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CHAPTER

Epilepsy

Michael A. Rogawski

1. lntraduction

Epilepsy, a condition of recurrent, paroxysmal seizures, is amajor, worldwide, health problem. The epileptic seizure representsan 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 a n epileptic patientcan be diminished or eliminated by drugs that target the basic excit-

ability mechanisms of neurons. Most presently used antiepilepticmedications were deveIoped by empirical drug screening or seren-

dipity. However, in the past several decades, as understanding of fhe

functional activity of brain circuits has increased, there has been an

intense effortto develop new antiepileptic medications on a rationalbasis.

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-conductingchannels--the fundamental mediators of electrical excitability in

neurons. There are two major classesofneuronal 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 StrategiesEds.: L. Pullan and 4 . Patel Hurnana Press Inc., Totowa, NJ



194 Xogawski

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 he seizure.

Voltage-dependentchannels contribute to the paroxysmal depolariz-ing shift, the single ceII correlate of the interictal (between seizure)

network discharge. Voltage-dependent channels also mediate actionpotentials 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 propagatebetween neurons within the neuronal aggregate and alsoparticipate

in the paroxysmal depolalizing shift. The voltage-dependent ion

channels include channels selective for Na+ and K+, that shape thesubthreshold behavior of neurons and mediate action potentials aswell as channels selective fox Ca2+hat 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-proteinsor 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, sothat there is an enormous variety of possible anticonvulsant strate-gies. The ultimate goal is, of course, to prevent the generation orpropagation 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; thereare some important leads along these lints as discussed in Section

3.1.)Given the critical role of the neuronal excitability mechanismsmediating seizures for normalbrain function, it would seem improb-

able thatan anticonvulsantdrugcould be developedwhich 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. Potentiationof Synaptic Inhibition

Enhancement of synaptic inhibition mediated by GABAA

receptors is an important anticonvulsant strategy. This can occur bytargeting 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 seizuremodels. Such drugs typically protect very effectively againstpentylenetetrazol seizures,but have far weaker activity in the maxi-

mal electroshock test.Despite the selectivityoftheir effects in animal

seizure models, GAB&-receptor potentiating anticonvulsants mayhave 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 anotable exception to this rule.)

2.1. GABA, Receptors as Targetsfor Anh'epilep t ic Drugs

Presently available drugs that potentiate GABAA eceptor

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, hat act similarly to

barbiturates, but probably interact with distinct sites on the GABAAreceptor 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 GABAA 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 statusepiiepticus (continuous seizures without return to consciousness),



196 Rogaws i

two significant limitations largely preclude their chronic use in epi-Iepsy therapy. The first limiting factor is that these benzodiazepinesproduce undesirable side effects, including sedation and muscle

relaxation,at doses comparable to those that protectagainst seizures.The second,andprobably more important,Iirnitation isthat tolerance

develops to the anticonvulsant activity ofbenzodiazepines receptoragonists with chronic use. In recent years, considerable effort has

been directed toward developing strategies for overcoming theselimitations. A variety o f benzodiazepines and nonbenzodiazepineshave been identified that interact with the benzadiazepine binding

site of the GABAn receptor complex in novel and potentially morefavorable ways. As yet, however, there is no convincing evidencethat any of these benzodiazepine receptor ligands will have practical.utility in the chronic treatment of epilepsy.

2.1.1.1. RENZODIAZEPINEECEPTOR ARTIALGONISTS.artialaganisrnatthe benzodiazepinereceptorhasbeen proposedby Haefely

(seeHaefely 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 lowintsinsic efficacy and induce smaller responses than do full agonists

at the same fractional receptor occupancy. Thus, at full receptoroccupancy, partial agonists produce submaximal biologicalresponses.However, the degreeof 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 receptoraga-

nistsmay 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 1296; tephens et al., 1990; etersen

et al., 1984),pyrozoloquinolines (CGS 9896;Bernard et al., 19851,imidazopyrimidines divaplon; Garhe r , 1988), and quinolizinones(Ro 19-8022;Jencketal.,1992).Samepartial 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., 1992; Facklam et a]., 1992a; Jensen et al., 1984).How-ever, Ro 19-8022 has lower eficacy for sedation and muscle relax-ation, and may also have reduced physical dependence liability.

Moreover, Ro 19-8022does not exhibit proconvulsant activity (likebenzodiazepine inverse agonists); nor is an abstinence syndrome(characterized by tremors and seizures) precipitated when chroni-cally treated animals are exposed to a benzodiazepine receptorantagonist. Interestingly, in line with the efficacy of conventionaI

benzodiazepines in human absence epilepsy, Ro 19-8022 has beenreported 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) anddivaplon (Feely et al., 1989;Deacon et al., 1991) similarly display

potent anticonvulsant activity, but less sedation and anticonvulsanttolerance than diazepam.Studies using recombinant GABAn recep-

tor subunits have demonstrated that bretazenil and divaplon possess35-58% and 2 1-28%, respectively, of the intrinsic efficacy of the

full agonist flunitrazepam (Knoflach et al., 1993;see also Facklamet al., 1992b).

How can w e understand the law propensity of benzodiazepinereceptor partial agonists to produce sedation, muscle relaxation, and

tolerance? It is now recognized that full benzodiazepine receptoragonists, such as df azepam,produce anticonvulsant effectswith low

receptor occupancy (Fig. I ) . For example, it has been estimated thatfull 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 forprotec-tion against tonic hindlimb extension in the maximal electroshock

test (Petersen et al., 1986). Similarly, sedative effects as assessedwith various tests of motor impairment also require high levels ofoccupancy (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 sameresponse as a full agonist, higher levels of receptor occupancy are



Xogawski

-

-

-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 ofmotor function in the rotarod test. In vivo radioligandmethod for determinationof receptoroccupancy is described in Petersen et al.(1984). Audiogenic = protection against sound-induced seizures in D B N 2mice; 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. ata obtained in the mouse(except as notedlare fromBraestmpand Nielsen ( 1 986), Petersenetal. (1984) ,

Jensen et al. (19X4), nd Petersen et al. ( 1 986).

required, The requirement for higher occupancy is presumablybecause the potentiation of each GABAAreceptor is smaller and

more GABAAreceptors must be recruited to obtain an equivalentresponse. For example, in one study, 50% protection in the penty-

lenetetrazol test was reported to occurwith 37% receptor occupancyby diazepam and 84%receptor occupancy by ZK 1296, a partial

agonist (Petersen et al., 1986). For benzodiazepine agonist effects

that require a high degree of GABAAreceptor potentiation (e.g.,



muscle relaxation, ataxia, amnesia, and respiratory depression), it

may not be possible to achieve the pharmacological effect with apartial 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 ofcertain GABAA receptor subunits, particularly the p subunit that

confersbenzodiazepine sensitivity (Zhao et a1., 1994).Nevertheless,

since the mechanism coupling chronic receptor activation tochangesin 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 asabecamil,may have a distinct spectrum of anticonvulsant activity in

comparison with conventional benzodiazepine receptor agonists suchas 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 differentGABAA eceptor subtypes,mainly dependent on the molecular formof the a-subunit (Knoflach et al., 1993; PribiIla et al., 1993).Thus,the unique ph m ac a lo gi ca l profile of abecarnil could arisefrom ts

actions as a partial agonist at some GABAA receptors and as a fu11

agonist at others. Animals treated chronically with abe~arn i l o not

exhibit a diazepam-like withdrawal syndrome on discontinuation ofthe drug (Loscheset al., 1990;Steppuhn et al., 1993;Llischer, 1993)-

Moreover, the development of anticonvulsant tolerance to abecarnilis dependent on he model used. Indeed, in some experimental para-digms no anticonvulsant tolerance is exhibited at the same time that

to1erancedevelops to the motor impairing activityof the dmg (Loscheret al., 1991b; Serra et aI., 1994).



200 Rogawski

2.1.1 -2.BENZODIAZEPTNEECEPTOR SUBTYPE-SELECTWEGONTSTS.

Abecamil is an exampIe ofa benzodiazepinereceptor ligand that actsas a partial agonist at some GABAA receptor subunit combinations

and not others. It therefore exhibits some subtype selectivity. Theimidazopyridines alpidem and zolpidem, in contrast, show nearIy

complete subtype selectivity. These compounds exhibit GABApotentiating activity at, for example, GABAA eceptors of composi-

tion a lBzyz , 2Ply2, lP lyz ,but not aSP2y2(Faun-Halley et al., 1993;Wafford et al., 1993; Horne et al., 1992). In binding to brain tissue,alpidernandzolpidem show selectivityfortheoli'BZ1subtype ofre ceptor (Benavides et al., 1988; Benavides etal., 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 asan anxiolytic have

been promising,with a very favorable side-effect profile in compari-sonwith conventional benzodiazepines.Alpidem binding exhibits a

differential regional: localization in brain that correlates roughly withthe presence of al/BZt binding sites. It has been suggested that the

regional selectivityofalpidemforw /BZl bindingsites,whichisdepen-

dent on the unique subunit specificity of the drug, could account forits favorable properties (Benavides et al., 1993).On he other hand,

the possibility that alpidern is a partial agonist at certain GABAP,

receptor subtypes couldalso explain its favorable profile (Perraultetal., 19933. However, recent studies with cloned subunitshave dem-

onstrated that alpidern (Imet al., 1993) and zolpidem (Horne et al.,1992) ase full agonistsat some, if not aII,GABAA eceptorsubtypes.

2.1.2. Barbifurafe-LikeDrugs

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,barbituratesare highly effective,

broad-spectrum anticonvulsants. Barbiturate-like compounds withimproved side-effectprofiles would provide interesting candidates

for clinical development.



