CHAPTER 6

Epilepsy

Michael A. Rogawski

1. lntraduction Epilepsy, a condition of recurrent, paroxysmal seizures, is a

major, worldwide, health problem. The epileptic seizure represents an abnormal synchronized discharge among a large population of central neurons. Although the cause of this synchronized activity is largely a mystery, the occurrence of seizures in an epileptic patient can be diminished or eliminated by drugs that target the basic excit- ability mechanisms of neurons. Most presently used antiepileptic medications were deveIoped by empirical drug screening or seren- dipity. However, in the past several decades, as understanding offhe functional activity of brain circuits has increased, there has been an intense effort to develop new antiepileptic medications on a rational basis. Although progress has been measured, some rationally devel- oped antiepileptic drugs are now entering the market.

The focus of attention for the rational design of antiepileptic drugs has been to target, either directly or indirectly, ion-conducting channels--the fundamental mediators of electrical excitability in neurons. There are two major classes of neuronal ion channels: volt- age-dependent channels that shape the subthreshold electrical behavior of the neuron, allow it to fire action potentials, and regulate

Neurotherapeutics: Emerging Strategies Eds.: L. Pullan and 4. Patel Hurnana Press Inc., Totowa, NJ

194 Xoga wski

its responsiveness EO synaptic signals; and neurotransmitter-rep- lated channels that mediate the synaptic signals, Each of these two classes of channel participates in the electrical events o f the seizure. Voltage-dependent channels contribute to the paroxysmal depolariz- ing shift, the single ceII correlate of the interictal (between seizure) network discharge. Voltage-dependent channels also mediate action potentials that allow transmission of the seizure discharge along axons to neighboring cells and distant brain sites. The neurotransmit- ter-regulated channels allow the abnormal discharge to propagate between neurons within the neuronal aggregate and also participate in the paroxysmal depolalizing shift. The voltage-dependent ion channels include channels selective for Na+ and K+, that shape the subthreshold behavior of neurons and mediate action potentials as well as channels selective fox Ca2+ that appear to play a critical, role in absence seizures (see Section 7.). Neurotransmitter receptor chan- nels include the fast-acting channels for the major excitatory and inhibitory neurotransmitters, glutamate and GABA, as well as far regionally specific transmitters such as monoamines (catechola- mines, serotonin, and histamine) and neuropeptides. In addition to rapid actions, the transmitters canmediate slower 'modulatory" sig- nals, where the receptor is coupled to an ion channel via G-proteins or other second messengers.

In principle, it may be possible to prevent the occurrence of seizures by targeting any one or a combination of these systems, so that there is an enormous variety of possible anticonvulsant strate- gies. The ultimate goal is, of course, to prevent the generation or propagation of seizure discharges without interfering with the nor- mal functioning of the nervous system. (An additional and perhaps more satisfying goal is to prevent the development of epilepsy; there are some important leads along these lints as discussed in Section 3.1 .) Given the critical role of the neuronal excitability mechanisms mediating seizures for normal brain function, it would seem improb- able that an anticonvulsantdrug could be developed which suppressed seizures without producing side effects. Nevertheless, several pres- ently available anticonvulsant drugs approach this goaI, indicating that it is attainable.

In this chapter, I consider a variety of approaches that are cur- rently under investigation for the rational development of new anti- epileptic drugs. The mechanisms of presently available antiepileptic drugs has been reviewed previously (Rogawski and Porter, 1990), and are not considered in detail here.

2. Potentiation of Synaptic Inhibition Enhancement of synaptic inhibition mediated by GABAA

receptors is an important anticonvulsant strategy. This can occur by targeting of the GABAA receptor itself, or by enhancement of synap- tic GABA levels. Drugs that enhance GABAA receptor-mediated inhibition show a distinctive spectrum o f activity in animal seizure models. Such drugs typically protect very effectively against pentylenetetrazol seizures, but have far weaker activity in the maxi- mal electroshock test. Despite the selectivity of their effects in animal seizure models, GAB&-receptor potentiating anticonvulsants may have a broad spectrum of activity in human seizure disorders. However, they are generally ineffective in absence seizures. (Benzo- diazepine receptor agonists that do have anti-absence activity are a notable exception to this rule.)

2.1. GABA, Receptors as Targets for Anh'epilep tic Drugs Presently available drugs that potentiate GABAA receptor

responses fa11 into two broad classes: benzodiazepine-like and bar- biturate-like modulators. In addition, there are a variety of agents, including neuroactive steroids and loreclezole, that act similarly to barbiturates, but probably interact with distinct sites on the GABAA receptor complex.

2.1.1. Novel Benzodiazepine Receptor Ligands Benzodiazepine receptor agonists, such as diazepam and

lorazepam, have powerful anticonwlsant properties as a result of their ability to enhance GABA A receptor-mediated neurotransmis- sion in the central nervous system (CNS). Although such benzodiaz- epine agonists have an impor.tant role in the acute treatment of status epiiepticus (continuous seizures without return to consciousness),

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two significant limitations largely preclude their chronic use in epi- Iepsy therapy. The first limiting factor is that these benzodiazepines produce undesirable side effects, including sedation and muscle relaxation, at doses comparable to those that protect against seizures. The second, and probably more important, Iirnitation is that tolerance develops to the anticonvulsant activity of benzodiazepines receptor agonists with chronic use. In recent years, considerable effort has been directed toward developing strategies for overcoming these limitations. A variety of benzodiazepines and nonbenzodiazepines have been identified that interact with the benzadiazepine binding site of the GABAn receptor complex in novel and potentially more favorable ways. As yet, however, there is no convincing evidence that any of these benzodiazepine receptor ligands will have practical. utility in the chronic treatment of epilepsy.

2.1.1.1. RENZODIAZEPINE RECEPTOR PARTIAL AGONISTS. Partial aganisrn at the benzodiazepine receptor has been proposed by Haefel y (see Haefely et al., 1990) as a promising approach to overcame the undesirable side effects and the propensity to the development of tolerance of full benzodiazepine agonists. Bartiat agonists have low intsinsic efficacy and induce smaller responses than do full agonists at the same fractional receptor occupancy. Thus, at full receptor occupancy, partial agonists produce submaximal biological responses. However, the degree of partial agonismmay vary depending on the subunit composition of the GAB& receptor W f l a c h et al., 1993). At anticonvulsant doses, partial benzodiazepine receptor aga- nists may produce less sedation and muscle relaxation than full ago- nists, and may also exhibit less tolerance and physical dependence.

Benzodiazepine receptor partial agonists have been identified within several structural classes, including me benzodiazepines (bretazenil, FG 8205; Martin et al., 1988; Tricklebank et al., 1990), P-carbolines (abecarnil, ZK 41 296; Stephens et al., 1990; Petersen et al., 1984), pyrozoloquinolines (CGS 9896; Bernard et al., 19851, imidazopyrimidines (divaplon; Garher, 1988), and quinolizinones (Ro 19-8022; Jencket al., 1992). Same partial agonist-for example, Ro 19-8022-have anticonvulsant potency and efficacy in animal seizure models comparable to full benzodiazepine receptor agonists

Epilepsy 197

(Jenck et al., 1 992; Facklam et a]., 1 992a; Jensen et al., 1 984). How- ever, Ro 19-8022 has lower eficacy for sedation and muscle relax- ation, and may also have reduced physical dependence liability. Moreover, Ro 19-8022 does not exhibit proconvulsant activity (like benzodiazepine inverse agonists); nor is an abstinence syndrome (characterized by tremors and seizures) precipitated when chroni- cally treated animals are exposed to a benzodiazepine receptor antagonist. Interestingly, in line with the efficacy of conventionaI benzodiazepines in human absence epilepsy, Ro 19-8022 has been reported to be effective in a rodent genetic model of absence epi- lepsy. Other benzodiazepine receptor partial agonists, including bretazenil (Haigh and Feely, 1988; Facklam et aI., 1992a) and divaplon (Feely et al., 1989; Deacon et al., 1991) similarly display potent anticonvulsant activity, but less sedation and anticonvulsant tolerance than diazepam. Studies using recombinant GABAn recep- tor subunits have demonstrated that bretazenil and divaplon possess 35-58% and 2 1-28%, respectively, of the intrinsic efficacy of the full agonist flunitrazepam (Knoflach et al., 1993; see also Facklam et al., 1992b).

How can we understand the law propensity of benzodiazepine receptor partial agonists to produce sedation, muscle relaxation, and tolerance? It is now recognized that full benzodiazepine receptor agonists, such as df azepam, produce anticonvulsant effects with low receptor occupancy (Fig. I). For example, it has been estimated that full benzodiazepine receptor agonists protect against sound-induced seizures in mice at occupancy levels of 2-5% and against penty- lenetetrazol seizures at occupancy levels of 25% (Braestrzlp and Nielsen, 1986). In contrast, 70-90% occupancy is needed for protec- tion against tonic hindlimb extension in the maximal electroshock test (Petersen et al., 1986). Similarly, sedative effects as assessed with various tests of motor impairment also require high levels of occupancy (50-76%; Petersen et al., 1986). Thus, full benzodiaz- epine receptor agonists exhibit anticonvulsant activity (against sound-induced and pentylenetetrazol seizures) at low doses, and sedation at higher doses. For partial agonists to elicit the same response as a full agonist, higher levels of receptor occupancy are

Xogawski

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- SPONTANEOUS ABSENCE IRAT)

AUDIOGENIC

Fig. 1. The various actions of the full benzodiazepine receptor agonist diazepam occur at different levels of receptor occupancy. Thus, partial ago- nists may not produce behavioral effects requiring high IeveIs of occupancy, such as impairment of motor function in the rotarod test. In vivo radioligand method for determination of receptor occupancy is described in Petersen et al. (1984). Audiogenic = protection against sound-induced seizures in DBN2 mice; MES =maximal electroshock test; DMCM =protection against seizures induced by the benzodiazepine receptor inverse agonist merhyl 6,7- dimethoxy-4-ethyl-b-carboiine-3-carboxylate. Data obtained in the mouse (except as notedlare fromBraestmp and Nielsen ( 1 986), Petersen et al. (1 984), Jensen et al. ( 19X4), and Petersen et al. (1 986).

required, The requirement for higher occupancy is presumably because the potentiation of each GABAA receptor is smaller and more GABAA receptors must be recruited to obtain an equivalent response. For example, in one study, 50% protection in the penty- lenetetrazol test was reported to occur with 37% receptor occupancy by diazepam and 84% receptor occupancy by ZK 91296, a partial agonist (Petersen et al., 1986). For benzodiazepine agonist effects that require a high degree of GABAA receptor potentiation (e.g.,

muscle relaxation, ataxia, amnesia, and respiratory depression), it may not be possible to achieve the pharmacological effect with a partial agonist, even with full occupancy. Thus, partial agonists can have anticonvulsant activity and yet not show adverse side effects at: any dose. There is substantial evidence that benzodiazepine receptor tolerance occurs as a result of a downregulation in the expression of certain GABAA receptor subunits, particularly the p subunit that confers benzodiazepine sensitivity (Zhao et a1 ., 1994). Nevertheless, since the mechanism coupling chronic receptor activation to changes in the expressfan of the y2 subunit gene is not well understood, it is not yet possible to explain the reduced tendency ofpartial agonists to induce tolerance. Despite the very favorable properties af benzodi- azepine receptor partial agonists, as yet there is no clinical data sup- porting the utility of these compounds in epilepsy therapy.

Certain benzodiasepine receptor partial agonists, such as abecamil, may have a distinct spectrum of anticonvulsant activity in comparison with conventional benzodiazepine receptor agonists such as diazepam (Turski et al., 1990). For example, abecarnil is highly active in a variety of chernoconvulsant models (Sersa et al., 19921, but, unlike diazepam, is inactive in the maximal electroshock test. The differences in the spectrum of anticonvuEsant activity may relate to the unique GABAA receptor subunit specificity of abecarnil. Recent studies with cloned GABAA receptor subunits indicate that abecarnil can exhibit partial or fuI1 agonist properties at different GABAA receptor subtypes, mainly dependent on the molecular form of the a-subunit (Knoflach et al., 1 993; PribiIla et al., 1993). Thus, the unique phmacalogical profile of abecarnil could arise from its actions as a partial agonist at some GABAA receptors and as a fu11 agonist at others. Animals treated chronically with abe~arnil do not exhibit a diazepam-like withdrawal syndrome on discontinuation of the drug (Losches et al., 1 990; Steppuhn et al., 1 993; Llischer, 1 993)- Moreover, the development of anticonvulsant tolerance to abecarnil is dependent on the model used. Indeed, in some experimental para- digms no anticonvulsant tolerance is exhibited at the same time that to1 erance develops to the motor impairing activity of the dmg (Losc her et al., 1991b; Serra et aI., 1994).

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2.1.1 -2. BENZODIAZEPTNE RECEPTOR SUBTYPE-SELECTWE AGONTSTS. Abecamil is an exampIe of a benzodiazepine receptor ligand that acts as a partial agonist at some GABAA receptor subunit combinations and not others. It therefore exhibits some subtype selectivity. The imidazopyridines alpidem and zolpidem, in contrast, show nearIy complete subtype selectivity. These compounds exhibit GABA potentiating activity at, for example, GAB AA receptors of composi- tion a lBzyz , a2Ply2, alPlyz, but not aSP2y2 (Faun-Halley et al., 1993; Wafford et al., 1993; Horne et al., 1992). In binding to brain tissue, alpidern and zolpidem show selectivity for the oli'BZ1 subtype ofrecep tor (Benavides et al., 1988; Benavides et al., 1993). AIpidem has anti- convulsant activity comparnbIe to benzodiazepines, but exhibits much-reduced sedative and muscle relaxant activity (Garreau et al., 1992). In addition, the drug fails to induce tolerance or dependence (Perrault et al., 1993). Human trials of alpidem as an anxiolytic have been promising, with a very favorable side-effect profile in compari- son with conventional benzodiazepines. Alpidem binding exhibits a differential regional: localization in brain that correlates roughly with the presence of al/BZt binding sites. It has been suggested that the regional selectivity ofalpidem for w t/BZl binding sites, which isdepen- dent on the unique subunit specificity of the drug, could account for its favorable properties (Benavides et al., 1993). On the other hand, the possibility that alpidern is a partial agonist at certain GABAP, receptor subtypes could also explain its favorable profile (Perrault et al., 19933. However, recent studies with cloned subunits have dem- onstrated that alpidern (Im et al., 1993) and zolpidem (Horne et al., 1992) ase full agonists at some, if not aII, GABAA receptor subtypes.

