest to know which AEDs, if any, are substrates for PGP and MRPs. Currently, at least three strategies are used in this respect. One is to evaluate whether the brain penetration of AEDs can be affected by PGP or MRP inhibitors, a second is to study drug penetration into the brain of PGP or MRP knockout mice, a third is to use cell lines which overexpress PGP or MRPs. The first AED for which active transport was reported is valproate (Frey and Lo¨ scher, 1978). The MRP1/MRP2 inhibitor probenecid increases valproate levels in CSF (Frey and Lo¨ scher, 1978) and brain extracellular fluid (Shen, 1999), indicating that valproate is a substrate for MRPs in the blood– CSF barrier and BBB, which was recently substantiated in vitro by using brain microvessel endothelial cells (Huai-Yun et al., 1998). In addition to valproate, brain extracellular levels of phenytoin and carbamazepine can be increased by PGP and MRP inhibitors, indicating that PGP and MRPs physiologically limit brain penetration of these major AEDs (Potschka and Lo¨ scher, 2001a,b; Potschka et al., 2001). Furthermore, PGP inhibition increases brain extracellular levels of lamotrigine, felbamate, and phenobarbital (Lo¨ scher and Potschka, 2002). Tishler et al. (1995) found that intracellular phenytoin levels in a MDR1 -expressing neuroectodermal cell line were only one fourth that in MDR1 -negative cells, suggesting that PGP significantly contributes to cell export of phenytoin. Phenytoin was also transported by PGP in a kidney epithelial cell line transfected with mdr1a cDNA, which could be blocked by the PGP inhibitor PSC 833 (Schinkel et al., 1996). The transport of carbamazepine was studied in Caco-2

cells, an in vitro model of the intestinal epithelium known to express high PGP levels (Owen et al., 2001). In these cells, the transport of carbamazepine was PGP-independent, and was not affected by PSC 833 (Owen et al., 2001). In mdr1 knockout mice, in which PGP is absent in the BBB, brain levels of phenytoin and carbamazepine were not different from wildtype mice (Schinkel et al., 1996; Owen et al., 2001). In contrast, another recent study on mdr1 knockout mice found that brain/plasma ratios for carbamazepine, lamotrigine, gabapentin, and topiramate were significantly higher in knockout mice compared with wildtype controls (Sills and Kwan, 2001). Furthermore, Rizzi et al. (2001) recently reported that phenytoin levels in the hippocampus of mdr1 knockout mice are significantly higher compared with wildtype mice. However, use of knockout mice is limited in the study of drug resistance because of the redundancy of the transporters: another transport protein may take over the function of one that has been knocked out (Schinkel, 1999). Thus failure of knockout to affect AED kinetics cannot be taken to prove that the protein knocked out does not transport AEDs. A further point when considering different results on AED transport from different model systems are hereditary polymorphisms in the genes encoding PGP and MRPs, resulting in functional alterations in these drug transporters (Kerb et al., 2001). An open question is whether the overexpression of PGP and MRPs in epileptogenic brain tissue of patients with pharmacoresistant epilepsy is a consequence of epilepsy, of uncontrolled seizures, of chronic treatment with AEDs, or is constitutive, i.e. present before onset of epilepsy, for instance related to the initial insult that causes epilepsy. Because pharmacoresistant patients have the same extent of neurotoxic side effects under AED treatment as patients who are controlled by AEDs, the overexpression of drug transporters in pharmacoresistant patients is most likely not global, but restricted to the epileptic focus or circuit. In this respect, it is interesting to note that in patients in whom the epileptic focus has been resected during epilepsy surgery—resulting in seizure control under treatment with AEDs— seizures may reoccur

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after cessation of AED treatment and become pharmacoresistant again, suggesting that a ‘secondary focus’ has become activated and drug-resistant, most likely by the reoccurrence of seizures. In rats, kainate-induced seizures have been found to transiently overexpress PGP in capillary endothelial cells and astroglia in the hippocampus (Zhang et al., 1999), supporting the hypothesis that seizures rather than epilepsy are responsible for overexpression of drug transporters. This could explain the finding that one of the major clinical predictors of pharmacoresistance is high seizure frequency prior to initiation of treatment (Regesta and Tanganelli, 1999). In summary, there is increasing evidence that genes encoding multidrug transporters such as PGP or MRPs are involved in the generation of pharmacoresistance in epileptic patients. If so, systemic or local administration of inhibitors of these drug transporters or designing novel AEDs that are not substrates for transporters may prove useful in pharmacoresistant epilepsy. Inhibitors of PGP, and more recently, MRPs are currently being evaluated clinically for reversal or prevention of intrinsic and acquired multidrug resistance in human cancer (Litman et al., 2001) and may soon become available for clinical trials in epilepsy.