Epilepsy 201

The sedative and anticonvulsant activity of barbiturates is

believed to be mainly due to their potentiationofCNS GABA-mediatedinhibition (Scholfield, 1938). This occurs by prolongation of burst

openings of singleGABARreceptorchannels (Macdonaldetal., 1989).However, effects on voltage-activated CaZ+ hannels also appear to

play a role in the sedative-hypnotic activity of the barbiturates andcould participate in their therapeutic activity as anticonvulsants

(ffrench-Mullen et al., 1993). In addition, direct activation o f the

GABAn receptor could account for the sedative-hypnotic side effectsof certain barbiturates (Rho et al., 1994b). Finally, barbituratesappear to have actions an excitatory amino acid receptors, withparticular selectivity for AMPA (a-amino-3-hydroxy -5-methy1-4-isoxazalepmpionic acid) receptors (Tavernaet al., 1994;Donevan and

Rogawski, unpublished). In line with the diversity of barbiturate cel-lular actions, there are wide differences in the pharmacologicalprop-erties of structurally similar barbiturates. Pentobarbital is a powerfulsedative-anesthetic, whereas the less sedative phenobarbital is usedmainly as an anticonvulsant.Although still incompletely understood,the basis for the reduced sedation with phenobarbital may relate torelative affinities for different target sites and also to the magnitudes

of effects produced at clinically relevant concentrations.For example,ithasbeenproposed that the stronglydepressantaction ofpentobarbita1could relate to its activity both as a GABA potentiator and a Ca2+

channel blocker. Phenobarbital, in contrast,has a higher affinity forGABAA receptors than forCa2+ hannels (ffrench-Mullenet aI., 1993).At clinically relevant concentrations, it may mainly act as a GABApotentiator, 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 lowertoxicity. As the concentration of phenobarbital moves into thesupratherapeutic (toxic) range, it becomes more strongly sedative,

possibly because effects on Ca2' channels become prominent. Thetypes ofCa2+ hannels relevant to the action of barbiturates have notbeen well defined. One hypothesis is that these are presynaptic chan-nels involved in the regulationofneurotransmitter release, and that the

depressant action is Iargely due to a reduction of glutamate release.



202 Rogawski

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 effectprofile (causing agitation and insomnia more frequentlythan sedation; Faught et al., 19931, and appears to act, in part, as a

barbiturate-like modulator of GABAA eceptors (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. Meprobamatehas also recentlybeen shown

to have barbiturate-like activity. However, it is much more potentand, unlike felbamate, can directly stimulate the opening of GABAA

receptor coupled CI- hannels in the absence sfGABA (Rhoet

al.,1994a). This latter activity could contribute to the propensity ofmeprobamate 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 wouldcounterbalance the CNS depression resulting from GABA potentia-

tion. (NMDA antagonists typically have behavioral activating effects

at low doses;Willetts et al., 1990.)

It hasbeen known for over fivedecades that the steroidsproges-

terone and deoxycorticosterone acetate have anticonvulsant activity

against pentylenetetrazol seizures (Selye, 1942; Craig, 1966), In theintervening years, other structurally related steroids, including 3,20-

pregnenediones nd 3,20-pregnanediones (Craig and Deason, J968),2P-morpholino-5a,3a-pregnanoIonesHewett et al., 19641, and the

steroid anesthetic alphaxalone (Peterson, 1989), have been reportedto have similar activity. In 1984,Harrison and Simmonds made the

seminal discovery that alphaxalone could selectivelypotentiate cen-tral neuron responses to GABA (but not glycine). The 3P-hydroxy

isomerbetaxalone was inactive, indicating ahigh degree of structural

specificity. Morerecently, it has been recognized that certain endoge-

nousmetabolites of progesterone and deoxycorticosterone are also



Epilepsy 203

highly potent modulators of the GABAn receptor (Majewska et aE.,

1986). These metabolites have anticonvulsant activity (Belelliet al.,1989, 1990;Bogskildeet al., 1988),and their anticonvulsantpoten-

cies correlate strongly with their ability to modulate GABAA eceptorcurrent (Kokate et al., 1994).Studiesusing fluctuation (noise) analy-

sis and single channel recording have demonstrated that the steroidsproduce a barbiturate-likeprolongation ofburstopeningsofGABAA

receptors (Sirnmonds, 1991;Twyrnan and Macdonald, 1992).In addi-

tion, they may causean increaseinchannel-openingfrequency,an effectnot observed with barbiturates. Like barbiturates, neuroactive steroidscan directly activate the GABAA eceptor 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 steroidsare 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 seizuremodels suggests that their primargr utility may be in the treatment ofabsence epilepsy (Rogawski and Porter, 1990). Indeed, there is ananecdotal report ofthe effectiveness of deoxycorticosterone acetate

in six of eight absence seizure patients (Aird and Gordan, 1951).

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 separationbetween their anticonvulsant and toxic doses, but are relatively weak

anticonvulsants(Craig and Deason, 1968). However, alphaxalone, amare potent anticonvulsant, protects against seizures only at dosesthat produce neurological impairment (Peterson, 1989).In a compre-hensive study of progesterone and deoxycorticosteronemetabolites,

there was up to a several-fold separation between the protective andtoxic doses. Thus the therapeutic index (ratio of the ED50 values formotor 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 a11of the steroidswere 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 favorableneurosteroids (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 iswhether 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 anticonvulsantactivity ofpregnanolonein the pentylenetetrazol test;Kokate et aI., 19956.)However,even if

tolerance does develop over time, steroidsmay stiIl be of value in theacute treatment of status epilepticus,as are benzodiazepines. In fact,the steroids appear to be highIy effective in the kainate and piIo-

carpinemodelsofstatusepilepticusin themouse(KokateandRogawski,1994;Kokate etal., 1995a). Because the solubility of steroids is poor,

selection of an appropriate vehicle or identificationo f water soluble

prodrug forms will be critical.Recently, the synthetic 3P-methyl analog of epiallopregnano-

Ione (3a-hydroxy,3P-methyl-5a-pregnan-20-one; CCD 1042) as

been demonstrated to have anticonvulsant potency that is onlyslightly weaker than its parent.However,the 3P-methylation confersgood metabolic stability and oral activity. In Phase I cIinical trials,CCD 042was well-toleratedat levels substantiallyhigherthan hoseassociated with seizure protection in animals (K. . Gee,personal

communication, 1995).

2.1.5. y-Bufyrolactones and y-Thiobutyrolactones

Anadditional seriesofGABAA receptormodulatingcompoundshas 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



Ep i l e p s y 205

and y-thiobutyrolactones.Certain ligands ofthe GBL site antagonize

GABAA eceptorresponse andare convulsant,whereas otherspoten-tiate GABA responses and have anticonvulsantproperties (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 maximalelectroshock test and other seizure models (Ferrendelli et al., 1989;

Holland et al., 1992).a-EMTBL has a therapeutic index that is lowrelative to other GABA modulatinganticonvulsants,but i s similarto

valproate. Preliminary results indicate that a-EMTBL may demon-strate lesstolerancethanclanazepan,suggestingthat GBL siteligands

may be ofutiIity in chronicepiIepsytherapy (Ferrendellietal., 1993).

Loreclezole is a novel triazolederivativethat is protective againstpentylenetetrazolseizuresbut is lessactive in the maximal electroshock

test (Wauquier et al., 1990;Kulak and Sobaniec, 1994).In addition,atlow, 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 otherbenzodiazepines. A potential benzodiazepine-Iike interaction withGABA, receptors is suggested by the observation that the anticon-

wlsant effectsofloreclezolecanbe reversed bybenzodiazepine ecep-tor inverseagonists (Vaught and Wauquier, 1991; Ashton etal., 1992).

The benmdiazepineantagonistflumazenil,however, failed toaltertheanticonvulsantactivityofloreclezole, ndicating that loreclezole is not

a benzodiazepinereceptor agonist (Wauquier etal., 1990).Moreover, onchronic (5-7 d) administration, tolerance did not develop to th e anti-

conwlsant effects of Ioreclezole as it does with benzodiazepines.LorecEezole can indeed enhance the activity of GABAA ecep-

tors, but it interacts with a novel allosteric modulatory site distinctfrom that of benzodiazepines, barbiturates, or neuroactive steroids(Wafford et al., 1994).Using native rat and cloned human GABAA

receptors, loreclezole strongly potentiated GABA-activated C1- cur-rent. However, activityof the drug did not require the presenceof the



y-subunit and was not blocked by flumazenil, confirming that

loreclezoIe does not interact with the benzodiazepine recognitionsite. Interestingly, loreclezole had >300-fold activity an receptors

containing the P I - or p3-subunit n comparisonwith those containingthe I subunit.Usingchimeric P-subunitsand 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 acidsubstitutions 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 ofdrugs that modulateGABAAreceptors in novel ways.

2.2. Vigabatrr.'~~,GABA Transamitrase Inhibitor

In additionto the focusonpostsynaptic GABAnreceptors, there

has been considerable interest and, indeed, success in the develop-mentofnew antiepileptic drugs thatmodify GABA disposition in the

brain. Vigabatrin (y-vinyl-GABA),perhaps themostnotableachieve-

ment in this respect, is an enzyme-activated rreversible inhibitor ofGABA transaminase, the main degradative e n m e or GABA. The

drug elevatesbrain GABA levels, and exhibitsanticonvulsantactiv-

ity in a variety of animal seizure models. Moreover, there is nowsubstantial clinical data supporting its efficacy in the treatment of

human eizure disorders (Reynolds, 1992).Although the precisewayin which vigabatrinprevents seizures remainsunclear (Bernasconiet

al., 19881, GABA transaminase inhibition may increase neuronalGABA in a releasable pool, so that larger amounts of GABA aredischarged with stimulation (Gale, 1992).Thus, increased amounts

o f GABA are released in a use-dependem fashion,as needed, during

seizures, It had been believed that tolerance develops only to thesedative side effects ofvigabatrin,but not to its anticonvulsantactiv-

ity (Remy and Beaumont, 1989). Now, however, there is emergingevidence that tolerancemay alsodevelopto the therapeutic effects of

the drug (Martin and Millac, 1993).