2.1.2. Barbifurafe-Like Drugs

Barbiturates have fallen out of favor as anticonvulsant agents largely because of their propensity to cause sedation, behavioral impairment, and deleterious effects on cognitive performance (Fawell et al., 1990). Nevertheless, barbiturates are highly effective, broad-spectrum anticonvulsants. Barbiturate-like compounds with improved side-effect profiles would provide interesting candidates for clinical development.

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The sedative and anticonvulsant activity of barbiturates is believed to be mainly due to their potentiationofCNS GABA-mediated inhibition (Scholfield, 1938). This occurs by prolongation of burst openings of single GABARreceptor channels (Macdonald et al., 1989). However, effects on voltage-activated CaZ+ channels also appear to play a role in the sedative-hypnotic activity of the barbiturates and could participate in their therapeutic activity as anticonvulsants (ffrench-Mullen et al., 1993). In addition, direct activation of the GABAn receptor could account for the sedative-hypnotic side effects of certain barbiturates (Rho et al., 1994b). Finally, barbiturates appear to have actions an excitatory amino acid receptors, with particular selectivity for AMPA (a-amino-3-hydroxy -5-methy 1-4- isoxazalepmpionic acid) receptors (Taverna et al., 1994; Donevan and Rogawski, unpublished). In line with the diversity of barbiturate cel- lular actions, there are wide differences in the pharmacological prop- erties of structurally similar barbiturates. Pentobarbital is a powerful sedative-anesthetic, whereas the less sedative phenobarbital is used mainly as an anticonvulsant. Although still incompletely understood, the basis for the reduced sedation with phenobarbital may relate to relative affinities for different target sites and also to the magnitudes of effects produced at clinically relevant concentrations. For example, it has been proposed that the strongly depressant action ofpentobarbita1 could relate to its activity both as a GABA potentiator and a Ca2+ channel blocker. Phenobarbital, in contrast, has a higher affinity for GABAA receptors than for Ca2+ channels (ffrench-Mullen et aI., 1993). At clinically relevant concentrations, it may mainly act as a GABA potentiator, but the absolute magnitude of the potentiation may be smaller than that produced by pentobarbital. The lack of effect on Ca2+ channels and the less robust potentiation could account for its lower toxicity. As the concentration of phenobarbital moves into the supratherapeutic (toxic) range, it becomes more strongly sedative, possibly because effects on Ca2' channels become prominent. The types of Ca2+ channels relevant to the action of barbiturates have not been well defined. One hypothesis is that these are presynaptic chan- nels involved in the regulation ofneurotransmitter release, and that the depressant action is Iargely due to a reduction of glutamate release.

2 02 Roga wski

It may be possible to obtain even greater reductions in the seda- tive-hypnotic activity of barbiturate-like GABA modulators. Thus, the newly introduced anticonvulsant felbamate has a very favorable side effect profile (causing agitation and insomnia more frequently than sedation; Faught et al., 19931, and appears to act, in part, as a barbiturate-like modulator of GABAA receptors (Rho et aE., 1994~). (Felbarnate produces certain severe idiosyncratic toxicities, unre- lated to its anticonwlsant action, that limit its clinical utility.) Felbamate is a dicarbamate structuralIy related to the sedative- anxiolytic meprobamate. Meprobamate has also recently been shown to have barbiturate-like activity. However, it is much more potent and, unlike felbamate, can directly stimulate the opening of GABAA receptor coupled CI- channels in the absence sf GABA (Rho et al., 1994a). This latter activity could contribute to the propensity of meprobamate to cause greater CNS depression. In addition to its activity as a barbiturate-Iike modulator of GABAA receptors, felbamate also can inhibit NMDA receptors as discussed in Section 3.1.5. This effect could produce behavioral activation that would counterbalance the CNS depression resulting from GABA potentia- tion. (NMDA antagonists typically have behavioral activating effects at low doses; Willetts et al., 1990.)

It has been known for over five decades that the steroids proges- terone and deoxycorticosterone acetate have anticonvulsant activity against pentylenetetrazol seizures (Selye, 1942; Craig, 1966), In the intervening years, other structurally related steroids, including 3,20- pregnenediones and 3,20-pregnanediones (Craig and Deason, J 968), 2P-morpholino-5a,3a-pregnanoIones (Hewett et al., 19641, and the steroid anesthetic alphaxalone (Peterson, 1989), have been reported to have similar activity. In 1984, Harrison and Simmonds made the seminal discovery that alphaxalone could selectively potentiate cen- tral neuron responses to GABA (but not glycine). The 3P-hydroxy isomer betaxalone was inactive, indicating a high degree of structural specificity. Morerecently, it has been recognized that certain endoge- nous metabolites of progesterone and deoxycorticosterone are also

Epilepsy 203

highly potent modulators of the GABAn receptor (Majewska et aE., 1986). These metabolites have anticonvulsant activity (Belelli et al., 1989, 1990; Bogskilde et al., 1988), and their anticonvulsant poten- cies correlate strongly with their ability to modulate GABAA receptor current (Kokate et al., 1994). Studies using fluctuation (noise) analy- sis and single channel recording have demonstrated that the steroids produce a barbiturate-like prolongation ofburst openings of GABAA receptors (Sirnmonds, 199 1; Twyrnan and Macdonald, 1992). In addi- tion, they may cause an increase in channel-opening frequency, an effect not observed with barbiturates. Like barbiturates, neuroactive steroids can directly activate the GABAA receptor in the absence of GABA.

Do neuroactive steroids have potential in the treatment of sei- zure disorders? Although highly active against seizures induced by pentylenetetrazol and other GABA receptor antagonists including bjcuculline and picrotoxin, GABA-potentiating neuroactive steroids are inactive in the maximal electroshock test. This contrasts with phenobarbital which shows some activity in the maximal electroshock test (Belelli et al., 1990). The profile of the steroids in animal seizure models suggests that their primargr utility may be in the treatment of absence epilepsy (Rogawski and Porter, 1990). Indeed, there is an anecdotal report ofthe effectiveness of deoxycorticosterone acetate in six of eight absence seizure patients (Aird and Gordan, 195 1).

The extent to which neuroactive steroids can protect against seizures at nontoxic (nonsedating) doses is not clear. All GABA- potentiating neurosteroids cause impairment of motor performance. However, there may be differences among stmcturally similar neu- roactive steroids in relative toxicity. Progesterone and certain pregnene and pregnane 3,20-diones exhibit a degree of separation between their anticonvulsant and toxic doses, but are relatively weak anticonvulsants (Craig and Deason, 1968). However, alphaxalone, a mare potent anticonvulsant, protects against seizures only at doses that produce neurological impairment (Peterson, 1989). In a compre- hensive study of progesterone and deoxycorticosterone metabolites, there was up to a several-fold separation between the protective and toxic doses. Thus the therapeutic index (ratio of the ED50 values for motor impairment and for protection in the pentylenetetrazol seizure

test) ranged from 1.2-3.8 (Kokate et al., 1994). However, the thera- peutic indices of a11 of the steroids were less than that of the benzo- diazepine clonazepam (8.9). Recently, steroid-like substituted bentejindenes (tricyclic molecules containing only a portion of the steroid A-ring) have been described which potentiate GABA responses and may have toxicity comparable to the most favorable neurosteroids (Rodgers-Neame et a1 ., 1992). Further structure- activity studies may lead to the identification of steroids with improved toxicity profiles. However, a key-but as yet unan- swered-question is whether neurosteroids will exhibit tolerance as do benzodiazepines. If so, like benzodiazepines, they will have lim- ited utility in the chronic therapy of seizure. (A recent study failed to observe any tolerance to the anticonvulsant activity ofpregnanolone in the pentylenetetrazol test; Kokate et aI., 19956.) However, even if tolerance does develop over time, steroids may stiIl be of value in the acute treatment of status epilepticus, as are benzodiazepines. In fact, the steroids appear to be highIy effective in the kainate and piIo- carpine models ofstatus epilepticus in the mouse (Kokate andRogawski, 1994; Kokate et al., 1995a). Because the solubility of steroids is poor, selection of an appropriate vehicle or identification o f water soluble prodrug forms will be critical.

Recently, the synthetic 3P-methyl analog of epiallopregnano- Ione (3a-hydroxy,3 P-methyl-5 a-pregnan-20-one; CCD 1 042) has been demonstrated to have anticonvulsant potency that is only slightly weaker than its parent. However, the 3P-methylation confers good metabolic stability and oral activity. In Phase I cIinical trials, CCD 1 042 was well-tolerated at levels substantially higher than those associated with seizure protection in animals (K. W. Gee, personal communication, 1995).

2.1.5. y-Bufyrolactones and y-Thiobutyrolactones

An additional series of GABAA receptor modulating compounds has recently been described that appear to act at a site distinct f o m benzodiazepines, barbiturates, and steroids (Holland er aI., 1993). This novel recognition site, referred to as the y-butyrolactone (GBL) site, is the locus of action of a variety of substituted y-butyolactones

Epilepsy 205

and y-thiobutyrolactones. Certain ligands ofthe GBL site antagonize GABAA receptor response and are convulsant, whereas others poten- tiate GABA responses and have anticonvulsant properties (Holland et al., 1990). Anticonvulsant GBL site ligands, such as a-ethyl,a- methyl-thiobutyrolactone (a-EMTBL), are protective in the penty- lenetetrazol seizure model as are other GABA-potentiating drugs. However, GBL site ligands may also be effective in the maximal electroshock test and other seizure models (Ferrendelli et al., 1989; Holland et al., 1992). a-EMTBL has a therapeutic index that is low relative to other GABA modulating anticonvulsants, but i s similar to valproate. Preliminary results indicate that a-EMTBL may demon- strate less tolerance thanclanazepan, suggesting that GBL site ligands may be ofutiIity in chronic epiIepsy therapy (Ferrendelli et al., 1993).

Loreclezole is a novel triazole derivative that is protective against pentylenetetrazol seizures but is less active in the maximal electroshock test (Wauquier et al., 1990; Kulak and Sobaniec, 1994). In addition, at low, nontoxic doses, the drug has anti-absence activity in a genetic model of generalized absence epilepsy (Ates et al., 1992). Conse- quently, loreclezole has a profile of activity similar to that of other benzodiazepines. A potential benzodiazepine-Iike interaction with GABA, receptors is suggested by the observation that the anticon- wlsant effects of loreclezole can be reversed by benzodiazepine recep- tor inverse agonists (Vaught and Wauquier, 199 1; Ashton et al., 1992). The benmdiazepine antagonist flumazenil, however, failed to alter the anticonvulsant activity of loreclezole, indicating that loreclezole is not a benzodiazepine receptor agonist (Wauquier et al., 1990). Moreover, on chronic (5-7 d) administration, tolerance did not develop to the anti- conwlsant effects of Ioreclezole as it does with benzodiazepines.

LorecEezole can indeed enhance the activity of GABAA recep- tors, but it interacts with a novel allosteric modulatory site distinct from that of benzodiazepines, barbiturates, or neuroactive steroids (Wafford et al., 1 994). Using native rat and cloned human GABAA receptors, loreclezole strongly potentiated GABA-activated C1- cur- rent. However, activity of the drug did not require the presence of the

y-subunit and was not blocked by flumazenil, confirming that loreclezoIe does not interact with the benzodiazepine recognition site. Interestingly, loreclezole had >300-fold activity an receptors containing the P I - or p3-subunit in comparison with those containing the I subunit. Using chimeric P-subunits and site-directed mutagen- esis, it was demonstrated that a single amino acid, at the carboxyl- terminal end of the P-subunit putative channel-lining domain TMZ, confers sensitivity to loreclezole (Wingrove et al., 1994). The affin- ity for GABA and benzodiazepines was unaltered by amino acid substitutions at this site.

Loreclezole reduced seizuse occurrences in several small clini- cal trials and was rdatively free sf side effects (Rentmeester and Hulsman, 19921, demonstrating the potential of drugs that modulate GABAA receptors in novel ways.

2.2. Vigabatrr.'~~, a GABA Transamitrase Inhibitor

In addition to the focus on postsynaptic GABAn receptors, there has been considerable interest and, indeed, success in the develop- ment of new antiepileptic drugs that modify GABA disposition in the brain. Vigabatrin (y-vinyl-GABA), perhaps the most notable achieve- ment in this respect, is an enzyme-activated irreversible inhibitor of GABA transaminase, the main degradative e n m e for GABA. The drug elevates brain GABA levels, and exhibits anticonvulsant activ- ity in a variety of animal seizure models. Moreover, there is now substantial clinical data supporting its efficacy in the treatment of human seizure disorders (Reynolds, 1992). Although the precise way in which vigabatrin prevents seizures remains unclear (Bernasconi et al., 19881, GABA transaminase inhibition may increase neuronal GABA in a releasable pool, so that larger amounts of GABA are discharged with stimulation (Gale, 1992). Thus, increased amounts of GABA are released in a use-dependem fashion, as needed, during seizures, It had been believed that tolerance develops only to the sedative side effects ofvigabatrin, but not to its anticonvulsant activ- ity (Remy and Beaumont, 1989). Now, however, there is emerging evidence that tolerance may also develop to the therapeutic effects of the drug (Martin and Millac, 1993).