6. Strategies for developing drugs which prevent epilepsy or its progression At least one third of epilepsy is a result of known causes (Annegers et al., 1996). Thus, prevention of epileptogenesis, the process by which the brain becomes epileptic, would be an important task in patients at risk for developing epilepsy, e.g. after brain injury or stroke (Temkin et al., 2001). A number of AEDs, including phenytoin, carbamazepine, valproate, and phenobarbital, have been evaluated in clinical trials to test whether they prevent post-traumatic epilepsy after brain injury. Results have been disappointing, with none of the drugs exerting any significant antiepileptogenesis effect (Hernandez, 1997; Temkin et al., 2001). Interestingly, in apparent contrast to the clinical trials, valproate was shown

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to inhibit epileptogenesis in animal models, i.e. the kindling and kainate models of temporal lobe epilepsy (Silver et al., 1991; Bolanos et al., 1998). Rather than opposing the predictability of data from animal models, this may indicate that the mechanisms responsible for epileptogenesis differ after different initiating events, such as brain injury or status epilepticus. So, improved understanding of basic mechanisms of epilepsy may provide clues to development of drugs capable of preventing epilepsy by counteracting the processes involved in epileptogenesis. Both animal data and clinical data clearly demonstrate that the mechanisms involved in ictogenesis (i.e. initiation, amplification, and propagation of seizures) differ from those involved in epileptogenesis. In a broader sense, epileptogenesis is meant to involve both the processes which make the brain susceptible to spontaneous recurrent seizures and the processes involved in the progression of epilepsy to chronic, often difficult-to-treat epilepsy, so that drugs acting on these processes may be interesting both for prevention of epilepsy and for counteracting the progression of epilepsy after first diagnosis, thereby improving prognosis. With respect to prevention of epilepsy, it would be important to identify diagnostic and surrogate markers helping to predict which patient will develop epilepsy after an insult, i.e. who needs prophylaxis. A wide variety of approaches is used in studies on the basic mechanisms of epilepsy, including both clinical (e.g. brain imaging, studies of human brain tissue, studies on gene mutations in familial epilepsies) and preclinical approaches. New or improved animal models of epilepsy play an important role in this regard (Lo¨ scher, 1999). Common pathological features between the human condition and the animal models may indicate a fundamental involvement of the given pathology in the process of epileptogenesis (Dalby and Mody, 2001). A number of underlying mechanisms of epilepsy are postulated, including an imbalance between excitatory and inhibitory neurotransmission, alterations in neurotransmitter receptor expression and function, development of ‘epileptic’ ion channels (channelopathies), functional (intrinsic) changes of neurons, development of epileptic networks (circuits) within and between

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brain regions, morphological changes such as hippocampal sclerosis and axonal sprouting (leading to aberrant neuronal synchronisation), and genetic causes (McNamara, 1999; Olsen et al., 1999; Coulter, 2001; Dalby and Mody, 2001). With respect to genetic forms of epilepsy, a true cure of epilepsy may be possible by developing a rational approach to therapy that is based on the understanding of the functional effects of a mutation or acquired aberration (McNamara, 1999; Prasad et al., 1999). However, the molecular mechanisms linking genotype with phenotype are largely unknown (Prasad et al., 1999). Genetic factors may also explain why some individuals develop epilepsy after acquired insults, whereas others do not (Prasad et al., 1999). By using approaches mapping mutations underlying human epilepsies, a number of single mutant genes have been recently identified in rare forms of familial epilepsies (Prasad et al., 1999; Scheffer and Berkovic, 2000; Steinlein and Noebels, 2000). All these genes code for voltage-gated or ligandgated ion channels, underlining the important role of channelopathies in some types of idiopathic epilepsy. However, most idiopathic epilepsies are certainly due to inheritance of two or more mutant genes (McNamara, 1999). Genetic animal models, including both spontaneous epileptic mutants and genetically engineered transgenic mice, have contributed significantly to our understanding of epilepsy mechanisms, permitting dissection of the genetic, biochemical, and pathophysiological factors that predispose to enduring hyperexcitability defects (Noebels, 1999; Prasad et al., 1999). More than 50 epilepsy genes have been identified to date in rodents and/or humans, demonstrating the enormous genetic heterogeneity of epilepsy (Frankel, 1999; Noebels, 1999; Steinlein and Noebels, 2000). About 1/3 of the identified gene defects comprises channelopathies, including abnormal function of voltage-gated sodium, potassium, calcium, and a number of ligand-gated channels. Based on the frequency at which mouse mutants appear and the diversity of the proteins involved, it has been suggested that about 1000 genes could influence seizure susceptibility when appropriately mutated (Frankel, 1999). Many gene defects can result in a single