Epilepsy 207

2.3. GABA Uptake BlockersAn alternative approach to increasing synaptic GABA levels is

inhibition of GABA reuptake. A variety of cyclic amino acids,

includingnipecoticacidand guavacine, inhibitglialorneusonal GABAuptake carriers. In general, however, thcse compounds have only

modest potency in vitro and do not enter into the CNS afterparentedadministration. In recent years, n variety of lipophilic analogs ofnipecotic acid and guavacine have been described (Braestrup et al.,

1990;Suzdak, 1993;Andersenet al., 1993).These com po zan d~ wh ichinclude tiagabine, CI-966,NNC-7 11, 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. Unlikenipecotic acidwith its moderate selectivityfo r neuronalGABAuptake,lipophilic GABA uptake inhibitors such as tiagabine show a modestlygreater affinity for glial uptake, but they do inhibit uptake in both celltypes. These compounds are not transported via th e GABA uptake

carrier nor do they stimulate GABA release. Recently, it has been

demonstrated that tiagabine, CI-966, and related lipophilic GABAuptake 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 systemicadministrationofCI-966 (Tayloretal.,1990)or tiagabine(Fink-Jensenet al., 1992;Sybirska et al., 19933was shown to increase extracellular

GABA overflow. Similar results were obtained in awake human sub-jects with hippocampal microdialysis probes foIlowing acute oral

administrationof tiagabine (During eta]., 1992).Experiments inbrainslicepreparations have demonstrated that Iipephilic GABA reuptake

blockers can prolong the actionofappliedGABAand aEso increase the

amplitude and duration of inhib itoy GABA-mediatedsynaptic 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 seizuremodels that is similar to otherdrugs 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 inducedbyotherchemoconvulsants that interfere with GABA-

ergicneurotransrnission, such as the benzodiazepine receptorinverseagonist DMCM (methyl 6,7-dimethoxy-4-ethyl-barboline-3-car-

boxylate). They are far less potent (tiagabine: Nielsen etal., 199 1 ) or

inactive (SW 89976A and SKF 100330-A: Swinyard et al., 1991) in

the maximal electroshocktest. In addition, lipophilic GABA uptakeinhibitors have shown activity in various reflex seizure models(including the photosensitive baboon Papio papio), and in theamygdala and corneal kindIing models (Suzdak, 1993; Swinyardet 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 thattol-

erancemay develop to these sideeffects,but not to the anticonvulsantactivity (Suzdak, 1994). LipophiIicGABA uptake inhibitors have a

spectrum of anticonvulsant activity in animal seizure models that isdistinct from the important presently marketed antiepileptic drugs

(Swinyard et aE., 1991 ) . Therefore, it isdifficultto predict fromanimalstudieswhich farms of human epilepsy would be most amenableto therapy with these agents. However, their potency and rela-

tively broad spectrum of activity suggest that they are interestingcandidates 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 dizzinesswere obsmedwith increased doses.) Importantly, there was no evi-

denceof

tolerance to the drug's anticonwlsant activity with chronicdosing for up to 12mo. n contrast, in an nitial trial in healthy voIun-teers,CL-966nduced a varietyof severe neurologicaland psychiatricsymptoms (Sedman et al., 1990). There are differences between thepharmacologicalproperties oftiagabineand CI-966 that could accountfor 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 in fom ation 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 GABAanalog, it does not interact with GABAa or GABAR eceptors, isnot

metabolicalIy converted to GABA or a GABA receptor agonist, anddoes not inhibit GABA uptake or degradation (Taylor, 1994).Haw-ever, gabapentin does increase GABA turnover in some brain regions

(Loscher et al., 199 1a) and may increase the release of GABA frombrain slicesin 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 H o m o u , 1994).A similar effecthas been reported in the hippocampus (Honmou et aI., 1994).

Although these studies with gabapentin and GABA release areintriguing, they are as yet inconclusive. Nevertheless, they serve to

focus attention on the possibility that drugs that enhance GABArelease could have potential as antiepileptic agents.

2.5. Potentiation of GABA and Glycine:Propofol and Chlomethiazole

The anestheticpropofol(2,6-diisopropylphenol) s a structurally

novel barbiturate-like modulator of GABAn receptors (Peduto et aI.,1991). However, in contrast to barbiturates such aspentobarbital, sev-eral recent reportshave suggested that subanesthetic dosesofpropofol:can protectagainstseizures n animal rnodeIs,with minimal behavioralsideeffects (Hasanetal., 1992;al-Haderetal., 1992;Hasan andWolley,



210 Rogawski

1994).In humans,propofol may be effective in the treatmentof statusepilepticus (Wood et al., 1988), although the drug has also beenreported 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 GABAAreceptors

(Hales and Lambed, 1991) . In additionto thesebarbiturate-like prop-erties, propofol is also able to potentiate glycinc-evoked currents

(Hales and Larnbert, 1991 ; ut see,Dolin et al., 1992), an actionnotshared by barbiturates, neuroactive steroids, or benzodiazqines.

Another combined GABAA/glycinemodulator forwhich thereis even more clinical information is chlormethiazole, a thiamine

derivative with sedative, hypnotic, anxiolytic, and anticonvulsantproperties (~gren , 986).

ChlormethiazoIeis particularly effective

in the matment of statusepilepticus (even in cases failing torespondto benzodiazepines and barbiturates).Chlomethiazole is a1so used

inpreeclampsia(toxemiaofpregnancy), eclampsia (toxemiaofpreg-nancy with seizures), and the alcohol withdrawal syndrome. En both

animalsand humans, chlormethinzoIehas anticonvulsant activity at

doses that do not produce sedation. Like propofol, chlormethiazole

potentiates responses to bothGABA andglycine,and can also directly

activate GABAAreceptors (Wales and Larnbert, 1992).Propofol andchlonnethiazole uniquely potentiate GABA and glycine, and bothappeartobe effective anticonvuIsantswith low behavioral toxicity at

anticonwlsant doses. The favorable toxicity could be explained ifthe potentiationofGABA and glycine were additive (oreven syner-gistic) for seizure protection, but were not additive for sedative sideeffects. Indeed, it is not apparent that a glycine potentiating drugwould exhibit sedative-hypnotic activity.

3. Interactionswith ExcitatoryAminoAcid Receptor Systems

Acomplementaryapproachto facilitation of synaptic inhibition

is blockade of synaptic excitation. The bulk of fast synaptic excita-tion in the CNS ismediated 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 am ino 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 ofmany

types of epileptic seizures (Rogawski, 1995).Recently, it has been

elegantly shown using microdialysis that glutamate levels areeIevated prior to the onset and during the expression of complexpartial seizures in human subjects (During and Spencer, 1993).Thereis thus strong heoretical support for the potential utility of excitatoryamino acid antagonists in the treatmentof 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 seizuremodels. NMDA antagonistsareparticularlypotent

in the maximal electroshock test in rodents and against reflex sei-zures in rodentsand primates (Chapman and M eldrum, 1993).They

are also protective against pentylenetetrazol and otherchemoconvul-snnts. Thus, the resuIts from animal testing suggest that NMDAantagonists may have utility in the treatment of generalized tonic-

clonic (convulsive) and partial seizures. NMDA antagonists havelower potency against spontan eous 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 ~IicepreparationseeRogawski, 1995). This suggests that NMDA

antagonists may prevent the development or progression of some

types of seizures. This property could be of value in certain cIinicaIsituations, such as during the critical period after brain injury or inprogressive childhood epilepsies. The status of efforts to develop asafe and effective NMDA antagonist for use in epilepsy therapy hasbeen recentIy reviewed (Rogawski, 1992).



3.1.1.CompetitiveNMDA Recognition Site AntagonistsIn 1982, Croucher et al. reported that phosphonic acid competi-

tive NMDA recognition site antagonists were protective against

sound-inducedand minimalpentylenetetrazol eizures inmice. This

important observationstimulated anintensiveresearch effort into the

medicinal chemistry of NMDA receptor antagonists (Rowley and

Leeson, 1992). Numerous structural classes ofNMDA antagonists

havebeen investigated.Perhaps the most mature lineof investigation

is that evolving from the straight chain o-phosphono-a-amino cid

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 analogswith the amino nitrogen ina six-member ring, as in CGS 19755 (cis-

4-phosphonomethyl-2-piperidine carboxylate; Hutchison et al.,

1989; Lehrnann etal., 1988),or in a piperazine ring,as in CPP (3-[2-

caxboxypiperazin-4-yllpropyl- 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 foundthat uunsaturation sf the chain, as in CGP 37849 ([El-2-amino-4-methyl-5-phosphono-3-pentanoicacid;Fagg et al., 1990;Schmutzet

al., 1990;De Sarro and De S a m , 1992), or additionof a ketone as inMDL 100453 ~[R]-4-oxo-5-phosphonowaline;hitten et aE., 1990)also resulted in compounds with p o w e f i l systemic anticsnvulsant

activity. Moreover, the carboxyethylester of CGP 37849 (CGP3955 1) showed particularly prolonged oral anticonvulsant activity(De Sarro and De Sarro, 1992). (The reduced analog MDL 10453also is a potent NMDA antagonist;Bigge et al., 1992.) Unsaturation

of the side chain of CPP to form CPPene (Aebischer et al., 1989)

similarly producedan anticonvulsant compound with a long durationof action, The D(-)-enantiorner is the active species of CPPene andhas 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 forother NMDA antagonists,protects against the

development of kindled seizures (Durmurnuller et al., 1994). Most

competitive NMDA recognition site antagonists have a phosphonic



Ep i l e p sy 223

acid group as in the original structuresproduced by Watkins (1 991).However, this is not essential to activity as demonstrated byLY233053 and LY275059, n which she phosphonic acid group is

replaced by the bioisosteric tetrazol functionality. LY233053offersgood protection against NMDA and maximal electroshock seizures,

but its duration of action is short (Schoepp et al., 1990a,b;Ornsteinet al,, 1992). The structurally novel bicyclic phosphonic acidLY2746 14has long lasting oral anticonvulsantactivity (Schoepp

e t al., 19913,whereas the corresponding tetmzol LY233536 (Leanderand Ornstein, 1990;Omstein et al., 19901, like LY233053, has amore sapid time course.

CompetitiveNMDA recognition site antagonistsexhibita broadspectrum of activity in animal seizure models. Not unexpectedly,these compounds are highly effective in protecting against seizures

and lethality induced by systemic or intracerebroventricularNMDA.

