New Antiepileptic Drugs: From Serendipity to Rational Discovery

Summary: Antiepileptic drug discovery has made enormous progress from the serendipity and screening processes of earlier days to the rational drug development of today. The modern era of research began with the recognition that enhancement of inhibitory processes in the brain might favorably influence the propensity for seizures, γ-aminobutyric acid (GABA) being the main inhibitory transmitter. Work in this field led to the development of vigabatrin, which inhibits the enzyme responsible for the degradation of GABA. More recently, research has focused on the therapeutic potential of blocking excitatory amino acids—in particular glutamate. Of the three receptors for glutamate, the N-methyl-d -aspartate (NMDA) receptor is considered the one of most interest in epilepsy, and research on a series of competitive NMDA receptor antagonists—especially those that are orally active—is in the forefront of antiepileptic drug development today. A further alternative for diminishing neuronal excitability is to modulate sodium, potassium, or calcium channels. The latter are especially implicated in absence seizures. Antiepileptic drug discovery has evolved from serendipity through random screening to a scientific era where drugs are designed rationally according to modern principals of neuroscience and the art of medicinal chemistry. Of the research directions currently being pursued, the following appear to be particularly promising: enhancement of inhibition, reduction in excitation, and modulation of the ionic channels that are the fundamental mediators of neuronal excitability. The application of modern approaches to drug discovery provides some optimism that effective new compounds will be marketed in the coming decade, with the promise of diminished suffering by persons with uncontrolled epilepsy.     

Choosing the correct antiepileptic drugs: from animal studies to the clinic

Epilepsy is a chronic condition caused by an imbalance of normal excitatory and inhibitory forces in the brain. Antiepileptic drug therapy is directed primarily toward reducing excitability through blockage of voltage-gated Na(+) or Ca(2+) channels, or increasing inhibition through enhancement of gamma-aminobutyric acid currents. Prior to clinical studies, putative antiepileptic drugs are screened in animals (usually rodents). Maximal electrical shock, pentylenetetrazol, and kindling are typically used as nonmechanistic screens for antiseizure properties, and the rotorod test assesses acute toxicity. Whereas antiseizure drug screening has been successful in bringing drugs to the market and improving our understanding of the pathophysiology of seizures, it merits emphasis that the vast majority of drug screening occurs in mature male rodents and involves models of seizures, not epilepsy. Effective drugs in acute seizures may not be effective in chronic models of epilepsy. Seizure type, clinical and electroencephalographic phenotype, syndrome, and etiology are often quite different in children with epilepsy than in adults. Despite these age-related unique features, drugs used in children are generally the same as those in adults. As awareness of the unique features of seizures during development increases, more drug screening in the immature animal will likely occur.     

Neurotransmission in Epilepsy

Summary: Some evidence indicates that in some types of focal epilepsy the enhanced excitability is due in part to impaired γ-aminobutyric acid (GABA)ergic inhibitory feedback. One form that this can take is impaired excitatory input to GABAergic interneurons. Enhanced excitatory receptor sensitivity, most characteristically involving N-methyl-D-aspartate (NMDA) receptors, has been identified in kindled rodents and in focal epilepsy in humans. Drugs that enhance GABA-mediated inhibition are anticonvulsant in many syndromes of generalized and focal epilepsy. Mechanisms through which this occurs include direct interaction with the GABAhenzodiazepine (BZD) receptor (BZDs, barbiturates, chlormethiazole), inhibition of GABA-transaminase (vigabatrin, VGB) and blocking GABA uptake (tiagabine, TGB). Glutamate receptor antagonists (both NMDA and non-NMDA antagonists) are potent anticonvulsants in many animal models of epilepsy. Whether pure glutamate receptor antagonists will have a clinical role as antiepileptic drugs (AEDs) remains to be established.     

GABA and epilepsy: their complex relationship and the evolution of our understanding.

Because of its abundance in the brain, its ability to produce hyperpolarizing inhibition of almost all neurons, its association with benzodiazepines, and the discovery that many convulsants inhibited its synthesis, gamma-aminobutyric acid (GABA) has often appeared to be the key to epilepsy. Many assumed that "primary" or "genetic" epilepsy must be a disorder of GABA synapses and that GABA agonists would be universal anticonvulsants if permeability and drug metabolism were controlled. The GABA synthetic gene was a logical "candidate gene" for epilepsy. However, the GABA-deficiency theory of epilepsy is less convincing today. GABA agonists were found to intensify seizures in some rodent and human cases. Absence and other generalized seizures in humans often worsened when treated with GABA transaminase inhibitors such as gamma-vinyl-GABA. Surprisingly, the GABA transaminase inhibitors appear to be more useful in partial than in generalized epilepsies. Neuronal GABA uptake blockers are proconvulsant. GABA agonists aggravate seizures in several mutants, ranging from the photosensitive baboon to the genetically epilepsy-prone rat. How can this be understood? Muscimol injections into the pedunculopontine nucleus increase seizures due to systematically administered convulsants, while the receptor blocker bicuculline suppresses seizures after injection into several brain regions, including the striatum. The result of inhibiting inhibitory circuits is excitation. Studies with GABA uptake blockers and the GABAB agonist baclofen are presented in which their combined administration provoked seizures in rats. Baclofen was shown also to increase the incidence of seizures evoked by pentylenetetrazole without increasing seizures due to local injections of excitatory amino acids. Baclofen antagonized the myoclonic effect of 5-hydroxytryptophan in rats with serotonin lesions. Baclofen augments some seizures and inhibits others. Selective inhibition of a particular tract, whether GABAergic or not, may have convulsant or anticonvulsant effects, depending on its connections and the state of the organism. GABAA receptor stimulation is usually but not always anticonvulsant. GABAB receptor stimulation may facilitate absence seizures and related primary generalized seizures. GABAB receptors may be abnormal in some forms of nonfocal epilepsy seen in childhood. It is likely that mutations of GABA transporter and GABAA receptor genes will be found in humans but they will probably not be patients with "pure epilepsy."     