epileptic phenotype, whereas many differing phenotypes may result from a single gene defect (Prasad et al., 1999). Experimental manipulations that target the mutant gene product and patterns of secondary cellular plasticity entrained by mutant genes during brain development may provide a basis for novel strategies to reverse the epileptogenic process (Noebels, 1999). Furthermore, on the basis of genetic and electrophysiologic studies of channelopathies, novel therapeutic strategies can be developed, as has been shown recently for the AED retigabine activating neuronal KNQC2/ 3 potassium channels which show functional abnormalities in an inherited form of juvenile epilepsy (Lerche et al., 2001). A powerful technique for identification of new drug targets for prevention or modification of epilepsy is gene expression analysis using either brain tissue from epilepsy models or from patients undergoing epilepsy surgery. The functional consequences of gene alterations thus found can subsequently be studied in transgenic mice. This technology has recently been used in the kindling model of temporal lobe epilepsy (Hiemisch et al., 2001), employing the novel technique of Massively Parallel Signature Sequencing (MPSS) for expression analysis (Brenner et al., 2000a,b). It was found that one of the most strongly regulated genes in the hippocampus of kindled rats is Homer 1A (Hiemisch et al., 2001), the founding member of a new gene family encoding the Homer group of synaptic proteins (Kammermeier et al., 2000; Xiao et al., 2000). Upregulation of Homer 1A is thought to uncouple crosslinked metabotropic glutamate receptors (mGluR) and inositol phosphate (IP3)-receptors and to enhance mGluR-activated inhibition of voltage-sensitive calcium channels leading to reduced and delayed calcium responses (Kammermeier et al., 2000; Xiao et al., 2000). This prompted experiments aimed to evaluate whether induced Homer 1A expression is causally linked to epileptogenesis (Krupp et al., 2000). In transgenic mice constitutively overexpressing Homer 1A protein in the brain kindling was retarded (Krupp et al., 2000). Thus, Homer 1A could constitute an interesting novel target for counteracting epileptogenesis.

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An important and still not resolved question is whether brain lesions such as hippocampal sclerosis, which are a hallmark of many localisation-related epilepsies, are the cause or consequence of spontaneous recurrent seizures. If brain lesions, which for instance can result from febrile convulsions, are a cause of epilepsy, then administration of neuroprotective drugs would form a potent means of preventing epilepsy. To address this important question, different neuroprotective drugs, including the NMDA antagonist MK-801 (dizocilpine), have been administered to rats after a kainate-induced status epilepticus in the latent (‘silent’) period before the development of spontaneous recurrent seizures in the kainate model of temporal lobe epilepsy (Ebert et al., 2002). The massive sclerosis in the hippocampus and piriform cortex induced by status epilepticus was prevented by administration of MK-801 after the status, indicating that delayed cell death involving NMDA-receptor-mediated events is mainly responsible for the brain damage. However, despite this neuroprotective effect of MK-801, all rats developed spontaneous recurrent seizures, indicating that damage in limbic brain regions is not critically involved in limbic epileptogenesis (Ebert et al., 2002).