They are also protective in the maxima1 electroshock test with corn-

parabIe or slightly lower potency (Ferkany et nl . , 1989). Protectionis 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 cid) has been describedwhich isless

potent in themaximal electroshocktest than againstNMDA or penty-lenetetrasol seizures (Ferkany et al., 1989). Recently, the highest

potency isomer of NPC 12626 has been identified as the ZR,4R,5Sform (Hamilton et al., 1993). It too shows a reverse ordering ofpotencies in animal seizure models (Ferkany et al., E993).Whetherthis unique profile is related to an unusual selectivity fo r NMDA

receptor subtypes or some other property remains tobe determined.Although diverse NMDA recognitionsiteantagonistswith good

bioavailability and favorable pharmacokinetic properties are now athand, the problemofside effectshas not been so readily solved.Since

NMDA receptors are critical to numerous brain functions, NMDAantagonists can produce a variety of behavioral and neurologicaltoxicities. There are, however, certain encouraging features of thepreclinical 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

otheranticotkvulsantdrugs.Drug discriminationstudies indicate that

the subjective effects ofcompetitive NM DA recognition site antago-

nists in rodents and primates are distinct from those ofdissociativeanesthetic-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 sideeffects such as stereotypiesand disturbancesof locomotor behavior

common with dissociative anesthetic-like NMDA antagonists (see

Section 3.1.2.), these stiIl occur.The side effectsmay be especiallyprominent in kindled animals (Loscher and H ~ n a c k , 991a,b).

Tol-erance does not seem to develop to either the therapeutic activity orthe side effects (Smith and Chapman, 1993)-

Despite the various favorable properties ofNMDA recognitionsite antagonists,reports of clinical trials have been disappointing. In

an open-Iabel study of D-CPPene in eight patients with intractablecomplexpartial seizures, a 1patientswithdrew prematurely becauseof side effects. The side effects included poor concentration, seda-

tion, ataxia, depression, dysarthrta (impairment of speech due tomotor 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 mayhave greater sensitivity to the side effects ofNMDA antagonists.Afinal problem is that competitive antagonists (like the dissociative

anesthetic-like compounds discussed in Section 3.1.2.) can produceneuronal vacuolization and morphological damage in certain brainregions (Olney et al., 1991).

Until recently, it was not appreciated that competitive NM 9Arecognition 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 appearsto have modestly higher affinity for NMDA receptors composed of

W D A R l - lb subunits that contain a 2 1 -amino acid insert generated



by alternative splicing of exon 5 (seeMcBain and Mayer, 1994). In

addition to sensitivity to competitive antagonists, this N-terminalvariation determines a variety of functional properties of the NMBA

receptor, including potentiation by ZnZ+nd polyamines and inhibi-tion by protons (HolImann et al., 1993). In the future, it may bepossible to more selectively target specific NMDA receptor subunit

combinations, thus potentially providing for the developmentof less

toxic anticonvulsants.

3.1.2. Channel-BlockingNMDA Receptor Antagonists

The NMDA receptor channel blockers are often referred to as

'kncompetitive" antagonists, because their binding and blockingaction requires the receptor-channel to be gated in the open state.

Occupancy of a binding site within the ionophore of the NMDAreceptor 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 antagonistsshare with other noncompetitive antago-

nists of the NMDA receptorthe theoreticaI advantage that their block-ing actionwouldnotbe overcome by high synaptic levelsofgIutamateasmayoccur during seizures. In addition,uncompetitiveantagonistshave the additional theoretical advantage of use-dependence, imply-ing that their inhibitory action may specificallybe potentiatedatsites

of excessive receptor activation. The current status and potentialclinical utiIityofchannel-blocking NM DAreceptor antagonistshaverecently been reviewed (Rogawski, 1992, 1993).

Channel-blocking NMDA receptor antagonists fa11 into two

broad categories:dissociativeanesthetic-like agents and Iow affinityantagonists. Shortly after the discovery of selective competitive

NMDA recognition site antagonists in the early 1980s, the dissocia-tive anestheticsphencyclidine andketamineweredemonstrated to beeffective NMDA receptor antagonists (Anis et al., 1983). The struc-turally dissimilardissociativeanesthetic-likeagentdizocilpine (MK-

801) is also an extremely potent NMDA receptor antagonist. Thesecompounds exert their block in a use-dependentand voltage-depen-dent fashion, indicating that they act by an open-channel mechanism



216 Rogawski

(Huettner and Bean, f 9881, Like competitive NMDA recognitionsite antagonists, dissociative anestheticsare potent broad spectrumanticonvulsants in a wide variety of animal seizure models (see

Rogawski, 1992).However, at anticonvulsant doses, they producesevere neurobehavioral side effect-including cognitive, sensory,

and various schizophrenia-likedeficits (Krystal et al., 1994)-thatlimit 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 franknecrosis 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 notbeen documented in primates. Moreover, it is now apparent thatsomeNMDA antagonists may produce only transitory vacuoIization

with no necrosis, or no changes at all.The anticonvuIsant activityof dissociative anesthetics is shared

by a variety ofchannel-blocking NMDA antagonists, someofwhich

are analogs of the dissociative anesthetics and others that are struc-turally distinct. Certain of these compounds have substantiallyreduced behavioral toxicity in animals. For several, there is humanclinical experience documenting an acceptable level of safety. Thesereduced-toxicity channel-blocking NMDA antagonists have beenreferred to as "low affinity" uncompetitive antagonists. However,low affinityper-semay not be the only factor that accounts for their

more favorab le toxicity characteristics(see below).

The existenceof low-toxicity, channel-blocking NMDA antag-onistswas first recognized with the discovery that certain l-phenyl-

cycloalkylamines uch as 1-phenylcyclahexylamine the analog ofphencyclidine substituting a primary amine for the piperidine ring)

and 1-phenylcyclopentylaminewere effectiveanticonvulsants, 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 similarproperties, 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]-[l-(2-thienyl)cyclohexyl]piperidine and [3H]dizocilpine) to

brain membranesandblock NMDA receptor currents in a use-depen-dent and voltage-dependentmanner, ndicating that they are chan-

nel-blocking NMDA antagonists (Rogawski et aI., 1992; Jones

and Rogawski, 1992). However, the binding affinities of thesereduced-toxicity uncompetitiveNMDA receptor antagonists are lower

than thatofphencyclidine and dizocilpine. At equieffective concen-trations, these lower affinity ligands would exhibit faster apparent

rates of block and unblock, and this may, t least in part, account for

the lower toxicity (see Rogawski, 1993). However, reduced bindingaffinity 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 isnow evidence that lower toxicity Iigandsmay bind selec-

tivelytoadifferentpopulationofNMDAreceptors thando dissociative

anesthetics (Porter and Greenmyre, 1995).

Recent work with cloned NMDA receptor subunits suggests

that there may be differences among subunit combinations withrespect to their sensitivity to channel-blocking agents (McBain and

Mayex, 19943.For example,assembly of the NMDAR1 subunit withNR2C 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- 1b splice variant; see

Section3.1.1.) confers higher sensitivity to block by dizocilpine andketarnine (Hollmann et al., 1993;Rodriguez-Pazet al., 1995).Moresubtle variations in the structure of NMDA receptors could also

modify the actionsof channel bIockers. A particularlycritical site ofthe NMDARl subunit is the neutral asparagine at position 598. Thiscorrespondstothe editedarginineJgIutamine(QIR) ite in membrane

segment 2 (M2) of AMPA receptors, where a positively charged



arginine residue in GluR2confers low Ca2+pemeabilityseeSection3.2.2.). Similarly,mutations in NMDAR1 that switch the asparagine

to a positively charged arginine generate receptors resistantto blockby dissociative anesthetics (Kawajiri and Dingledine, 1993;Mori

et al., 1992; Sakurda et a]., 1993). Although there is currently no

evidence forRNA editing ofNMDA receptors at this site (as occursfor AMPA receptor subunits), variations in channel structure inthis or a nearby region of the receptor could have important conse-

quences for the sensitivityto channel blockers.Of course, it remainsto be determined which, if any, structural variation in the NMDAreceptor 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 otherthan binding affinity and subunit selectivity might also contribute tothe more favorable side-effect profilesofcertain low-affinity NMDA

antagonists (Rogawski, 1993). In particular, synergism betweeneffectson NM DA receptorsandotheranticonvulsant argets couldbe

a factor in some circumstances. For example, ADCI has Na* channelblocking activity similarla carbamazepine (see Section4.1,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 (formerlyFPL 12495; ,2-diphenyl-2-

propylamine), Ithe metaboliteof the anticonvulsant remacemide; and

the adamantane analog memantine. In contrast to dissociative anes-

thetics, low-affinity channel-blockingNMDA antagonists substitutepoorly, or not at all, for dizocilpine in drug discrimination testing.This indicates that they may not producedissociativeanesthetic-likesubjective effects (Grant, Colombo, Grant, and Rogawski, unpwb-

lished).Moreover, fordextrornethorphan, dextrorphan,remacemide,and memantine, substantial clinical experience indicates that the

compounds can be well tolerated in humans. However, adverseexperiences with all of these compounds have been reported (see

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



Epilepsy 219

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 classof 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 ;

Morrisettetal., 1990).Thismay be in part because of a compensatory

upregulation in the expression of NMDA receptors after chronic

ethanol exposure (Sanna et al., 1993).ADCI isparticularly effectivein this regard (Grant et al., 1992).