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2.3. GABA Uptake Blockers An alternative approach to increasing synaptic GABA levels is

inhibition of GABA reuptake. A variety of cyclic amino acids, including nipecotic acid and guavacine, inhibit glial orneusonal GABA uptake carriers. In general, however, thcse compounds have only modest potency in vitro and do not enter into the CNS after parented administration. In recent years, n variety of lipophilic analogs of nipecotic acid and guavacine have been described (Braestrup et al., 1990; Suzdak, 1993; Andersenet al., 1993). These compozand~which include tiagabine, CI-966, NNC-7 1 1, and SKF lQ0330A---are highly potent and specific inhibitors of GABA uptake into glia and neurons, but have essentially no activity on other uptake transporters. Unlike nipecotic acidwith its moderate selectivity for neuronal GABAuptake, lipophilic GABA uptake inhibitors such as tiagabine show a modestly greater affinity for glial uptake, but they do inhibit uptake in both cell types. These compounds are not transported via the GABA uptake carrier nor do they stimulate GABA release. Recently, it has been demonstrated that tiagabine, CI-966, and related lipophilic GABA uptake inhibitors are highEy selective for the GAT-1 cIoned GABA transporter, the predominant form in rat brain (Burden et al,, 1994). Using in vivo microdialysis in rodents and primates, the systemic administration of CI-966 (Taylor et al., 1990) or tiagabine (Fink-Jensen et al., 1992; Sybirska et al., 19933 was shown to increase extracellular GABA overflow. Similar results were obtained in awake human sub- jects with hippocampal microdialysis probes foIlowing acute oral administration of tiagabine (During eta]., 1992). Experiments in brain slice preparations have demonstrated that Iipephilic GABA reuptake blockers can prolong the action of applied GABA and aEso increase the amplitude and duration of inhibitoy GABA-mediated synaptic poten- tials (Reckling et aI., 1990; Thompson and Gahwiler, 1992). In addi- tion, these compounds inhibit epileptiform activity in the slice.

Lipsphilic GABA uptake inhibitors have a spectrum s f anti- convulsant activity in animal seizure models that is similar to other drugs which act on brain GABA systems, such as the benzodiaz- epines. These compounds are highly effective in protecting against pentylenetetrazol seizures and may also have activity against sei-

zures induced by other chemoconvulsants that interfere with GABA- ergic neurotransrnission, such as the benzodiazepine receptor inverse agonist DMCM (methyl 6,7-dimethoxy-4-ethyl-barboline-3-car- boxylate). They are far less potent (tiagabine: Nielsen et al., 199 1) or inactive (SW 89976A and SKF 100330-A: Swinyard et al., 199 1) in the maximal electroshock test. In addition, lipophilic GABA uptake inhibitors have shown activity in various reflex seizure models (including the photosensitive baboon Papio papio), and in the amygdala and corneal kindIing models (Suzdak, 1993; Swinyard et al., 1991; Smith et al., 1995). At doses higher than those required to protect against seizures, lipophilic GABA uptake inhibitors pro- duce neurological impairment, as do other drugs that enhance GABAergic neurotransrnission. However, there is evidence that tol- erance may develop to these side effects, but not to the anticonvulsant activity (Suzdak, 1994). LipophiIic GABA uptake inhibitors have a spectrum of anticonvulsant activity in animal seizure models that is distinct from the important presently marketed antiepileptic drugs (Swinyard et aE., 199 1). Therefore, it is difficult to predict fromanimal studies which farms of human epilepsy would be most amenable to therapy with these agents. However, their potency and rela- tively broad spectrum of activity suggest that they are interesting candidates for further development.

In human clinical trials, tiagabine has shown activity against complex partial seizures, with an overall incidence of side effects no different fiom placebo at therapeutic doses (Mengel, 1994). v o w - ever, adverse side effect such as headache, tiredness, and dizziness were obsmed with increased doses.) Importantly, there was no evi- dence of tolerance to the drug's anticonwlsant activity with chronic dosing for up to 12 mo. In contrast, in an initial trial in healthy voIun- teers, CL-966 induced a variety of severe neurological and psychiatric symptoms (Sedman et al., 1990). There are differences between the pharmacological properties oftiagabine and CI-966 that could account for the striking differences in the clinical responses to the two drugs (see Suzdak, 1993). AItematively, it may simply be that the initial doses chosen for CI-966 were excessive. It is uncertain whether the favorable safety record of tiagabine will be maintained in larger tri-

Epilepsy 209

als. The available infomation suggests that cautious optimism about the potential of lipophilic GABA uptake inhibitors is warranted.

2.4. Potenfintion, of GABA Release Although drugs that elevate synaptic GABA levels by interfer-

ing with GABA removal mechanisms have anticonvulsant activity, the continuously elevated GABA levels may have untoward effects. A theoretically more attractive approach would be to increase the amount of GABA released with each synaptic stimulus. At present, no selective GABA releasing agents have been described. However, there is some preliminary evidencc that the clinically effective anti- epileptic drug gabapentin could act by such a mechanism.

Although gabapentin was originally synthesized as a GABA analog, it does not interact with GABAa or GABAR receptors, is not metabolicalIy converted to GABA or a GABA receptor agonist, and does not inhibit GABA uptake or degradation (Taylor, 1994). Haw- ever, gabapentin does increase GABA turnover in some brain regions (Loscher et al., 1 99 1 a) and may increase the release of GABA from brain slices in vitro (Gdtz et al., 1993). Recently, it has been observed that gabapentin can potentiate electrophysiological responses to released GABA in rat neonatal optic nerve, presumably by increas- ing release from gIia (Kocsis and Homou, 1994). A similar effect has been reported in the hippocampus (Honmou et aI., 1994). Although these studies with gabapentin and GABA release are intriguing, they are as yet inconclusive. Nevertheless, they serve to focus attention on the possibility that drugs that enhance GABA release could have potential as antiepileptic agents.

2.5. Potentiation of GABA and Glycine: Propofol and Chlomethiazole

The anesthetic propofol(2,6-diisopropylphenol) is a structurally novel barbiturate-like modulator of GABAn receptors (Peduto et aI., 199 1). However, in contrast to barbiturates such as pentobarbital, sev- eral recent reports have suggested that subanesthetic doses ofpropofol: can protect against seizures in animal rnodeIs, with minimal behavioral side effects (Hasanetal., 1992; al-Haderetal., 1992; Hasan and Wolley,

210 Roga wski

1 994). In humans, propofol may be effective in the treatment of status epilepticus (Wood et al., 1988), although the drug has also been reported to cause seizures under certain circumstances (Makela et a]., 1993). Propof01 produces an enhancement of GABA-activated C1- current and, at high doses, can directly activate GABAA receptors (Hales and Lambed, 199 1). In addition to these barbiturate-like prop- erties, propofol is also able to potentiate glycinc-evoked currents (Hales and Larnbert, 199 1 ; but see, Dolin et al., 1992), an action not shared by barbiturates, neuroactive steroids, or benzodiazqines.

Another combined GABAA/glycine modulator for which there is even more clinical information is chlormethiazole, a thiamine derivative with sedative, hypnotic, anxiolytic, and anticonvulsant properties (~gren , 1986). ChlormethiazoIe is particularly effective in the matment of status epilepticus (even in cases failing to respond to benzodiazepines and barbiturates). Chlomethiazole is a1 so used inpreeclampsia (toxemia of pregnancy), eclampsia (toxemia of preg- nancy with seizures), and the alcohol withdrawal syndrome. En both animals and humans, chlormethinzoIe has anticonvulsant activity at doses that do not produce sedation. Like propofol, chlormethiazole potentiates responses to both GABA and glycine, and can also directly activate GABAA receptors (Wales and Larnbert, 1992). Propofol and chlonnethiazole uniquely potentiate GABA and glycine, and both appear to be effective anticonvuIsants with low behavioral toxicity at anticonwlsant doses. The favorable toxicity could be explained if the potentiation of GABA and glycine were additive (or even syner- gistic) for seizure protection, but were not additive for sedative side effects. Indeed, it is not apparent that a glycine potentiating drug would exhibit sedative-hypnotic activity.

3. Interactions with Excitatory Amino Acid Receptor Systems

A complementary approach to facilitation of synaptic inhibition is blockade of synaptic excitation. The bulk of fast synaptic excita- tion in the CNS is mediated by the excitatory amino acid glutamate, acting on AMPA (a-amino-3-hydroxy-5=methyl-4-i~0~au,lepropi0ni~

acid) and NMDA (N-methyl-D-aspartic acid) receptors. In recent years, there has been intense interest in the medicinal chemistry of excitatory amino acid antagonists because sf their potential in the treatment of neurological disorders, including epilepsy. The con- viction that excitatory amino acid antagonists have a roIe in epilepsy therapy is based on extensive in vitro and in vivo evidence that NMDA and AMPA receptors participate in the expression of many types of epileptic seizures (Rogawski, 1995). Recently, it has been elegantly shown using microdialysis that glutamate levels are eIevated prior to the onset and during the expression of complex partial seizures in human subjects (During and Spencer, 1993). There is thus strong theoretical support for the potential utility of excitatory amino acid antagonists in the treatment of human seizure disorders. However, as yet, this has not been verified in clinical trials.

3.1. NMDA Receptor Antagonists NMDA receptor antagonists have a broad spectrum of activity

in animal seizure models. NMDA antagonists are particularly potent in the maximal electroshock test in rodents and against reflex sei- zures in rodents and primates (Chapman and Meldrum, 1993). They are also protective against pentylenetetrazol and other chemoconvul- snnts. Thus, the resuIts from animal testing suggest that NMDA antagonists may have utility in the treatment of generalized tonic- clonic (convulsive) and partial seizures. NMDA antagonists have lower potency against spontaneous absence-like seizures in rodents, so that they are less likely to be of utility in human absence seizures. A particularly interesting feature of NMDA antagonists is their pow- erful ability to protect against the induction of kindled seizures in vivo and against kindling-like phenomena in the in vitro hippocam- pal ~Iicepreparation (see Rogawski, 1 995). This suggests that NMDA antagonists may prevent the development or progression of some types of seizures. This property could be of value in certain cIinicaI situations, such as during the critical period after brain injury or in progressive childhood epilepsies. The status of efforts to develop a safe and effective NMDA antagonist for use in epilepsy therapy has been recentIy reviewed (Rogawski, 1992).

3.1.1. Competitive NMDA Recognition Site Antagonists In 1982, Croucher et al. reported that phosphonic acid competi-

tive NMDA recognition site antagonists were protective against sound-induced and minimal pentylenetetrazol seizures in mice. This important observation stimulated an intensive research effort into the medicinal chemistry of NMDA receptor antagonists (Rowley and Leeson, 1992). Numerous structural classes of NMDA antagonists have been investigated. Perhaps the most mature line of investigation is that evolving from the straight chain o-phosphono-a-amino acid compounds that were the basis of the study by Crouchex et al. (see Watkins, 1991). The original compounds are very polar and as a consequence have poor brain penetration. However, cyclic analogs with the amino nitrogen in a six-member ring, as in CGS 19755 (cis- 4-phosphonomethyl-2-piperidine carboxylate; Hutchison et al., 1989; Lehrnann et al., 1988), or in a piperazine ring, as in CPP (3-[2- caxboxypiperazin-4-yllpropyl- 1 -phosphonate; Davies et al., 1986; Lehmann et al., 1987), had improved systemic activity, and were even effective orally (Pate1 et al., 1990). It was subsequently found that uunsaturation sf the chain, as in CGP 37849 ([El-2-amino-4- methyl-5-phosphono-3-pentanoic acid; Fagg et al., 1990; Schmutz et al., 1990; De Sarro and De Sam, 1992), or addition of a ketone as in MDL 100453 ~[R]-4-oxo-5-phosphonowaline; Whitten et aE., 1990) also resulted in compounds with powefil systemic anticsnvulsant activity. Moreover, the carboxyethylester of CGP 37849 (CGP 3955 1) showed particularly prolonged oral anticonvulsant activity (De Sarro and De Sarro, 1992). (The reduced analog MDL 10453 also is a potent NMDA antagonist; Bigge et al., 1992.) Unsaturation of the side chain of CPP to form CPPene (Aebischer et al., 1989) similarly produced an anticonvulsant compound with a long duration of action, The D(-)-enantiorner is the active species of CPPene and has been used in several studies. D(-)-CPPene has protective activity in a variety of reflex epilepsy models (Patel et al., 1990; Smith and Chapman, 1993; Smith et al., 1993; De Sarm and De Sarro, 1993) and, as is the case for other NMDA antagonists, protects against the development of kindled seizures (Durmurnuller et al., 1 994). Most competitive NMDA recognition site antagonists have a phosphonic

Epilepsy 223

acid group as in the original structures produced by Watkins (1 991). However, this is not essential to activity as demonstrated by LY233053 and LY275059, in which she phosphonic acid group is replaced by the bioisosteric tetrazol functionality. LY233053 offers good protection against NMDA and maximal electroshock seizures, but its duration of action is short (Schoepp et al., 1990a,b; Ornstein et al,, 1992). The structurally novel bicyclic phosphonic acid LY2746 14 has long lasting oral anticonvulsant activity (Schoepp et al., 199 13, whereas the corresponding tetmzol LY233536 (Leander and Ornstein, 1990; Omstein et al., 19901, like LY233053, has a more sapid time course.

Competitive NMDA recognition site antagonists exhibit a broad spectrum of activity in animal seizure models. Not unexpectedly, these compounds are highly effective in protecting against seizures and lethality induced by systemic or intracerebroventricular NMDA. They are also protective in the maxima1 electroshock test with corn- parabIe or slightly lower potency (Ferkany et nl., 1989). Protection is also conferred against pentylenetemzol-induced clonic seizures, but typically at somewhat higher doses. However, a structurally unique cyclic phosphonic acid NPC 12626 (2-amino-4,5-[1,2-cyclo- hexyll-7-phosphonoheptanoiG acid) has been described which is less potent in the maximal electroshock test than against NMDA or penty- lenetetrasol seizures (Ferkany et al., 1989). Recently, the highest potency isomer of NPC 12626 has been identified as the ZR,4R,5S form (Hamilton et al., 1993). It too shows a reverse ordering of potencies in animal seizure models (Ferkany et al., E 993). Whether this unique profile is related to an unusual selectivity for NMDA receptor subtypes or some other property remains to be determined.