7. Adjunctive treatment options to conventional pharmacotherapy of epilepsy Apart from systemic (usually oral) administration of AEDs for treatment of epilepsy, a number of usually adjunctive treatment procedures are being used or under development for clinical use, particularly for patients who do not achieve seizure control or have significant adverse effects under conventional treatment with AEDs (Aiken and Brown, 2000). Most of these adjunctive procedures have not been evaluated as stand alone treatment (with the exception of epilepsy surgery where a small number of patients have been withdrawn from antiepileptic drug (following successful surgery) (see below). Vagus nerve stimulation is currently the most common non-pharmacological adjunctive therapy that is available for pharmacoresistant partial epilepsy, and is less invasive

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and extensive (but also less effective) than resection surgery (Uthman, 2000). Other advancing types of neurostimulation involve transcranial magnetic stimulation (TMS) or direct current (DC) stimulation through the intact scalp, and electrical stimulation of brain regions via depth electrodes (Ziemann et al., 1998; Velasco et al., 2000a,b). A special diet, the ketogenic diet, which was first developed 80 years ago, has been rejuvenated for reducing seizure frequency in severe types of epilepsy in children (Lefevre and Aronson, 2000). Immune mechanisms are involved in the pathogenesis of some forms of epilepsy, so that immune therapy may be an approach in such cases (Aarli, 2000). Interesting findings in this respect are increased autoantibodies to glutamic acid decarboxylase (GAD), the enzyme responsible for GABA synthesis, antibodies to the glutamate receptor GluR3, and antibodies to phospholipids in patients with refractory epilepsy (Angelini et al., 1998; Aarli, 2000; Peltola et al., 2000). Another fascinating treatment option, which is currently undergoing clinical trial, is focus-targeted drug treatment, using a biosensor device that anticipates seizures and subsequently applies treatment via a minipump into the focus to prevent seizures (Le Van Quyen et al., 2001; Litt et al., 2001). Whereas all the afore-mentioned therapies provide only symptomatic relief, there are a number of non-pharmacological therapies that have the potential to cure epilepsy. Epilepsy surgery with resection of epileptogenic tissue is an important alternative treatment for patients with intractable partial epilepsy (Foldvary et al., 2001). The efficacy of resective surgery on drug-resistant temporal lobe epilepsy was recently assessed in the first ever randomised controlled trial comparing immediate temporal lobe surgery with continued medical treatment versus optimised medical treatment alone. At 1 year, 38% of patients were free of seizures with impaired awareness following surgery, compared with 8% in the medical group (PB 0.001). This trial shows that the combination of surgery with antiepileptic drug treatment is more effective than drug treatment without surgery (Wiebe et al., 2001). The outcome in this first controlled trial is lower than the 70% in

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uncontrolled clinical observations (Engel, 1993). Possible explanations for the discrepancy are (i) that the controlled trial design is a more stringent test than clinical observations, and (ii) that the surgical outcome may continue to improve during longer post-surgical follow up than that foreseen in the trial. Also, this trial did not address the important issue if epilepsy surgery is a cure for drug-resistant epilepsy. Most experts agree that in patients who become seizure-free after surgery and remain seizure-free after complete withdrawal of their medication for several years, the epilepsy is cured. Efforts to completely discontinue AEDs in patients who were seizure-free after surgery, resulted in a seizure relapse in 30 of 84 patients (36%) in one recent retrospective study (Schiller et al., 2000). When drugs were tapered but not withdrawn, seizures recurred in 13 of 96 patients (14%) and in 7% of 30 patients where drug treatment was not changed. This important finding suggests (i) that most patients continue to require a combination of both surgery and drug treatment, and (ii) that epilepsy surgery was a cure for epilepsy in approximately 25% (54 of 210) patients becoming seizure-free after surgery. The assumption that all patients become seizure-free following surgery is, however, too optimistic. Assuming that, at most, 70% of patients undergoing temporal lobe surgery will become seizure-free (Engel, 1993), the conservatively calculated cure rate would be in the range of 30 of 300 surgical patients (10%) with previously drug-resistant epilepsy. In an earlier study, AED treatment was discontinued in 39 of 104 patients after being seizure-free for at least 1 year following anterior temporal lobe surgery, and in 26 patients (25%) no seizures recurred within 3 years (Murro et al., 1991). Assuming again, that at most 70% of patients are becoming seizure-free following surgery, the conservatively calculated cure rate would be 18%. Clearly, further studies on the curative ability of temporal lobe epilepsy surgery are needed. Fortunately, reinstitution of medical treatment after seizure recurrence in patients withdrawn from AED treatment proved effective in all but two of 30 patients (Schiller et al., 2000). Intruigingly, some patients developed drug-resistancy during drug-withdrawal. If confirmed the data