3.1.3. GIycine Site Antagonists

It is now well recognized that glycine is a required co-agonistfor activation of the NMDA receptor (Johnson and Ascher, 1987;

KlecknerandDingledine, 1988).Consequently,agents thatcompeti-

tively antagonize the action of glycine would produce a functional

block ofthereceptor and could,therefore,have anticonvulsantactiv-

ity. Several classes of antagonists arenow known which more or lesssefectively 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.,

1991; Baron et al., 1990;Koek and Colpaert, 1990;Palfreyman andBaron, 199 1; Bisaga et al., 19933. Recently, Rundfeld et al. ( 1 994)

observed that intraventricular injection of glycine site antagonistsand partial agonistsincreases 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 siteof the NMDA receptor may be of utiIity in the treatment of seizuretypes 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 developmentof systemically active glycine site antago-nists came from the recognition that H A - 9 6 6 (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 electroshockseizure 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, andmore favorable toxicity properties, such asL-687,414 (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 cid (ACPC), a particularly highaffinity ligand. ACPC is protective in several seizure models

(SkoEnick eta]., 1989; Witkin and TorEeIla, 1991; 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 unusualspectrum of activity. Indeed, it is surprising that the drug is anti-

convulsant at all.

Recently, certain 5-nitro quinoxaline-2,3-dioneswere demon-

strated to have highly potent (KI, 10 Rn?l) glycine site blocking

activity and good central availability when administered systemi-cally.These compoundsalso block AMPA receptors, but do sowith

several hundred-fold lower potency (Woodward et al., 1993). Twosuch compounds, 5-nitro-6-rnethyl-7-chloro-2,3-quinoxali~eW C Q X ) and 5-nitm-6,7-dimethyl-2,3-quinoxalinedioneMMDX),have been reported to have activity in the maximal electroshock testat reIatively low systemic doses (Tran et al., 1994). However, theextent to which the weak AMPA receptorblocking activity contrib-

utes to the compounds' anticonvulsant effects i sunclear. The related

quinoxalinedione ACEA-1021 (5-nitro-6,7-dichIoro- 1,4-dihydro-



Epilepsy 221

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

A series ofrelated structures-the 3-nitro-3,4-dihydro-2-quino-lones-have been described with varying degrees of glycine site orAMPA receptor selectivity. These compounds are based on 7-chloro-kynurenicacid, apotent glycine site antagonist in vitro, but eliminatethe carboxylic acid group that seems to prevent blood-brain barrierpenetration. One of these, L-698,544, exhibited good systemic activ-

ity as an anticonvulsant(EDSO,3.2 mg/kg? p) in the DBA/2 mouse(Carling et al., 1993).Although L-698,544 interacts with the NMDA

(Kh, .7 pkf)and AMPA (Kk,,.2 pM)recognition sites, these inter-actions occur with somewhat lower affinity than for the glycine site(ICso,0.4 1w . prodrug ester of the potent glycine site antagonist5,T-dichlorokynurenic acid has also been reported to have systemicnnticonvulsant activity in the DBAI2 mouse (EDSO,2 mglkg, ip;

Moore etal., 1993).Similarly,another quinolinoneglycine siteantag-onist MDL 104,653(3-phenyl4-hydroxy-T-chlor~-quino~in-2[1H]-

one; McQuaid et al., 1992)has been observed to have anticonvulsantactivity, when administered either intraperitoneally or orally, in

DBAJ2 mice (Chapman et al., 1993).Interestingly, high doses of thecompound 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 ofL-70 1,273 (3-cyclopropyl-ketone-4-

hydroxyquinolin-2[ 1H]-one), with high potency against audiogenicseizures in D B N 2 mice (4.1rngkg, ip; Rowley et al., 1993).This com-pound has moderately high activity at the glycinesite (IC50,0.42m,but also interacts with the NMDA recognition site (Kbr 3.4 pi%!),

although it is inactive at;AMPA receptors. More recently, a seriesof3'-substituted-3-phenyl analogs of the 4-hydroxyquinolinones were



222 Rogawski

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 showactivity at the NMDA recog-

nition site but do not block AMPA receptors. These compoundsappear to be the most potent systemicallyactive glycine site antago-

nists described to date, with El350 values <1 mg/kg in the D B N 2mouse modelwhenadministeredeither in~aperitoneallyrorally. It

would appear that these compoundshave favorable toxicity charac-teristics,producingmotor impairmentonly at doses that are substan-tially higher than those that protect against seizures, but this wiHrequire further confirmation. Thus, a variety of potent, and more or

less selective, glycine site antagonists are now available, some o f

which have high systemic potency and excellentoral bioavailability.T remains to be determined if these compounds will be superior toother types of M D A antagonists for seizure therapy. Of particular

interest is thepossibility that the additionalAMPA receptor blockingactivity ofsome of the glycine site antagonists may actually provide

enhanced anticonvulsant activity. As discussed in Section 3 . 2 ,

AMPA receptorblockade can confer apowerful anticondsant effect.

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

amines spermhe and permidine enhanced binding of ~HJdizocilpine

to NMDA receptors in brain homogenatessuggestedthatpolyarnines

could facilitate gatingof the NMDA receptor.Although polyamineshave 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-independentmechanism (Williamset al., 1994; Zhang et al., 1994b3.

Therehas been interest in developingpolyaminesite antagonists,but

success to date has been limited.

Two related compoundsproposed aspolyamine site antagonists

are theneuroprotective agents ifenprodil and eliprodil (SL 2.0715)



Epilepsy 223

(Scatton et al., 1994). Ifenprodil and eliprodil are now we11 recog-

nized to be potent noncompetitive NMDA antagonistsboth in vitro

and in vlvo. Electrophysiological studies in cultured hippocampal

neurons havedemonstrated that ifenprodil produces a complex effecton 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 considerableevidence that ifenprodil and eliprodil

can functionally interact with polyarnine effects on the NMDAreceptor, it is unlikely that this occurs at a common recognition site

(Ogita et al., 1992). It has instead been proposed that the polyamineand ifenprodiEJeliprodi1binding sitesmay shareoverlapping domainsan 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 receptorscomposedof NRlA and NR2B subunits, although it can also block NRlA/

NR2A receptors at substantially higher concentrations (Williams,1993). Therefore, at ordinary doses, ifenprodil m ay interact with

only a subpopulation of NMDA receptors. Interestingly, it nowappears that polyamine facilitation is variably expressed depending

on the subunit composition of the NMDA receptor. The 2B subunitisa key subunitin 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 toxicityprofiles than other NMDA antagonists (Scatton et al., 1994).At

neuroprotective doses, they do not produceneurological impairmentand 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. n addi-

tion, eliprodil does not appear to produce pathomorphologicalchanges, 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 selectivityof 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 (D e Sarro and De Sarro, 1993; Scatton et al., 1994).These effects, however, occur at doses that are higher than theneuroprotective doses and are nearer to the doses that cause toxiceffects. For example, in the case o f ifenprodil, there is little separa-

tion in the doses that protect against seizures (mouse maximal elec-troshock test ED50,29mgkg, p) and induce motor impairment. For

eliprodil the therapeutic index issomewhatbetter (e lec tmshockED~~,

15 mgikg, ip; rotarodED501

59 rng/kg). (Interestingly, however,eliprodil has been shown o potentiate the anticonvulsantactivity ofthe competitive NMDA recognition site antagonist CGP 37849;

Deren-Wesolek and Maj, 1993.) Thus, t may be that the NMDAreceptor subtype selectivity of ifenprodil/eliprodil s not optimally

suited for seizure protection, at least in the animal models used for

screening.Whetherthis is a characteristic of all functionalpolyamine

antagonists,remains to be determined.

A varietyofothercompoundshave been proposed aspolyamineantagonists, including several polyarnines (Williams et al., 1990)andcertain peptide componentsofmarine 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, includingchannel-blockingactivity, which eonfound any interpretationoftheir

specific polynmine site effects (Donevan et al., 1992;Submmaniamet 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 ofNMDA-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

increasewith kindling, either produced electrically (Hayashi et al.,1989)or by repeated treatment with pentylenetetrazol (Hayashi et

al., 1993).However,it remainsundeterminedwhether these increasedlevels contribute to the hyperexcitability of the kindled state. Should

this be the case, the argument supporting the potential use ofpolyamine siteantagonists in seizuretherapy would be strengthened.

3.1.5.Anfagovtists

rat Ofer Sites on heNMDAReceptor Complexand the Combined Acf ons ofFelbamate

on NMDA/GABA, Recep fors

In recent years, a classofNMDA antagonisthas been identifiedin which inhibition is exerted at sites that are distinct from those of

known N M D A receptor coagonists or facilitatory modulators.Indeed, ifenprodiI and eIiprodil are probably best considered amongthis class of NMDA antagonist. Other examples include a series

of n-alkyl diamines that inhibit NMDA receptors by acting at a

nonvoltage dependent, hydrophobicbinding site as well as in n chan-nel-blocking fashion (Subramaniam et al., 1994).Similarly, the anti-convulsantrernacemide may also block NMDA receptors in part bysuch an allosteric action (Submm aniamet aI., 1995a). Felbamate too

is an example of an anticonvulsant that may antagonize NMDAreceptors 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 felbamatedoesnot 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 GABAAreceptor responses within the same range

of concentrations hat it blocks NMDA responses (Rho et al., 1994b3.Both of these effectsoccur at concentrationsof felbamatelikely tobe

present in the brain during antiepileptic therapy. Felbamate exhibitsa 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 againstseizures induced by pentylenetetrazol; Swinyard et al., 1986.) Clini-cally,the drug has activity in the treatment of partial and secondarilygeneral ized tonic4onic seizures and it is also effective againstrefractory seizures of the Lennox-Gastaut syndrome in children,

which are notoriously difficult to treat. The combination of actionsof felbamateon excitatoryand inhibitory synaptic transmissioncouldaccount for its broad spectrum of activity and favorable side-effectprofile. More specificaIly, it can be postulated that the distinct

actions on NMDA and GABAAreceptors are by themselves belowthe threshold for side effects, but that the actions at the two sitesare

additive or even synergistic against seizures. That a drug such as

felbamate with low neurological toxicity may exert its therapeuticactivity v i a low-affinity interactions with more than one receptorsystem contradicts the conventional view that side-effect reduction

is best achieved by high selectivity and potency. However, foranticonvulsantdrugs that modulate excitability mechanisms criti-

cal to normal brain function, agents with high potency and efficacymay have unacceptable toxicity. (See Section 2.1.1.1. for a moredetailed discussion with reference to partialbenzodiazepine receptor

agonists.) Therefore, the search for drugs that exert low efficacyeffects on more than one receptor seemsto 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 ofmodel sys-

tems (Rogawski, 1994). Consequently, the observation that AMPA

receptor antagonistshave anticonvulsant activity isnot unexpected.