Although diverse NMDA recognition site antagonists with good bioavailability and favorable pharmacokinetic properties are now at hand, the problemof side effects has not been so readily solved. Since NMDA receptors are critical to numerous brain functions, NMDA antagonists can produce a variety of behavioral and neurological toxicities. There are, however, certain encouraging features of the preclinical profile of competitive NMDA recognition site antag- onists. AnticonvuIsant effects typically occur at doses below those

that produce motor impairment (De Sarro and De Sarro, 1993; Ferkany et al., 1993). However, the separation between anti- conwlsant activity and motor toxicity is typically smaller than for other anticotkvulsant drugs. Drug discrimination studies indicate that the subjective effects of competitive NMDA recognition site antago- nists in rodents and primates are distinct from those of dissociative anesthetic-like NMDA antagonists (Gold and Balster, 1993; Bobelis and Ralster, 1993). While competitive antagonists do not seem to have the same propensity for producing certain behavioral side effects such as stereotypies and disturbances of locomotor behavior common with dissociative anesthetic-like NMDA antagonists (see Section 3.1.2.), these stiIl occur. The side effects may be especially prominent in kindled animals (Loscher and H~nack, 1 99 1 a, b). Tol- erance does not seem to develop to either the therapeutic activity or the side effects (Smith and Chapman, 1993)-

Despite the various favorable properties ofNMDA recognition site antagonists, reports of clinical trials have been disappointing. In an open-Iabel study of D-CPPene in eight patients with intractable complex partial seizures, a 1 patients withdrew prematurely because of side effects. The side effects included poor concentration, seda- tion, ataxia, depression, dysarthrta (impairment of speech due to motor impairment), and amnesia (Sveinbjornsdottir et al., 1993). None of the patients experienced a reduction in seizure frequency. Interestingly, healthy volunteers tolerated much higher doses than the patients. Thus, as in kindled animals, patients with epilepsy may have greater sensitivity to the side effects of NMDA antagonists. A final problem is that competitive antagonists (like the dissociative anesthetic-like compounds discussed in Section 3.1.2.) can produce neuronal vacuolization and morphological damage in certain brain regions (Olney et al., 1991).

Until recently, it was not appreciated that competitive NM9A recognition site antagonists exhibit differing activities at NMDA receptors composed of different subunit combinations. The informa- tion now available indicates that this is the case. Thus, CPP appears to have modestly higher affinity for NMDA receptors composed of W D A R l - l b subunits that contain a 2 1 -amino acid insert generated

by alternative splicing of exon 5 (see McBain and Mayer, 1994). In addition to sensitivity to competitive antagonists, this N-terminal variation determines a variety of functional properties of the NMBA receptor, including potentiation by ZnZ+ and polyamines and inhibi- tion by protons (HolImann et al., 1993). In the future, it may be possible to more selectively target specific NMDA receptor subunit combinations, thus potentially providing for the development of less toxic anticonvulsants.

3.1.2. Channel-Blocking NMDA Receptor Antagonists The NMDA receptor channel blockers are often referred to as

'kncompetitive" antagonists, because their binding and blocking action requires the receptor-channel to be gated in the open state. Occupancy of a binding site within the ionophore of the NMDA receptor prevents cation flux through the channel, thus producing a functional block of NMDA receptor responses (MacDonald et al., 199 1 ). Unlike competitive NMDA recognition site antagonists, uncompetitive antagonists share with other noncompetitive antago- nists of the NMDA receptor the theoreticaI advantage that their block- ing action wouldnot be overcome by high synaptic levels of gIutamate asmay occur during seizures. In addition, uncompetitive antagonists have the additional theoretical advantage of use-dependence, imply- ing that their inhibitory action may specifically be potentiated at sites of excessive receptor activation. The current status and potential clinical utiIity of channel-blocking NMDAreceptor antagonists have recently been reviewed (Rogawski, 1992, 1993).

Channel-blocking NMDA receptor antagonists fa11 into two broad categories: dissociative anesthetic-like agents and Iow affinity antagonists. Shortly after the discovery of selective competitive NMDA recognition site antagonists in the early 1980s, the dissocia- tive anesthetics phencyclidine and ketamine were demonstrated to be effective NMDA receptor antagonists (Anis et al., 1983). The struc- turally dissimilar dissociative anesthetic-like agent dizocilpine (MK- 801) is also an extremely potent NMDA receptor antagonist. These compounds exert their block in a use-dependent and voltage-depen- dent fashion, indicating that they act by an open-channel mechanism

21 6 Roga wski

(Huettner and Bean, f 9881, Like competitive NMDA recognition site antagonists, dissociative anesthetics are potent broad spectrum anticonvulsants in a wide variety of animal seizure models (see Rogawski, 1992). However, at anticonvulsant doses, they produce severe neurobehavioral side effect-including cognitive, sensory, and various schizophrenia-like deficits (Krystal et al., 1994)-that limit their potential clinical utility. In addition, dissociative anes- thetics have been reported to cause reversible vacuolization in rat cortical neurons at low doses (Olney et al., 1989, 19911, and frank necrosis at higher doses (Fix et al., 1993). The propensity of dissocia- tive anesthetics to cause neuronal injury is shared by many NMDA antagonists, raising doubts as to overall safety of such compounds. However, caution must be exercised in extrapolating the histo- pathdogical findings obtained in rats to other species. Indeed, NMDA antagonist-induced pathomorphological changes have not been documented in primates. Moreover, it is now apparent that some NMDA antagonists may produce only transitory vacuoIization with no necrosis, or no changes at all.

The anticonvuIsant activity of dissociative anesthetics is shared by a variety of channel-blocking NMDA antagonists, some of which are analogs of the dissociative anesthetics and others that are struc- turally distinct. Certain of these compounds have substantially reduced behavioral toxicity in animals. For several, there is human clinical experience documenting an acceptable level of safety. These reduced-toxicity channel-blocking NMDA antagonists have been referred to as "low affinity" uncompetitive antagonists. However, low affinityper-se may not be the only factor that accounts for their more favorable toxicity characteristics (see below).

The existence of low-toxicity, channel-blocking NMDA antag- onists was first recognized with the discovery that certain l-phenyl- cycloalkylamines such as 1-phenylcyclahexylamine (the analog of phencyclidine substituting a primary amine for the piperidine ring) and 1-phenylcyclopentylamine were effective anticonvulsants, but had a reduced propensity to produce motor impairment at anticon- vulsant doses (Rogawski et nl., 1988, 1989; Thurkauf et al., 1990; Blake et nl., 1992). Later, an analog of dizocilpine ADCI (5-amino-

Epilepsy 217

carbonyl-l0,11-dihydro-5H-dibenzo[a,~cyclohepten-5,IO-imine) was observed to have similar properties, and also to retard the devel- opment of kindled seizures (Rogawski et al., 1991). These com- pounds displace binding of dissociative anesthetic receptor Iigands (pH] 1 -[l-(2-thienyl)cyclohexyl]piperidine and [3H]dizocilpine) to brain membranes and block NMDA receptor currents in a use-depen- dent and voltage-dependent manner, indicating that they are chan- nel-blocking NMDA antagonists (Rogawski et aI., 1992; Jones and Rogawski, 1992). However, the binding affinities of these reduced-toxicity uncompetitive NMDA receptor antagonists are lower than that of phencyclidine and dizocilpine. At equieffective concen- trations, these lower affinity ligands would exhibit faster apparent rates of block and unblock, and this may, at least in part, account for the lower toxicity (see Rogawski, 1993). However, reduced binding affinity alone cannot completely explain the lower toxicity since there is an imperfect correlation between binding affinity and thera- peutic index (Rogawski et al., 1989). Moreover, ketamine, a drug with strong dissociative anesthetic properties, has a binding affinity comparable to many of the less toxic channel-blocking agents. In fact, there is now evidence that lower toxicity Iigands may bind selec- tively to a different population ofNMDAreceptors than do dissociative anesthetics (Porter and Greenmyre, 1995).

Recent work with cloned NMDA receptor subunits suggests that there may be differences among subunit combinations with respect to their sensitivity to channel-blocking agents (McBain and Mayex, 19943. For example, assembly of the NMDAR1 subunit with NR2C generates receptors with lower affinity for some channel blockers than is the case with NR2A orNR2B. In addition, the pres- ence of exon 5 in NMDAR1 (the NMDARl- 1 b splice variant; see Section 3.1. 1.) confers higher sensitivity to block by dizocilpine and ketarnine (Hollmann et al., 1993; Rodriguez-Paz et al., 1995). More subtle variations in the structure of NMDA receptors could also modify the actions of channel bIockers. A particularly critical site of the NMDARl subunit is the neutral asparagine at position 598. This corresponds to the edited arginineJgIutamine (QIR) site in membrane segment 2 (M2) of AMPA receptors, where a positively charged

arginine residue in GluR2 confers low Ca2+pemeability (see Section 3.2.2.). Similarly, mutations in NMDAR1 that switch the asparagine to a positively charged arginine generate receptors resistant to block by dissociative anesthetics (Kawajiri and Dingledine, 1993; Mori et al., 1992; Sakurda et a]., 1993). Although there is currently no evidence for RNA editing of NMDA receptors at this site (as occurs for AMPA receptor subunits), variations in channel structure in this or a nearby region of the receptor could have important conse- quences for the sensitivity to channel blockers. Of course, it remains to be determined which, if any, structural variation in the NMDA receptor accounts for the selective binding of low-affinity channel- blocking NMDA antagonists, and, moreover, whether this contrib- utes to their more favorable toxicity characteristics. Factors other than binding affinity and subunit selectivity might also contribute to the more favorable side-effect profiles ofcertain low-affinity NMDA antagonists (Rogawski, 1993). In particular, synergism between effects on NMDA receptors and other anticonvulsant targets could be a factor in some circumstances. For example, ADCI has Na* channel blocking activity similar la carbamazepine (see Section 4.1, although the extent to which this occurs at concentrations relevant to the anticonvulsant activity is not clear (White, 1994).

In recent years, several anticonvulsants of potential clinical importance have been recognized as low-affinity NMDA receptor channel-blockers. These include dextrornethorphan and its metabo- lite dextrorphan; ARL 12495 (formerly FPL 12495; 1,2-diphenyl-2- propylamine), Ithe metabolite of the anticonvulsant remacemide; and the adamantane analog memantine. In contrast to dissociative anes- thetics, low-affinity channel-blocking NMDA antagonists substitute poorly, or not at all, for dizocilpine in drug discrimination testing. This indicates that they may not produce dissociative anesthetic-like subjective effects (Grant, Colombo, Grant, and Rogawski, unpwb- lished). Moreover, for dextrornethorphan, dextrorphan, remacemide, and memantine, substantial clinical experience indicates that the compounds can be well tolerated in humans. However, adverse experiences with all of these compounds have been reported (see

Rogawski, 1993). Controlled therapeutic trials will be necessary to

Epilepsy 21 9

verify the efficacy and safety of these low-affinity, uncompetitive NMDA antagonists in the treatment of seizure disorders. Neverthe- less, there is cause for optimism with this class of compounds.

In addition to their potential in chronic epilepsy therapy, NMDA antagonists may have utility in the treatment of seizures that occur during ethanol withdrawal (Grant et a!., 1990; Liljequist, 199 I ; Morrisett et al., 1990). This may be in part because of a compensatory upregulation in the expression of NMDA receptors after chronic ethanol exposure (Sanna et al., 1993). ADCI is particularly effective in this regard (Grant et al., 1992).

3.1.3. GIycine Site Antagonists It is now well recognized that glycine is a required co-agonist

for activation of the NMDA receptor (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). Consequently, agents that competi- tively antagonize the action of glycine would produce a functional block of the receptor and could, therefore, have anticonvulsant activ- ity. Several classes of antagonists are now known which more or less sefectively block the glycine site (Huettner, 199 I ; Carter, 1992). With intracerebroventricular administration, several of these com- pounds protect against various types of seizure activity (Singh et al., 199 1; Baron et al., 1990; Koek and Colpaert, 1990; Palfreyman and Baron, 1 99 1; Bisaga et al., 19933. Recently, Rundfeld et al. ( 1 994) observed that intraventricular injection of glycine site antagonists and partial agonists increases seizure threshold in arnygdala-kindIed rats, an effect not observed with other types of NMDA antagonists. A similar positive effect of a glycine site partial agonist (D-cyclos- erine) was observed against kainate-induced seizures (Baran et al., 1994). These authors suggested that agents active at the glycine site of the NMDA receptor may be of utiIity in the treatment of seizure types that are refractory even to other NMDA antagonists.

A significant impediment to the evaluation of glycine site antagonists in conventional seizure models has been their lack of CNS accessibility. This problem now seems to have been largely overcome, but complete studies of the activity of available com- pounds in animal seizure models have not yet been reported. Initial

220 Rogawski

clues to the development of systemically active glycine site antago- nists came from the recognition that HA-966 (3-amino-I- hydroxypyrmlidin-2-one) could be resolved into enantiorners with distinct pharmacological activities. One enantiomer was a partial, low-affinity antagonist of the glycine site (Singh et al., 1990; Pullan et nl., 1990). This R(+)-enantiomer has activity in the electroshock seizure test when administered intravenously, but its therapeutic index is poor (Vartanian and Taylor, 1991). However, several ana- logs have been synthesized with greater potency, selectivity, and more favorable toxicity properties, such as L-687,4 14 (3R-amino- 1 - hydraxy-4R-methylpysrolidin-2-one) (Leeson et al., 1990, 1993; Smith and Meldrum, 1992). Nevertheless, despite intense effort, it was not possible to produce a compound in this series with suffi- ciently favorable properties to warrant clinical development.