suggest that drug-resistancy is a dynamic process in these patients, which can transiently be attenuated by adjunctive surgery in some patients, but cannot be stopped by surgery alone and resurfaces during withdrawal. However, surgery has risks and costs that have to be considered. In contrast to resective surgery, the use of neurotransplantation as a potential treatment in drug-resistant epilepsy is still in its infancy (Bjo¨ rklund and Lindvall, 2000). Because the adult brain has only a limited capacity for self-repair, there is great interest in the possibility of neuronal replacement and partial reconstruction of neuronal circuitry by transplanting new cells. The effects of cell transplantation have been investigated in a variety of animal models of seizures or epilepsy, but the majority of studies have been done in the kindling model of temporal lobe epilepsy (Bjo¨ rklund and Lindvall, 2000). Different strategies for seizure suppression have been used: cell transplantation into the epileptic focus (e.g. the hippocampus) in an attempt to restore function, or cell transplantation into sites that contribute to the spread and generalisation of seizures, such as the substantia nigra pars reticulata (SNR). Grafts of foetal striatal GABAergic neurons transplanted into the SNR suppressed the spread and generalisation of seizure activity in already kindled rats, but these effects were only transient, either because GABA release from the grafts declined over time, or because the host target neurons downregulated their own GABA receptors following transplantation (Lo¨ scher et al., 1998). Transplantation of conditionally immortalised cells, engineered to produce and release GABA, were shown to significantly retard the kindling process when injected into the SNR before the onset of kindling (Thompson et al., 2001). Grafting of encapsuled adenosine-releasing fibroblasts into the brain ventricles of kindled rats provided a nearly complete protection from seizures, but seizure protection declined during subsequent weeks, most likely from a reduction of cell viability (Huber et al., 1998). As an alternative to the use of foetal neurons or engineered cell lines, stem and progenitor cells may constitute a novel encouraging approach for cell transplantation (Bjo¨ rklund and Lindvall, 2000), but to our

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knowledge no data on this approach in epilepsy models are available as yet. Although the scientific basis for clinical neurotransplantation trials in epilepsy is currently weak, intracerebral implantation of porcine foetal GABA-rich tissue has been performed in a group of patients with pharmacoresistant focal epilepsy (Schachter et al., 1998). Gene therapy is considered an area full of promise, but using genetic information to develop gene therapy for epilepsy has just begun. Gene therapy requires identification of genetic defects in specific epilepsies and an understanding of the functional consequences of those defects. Only then can methods be developed to correct the genetic defects or their downstream consequences. Because most of the common inherited epilepsies are not transmitted through simple (monogenic) inheritance, their complex (polygenic) transmission complicates attempts to isolate the specific defects and to develop appropriate therapies (Scheffer and Berkovic, 2000). As a first step in the development of a gene therapy approach to epilepsy, virus vectors have been successfully used to protect neurons from damage after a kainateinduced status epilepticus in rats, for instance by over-expression of an antiapoptotic gene such as Bcl-2 (McLaughlin et al., 2000).

8. Conclusions and goals for future developments of antiepileptic drugs Current antiepileptic therapies are unsatisfactory as they provide only symptomatic relief, are not effective in a significant percentage of epileptic patients, and are often accompanied by persistent adverse effects. The emerging insights into the cellular, molecular and genetic mechanisms of ictogenesis and epileptogenesis are likely to lead to new therapies, prevention, or even a cure. Apart from conventional pharmacotherapy, there are several adjunctive and promising strategies for treatment of epilepsy that are likely to gain importance. The rapidly expanding information about the mechanisms of epilepsy and its treatment provides the basis for reevaluating some of the commonly accepted paradigms that guide ap-

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proaches to treatment. Apart from improved treatment of epilepsy, the field of epilepsy research produces many important insights into normal brain functions, such as processes involved in different forms of synaptic plasticity, brain networks involved in motor control, and regulation of receptors and signal transduction. The primary goal of epilepsy research, namely, understanding the mechanisms of human epilepsies, remains a most challenging and interdisciplinary task, but one that will hopefully open up new opportunities for improved treatment of patients with epilepsy. Immediate goals for future development include (i) drugs with improved tolerability, (ii) drugs effective for pharmaco-resistant epilepsies, (iii) disease-modifying compounds, and finally (iv) drugs which prevent epilepsy in patients at risk prior to their first seizure.

Acknowledgements We wish to thank S.M. Sisodiya (Institute of Neurology, University College London, UK) and H.S. White (Department of Pharmacology and Toxicology, Salt Lake City, Utah, USA) for fruitful discussions and constructive criticisms during the preparation of this manuscript.

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