However, at present, it is unclear if AMPA receptor antagonistswill be sufficiently free of toxicity to be clinically useful anti-

convulsants.Nevertheless,thepossibility of selectively targeting thevarious AMPA receptor subtypes provides enormous opportunities

for drug development.



Epilepsy 227

3.2.1. CornpstifiveAMPA Receptor Anfagonisfs

During the early period of excitatory amino acid receptor

research, AMPA receptor antagonists were unavailable. However, inthe late 1980sthe situation changed: Hanore et al. (1988) deveIapeda series of quinoxaiinediones as selective and relatively potentcompetitive AMPA recognition site antagonists (see Davies and

CoIlingridge, 1990). Although the first quinoxalinediones to be

described were not systemically active, compounds with goodsystemic bioavailabilityarenow available.These include the benzo-

h s e dquinoxnlinedioneNBQX 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;hmori et al., 1994) that mayhave improved CNS penetration in comparisonwith NBQX. A seriesof isatin oxamines has alsobeen described which are highly selective

AMPA receptor antagonists (Watjen et al., 1993).These compoundshave anticonvulsant activity against AMPA-induced seizuresfollowing intravenousand oral administration. In addition,a substi-

tuted decahydroisoquinoline-3-carboxyliccid has been recently

described with good systemic AMPA receptor blocking activity

(Omstein et al,, 1993). This compound, LY215490 ([3SR,4aRS,

4RS,8aRS]-6-[2-(1H-tetrazol-5-yl)ethyl]decahydroisoquinoline-3-

carboxylic acid), also interacts with the NMDA recognition site, butwith 5- to 10-fold lower potency.

Competitive AMPA recognition site antagonists are protectivein the maximal electroshock test and against seizures induced byvarious 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., 1991;Smith et al.,1991;Meldrum et al,, 1992; Ohmori et al., 1994). In the kindling

model,NBQX has been reported to haveprotectiveactivity, 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 activityagainst kindled seizures,NBQX has no effecton he developmentof



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 ggnerallybeen found that AMPA receptor antagonists haveneurological, toxicity at anticonvulsant doses, although this varies

depending on the seizuremode1 and test conditions <we Chapmanet al., 199I). Thus, NBQX produced motor impairment at doses that

are similar to those that are protective in the maximal electroshocktest (Yamaguchi et al., 1993).The LY215490 has only a modestlybetter therapeutic ra tio (=2.2) (Omstein et al., 1993).Nevertheless,it would be premature to conclude that AMPA receptor antagonistsare unlikely to have clinical utility in seizure therapy, since block-

ade of AMPA receptorsis

an entirely new therapeutic strategy. Onlythrough clinical trials will it be possible to assess the significanceof the therapeutic ratio values determined from animal testing.

Lascheret al. (1993) observed that low doses of NMDA receptorantagonists synergistically enhance the anticonvulsant ac tivity of

NBQX in the kindlingmodel, withoutproducing anincrease n adverseeffects. Although the basis of thisphenomenon s poorly understood,

it seems reasonable that a combination of NMDA and AMPA receptor

blockade may ultimatelyprove to be the optima1approach forepilepsytherapy. In fact, Iow levels ofNMDA receptor blockade may providean antiepileptogenic effect and may also protect against seizure-induced brain damage, inamanner not obtainedwith AMPA receptorantagonists (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 superiortherapeutic activity with acceptable side-effect properties.

Another potential approach to obtaining improved anticon-vulsant activity with a reduction in side effects would be to targetAMPA 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 thatnon-NMDA receptors can be selectively targeted with antagonists.

Thus, he isatin oxime NS- 102 (5-nitro-6,7,8,9-tetmhydrobenzo-



Epilepsy 229

k]indale-2,3-dione-3-oxime)sa non-NMDA antagonistwith 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.NoncornpefitiveAMPA 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 siteantagonists.These antagonists-typified by the 2,3-benzodiazepine(homophthalazine) G Y M 52466 (1-14-aminophenyll-4-methyl-7,8-

methylenedioxy-5H-2,3-benzsdiazepine)-offer an alternativeapproach for blockade of AMPA receptors with potentially distinc-

tive consequences (Tarnawa et al., 1990; Ouardouz and Durand,

1991;Jones et al., 1992).GYKl52466, unlike the quinoxalinedioneAMPA antagonists, does not interact with the AMPA recognition

site, nor is it a channel blocker. Rather, it appears to block AMPA

receptors in a noncompetitivefashionvia anovel allostericsiteon the

AMPAreceptorcomplex (Doncvanand Rogawski, 1993). The block-ingactionofGYKl52466 is highlyselectiveinvitro, withessentially

no effect on NMDA, metabotropic glutamate, or GABAAreceptorresponses, The lack sf effect on GABAAreceptors is particularlynotable in view of the structural similarity of GYKI 52466 with

conventional 1,4-benzodiazepines see Section 2.1. .).Like competitive AMPA recognition site antagonists, GYKl

52466 has anticonvulsant activity in a broad spectrum of animalseizure models (Chapman et al., 1991; Smith et al., 1991;Yamaguchi

et al., 1993; Steppuhn and Turski, 1993). In some experimentalmodels, 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

synapticallyreleased glutaman would surmount the blocking actionofa competitiveantagonistso that high doses of the antagonistwould



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

A series of N-substituted GYKE 52466 analogs has been

described with varying degrees ofAMPA receptor blocking activity(Tarnawa et al., 1993). Two of these analogs with greater in vitroAMPA receptorblocking potencies were found to have correspond-ingly greater invivo anticonvulsantpotencies Donevanetal., 1994).

This supports the view that th e 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 havea linear

or outwardly rectifyingcurrent-voltage relationship and a low permeability for Ca2+.Com-binations lacking GluR2 are inwardlyrectifyingand have a high Ca2+

permeability (Hume et al., 1991; Hollrnann et al., 1991; Verdoornetal., 199I). GYKI 52466 appearstoblock Ca2'-permeable and Ca2+-

impermeable AMPA receptors with equal potency (Donevan andRogawski ,unpublished). This is in contrast to the barbiturate pento-barbital that selectivelyblocks Ca2+-impermeableAMPA receptors

(Taverna et al., 1994)and certain arthropod toxins that are selectivefor Ca2+-permeableAMPA receptors (see Section 3.2.3 .). Interest-ingly,GYKI 52466and itsanalogsappeartohavemuchhigherblock-ing potency for AMPA-sel ective non-NMD A receptor subunits thanforkainatep r e f m g subunits (Patemain et a]., 1995).The mplications

of these various selectivity differences arenot yet well understood.

3.2.3.Channel-BlockingAMPA RecepforAntagonists

At present, no selective channel-blocking AMPA receptorantagonists have been identified. However, certain polyaminearthropod toxins could provide clues to the development of such

compounds.The toxins (such as argiotoxin636,philanthotoxin,and

Joro spider toxin) act as potent noncompetitive antagonistsof verte-

brate AMPA receptors (Brackley et al., 1993;Blaschke et al., 1993).These toxins act in a use- and voltage-dependentmanner suggesting

that they are channel blockers (Herlitze et al., 1993). However, the



Ep i l e p s y 231

toxins are not selective for AMPA receptors, and indeed their block

ofNMDA receptors is more reliable and oftenmore potent (Priestleyet al., 1989;Ragsdale et al., 1989).A likely reason for the inconsis-

tency ofthe AMPA blocking action is suggestedby the recent discov-ery that th e polyamine toxins selectively block inwardly rectifying

AMPAreceptors(that is,those that lack the G l u m subunit)(Blaschkeet al., 1993; Herlitze et al., 1993).

From the point ofview oftheir use as pharmacological tools, the

fact that the toxins block both NMDA and AMPA receptors is adisadvantage. How ever, 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 toxinsfor inwardly rectifying AMPA receptors may be a distinct advantage.

Sincerectifying AMPA receptors are Ca2+ ermeable, 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, ithas recently been reported that amygdaloidkindling

is associated with a 25-30% reduction in G l u m 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 tonormal at 1wk and1 mo. imilar changes were not obtained after induction of seizures

with the convulsant pentylenetetrazol. It is conceivable that thedecrease in Glum could be a factor in th e induction, but not themain-

tenance, of kindled seizures. If this is the case, agents such as thepolyarnine toxins which selectively block AM PAreceptors lacking the

G l u m subunit could have interesting antiepileptogenic potential.An additional Iine of experimentation is consistent with the

possibility that GluR2 subunit downreguIation could play a role inseizure-inducedneuropathology.Thus, riedman etal.(19943recentlyreported that persistent seizure activity (status epilepticus) can result

in reduced expression of the GluR2 subunit in hippocampal regionsmost vulnerable to neurodegeneratian. This reduction occurs with a



time course that parallels theneuronal cell loss,supporting a role for

Ca2+ ermeable AMPA receptors in the seizure-induced pathology.

Polyamine toxin-like agents with specificity for Ca2+-permeable

AMPA receptors might, therefore,be well suitedasneuroprotectants

to ameliorate seizure-induced pathological changes.

Although only limited information isavailable, polyarnine tox-ins and a series of smaI1 mol wt analogs referred to as "araxins" dohave anticonvulsantand neuroprotective activity when administeredintracerebroventricularl or systemically (Seymour andMena, 198 9;

Mueller, personal communication).At therapeutically effectivedoses,the compounds donot appear to produce dissociative anesthetic-likebehavioral or histopathological effects, and do not generalize to

phencyclidine in drug discrimination studies. However,with systemic

administmtion,rhereis poor separationbetween the dosesthatconferseizure protection and induce neurological impairment. In addition,

the toxins may produce significant cardiovascular side effects.