Another gIycine site partial agonist with anticonwlsant activity is 1 -aminocyclopropanecarboxyliG acid (ACPC), a particularly high affinity ligand. ACPC is protective in several seizure models (SkoEnick eta]., 1989; Witkin and TorEeIla, 199 1; Smith et al., 1993; Bisaga et al., 19931, but may not be active in the maximal elec- troshock test. Unlike other glycine site partial agonists with anticonwlsant properties, ACPC has nearIy full efficacy (about 85%; Watson and Lanthorn, 19901, thus perhaps accounting for its unusual spectrum of activity. Indeed, it is surprising that the drug is anti- convulsant at all.

Recently, certain 5-nitro quinoxaline-2,3-diones were demon- strated to have highly potent (KI, < 10 Rn?l) glycine site blocking activity and good central availability when administered systemi- cally. These compounds also block AMPA receptors, but do so with several hundred-fold lower potency (Woodward et al., 1993). Two such compounds, 5-nitro-6-rnethyl-7-chloro-2,3-quinoxali~e W C Q X ) and 5-nitm-6,7-dimethyl-2,3-quinoxalinedione (MMDX), have been reported to have activity in the maximal electroshock test at reIatively low systemic doses (Tran et al., 1994). However, the extent to which the weak AMPA receptor blocking activity contrib- utes to the compounds' anticonvulsant effects i s unclear. The related quinoxalinedione ACEA- 102 1 (5-nitro-6,7-dichIoro- 1,4-dihydro-

Epilepsy 221

2,3-quinoxalinedione) has been reported not to substitute for phencyclidine in drug discrimination testing, indicating that this class of compounds may not produce dissociative anesthetic-like subjec- tive effects (Balster et al., 1993). It is possible that selective actions of these compounds at different NMDA receptor subtypes could, in part, account for the favorable behavioral profile. However, to date, glycine site antagonists have not been reported to have substantial subunit selectivity.

A series ofrelated structures-the 3-nitro-3,4-dihydro-2-quino- lones-have been described with varying degrees of glycine site or AMPA receptor selectivity. These compounds are based on 7-chloro- kynurenic acid, a potent glycine site antagonist in vitro, but eliminate the carboxylic acid group that seems to prevent blood-brain barrier penetration. One of these, L-698,544, exhibited good systemic activ- ity as an anticonvulsant (EDSO, 13.2 mg/kg? ip) in the DBA/2 mouse (Carling et al., 1993). Although L-698,544 interacts with the NMDA (Kh, 6.7 pkf) and AMPA (Kk,, 9.2 pM) recognition sites, these inter- actions occur with somewhat lower affinity than for the glycine site (ICso, 0.4 1 w. A prodrug ester of the potent glycine site antagonist 5,T-dichlorokynurenic acid has also been reported to have systemic nnticonvulsant activity in the DBAI2 mouse (EDSO, 62 mglkg, ip; Moore et al., 1993). Similarly, another quinolinone glycine site antag- onist MDL 104,653 (3-phenyl4-hydroxy-T-chlor~-quino~in-2[1H]- one; McQuaid et al., 1992) has been observed to have anticonvulsant activity, when administered either intraperitoneally or orally, in DBAJ2 mice (Chapman et al., 1993). Interestingly, high doses of the compound produced ataxia and sedation, and the therapeutic ratio was no better than for other NMDA antagonists.

Study of a further series of noncarboxylic acid quinoIinones resulted in the identification of L-70 1,273 (3-cyclopropyl-ketone-4- hydroxyquinolin-2[ 1H]-one), with high potency against audiogenic seizures in DBN2 mice (4.1 rngkg, ip; Rowley et al., 1993). This com- pound has moderately high activity at the glycine site (IC50, 0.42 m, but also interacts with the NMDA recognition site (Kbr 3.4 pi%!), although it is inactive at; AMPA receptors. More recently, a series of 3'-substituted-3-phenyl analogs of the 4-hydroxyquinolinones were

222 Roga wski

discovered with nanomolar binding affinities for the glycine site (Kulagowski et al., 1994). As i s the case for the parent 3-ketones, the 3-phenyl substituted analogs show activity at the NMDA recog- nition site but do not block AMPA receptors. These compounds appear to be the most potent systemically active glycine site antago- nists described to date, with El350 values <1 mg/kg in the DBN2 mouse model when administered either in~aperitoneally or orally. It would appear that these compounds have favorable toxicity charac- teristics, producing motor impairment only at doses that are substan- tially higher than those that protect against seizures, but this wiH require further confirmation. Thus, a variety of potent, and more or less selective, glycine site antagonists are now available, some of which have high systemic potency and excellent oral bioavailability. Tt remains to be determined if these compounds will be superior to other types of M D A antagonists for seizure therapy. Of particular interest is the possibility that the additional AMPA receptor blocking activity of some of the glycine site antagonists may actually provide enhanced anticonvulsant activity. As discussed in Section 3 . 2 , AMPA receptor blockade can confer a powerful anticondsant effect.

3.1 -4. Polyamine Site Antagonists The demonstration by Ransom and Stec (1988) that the poly-

amines spermhe and spermidine enhanced binding of ~HJdizocilpine to NMDA receptors in brain homogenates suggested that polyarnines could facilitate gating of the NMDA receptor. Although polyamines have multiple effects on NMDA receptors (Rock and Macdonald, 1992; Benveniste and Mayer, 19931, a major action is to increase channel opening frequency via an interaction with a distinct recog- nition site en the NMDA receptor complex (Williams et al., 1991). The polyarnine facilitatory effect appears to be mediated by an increase in the affinity for glycine and also by a distinct glycine- independent mechanism (Williams et al., 1994; Zhang et al., 1994b3. There has been interest in developing polyamine site antagonists, but success to date has been limited.

Two related compounds proposed as polyamine site antagonists are the neuroprotective agents ifenprodil and eliprodil (SL 82.07 15)

Epilepsy 223

(Scatton et al., 1994). Ifenprodil and eliprodil are now we11 recog- nized to be potent noncompetitive NMDA antagonists both in vitro and in vlvo. Electrophysiological studies in cultured hippocampal neurons have demonstrated that ifenprodil produces a complex effect on gating of the NMDA receptor channel (Legendre and Westbrook, 1991). This action occurs in a manner that is distinct from that of other NMDA antagonists, but is otherwise poorIy characterized. Although there is considerable evidence that ifenprodil and eliprodil can functionally interact with polyarnine effects on the NMDA receptor, it is unlikely that this occurs at a common recognition site (Ogita et al., 1992). It has instead been proposed that the polyamine and ifenprodiEJeliprodi1 binding sitesmay share overlapping domains an the NMDA receptor complex or that the apparent antagonism occurs because of a functional interaction at distinct sites.

Recently, it has been observed that the distribution of PWIifen- prodil binding sites in brain closely matches the expression of NMDAR-2B subunit mRNA (Dana et al., 1991). Moreover, ifen- prodil appears to selectively antagonize NMDA receptors composed of NRlA and NR2B subunits, although it can also block NRl A/ NR2A receptors at substantially higher concentrations (Williams, 1993). Therefore, at ordinary doses, ifenprodil may interact with only a subpopulation of NMDA receptors. Interestingly, it now appears that polyamine facilitation is variably expressed depending on the subunit composition of the NMDA receptor. The 2B subunit is a key subunit in conferring the receptor with polyarnine sensitivity (Zhang et al., 1994b).

Despite their relatively potent NMDA receptor blocking activ- ity, ifenprodil and eliprodil appear to have more favorable toxicity profiles than other NMDA antagonists (Scatton et al., 1994). At neuroprotective doses, they do not produce neurological impairment and do not substitute for phencyclidine in drug discrimination studies or induce amnestic effects in passive avoidance testing. Moreover, clinical trials have indicated that the drugs do not produce psycho- stimulant, amnestic, or psychotomimetic effects in humans. In addi- tion, eliprodil does not appear to produce pathomorphological changes, that, as has been noted in Section 3.1.2., are characteristic

of other NMDA antagonists (Duval et al., 1992). Tt has been sug- gested that the unique subunit selectivity of ifenprodil and eliprodil may account for their favorable toxicity characteristics.

As is the case for other M D A antagonists, ifenprodil and eliprodil have a broad spectrum of anticonvulsant activity in animal seizure models (De Sarro and De Sarro, 1993; Scatton et al., 1994). These effects, however, occur at doses that are higher than the neuroprotective doses and are nearer to the doses that cause toxic effects. For example, in the case of ifenprodil, there is little separa- tion in the doses that protect against seizures (mouse maximal elec- troshock test ED50,29 mgkg, ip) and induce motor impairment. For eliprodil the therapeutic index is somewhat better (electmshockED~~, 15 mgikg, ip; rotarod ED501 59 rng/kg). (Interestingly, however, eliprodil has been shown to potentiate the anticonvulsant activity of the competitive NMDA recognition site antagonist CGP 37849; Deren-Wesolek and Maj, 1993.) Thus, it may be that the NMDA receptor subtype selectivity of ifenprodil/eliprodil is not optimally suited for seizure protection, at least in the animal models used for screening. Whether this is a characteristic of all functionalpolyamine antagonists, remains to be determined.

A variety of other compounds have been proposed as polyamine antagonists, including several polyarnines (Williams et al., 1990) and certain peptide components of marine snail toxins (Skolnick et al., 1994). Nevertheless, few of these proposed antagonists are suitable for evaluating the idea that polyamine antagonism is a viable anti- convulsant strategy. Indeed, several putative polymine site antago- nists have complex effects on the NMDA receptor, including channel-blocking activity, which eonfound any interpretation of their specific polynmine site effects (Donevan et al., 1992; Submmaniam et al., 1992). However, one interesting candidate antagonist is N-(3- aminopropy1)cyclohexylamine (APCHA), a cyclohexyl diamine. It has been demonstrated to block the facilitation by spermidine of NMDA-induced seizures in mice (Chu et al., 1994). Unfortunately, APCHA may not be active with systemic administration and it has yet to be demonstrated that this compound acts via a specific inter- action with the polyamine site.

Epilepsy 225

There is some evidence that brain polyamine concentrations may increase with kindling, either produced electrically (Hayashi et al., 1989) or by repeated treatment with pentylenetetrazol (Hayashi et al., 1 993). However, it remains undetermined whether these increased levels contribute to the hyperexcitability of the kindled state. Should this be the case, the argument supporting the potential use of polyamine site antagonists in seizure therapy would be strengthened.

3.1.5. An fagovtists rat Of her Sites on the NMDA Receptor Complex and the Combined Acf ions of Felbamate on NMDA/GABA, Recep f ors

In recent years, a class ofNMDA antagonist has been identified in which inhibition is exerted at sites that are distinct from those of known NMDA receptor coagonists or facilitatory modulators. Indeed, ifenprodiI and eIiprodil are probably best considered among this class of NMDA antagonist. Other examples include a series of n-alkyl diamines that inhibit NMDA receptors by acting at a nonvoltage dependent, hydrophobic binding site as well as in n chan- nel-blocking fashion (Subramaniam et al., 1994). Similarly, the anti- convulsant rernacemide may also block NMDA receptors in part by such an allosteric action (Submmaniam et aI., 1995a). Felbamate too is an example of an anticonvulsant that may antagonize NMDA receptors by an allosteric effect on channeI gating, although a fast channel-blocking action of the drug may be more important. Although glycine and the glycine site agonist d-serine are able to functionally reverse the anticonwlsant effects of felbamate in vivo (Hamsworth et al., 1993; Coffin et al., E 994), there is strong evi- dence that felbamate does not act directly at the glycine site (Rho et aI., 1994d; Subramaniam et al., 1995b).

As discussed in Section 2. I .2., felbamate also produces a mod- est potentiation of GABAA receptor responses within the same range of concentrations that it blocks NMDA responses (Rho et al., 1994b3. Both of these effects occur at concentrations of felbamate likely to be present in the brain during antiepileptic therapy. Felbamate exhibits a broader spectrum of anticonvulsant activity than conventiona1 antiepileptic agents such as phenytoin and carbamazepine. (It con-

226 Rogawski

fers protection in the maximal electroshock test and also against seizures induced by pentylenetetrazol; Swinyard et al., 1986.) Clini- cally, the drug has activity in the treatment of partial and secondarily generalized tonic4onic seizures and it is also effective against refractory seizures of the Lennox-Gastaut syndrome in children, which are notoriously difficult to treat. The combination of actions of felbamate on excitatory and inhibitory synaptic transmission could account for its broad spectrum of activity and favorable side-effect profile. More specificaIly, it can be postulated that the distinct actions on NMDA and GABAA receptors are by themselves below the threshold for side effects, but that the actions at the two sites are additive or even synergistic against seizures. That a drug such as felbamate with low neurological toxicity may exert its therapeutic activity v i a low-affinity interactions with more than one receptor system contradicts the conventional view that side-effect reduction is best achieved by high selectivity and potency. However, for anticonvulsant drugs that modulate excitability mechanisms criti- cal to normal brain function, agents with high potency and efficacy may have unacceptable toxicity. (See Section 2.1.1.1. for a more detailed discussion with reference to partial benzodiazepine receptor agonists.) Therefore, the search for drugs that exert low efficacy effects on more than one receptor seems to be a worthwhile strategy.

3.2. RMPA Receptor Antagonists AMPA (non-NMDA) receptors play a key roIe in CNS integra-

tion as the prime mediators of fast synaptic excitation. The critical nature of AMPA receptors is hard to overstate inasmuch as they participate in virtually every central circuit and, of particular impor- tance here, mediate epileptiform activity in a variety of model sys- tems (Rogawski, 1994). Consequently, the observation that AMPA receptor antagonists have anticonvulsant activity is not unexpected. However, at present, it is unclear if AMPA receptor antagonists will be sufficiently free of toxicity to be clinically useful anti- convulsants. Nevertheless, the possibility of selectively targeting the various AMPA receptor subtypes provides enormous opportunities for drug development.

Epilepsy 227

3.2.1. Cornpsti five AMPA Receptor Anf agonisfs

During the early period of excitatory amino acid receptor research, AMPA receptor antagonists were unavailable. However, in the late 1980s the situation changed: Hanore et al. ( 1988) deveIaped a series of quinoxaiinediones as selective and relatively potent competitive AMPA recognition site antagonists (see Davies and CoIlingridge, 1990). Although the first quinoxalinediones to be described were not systemically active, compounds with good systemic bioavailability are now available. These include the benzo- h s e d quinoxnlinedione NBQX C2,3 -dihydroxy-6-nitro-7-sul famoyl- benzo[F]quinoxaline; Sheardown et al., 1990) and the 6-irnidazol substituted quinoxaIinedione YM90K (6-[lH-imidazal-1-yll-7- nitro-2,3-[1H-4~-quinoxalinedione; Ohmori et al., 1 994) that may have improved CNS penetration in comparison with NBQX. A series of isatin oxamines has also been described which are highly selective AMPA receptor antagonists (Watjen et al., 1 993). These compounds have anticonvulsant activity against AMPA-induced seizures following intravenous and oral administration. In addition, a substi- tuted decahydroisoquinoline-3-carboxylic acid has been recently described with good systemic AMPA receptor blocking activity (Omstein et al,, 1993). This compound, LY 2 15490 ([3SR,4aRS, 4RS,8aRS]-6-[2-(1H-tetrazol-5-yl)ethyl]decahydroisoquinoline-3- carboxylic acid), also interacts with the NMDA recognition site, but with 5- to 10-fold lower potency.

Competitive AMPA recognition site antagonists are protective in the maximal electroshock test and against seizures induced by various chemoconvulsants (Yarnaguchi et al., 1993; Omstein et al., 1993). In addition, these compounds have activity against reflex seizures in rodents and primates (Chapman et al., 199 1 ; Smith et al., 1991 ; Meldrum et al,, 1992; Ohmori et al., 1994). In the kindling model, NBQX has been reported to have protective activity, although it is less potent than in other seizure models (Lbscher et al., 1993). This is in sharp contrast to NMDA antagonists which are weak or ineffective against established kindled seizures. Despite its activity against kindled seizures, NBQX has no effect on the development of

kindling (DiirmiiIler et aI., 1994). This is another important differ- ence from NMDA antagonists, which, as has already been noted (Section 3.1 .), do protect against kindling devejoprnent.

It has ggnerally been found that AMPA receptor antagonists have neurological, toxicity at anticonvulsant doses, although this varies depending on the seizure mode1 and test conditions <we Chapman et al., 199 I). Thus, NBQX produced motor impairment at doses that are similar to those that are protective in the maximal electroshock test (Yamaguchi et al., 1993). The LY215490 has only a modestly better therapeutic ratio (=2.2) (Omstein et al., 1993). Nevertheless, it would be premature to conclude that AMPA receptor antagonists are unlikely to have clinical utility in seizure therapy, since block- ade of AMPA receptors is an entirely new therapeutic strategy. Only through clinical trials will it be possible to assess the significance of the therapeutic ratio values determined from animal testing.

Lascher et al. (1 993) observed that low doses of NMDA receptor antagonists synergistically enhance the anticonvulsant activity of NBQX in the kindling model, without producing anincrease in adverse effects. Although the basis of this phenomenon is poorly understood, it seems reasonable that a combination of NMDA and AMPA receptor blockade may ultimately prove to be the optima1 approach for epilepsy therapy. In fact, Iow levels of NMDA receptor blockade may provide an antiepileptogenic effect and may also protect against seizure- induced brain damage, in a manner not obtained with AMPA receptor antagonists (Berg et al., 1993). As discussed in Section 3.1.3., many of the recently described gIycine site antagonists also have various degrees o f AMPA receptor blocking activity. It will be of interest to determine if the combined activities provide cEinically superior therapeutic activity with acceptable side-effect properties.

Another potential approach to obtaining improved anticon- vulsant activity with a reduction in side effects would be to target AMPA receptor subtypes specifically involved in seizure genera- tion or propagation. Such putative seizure-related AMPA receptor subunits have yet to be identified. However, there is evidence that non-NMDA receptors can be selectively targeted with antagonists. Thus, the isatin oxime NS- 1 02 (5-nitro-6,7,8,9-tetmhydrobenzo-

Epilepsy 229

k]indale-2,3-dione-3-oxime) is a non-NMDA antagonist with selec- tivity for the kainate binding subunit GluR6 (Johansen et aI., 1993; Verdoorn et al., 1994). However, the anticonvulsant profile of this compound remains to be determined.

3.2.2. Noncornpefitive AMPA Receptor Antagonists A novel class of selective AMPA receptor antagonists has

recently been identified that exert their blocking action in a mecha- nistically distinct fashion from competitive AMPA recognition site antagonists. These antagonists-typified by the 2,3-benzodiazepine (homophthalazine) GYM 52466 (1-14-aminophenyll-4-methyl-7,8- methylenedioxy-5H-2,3-benzsdiazepine)-offer an alternative approach for blockade of AMPA receptors with potentially distinc- tive consequences (Tarnawa et al., 1990; Ouardouz and Durand, 199 1 ; Jones et al., 1992). GYKl52466, unlike the quinoxalinedione AMPA antagonists, does not interact with the AMPA recognition site, nor is it a channel blocker. Rather, it appears to block AMPA receptors in a noncompetitive fashion via anovel allosteric site on the AMPAreceptor complex (Doncvan and Rogawski, 1993). The block- ing action of GYKl52466 is highly selective in vitro, with essentially no effect on NMDA, metabotropic glutamate, or GABAA receptor responses, The lack sf effect on GABAA receptors is particularly notable in view of the structural similarity of GYKI 52466 with conventional 1,4-benzodiazepines (see Section 2.1. I .).

Like competitive AMPA recognition site antagonists, GYKl 52466 has anticonvulsant activity in a broad spectrum of animal seizure models (Chapman et al., 199 1; Smith et al., 199 1 ; Yamaguchi et al., 1993; Steppuhn and Turski, 1993). In some experimental models, GYKI 52466 has improved anticonvulsant activity corn- pared with NBQX. In others, NBQX appears to have greater selectivity and potency. One situation where the noncompetitive antagonists would theoretically be preferable to competitive antago- nists is in protection against seizures associated with high synaptic glutamate levels (see Rogawski, 1993). Under these conditions, the synaptically released glutaman would surmount the blocking action of a competitive antagonist so that high doses of the antagonist would

be required to confer seizure protection. However, these high doses would be more likely to induce side effects. In contrast, minimal doses of drugs Iike GYKX 52466 may be effective, since equivalent degrees of block are achieved whatever the glutamate level.

A series of N-substituted GYKE 52466 analogs has been described with varying degrees of AMPA receptor blocking activity (Tarnawa et al., 1993). Two of these analogs with greater in vitro AMPA receptor blocking potencies were found to have correspond- ingly greater invivo anticonvulsantpotencies (Donevanet al., 1994). This supports the view that the anticonvulsant activity of the 2,3- benzodiazepines is due to AMPA receptor blockade.

Recently, it has been demonstrated that AMPA receptors con- taining the GluR2 subunit have a linear or outwardly rectifying current-voltage relationship and a low permeability for Ca2+. Com- binations lacking GluR2 are inwardly rectifying and have a high Ca2+ permeability (Hume et al., 1991; Hollrnann et al., 1991; Verdoorn et al., 1 99 I). GYKI 52466 appears to block Ca2'-permeable and Ca2+- impermeable AMPA receptors with equal potency (Donevan and Rogawski ,unpublished). This is in contrast to the barbiturate pento- barbital that selectively blocks Ca2+-impermeable AMPA receptors (Taverna et al., 1994) and certain arthropod toxins that are selective for Ca2+-permeable AMPA receptors (see Section 3.2.3 .). Interest- ingly, GYKI 52466 and its analogs appear to have much higher block- ing potency for AMPA-sel ective non-NMD A receptor subunits than for kainate p r e f m g subunits (Patemain et a]., 1995). The implications of these various selectivity differences are not yet well understood.

3.2.3. Channel-Blocking AMPA Recepfor Antagonists At present, no selective channel-blocking AMPA receptor

antagonists have been identified. However, certain polyamine arthropod toxins could provide clues to the development of such compounds. The toxins (such as argiotoxin 636, philanthotoxin, and Joro spider toxin) act as potent noncompetitive antagonists of verte- brate AMPA receptors (Brackley et al., 1993; Blaschke et al., 1993). These toxins act in a use- and voltage-dependent manner suggesting that they are channel blockers (Herlitze et al., 1993). However, the

Epilepsy 231

toxins are not selective for AMPA receptors, and indeed their block ofNMDA receptors is more reliable and often more potent (Priestley et al., 1989; Ragsdale et al., 1989). A likely reason for the inconsis- tency ofthe AMPA blocking action is suggested by the recent discov- ery that the polyamine toxins selectively block inwardly rectifying AMPAreceptors (that is, those that lack the Glum subunit) (Blaschke et al., 1993; Herlitze et al., 1993).

From the point ofview of their use as pharmacological tools, the fact that the toxins block both NMDA and AMPA receptors is a disadvantage. However, this may not be an obstacle to the use of such compounds as therapeutic agents. As has. been noted previously, combined NMDAIAMPA receptor blockade may produce a syner- gistic anticonvulsant effect. Moreover, the seIectivity of the toxins for inwardly rectifying AMPA receptors may be a distinct advantage. Since rectifying AMPA receptors are Ca2+ permeable, it is not hard to imagine that they could play a role in seizure generation or in the neurotoxicity associated with prolonged seizures. In fact, there is sug- gestive, but by no means conclusive, evidence in support of both pos- sibilities. Thus, it has recently been reported that amygdaloid kindling is associated with a 25-30% reduction in Glum levels in the limbic forebrain and pirifom coflexlamygdala (but not in the entorhinal cor- tex or amygdala; Prince et al., 1995). The decrease-which was spe- cific for the GluW subunit-was observed in kindled animals at 24 h after the last seizure; GluR2 levels had returned to normal at 1 wk and 1 mo. Similar changes were not obtained after induction of seizures with the convulsant pentylenetetrazol. It is conceivable that the decrease in Glum could be a factor in the induction, but not the main- tenance, of kindled seizures. If this is the case, agents such as the polyarnine toxins which selectively block AMPAreceptors lacking the Glum subunit could have interesting antiepi leptogenic potential.

An additional Iine of experimentation is consistent with the possibility that GluR2 subunit downreguIation could play a role in seizure-induced neuropathology. Thus, Friedman et al. ( 1 9943 recently reported that persistent seizure activity (status epilepticus) can result in reduced expression of the GluR2 subunit in hippocampal regions most vulnerable to neurodegeneratian. This reduction occurs with a

time course that parallels the neuronal cell loss, supporting a role for Ca2+ permeable AMPA receptors in the seizure-induced pathology. Polyamine toxin- li ke agents with specificity for Ca2+-permeable AMPA receptors might, therefore, be well suited as neuroprotectants to ameliorate seizure-induced pathological changes.

Although only limited information is available, polyarnine tox- ins and a series of smaI1 mol wt analogs referred to as "araxins" do have anticonvulsant and neuroprotective activity when administered intracerebroventricularl y or systemically (Seymour and Mena, 1 98 9; Mueller, personal communication). At therapeutically effective doses, the compounds do not appear to produce dissociative anesthetic-like behavioral or histopathological effects, and do not generalize to phencyclidine in drug discrimination studies. However, with systemic administmtion,rhere is poor separation between the doses that confer seizure protection and induce neurological impairment. In addition, the toxins may produce significant cardiovascular side effects.

3.3. Metaboiropic Glutamate Receptors Metabotropic glutamate receptors are a diverse group of G-

protein-coupled receptors that are structurally and functionally dis- tinct from other glutamate receptors (Suzdak et al., 1994). In contrast to NMDA and AMPA receptors which are themselves ion channels, rnetabotropic glutamate receptors act via various second messengers to indirectly regulate a wide variety of ion channels. The potentia1 role of metabotropic glutamate receptors as targets foranticonvulsant drugs is not well defined. Nevertheless, activation of rnetabotropic glutamate receptors with selective agonists can produce, in adult rodents, limbic-like seizures similar to those observed in kindled animals (Sacaan and Schoepp, 1992; Tizzano et al., P 994). In neona- tal rats, metabotropic agonists can produce frank convulsions (McDonald et al., 1993). Interestingly, however, low doses ofcertain metabotropic glutamate receptor agonists can have weak anti- convulsant effects (see Thornsen et al., 1994). The anticonvulsant effects presumably arise because the rnetabotropic glutamate recep- tor agonists reduce glutamate release by a presynaptic action on inhibitory autoreceptors (Glaum and Miller, 1994).

Recently, a novel series of conformationally restricted phenyl- gIycine analogs have been described which act as competitive antag- onists of at least some metabotropic glutamate receptors (Birse et ai., 1993; Thornsen and Suzdak, 1993; Ferraguti et al., 1994). One of these compounds, IS)-4-carboxy-3 -hydroxyphenylglycine ([SJ- 4C3HPG), has been reported to protect against audiogenic seizures in DBA/2 mice at doses that do not produce neurological impaiment (Thornsen et al., 1994). This effect occurred with intraventricdar, but not systemic, administration of the drug. In addition, (S)-4C3HPG diminished the epileptiform activity observed in rat cortical slices exposed to Mg2+-free conditions. L-AP3 (L-2-amino-3-phosphono- propionate), a low potency noncompetitive metabotropic receptor antagonist (Schoepp, 19941, has also produced a weak anticonvulsant effect folIowing intraventricular administration (Klitgaard and Jack- son, 1993). Bath IS)-4C3HPG and L-AP3 can have rnetabotropic glutamate receptor agonist properties under some circumstances (Birse et al., 1993). It is therefore conceivable that the anticonwlsant activity of these compounds relates, at least in part, to this agonist activity, possibly by presynaptic effects an glutamate release. Indeed, it is not clear at present whether metabotropic receptor agonism, antagonism, or a combination of agonist and antagonist effects at dis- tinct metabotropic receptor types represents the most effective anticonvulsant approach. NevertheIess, targeting of the various metabotropic receptor types with selective agonists and antagonists provides interesting opportunities for anticonvulsant drug development.