3.3. Metaboiropic G lutam ate Receptors

Metabotropic glutamate receptors are a diverse group o f G-protein-coupled receptors that are structurally and functionally dis-

tinct from other glutamate receptors (Suzdak etal., 1994).In contrastto NMDA and AMPA receptors which are themselves ion channels,rnetabotropicglutamate receptorsact via varioussecondmessengersto indirectly regulate a wide variety of ion channels. The potentia1

roleofmetabotropicglutamatereceptorsas targets foranticonvulsantdrugs is not well defined. Nevertheless, activation of rnetabotropicglutamate receptors with selective agonists can produce, in adult

rodents, limbic-like seizures similar to those observed in kindled

animals (Sacaanand Schoepp, 1992;Tizzano et al., P994). In neona-tal rats, metabotropic agonists can produce frank convulsions(McDonald et al.,1993).Interestingly,however,low doses ofcertainmetabotropic glutamate receptor agonists can have weak anti-convulsant effects (see Thornsen et al., 1994). The anticonvulsanteffectspresumably arise because the rnetabotropic glutamaterecep-tor agonists reduce glutamate release by a presynaptic action on

inhibitory autoreceptors (Glaum and Miller, 1994).



Recently, a novel seriesof 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 seizuresin DBA/2 mice at doses that do not produce neurological im pa im en t

(Thornsen et al., 1994). This effect occurred with intraventricdar,

but not systemic, administration of the drug. In addition,(S)-4C3HPGdiminished the epileptiform activity observed in rat cortical slicesexposed to Mg2+-free onditions. L-AP3 (L-2-amino-3-phosphono-

propionate), a low potency noncompetitive metabotropic receptorantagonist(Schoepp, 19941,has alsoproduced a weak anticonvulsanteffectfolIowing intraventricular administration (Klitgaard and Jack-

son, 1993). Bath IS)-4C3HPG and L-AP3 can have rnetabotropicglutamate 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 agonistactivity, possibly by presynaptic effectsan glutamate release. Indeed,

it is not clear at present whether metabotropic receptor agonism,

antagonism, or a combination ofagonist and antagonist effectsat dis-tinct metabotropic receptor types represents the most effectiveanticonvulsant approach. NevertheIess, targeting of the various

metabotropic receptor types with selective agonists and antagonistsprovides interestingopportunitiesfor anticonvulsant drug development.

3.4. GlutanrateRelease Inhibitors

Metabotropic glutamate receptorsaxejust one exampleof a host

of receptors and ion channels of the glutamatergic nerve terminal

that are attractive anticonvulsant targets. It has aiready been notedthat barbiturates might in part exert their anticonvulsant activity viainhibition sf glutamate release (through effects on voltage-depen-

dent Ca2+ hannels). In addition, as discussed in the next section, the

therapeutic effectsof anticonvulsant drugs that interact with voltage-

dependent Na' channels is likely due to some extent to inhibition ofglutamate release. It appears that certain toxins including o-Agn



IVA and cs-Aga MVIIC, which block Ca2+ hannels in presynapticnerve terminals can have anticonvulsant effectswhen administered

inbacerebroventricularly (Jackson et al., 1994). 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 ofaction s not well understood (Reddy et aI., 1994).Thereare

thus a number of interesting leads to glutamate release antagonists ofpotential 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 NatChannels

A wide variety of anticonvulsant drugs are believed to act, atleast 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. Therapeuticconcentrationsof the drughave little activity on resting Na'channelsat hyperpolarized membrane potentials. Thus, at a potential of -80

mV, 10 p M henytoin, a concentration at the high end of the range

of free therapeutic plasma levels, would produce no more than 15%block of Na' current (Lang et a!,, 1993). However, there is moresignificant block when Na' channels are repetitively activated or

when the initial membrane potential is a t a more depolarized level.

These properties are believed to reflect the higher affinity ofphenyrain for activated and inactivated channels than for resting

channels (Ragsdale et al., 1991). During high frequency firing, thereis aprogressive accumulationof 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 accountingfor 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.



Epi lepsy 235

The precise way in which a modification of Na+channel gatingresults in seizure protection is not we11 understood. However, thefollowing hypothesishas often been proposed. During seizures, neu-

rons fire more rapidIy and are more depolarized than under normalconditions, 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 andMacdonald, 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 therelease 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

anticon wlsant 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 anticanvuEsanteffective in the treatment of refractory partial seizures (Yuen, 1994),

has a similar profile to phenytoin; it is protective in the maximalelectroshock 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 restingmembrane potential, in a manner similar to that ofphenytoin (Cheunget aI., 1992). In addition, the drug suppresses burst firing m corticalneurons(Leesand Leach, 1993),and inhibits binding o f [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, Iamotsigineproduced a use-dependent inhibition of Na+channels and slowed therate of recovery from inactivation, effects similar to those produced

by phenytoin (Lang et al., 1993; Taylor, 1993). Thus, he similaritybetween larnotrigine and phenytoin in animal seizure modeIs is par-alleled by corresponding actions on Na* channels.



236 Roguwski

Riluzole is another anticonvulsant drugwith a phenytoin-likespectrumofactivity (Mizouleet al., 1985).(Riluzole also hasneuro-protective properties [see,for example, Wahl et al., 19933 as doesphenytoin [Stanton and Moskal, 1991; Hayakawa et al., 19941.)Among tsvariouspharmacological actions, riluzole is a blocker of

voltage-dependent Na+ channels. Interestingly, however, unlikephenytoin, the block produced by riluzole does not occur in a use-dependentfashion, suggesting that it doesnot preferentially bind toactivated Na+ channels. Rather, riluzole has an exceptionally

high (>150-300-foId) selectivity for the inactivated state of theNat channel (Benoit and Escande, 1991). These results withriluzole indica te that stabilization of the inactivatedstateo f he Na+

channel may be a particularly critical factor in the anticonvulsantactivity of phenytoin.Severalother anticonvulsant drugs with a phenytoin-Iike spec-

trum of anticonvulsantactivity interactwith Na+channelsin a fash-ionsimilartophenytoin. These include carbamaqine (Ragsdale etal.,

1991;Lang et al., 1993),ralitoline (Rock t 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-(1yrraIidiny1)-

cyclohexylJbenzarnide)(Zhu and Im,1992), its two major activemetabolites (Zhu et al., 1993);and several other structurally relatedbenzarnides with anticonvulsant properties (Zhu et al., 1992) have

been s h o w to interact with Na' channels in a voltage- and use-

dependentfashion, similarlyto phenytoin. The success in identifyingphenytoin-Iike anticonvulsantsisprobably a result of thewidespread

use of the maximal electroshock test as a screening method (inas-

much as the test is highly sensitive to such agents). Since there arealready a large number of safeand effective anticonvu1santdrugs ofthis 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 combinationwith other pharmacological activities hat result in seizureprotection

(forexample,potentiation of GABA or inhibitionof excitatoryaminoacid transmission). The major acute neurologica1 doxicity of

phenytoin is xeferabIe to cerebellar dysfunction, possibly because







Epilepsy 239

ATP levels in substantia n i p eurons that would activateGTPchan-

nels and depressGABA release. The overall effectwould be to obvi-ate the critical role played by the substantia nigra in the control of

seizure spread (Gale, 1985, 1992). In hypoglycemia, reduced blood

glucose would also lead to diminished intraceilular ATP levels and

a similar inactivationof 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 seizureactivity. Thus, cromakalimreduced bursting ofhippocampa1 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 failedto affect hippocampal epileptiformdischarges,although it was able

to inhibit the response to anoxia (Mattia et al., I 994). There are afew reports that intraventricnEarly administered cromakalim canprotect against seizures in certain in vivo chemoconvulsantmodels

(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 hasbeen demonstrated to have CNS ctivity following systemic adrnin-

istration. Recently, however, benzopyrans structuraIly related tocromakalim 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+ hannelsremains to be determined.

TheKt hannel opener drugs represent an interesting directionfor anticonvulsant drug development. However, there are a number

of impediments that must be overcome before this strategy can beconsidered seriously. As noted, blood-brain barrier permeable ana-

logs are required. Assuming such compounds become available,

nonselective K+ hannel opener drugs could theoretically haveproconvulsant effectsmediated, for example, by the substantia nigra.In addition, there isevidence that some K+channel openerscan block

certain voltage-dependent K+channel types that might predispose to

seizures (Politiet al., 1993).Atpresent, thepotential 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 availableanticonvulsant drugs may act byenhancing K* channel function. For example, it has been proposedthat the tetronic acid derivative losigamone could protect against

seizures by such a mechanism (Klihr and Heinemann, 1990). In

addition,there is a report indicating thatcarbamazepinemay 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

andRelease-EnhancingAgents

Adenosine is a powerful endogenous inhibitory neuro-modulator that isnormally present in the extracellularenvironment

at a concentration of about 1 ph4 Zetterstrorn etal., 1982; Greeneand Haas, 1991). Adenosine exerts its inhibitory action on CNSexcitability by direct (postsynaptic) hyperpolarization of neuronsand by inhibition of synaptic release,particularly of glutamate, viaadenosine 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 stimulateadenosine 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 againstseizure activity is the well-known proconvuIsant activity o fadenosine A I 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 preparationhave demonsbatedthat adenosine can suppress epileptifom activity(Dunwiddie, 1980; 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; Alzheimeret al., 1993). There is evidence that adenosine selectively depresses

excitatory but not inhibitory synaptic transmission (Yoon and

Rothman, 1991; 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, GABABagonists)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 anticonvulsantpropeaies because it hasan extremeIy short biological half-life (5-6 ) and probably pen-etratesthe blood-brain barrier poorly (Rudolphi et al.. 1992).Never-theless, adenosine can protect against audiogenic seizures in mice(Maitreet al., 1974)and alsoprolongs the latency to pentyleneeetrazol

seizures (Dunwiddie and Worth, 1982). Selective adenosine A 1

receptor agonis t s - such a s 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, 1991);

hornocysteine,a putative adenosine sequestering agent (hlarangsset aI., 1990); and 4-aminopyridine (Jackson et aI., 1994). In addition,

adenosine agonists prevent the development of kindling induced byelectrical 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 againstseizures in fully kindled animals (Barraco et al., 1984; Rosen andBerman, 1985). Conventional anticonvulsant drugs generally do not



interfere with kindling development, although NMDA antagonistshave a p a w e A l antiepileptogeniceffect (seeSection 3.1 .).Thus, theability of adenosine agonists to prevent kindling developmentcouldbe 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 animalseizure models, it is unlikely that these compounds would be clini-cally useful anticonvulsants. Adenosine A l agonistshave powerfuIcardiovascular effects as a result of their actions on peripheral

adenosinereceptors in the heart and vascufature. Even iftheseperiph-era1side effectscouldbe overcome(for example by coadministratian

of a blood-brain barrier impermeable adenosine antagonist such as

8-p-sulfophenyltheophylline), adenosine agonists at anticonvulsantdoses may produce strong sedative effects that would limit theirclinical utility (Dunwiddie and Worth, 1982).Consequently,alterna-

tive strategies wilt need to be developed. One approach is to utilizeinhibitors of adenosine inactivation. These may specifically targetepileptic brain regions since seizure activity is accompaniedby localrelease 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 nucleosidetransport (uptake) systems in brain that inactivate released aden-

osine. Electrophysiological studies in the brain slice preparationshave indicated that adenosineuptakeinhibitors such as dipyridamole,dilazep, nitrobenzylthioguanosine, nitrobenzylthioinosine, hexa-

bendine, and soluflazine can potentiate the inhibitory effects ofexogenous adenosine (Sanderson and Scholfield, 1986;Ashton et at.,1987) and reduce epileptiform activity (Ashton et al., 1988). How-ever, adenosine uptake inhibitors generallyhave little effect on nor-mal synaptic transmission. Moreover, microinjection of severaladenosine 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 adenosineuptake 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 enzymeinhibitorshave anticonvulsant activity when njected locally (Zhang

et a!., 19931, there is no evidence that they are effective with systemicadministration. Finally, the purine precursor 5-amino-4-irnidazolecarboxarnide riboside (AICAr) (which increases adenosine levels

during ATP catabolism, as may occur in a seizure focus), has weakanticonvulsant 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 analogswith improved bioavailability before evaluating the potential of thisapproach. In sum, denosine release enhancement-whether by

adenosine uptake blockade, inhibition of adenosine metabolism, orpromotion of adenosine release (as may occur with the xanthine

oxidase inhibitor oxyputinal; Rudolphi et al., 1992)-would appear

to be a worthwhile investigational approach for anticonvulsant drugdevelopment.

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 (Penry et aI., 1975). A child exhibitingan absence attack becomes immobile, stares, and may display anautomatism such as eyelid fluttering or a slightmovement 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-wavebursts. The disorder israre (constitutingperhaps 8%of patients with childhood epilepsy) and usually self-limiting(absence seizures typically remit by adolescence). Absence seizuresare 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 ofabsence seizures. Ethosuxirnide, a drug with little activity in other



seizure types, is highIy effective in the treatmentofabsence 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 pharmacologicalevidence

provides strong support for the concept that the pathophysiological

mechanisms underlying absence seizures are distinct from those ofgeneralized convulsive or partial epilepsies. Indeed, absence sei-zures are beIieved to represent a disorder of corticothalarnicrhyth-

micity, as originalIy proposed in the corticoreticular hypothesis ofGloor (1968). Therefore, the brain systemsmeditating absence sei-

zures may be entirely distinct from those that participate in other

seizure types,

Current understanding of absence seizure mechanisms derivesfrornextensive studiesofGloorand his colleagueswith the penicillinmodel of generalized spike-and-wave in the cat (Gloor, E 984).Thesestudieswere based on the historic work of Penfield and Jasper in the

1940sand 1950s (see Jasper, 1991 ) on centrencephalicseizures(i.e.,

seizures hat arise in both hemispheres simultaneously and are asso-ciated with immediate Ioss ofconsciousness,as in absence seizures).Itwas hypothesized by Penfield and Jasper that centrencephalic sei-

zuresaregenerated by activity in subcortical structures,including thethalamus, with widespreadprojections 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,1987). Tn agreement with this ideawas the observation that both cortex and thalamus ate required for

spike-and-wave discharges: surgical removal or pharmacologicalinhibition of either eliminates epileptifom activity. The criticalinvolvement of cortex and thalamus has now been confirmed in a

brain slice preparation (Coulter and Lee, 1993) and in the geneticabsence 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 hascome 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., 1991;Huguenard and McCormick, 1992).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+ hannels (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+ hannelsthat are normally inactivated at resting potential. Activation of theLTS in thalamocorticalrelay neurons is, in turn, dependent on inhibi-tory drive from thalamic reticular nucleus neurons (Huguenard andPrince, 1992b$,These neuronshave intrinsic pacemaker activity that

generates rhythmic sequences of oscillatory bursts (Bal an dMcComick, 1993). Interestingly, this burst firing is also dependenton aT-typeCa2'current,but with somewhatdifferent properties fromthat present in relay neurons (Huguenard and Prince, 1992b). Tha-lamic reticularnucleus neurons are GABAergic. These neurons formrecurrent inhibitory connections among themselves, and also pro-vide a powerfuI inhibitory input necessary to de-inactivate T-typeCaZ+ hannelsof relay neurons. The prolonged inhibitory postsynap-

tic potential (IPSP) in relay neurons is probably mediated by GABAAand GABAB receptors (Thornson, 1988;Soltesz et al., 1989).Pres-

ently, the role of GABAB eceptors in regulating the norm al firingof relayneurons is poorly defined (McCormick, 1992; von Krosigk

et al., 1993). However, during seizure activity (as can be inducedwith 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 tudies ofthe thalamus have led to a mechanistic understanding of the selectiveantiabsence drug ethosuximide and have also suggested strategies



for the development ofnew medicationsforabsence seizures.Thus,Coulter et a!. (1989b) demonstrated that ethosuximide produces amodest (up to 40%)reduction in amplitudeof the T-type Ca2+ ur-rent in thalamicneurons see alsoKostyuk et al., 1992;Huguenard

and Prince, 1994).The ECso for this effect is 200 pI4and there is

a roughly 2&35% reduction in Ca2+ urrent at ethosuximide con-centrationswithin theclinically relevant concentration range. Corn-puter simulations of individual thalamic neurons have shown that

even smaller reductions in the T-type Ca2+ urrent could result in adiminutionof the LTS Lytton and Sejnowski, 1992).These resultssupport the hypothesis that ethosuximide's effects on the T-type

Ca2+ urrent selectivelyalter the dynamics of slow bursting in tha-

lamic relay neurons that could lead to protection against absence

seizures. Additionally, however, ethosuximide probably also actson the T-type Ca" current in nucleus reticularis neurons to reducetheir burst firing (Huguenard and Prince, 1992a). The effect onreticularis neurons would diminish the hyperpolarizing influence

that these cells generate in relay neurons and that is necessary fortheir 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 synchronizationduring absence seizures.

In additionto ethosuximide,dimethadione (the active metab-

olite of the anti-absence drug trirnethadione) and methylphenyl-succinimide (the active metabolite o f another anti-absence drugmethsuxirnide) have been shown to cause a reduction in theT-type Ca2+current in both thalamic relay-neurons and nucleusreticularis neurons (Coulter et al., 1990; 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 t o

affect T-type Ca2+currents in thalamic neurons at clinically rel-evant concentrations. Nonetheless, the drug has been reported toaffecta similar current in a peripheral neuron (Kelly et al., 1 990).Whether valproate's activity as an absence agent relates to

effects on Ca2+ hannels or to effects on another molecular target

is unknown.



Epilepsy

7.2. GABA, Receptor Antagonists

The critical role of GABAR receptors in the generation of

absence seizureshas been demonstrated in several animal models bythe use of selective GABAs agonists and antagonists. Thus, the

GABAs agonist baclofen enhanced, and the GABABantagonistCGP35348 inhibited, absence-like seizures in y-hydroxybutyrate-treated

rats (Snead, 1992), he GAERS rat (Liu et al., 1992;Marcscaux et al.,

1992a), and the lethargic mouse (Hosford etal., 1992). nterestingly,

in the lethargic mouse there is evidence for a genetic defect resultingin the overexpression o f GABABreceptors. 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

receptorswasobserved (Spreaficoet:al., 19933.Thus,while absence-

like seizures can occur in animal models as a result of a variety ofunderlying pathophysiological mechanisms, GABAe receptorantagonists appear to be effective in all instances .

The precise site at which GABAAantagonists exert their anti-

absence activity has not been defined with certainty. However, elec-

trophysiological studies in thalamic slices hav e supported thehypothesis that,during eizure activity, synapticactivationofGABAR

receptors generates the hyperpolarization necessary to de-inactivate

the T-type CaZ+urrent (vonKrosigk et al., 1993).Interruptionofthishyperpolarizing influence by antagonists would prevent the seizureactivity, but would not affect normal ongoing activity, which seems

Iessdependent on GABAB eceptor activation. Although there isnowextensive experimental evidence supporting the potentiaI cIinicaZutility of GABABantagonists as anti-absence agents, this will needto be confirmed in clinical trials, now in progress.

7.3.Potential Anti-A bsence Drugsif UnknownMecha~ i sm

There are a variety of compounds that have an anticonvulsant

profile in animal seizure models simiIar to that ofexthosuximide,butwhose mechanism of action is unknown. One uch compound is the



imidazopyridine AHR- 12245 (2-[4-chEorophenyl]-3H-imadam[4,5-

blpyidine-3-acetamid) (Johnson et al., 1991). Like ethosuxirnide,

AHR- 12245 isactive orallyagainstpentylenetetmzal seizuresinbothmice and rats, but is ineffective in the maximal electroshock test.However, AHR- E 2245 has a substantially greater therapeutic index

than ethosuxirnideand is thereforean interestingcandidate for evalu-ation in the treatment of generalized absence seizures.Asnotedin Sections2.1.4.and 2.2.6.,GABA-potentiating m u -

roactive steroids and IoreclezaEe have profiles of activity similar tothatofethosuxirnide.Whether these compoundswill prove useful in

the treatment of absence seizures remains unknown.

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