3.4. Glutanrate Release Inhibitors

Metabotropic glutamate receptors axe just one example of a host of receptors and ion channels of the glutamatergic nerve terminal that are attractive anticonvulsant targets. It has aiready been noted that barbiturates might in part exert their anticonvulsant activity via inhibition sf glutamate release (through effects on voltage-depen- dent Ca2+ channels). In addition, as discussed in the next section, the therapeutic effects of anticonvulsant drugs that interact with voltage- dependent Na' channels is likely due to some extent to inhibition of glutamate release. It appears that certain toxins including o-Agn

IVA and cs-Aga MVIIC, which block Ca2+ channels in presynaptic nerve terminals can have anticonvulsant effects when administered inbacerebroventricularly (Jackson et al., 1 994). Moreover, a series s f novel N,N'-diarylguanidines have been reported to be potent, sys- temically active glutamate release blockers although their mecha- nism of action i s not well understood (Reddy et aI., 1994). There are thus a number of interesting leads to glutamate release antagonists of potential clinical utility. The challenge will be to develop agents that have sufficiently low toxicity to be useful in seizure therapy.

4. Interactions with Voltage-Dependent Nat Channels

A wide variety of anticonvulsant drugs are believed to act, at least in part, via their effects on voltage-dependent Na* channels. Phenytoin is perhaps the best studied example. Phenytoin is a reia- tively weak Na* channel blocker and its ability to inhibit Na+ channel activity is highly dependent on the state of the channel. Therapeutic concentrations of the drug have little activity on resting Na'channels at hyperpolarized membrane potentials. Thus, at a potential of -80 mV, 10 p M phenytoin, a concentration at the high end of the range of free therapeutic plasma levels, would produce no more than 1 5% block of Na' current (Lang et a!,, 1993). However, there is more significant block when Na' channels are repetitively activated or when the initial membrane potential is at a more depolarized level. These properties are believed to reflect the higher affinity of phenyrain for activated and inactivated channels than for resting channels (Ragsdale et al., 199 1). During high frequency firing, there is aprogressive accumulation of drug binding as Na* channels cycle through the activated and inactivated states with each action poten- tial. In addition, a greater proportion of Na+ channels is in the inacti- vated state at depolarized membrane potentials, thus accounting for the voltage dependence of block. Thc rate at which phenytoin- bound Na+ channels recover from inactivation is markedly slowed, particularly at depolarized potentials (Schwartz and Grigat, 1 989; Kuo and Bean, 1994). This property accounts for the stabilization of Na+ channels in the inactivated state.

Epilepsy 235

The precise way in which a modification of Na+ channel gating results in seizure protection is not we11 understood. However, the following hypothesis has often been proposed. During seizures, neu- rons fire more rapidIy and are more depolarized than under normal conditions, so that their Na* channels would be more susceptible to block. This would allow phenytoin to selectively suppress high fre- quency firing. Indeed, at low concentrations, phenytoin is well known to produce a selective inhibition of repetitive action potential firing, whereas single action potentials are resistant (McLean and Macdonald, 1983). The effects of phenytoin an Na* channels may ultimately translate into reduced glutamate-mediated synaptic exci- tation, and this cousd play an important role in its anticonvulsant activity. In fact, phenytoin inhibits the release of various neurotrans- mitters, including glutamate from brain slices stimulated by the Wa* channeI activator veratrine (Leach et al., 1986).

A wide variety of anticonvulsant drugs have a spectrum of anticonwlsant activity in animal seizure models simiIar to that of phenytoin (Rogawski and Porter, 1990). Many of these may also act via an interaction with Na+ channels, and in several cases this has been confirmed in vitro. For example, lamotrigine, an anticanvuEsant effective in the treatment of refractory partial seizures (Yuen, 1994), has a similar profile to phenytoin; it is protective in the maximal electroshock test but not against pentylenetetrazol-induced clonic seizures (Miller et al., 1986). At clinically relevant concentrations, lamatrigine has been shown to block repetitive firing of CNS neu- rons. This effect can be reversed by hyperpolarization of the resting membrane potential, in a manner similar to that of phenytoin (Cheung et aI., 1 992). In addition, the drug suppresses burst firing m cortical neurons (Lees and Leach, 1993), and inhibits binding of [3M]batracho- toxinin A 20-a-benzoate, a Iigand for Na* channels, to rat brain synap- tosomes (Cheung et al,, 1992). In voltage clamp studies, Iamotsigine produced a use-dependent inhibition of Na+ channels and slowed the rate of recovery from inactivation, effects similar to those produced by phenytoin (Lang et al., 1993; Taylor, 1 993). Thus, the similarity between larnotrigine and phenytoin in animal seizure modeIs is par- alleled by corresponding actions on Na* channels.

236 Rogu wski

Riluzole is another anticonvulsant drug with a phenytoin-like spectrum of activity (Mizoule et al., 1985). (Riluzole also has neuro- protective properties [see, for example, Wahl et al., 19933 as does phenytoin [Stanton and Moskal, 1991; Hayakawa et al., 19941.) Among its various pharmacological actions, riluzole is a blocker of voltage-dependent Na+ channels. Interestingly, however, unlike phenytoin, the block produced by riluzole does not occur in a use- dependent fashion, suggesting that it does not preferentially bind to activated Na+ channels. Rather, riluzole has an exceptionally high (>150-300-foId) selectivity for the inactivated state of the Nat channel (Benoit and Escande, 1991). These results with riluzole indicate that stabilization of the inactivated state of the Na+ channel may be a particularly critical factor in the anticonvulsant activity of phenytoin.

Several other anticonvulsant drugs with a phenytoin-Iike spec- trum of anticonvulsant activity interact with Na+ channels in a fash- ion similar to phenytoin. These include carbamaqine (Ragsdale et al., 1991; Lang et al., 1993), ralitoline (Rock et al., 1991; Fischer et al., 1992), and flunarizine (Kfskin et al., 1993). In addition, the antican- vulsant U 54494A (3,4-dichloro-N-methyl-N-[2-(1 pyrraIidiny1)- cyclohexylJbenzarnide) (Zhu and Im, 1992), its two major active metabolites (Zhu et al., 1993); and several other structurally related benzarnides with anticonvulsant properties (Zhu et al., 1992) have been s h o w to interact with Na' channels in a voltage- and use- dependent fashion, similarly to phenytoin. The success in identifying phenytoin-Iike anticonvulsants is probably a result of the widespread use of the maximal electroshock test as a screening method (inas- much as the test is highly sensitive to such agents). Since there are already a large number of safe and effective anticonvu1sant drugs of this type, the development of additional phenytoin-like compounds may not seem be a high priority. However, it would be reasonable to search for drugs that produce phenytoin-like effects in combination with other pharmacological activities that result in seizure protection (for example, potentiation of GABA or inhibition of excitatory amino acid transmission). The major acute neurologica1 doxicity of phenytoin is xeferabIe to cerebellar dysfunction, possibly because

Epilepsy 237

cerebellar neurons are among the most rapidly firing in the CNS and are therefore particularly susceptible to use-dependent block. Differ- ent types of acute toxicities are produced by anticonvulsant drugs acting on other targets. For example, GABA-potentiating drugs typi- cally produce sedation, an effect not usually seen with phenytoin. Thus, by combining Na'channel blocking activity with another action it may be possible to produce enhanced anticonvulsant effects with- out a corresponding increase in toxicity,

5. K+ Channel Opener Drugs Like GABAA receptors, the fundamental role of Ki channels is

to dampen excitability. Consequently, drugs that enhance the activity of K* channels are reasonable antiepileptic drug candidates. Activa- tion of K+ channels would be expected either Ea hyperpolarize neu- rons and thus inhibit them or to limit action potential firing by increasing the opposing influence that K+ currents have on depalas- izing Na* currents.

In recent years, a diverse group of molecules have been identi- fied that are capable of opening K' channels in excitable cells. Research in this area began with the discovery that the antihyper- tensive benzopyran cromakalim relaxed vascular smooth muscle by opening K+ channels (Weston and Edwards, 1992). Subsequently, it was recognized that several other structurally dissimilar antihyper- tensive agents, including diazoxide, minoxidil, and pinacidil, were also K+ channel openers. These studies with existing compounds led to the synthesis of a large number of novel K+ channel openers (Robertson and Steinberg, 1990).

There are a great variety of K+ channels in neuronal cells and a corresponding diversity in their functional roles. Consequently, in characterizing the pharmacological effects of a K+ channel opener drug, it is of critical importance to define the type of K+ channels modulated by the drug. The range of K+ channel types sensitive to K+ channel opener drugs is still incompletely defined. It may be that the channel types affected depend on the specific drug and tissue exam- ined. For example, the benzimidazolone NS 004 (1 -[2-hydroxy-5- chlorophenyl]-5-trifluaromethyl-2-benzimidazolone) is said to

Epilepsy 239

ATP levels in substantia n i p neurons that would activate GTPchan- nels and depress GABA release. The overall effect would be to obvi- ate the critical role played by the substantia nigra in the control of seizure spread (Gale, 1 985, 1992). In hypoglycemia, reduced blood glucose would also lead to diminished intraceilular ATP levels and a similar inactivation of the substantia nigm seizure-control mecha- nism, perhaps contributing to hypoglycemic seizures.

In other brain regions such as the hippocampus, Knw channel activation could serve to inhibit seizure activity. Thus, cromakalim reduced bursting of hippocampa1 neurons in the in vitro slice prepa- ration (Alzheirner and ten Bruggencate, 1988) and in cell culture (Simon and Lin, 1993). However, in other situations the drug failed to affect hippocampal epileptiform discharges, although it was able to inhibit the response to anoxia (Mattia et al., I 994). There are a few reports that intraventricnEarly administered cromakalim can protect against seizures in certain in vivo chemoconvulsant models (Gandolfo et al., I989a,b; Del Pozo et al., 1990; Popoli et al., 1991), but these reports require confirmation with more traditional anti- convulsant screening tests. As of yet, no K* channel opener drug has been demonstrated to have CNS activity following systemic adrnin- istration. Recently, however, benzopyrans structuraIly related to cromakalim have been described with oral anticonvulsant activity in the maximal electroshock test (Evans et al., 1992). Whether this anticonvulsant activity occurs as a result o f effects on K+ channels remains to be determined.

The Kt channel opener drugs represent an interesting direction for anticonvulsant drug development. However, there are a number of impediments that must be overcome before this strategy can be considered seriously. As noted, blood-brain barrier permeable ana- logs are required. Assuming such compounds become available, nonselective K+ channel opener drugs could theoretically have proconvulsant effects mediated, for example, by the substantia nigra. In addition, there is evidence that some K+ channel openers can block certain voltage-dependent K+ channel types that might predispose to seizures (Politi et al., 1993). Atpresent, the potential utility of K+ chan- nel opener drugs in epilepsy therapy is uncertain.

Interestingly, however, there are a few reports in the literature that suggest that presently available anticonvulsant drugs may act by enhancing K* channel function. For example, it has been proposed that the tetronic acid derivative losigamone could protect against seizures by such a mechanism (Klihr and Heinemann, 1990). In addition, there is a report indicating that carbamazepine may enhance a K+ current in neocortical neurons (Zona et al., 1990), However, until further confirmatory data is obtained, these observations must be considered preliminary.

6. Adenosine Agonists and Release-Enhancing Agents

Adenosine is a powerful endogenous inhibitory neuro- modulator that is normally present in the extracellular environment at a concentration of about 1 ph4 (Zetterstrorn et al., 1982; Greene and Haas, 1991). Adenosine exerts its inhibitory action on CNS excitability by direct (postsynaptic) hyperpolarization of neurons and by inhibition of synaptic release, particularly of glutamate, via adenosine receptors on excitatory nerve terminals (Proctor and Dunwiddie, 1987). These actions are primarily mediated by aden- osine receptors of the A1 type (McCabe and Scholfield, 1985). Dur- ing seizure activity, adenosine levels increase dramatically, which has led to the proposal that the adenosine may mediate seizure arrest and postictal refractoriness (Lewin and Bleck, 1981; Dragunow, 1986; Whitcomb et al., 1990; During and Spencer, 1992). A recent study in the hippocampal dice preparation has suggested that glutamate acting on NMDA receptors can stimulate adenosine release from interneurons (Manzoni et al., 1994). How- ever, the extent to which this accounts for the adenosine released during seizures in the hippocampus or elsewhere remains uncer- tain. Evidence that endogenous adenosine release protects against seizure activity is the well-known proconvuIsant activity of adenosine AI antagonists (Albertson et al., 1983; Murray et al., 1985). Conversely, treatments that increase adenosine levels or its actions on inhibitory mechanisms may be effective in preventing

Epilepsy

seizures. For example, studies in a hippocampal slice preparation have demonsbated that adenosine can suppress epileptifom activity (Dunwiddie, 1 980; Lee et al., 1984; Ault and Wang, 19X6), whereas blockade of adenosine receptors induces sustained interictal dis- charge and seizure-like activity (Thompson et al., I 992; Alzheimer et al., 1993). There is evidence that adenosine selectively depresses excitatory but not inhibitory synaptic transmission (Yoon and Rothman, 199 1; Thompson ct a]., 1992; Katchman and Hershkowitz, 1993). The selectivity for excitatory transmission provides a mecha- nism for adenosine's superior anticonvulsant activity in comparison with other presynaptic modulators (for example, GABAB agonists) that may reduce inhibitory as well as excitatory synaptic events.

Much evidence from in vivo experimental models demonstrates that adenosine receptor stimulation can modulate seizure activity. Adenosine itself has weak anticonvulsant propeaies because it has an extremeIy short biological half-life (5-6 s) and probably pen- etrates the blood-brain barrier poorly (Rudolphi et al.. 1992). Never- theless, adenosine can protect against audiogenic seizures in mice (Maitre et al., 1974) and also prolongs the latency to pentyleneeetrazol seizures (Dunwiddie and Worth, 1982). Selective adenosine A 1 receptor agonists-such as L-phenylisopropyladenosine, cyclo- hexyladenosine, and 2-chloroadenosine-have much greater anti- convulsant potency. These compounds have been reported to have activity in the maximal electroshock test (Wiesner and Zimring, 1994). In addition, they protect against seizures induced by pentylene- telrazol (Dunwiddie and Worth, 1982; Murray et al., 1985); bicu- cullfne (Zhang et al., 1994); penicillin (Niglio eta]., 1988); DMCM, a benzodiazepine receptor inverse agonist (Petersen, 199 1); hornocysteine, a putative adenosine sequestering agent (hlarangss et aI., 1990); and 4-aminopyridine (Jackson et aI., 1994). In addition, adenosine agonists prevent the development of kindling induced by electrical stimulation of the amygdala (Dragunow and Goddard, 1984) or by the j3-carboline FG 7142 (Stephens and Weidmann, 1989). In contrast, adenosine agonists have variable activity against seizures in fully kindled animals (Barraco et al., 1984; Rosen and Berman, 1985). Conventional anticonvulsant drugs generally do not

interfere with kindling development, although NMDA antagonists have a paweAl antiepileptogenic effect (see Section 3.1 .). Thus, the ability of adenosine agonists to prevent kindling development could be related to their inhibition of excitatory amino acid release and a consequent reduction in the activation of NMDA receptors.

Despite the effectiveness of adenosine A1 agonists in animal seizure models, it is unlikely that these compounds would be clini- cally useful anticonvulsants. Adenosine Al agonists have powerfuI cardiovascular effects as a result of their actions on peripheral adenosine receptors in the heart and vascufature. Even iftheseperiph- era1 side effects could be overcome (for example by coadministratian of a blood-brain barrier impermeable adenosine antagonist such as 8-p-sulfophenyltheophylline), adenosine agonists at anticonvulsant doses may produce strong sedative effects that would limit their clinical utility (Dunwiddie and Worth, 1982). Consequently, alterna- tive strategies wilt need to be developed. One approach is to utilize inhibitors of adenosine inactivation. These may specifically target epileptic brain regions since seizure activity is accompanied by local release of adenosine. Such agents would theoretically potentiate the protective effects of adenosine where needed, without producing untoward side effects (since release of adenosine may be less signifi- cant under nonepileptic conditions). There are multiple nucleoside transport (uptake) systems in brain that inactivate released aden- osine. Electrophysiological studies in the brain slice preparations have indicated that adenosine uptake inhibitors such as dipyridamole, dilazep, nitrobenzylthioguanosine, nitrobenzylthioinosine, hexa- bendine, and soluflazine can potentiate the inhibitory effects of exogenous adenosine (Sanderson and Scholfield, 1986; Ashton et at ., 1987) and reduce epileptiform activity (Ashton et al., 1988). How- ever, adenosine uptake inhibitors generally have little effect on nor- mal synaptic transmission. Moreover, microinjection of several adenosine uptake inhibitors into the rat prepiriform cortex reduced bicuculline-induced seizures (Zhang et al., 1993). n u s , adenosine uptake inhibition is a potentially promising anticonwlsant strategy, However, the available adenosine uptake inhibitors are generally of low potency or do not penetrate the blood-brain barrier. Another

Epilepsy 243

approach to selectively enhance endogenous adenosine levels is to inhibit adenosine metabolism with, for example, inhibitors of aden- osine kinase (such as 5'-amino-5'-deoxyadenosine) or adenosine deaminase (such as 2'-deoxycoformycin). Although such enzyme inhibitors have anticonvulsant activity when injected locally (Zhang et a!., 19931, there is no evidence that they are effective with systemic administration. Finally, the purine precursor 5-amino-4-irnidazole carboxarnide riboside (AICAr) (which increases adenosine levels during ATP catabolism, as may occur in a seizure focus), has weak anticonvulsant activity that can be enhanced by the adenosine uptake blocker mioflazine (Marangos et al., 1990). AICAr has low bloo& brain barrier permeability; it will be necessary to develop analogs with improved bioavailability before evaluating the potential of this approach. In sum, adenosine release enhancement-whether by adenosine uptake blockade, inhibition of adenosine metabolism, or promotion of adenosine release (as may occur with the xanthine oxidase inhibitor oxyputinal; Rudolphi et al., 1 992)-would appear to be a worthwhile investigational approach for anticonvulsant drug development.

7. The Special Problem of Absence Epilepsy Absence epilepsy, commonly referred to aspetit mal, is a child-

hood neurologicaI disorder characterized by episodes of sudden loss of awareness lasting 3-10 s (Penry et aI., 1975). A child exhibiting an absence attack becomes immobile, stares, and may display an automatism such as eyelid fluttering or a slight movement of the face. There is no loss of body tone (falling), as in generalized tonic-clonic seizures. Absence seizures have a characteristic electroencephalo- graphic signature consisting of bilateral, symmetrical, synchronous, 3 Hz spike-wave bursts. The disorder is rare (constituting perhaps 8% of patients with childhood epilepsy) and usually self-limiting (absence seizures typically remit by adolescence). Absence seizures are fundamentally different from other types of human epilepsies in several respects. For the purposes of this discussion, the most impor- tant difference is the unique pharmacologica1 responsiveness of absence seizures. Ethosuxirnide, a drug with little activity in other

seizure types, is highIy effective in the treatment of absence seizures. Conversely, absence seizures are worsened by antiepif eptic drugs, such as phenytoin and carbamazepine, that are effective in general- ized convulsive and partial epilepsies, This pharmacological evidence provides strong support for the concept that the pathophysiological mechanisms underlying absence seizures are distinct from those of generalized convulsive or partial epilepsies. Indeed, absence sei- zures are beIieved to represent a disorder of corticothalarnic rhyth- micity, as originalIy proposed in the corticoreticular hypothesis of Gloor (1 968). Therefore, the brain systems meditating absence sei- zures may be entirely distinct from those that participate in other seizure types,

Current understanding of absence seizure mechanisms derives frornextensive studies of Gloor and his colleagues with the penicillin model of generalized spike-and-wave in the cat (Gloor, E 984). These studies were based on the historic work of Penfield and Jasper in the 1940s and 1950s (see Jasper, 199 1) on centrencephalic seizures (i.e., seizures that arise in both hemispheres simultaneously and are asso- ciated with immediate Ioss of consciousness, as in absence seizures). It was hypothesized by Penfield and Jasper that centrencephalic sei- zures are generated by activity in subcortical structures, including the thalamus, with widespread projections to the cortex. This hypothesis was refined by Gloot, who proposed that the cortex and thalamus acted in concert during generalized seizures. Simultaneous record- ings in the cortex and thalamus of cats treated with penicillin dem- onstrated a pattern of firing during each burst consistent with the circulation of impulse flow within a loop between thalamus and cortex (Avoli et al., 1983; Avoli, 1 987). Tn agreement with this idea was the observation that both cortex and thalamus ate required for spike-and-wave discharges: surgical removal or pharmacological inhibition of either eliminates epileptifom activity. The critical involvement of cortex and thalamus has now been confirmed in a brain slice preparation (Coulter and Lee, 1993) and in the genetic absence epilepsy rat from Strasbourg (GAERS), a more realistic model of human absence epilepsy than the penicillin model in the cat (Marescaux et al., 199213).

Epilepsy 245

A more detailed understanding of the cellular events in genera- tion of the spike-wave discharges by the thalamocortical circuit has come from electrophysiological (voltage clamp) studies of thalamic neurons and, more recently, from modeling of the membrane poten- tial of these cells using parameters derived from voltage clamp experiments (Wang et al., 199 1 ; Huguenard and McCormick, 1 992). Burst firing in thalamic neurons is dependent on a slow potential, referred to as the "low-threshold spike" (LTS), mediated by T-type voltage-dependent Ca2+ channels (Jahnsen and LlinBs, 1984a,b; Suzuki and Rogawski, 1989; CouIter et al., 1989a). Strong hyperpo- larization is required to prime (de-inactivate) T-type Ca2+ channels that are normally inactivated at resting potential. Activation of the LTS in thalamocortical relay neurons is, in turn, dependent on inhibi- tory drive from thalamic reticular nucleus neurons (Huguenard and Prince, 1 992b$, These neurons have intrinsic pacemaker activity that generates rhythmic sequences of oscillatory bursts (Bal and McComick, 1 993). Interestingly, this burst firing is also dependent on a T-type Ca2' current, but with somewhat different properties from that present in relay neurons (Huguenard and Prince, 1992b). Tha- lamic reticularnucleus neurons are GABAergic. These neurons form recurrent inhibitory connections among themselves, and also pro- vide a powerfuI inhibitory input necessary to de-inactivate T-type CaZ+ channels of relay neurons. The prolonged inhibitory postsynap- tic potential (IPSP) in relay neurons is probably mediated by GABAA and GABAB receptors (Thornson, 1988; Soltesz et al., 1 989). Pres- ently, the role of GABAB receptors in regulating the normal firing of relay neurons is poorly defined (McCormick, 1 992; von Krosigk et al., 1993). However, during seizure activity (as can be induced with GABAA receptor antagonists in thalamic slices), GABAB receptors appear to play a critical role in synchronizing the firing (von Krosigk et al., 1993).

7.1. T-Type Vo liage-Dependent Ca2+ Channel Antagonists The fundamental insights from elech-ophysiological studies of

the thalamus have led to a mechanistic understanding of the selective antiabsence drug ethosuximide and have also suggested strategies

for the development of new medications for absence seizures. Thus, Coulter et a!. (1989b) demonstrated that ethosuximide produces a modest (up to 40%) reduction in amplitude of the T-type Ca2+ cur- rent in thalamic neurons (see also Kostyuk et al., 1992; Huguenard and Prince, 1994). The ECso for this effect is 200 pI4 and there is a roughly 2&35% reduction in Ca2+ current at ethosuximide con- centrations within the clinically relevant concentration range. Corn- puter simulations of individual thalamic neurons have shown that even smaller reductions in the T-type Ca2+ current could result in a diminution of the LTS (Lytton and Sejnowski, 1992). These results support the hypothesis that ethosuximide's effects on the T-type Ca2+ current selectively alter the dynamics of slow bursting in tha- lamic relay neurons that could lead to protection against absence seizures. Additionally, however, ethosuximide probably also acts on the T-type Ca" current in nucleus reticularis neurons to reduce their burst firing (Huguenard and Prince, 1992a). The effect on reticularis neurons would diminish the hyperpolarizing influence that these cells generate in relay neurons and that is necessary for their burst firing. The dual action of exthosuximide an nucleus reticularis and relay neurons would act in concert to interrupt thalamic bursting, and in turn thalarnocortical synchronization during absence seizures.

In addition to ethosuximide, dimethadione (the active metab- olite of the anti-absence drug trirnethadione) and methylphenyl- succinimide (the active metabolite of another anti-absence drug methsuxirnide) have been shown to cause a reduction in the T-type Ca2+ current in both thalamic relay-neurons and nucleus reticularis neurons (Coulter et al., 1 990; Huguenard and Prince, 1992a). In contrast, valproate, an agent that is also highly effec- tive in the treatment of absence seizures, has not been found to affect T-type Ca2+ currents in thalamic neurons at clinically rel- evant concentrations. Nonetheless, the drug has been reported to affect a similar current in a peripheral neuron (Kelly et al., 1 990). Whether valproate's activity as an absence agent relates to effects on Ca2+ channels or to effects on another molecular target is unknown.

Epilepsy

7.2. GA BA, Receptor Antagonists The critical role of GABAR receptors in the generation of

absence seizures has been demonstrated in several animal models by the use of selective GABAs agonists and antagonists. Thus, the GABAs agonist baclofen enhanced, and the GABAB antagonist CGP 35348 inhibited, absence-like seizures in y-hydroxybutyrate-treated rats (Snead, 1992), the GAERS rat (Liu et al., 1992; Marcscaux et al., 1992a), and the lethargic mouse (Hosford et al., 1992). Interestingly, in the lethargic mouse there is evidence for a genetic defect resulting in the overexpression of GABAB receptors. These animals show a modest increase in GABAe receptor density as assessed with radioligand binding and also exhibit evidence of enhanced GABAR receptor functional activity in brain slice recordings (Hosford et al., 1992). In the GAERS rat, however, no such alteration in GABAB receptors was observed (Spreafico et: al., 19933. Thus, while absence- like seizures can occur in animal models as a result of a variety of underlying pathophysiological mechanisms, GABAe receptor antagonists appear to be effective in all instances.

The precise site at which GABAA antagonists exert their anti- absence activity has not been defined with certainty. However, elec- trophysiological studies in thalamic slices have supported the hypothesis that, during seizure activity, synaptic activation of GABAR receptors generates the hyperpolarization necessary to de-inactivate the T-type CaZ+ current (von Krosigk et al., 1993). Interruption ofthis hyperpolarizing influence by antagonists would prevent the seizure activity, but would not affect normal ongoing activity, which seems Iess dependent on GABAB receptor activation. Although there is now extensive experimental evidence supporting the potentiaI cIinicaZ utility of GABAB antagonists as anti-absence agents, this will need to be confirmed in clinical trials, now in progress.

7.3. Potential Anti-A bsence Drugs if Unknown Mecha~ism

There are a variety of compounds that have an anticonvulsant profile in animal seizure models simiIar to that of exthosuximide, but whose mechanism of action is unknown. One such compound is the

imidazopyridine AHR- 1 2245 (2-[4-chEorophenyl]-3H-imadam[4,5- blpyidine-3-acetamid) (Johnson et al., 1991). Like ethosuxirnide, AHR- 12245 is active orally against pentylenetetmzal seizures in both mice and rats, but is ineffective in the maximal electroshock test. However, AHR- E 2245 has a substantially greater therapeutic index than ethosuxirnide and is therefore an interesting candidate for evalu- ation in the treatment of generalized absence seizures.

As noted in Sections 2.1.4. and 2.2.6., GABA-potentiating m u - roactive steroids and IoreclezaEe have profiles of activity similar to that of ethosuxirnide. Whether these compounds will prove useful in the treatment of absence seizures remains unknown.

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