Neuronal sensitivity to GABA and glutamate in generalized epilepsy with spike and wave discharges

In awake but painlessly immobilized cats the extracellular activity of the same cortical neurons was recorded before and for 2 to 5 h after the injection of penicillin G (350,000 IU/kg, i.m.) during the development of generalized epilepsy with bilaterally synchronous spike and wave discharges. Possible changes in their sensitivity to microiontophoretically applied glutamate and GABA during this period were searched for using computer-generated periejection histograms at intervals of about 30 min. In contrast to reported studies in other models of epilepsy, glutamate excited and GABA depressed virtually all neurons tested during fully developed spike and wave epilepsy. Spike height was not apparently affected either by the amino acids or by the development of epilepsy. Comparison of relative thresholds for the above effects on rhythmical neuronal activity associated with spike and wave discharge versus effects on random neuronal activity during the interburst periods, supported the idea that spikes and waves result from strong excitatory and inhibitory synaptic drives of the neurons. In all neurons until the appearance of spike and wave discharges, changes in the effect of amino acids, if observed, were small and statistically nonsignificant. This suggests that the hyperexcitability of cortical neurons which reportedly leads to the appearance of spike and wave discharges depends on mechanisms other than an increase in sensitivity to glutamate or a desensitization to GABA. Sometimes the sensitivity to GABA decreased later in this experimental model when the very frequent appearance of spike and wave discharges eventually led to EEG tonic-clonic seizures.     

Basic Science

The hormone melatonin has been reported to exhibit antiepileptic properties in clinical trials. However, recent animal studies have demonstrated that melatonin can have opposite effects on brain function, depending on the dose and timing of melatonin administration. In other words, although high pharmacologic doses are able to decrease brain excitability and suppress seizures, smaller doses of melatonin (administered at night when melatonin levels in the brain are highest), similar in amount to what is produced by the brain, can actually increase the excitability of neurons, making them more susceptible to seizure activity. In this study, we used an animal model of epilepsy to study the effects of melatonin on seizure development. We made two important observations: (a) seizures induced by the drug pilocarpine occurred with a shorter latency at night (when brain melatonin levels are highest) than during the day, and (b) when small doses of drug that block melatonin receptors are injected directly into the hippocampus, an area of the brain important for the development and spread of seizures, then seizures during the night were delayed. Furthermore, this effect was reversed by a drug that blocks the activity of GABA, the major inhibitory neurotransmitter in the brain, suggesting that melatonin may decrease GABA-receptor function in the hippocampus. Although we did not study the effects of melatonin directly, our data suggest that endogenous melatonin may enhance brain excitability and contribute to the development of epileptic seizures. This process may be involved with certain forms of nocturnal epilepsy and may raise a caution for persons with epilepsy who take melatonin. Epilepsia 2005;46(4).     

Extrasynaptic GABA and glutamate receptors in epilepsy

Epilepsy is a neurological disorder in which normal brain function is disrupted as a consequence of intensive and synchronous burst activity from neuron assemblies. Epilepsies result from long-lasting plastic changes in the brain affecting neurotransmitter release, the properties of receptors and channels, synaptic reorganization and astrocyte activity. There is considerable evidence for alterations in glutamatergic and GABAergic synaptic transmission in the origin of the paroxysmal depolarization shifts that initiate epileptic activity. However, recent studies on non-synaptic transmission, receptor mobility and glia-neuron signaling pathways suggest that extrasynaptic GABA and glutamate receptors may play an important role in seizure initiation, maintenance and arrest. Extracellular aminoacids such as glutamate, aspartate, glycine and GABA seem to communicate neurons and glial cells acting primarily on extrasynaptic receptors. Synaptic and extrasynaptic glutamate and GABA receptors have been show to play different roles in neuronal excitability. NMDA and GABAA receptors expressed in a single neuron can be differentially regulated based on subcellular localization, and it has been proposed that distinct regulation of synaptic versus extrasynaptic receptors provides a mechanism for receptor adaptation in response to a variety of stimuli. Furthermore, glutamate and GABA receptors are highly mobile, and the number and composition of extrasynaptic receptors can be modulated by several factors. This review addresses recent advances in our understanding of the role of extrasynaptic receptors in epilepsy, suggesting that extrasynaptic receptors and their mechanisms of regulation are expected to be important pharmacological targets.     

Recurrent neonatal seizures result in long‐term increases in neuronal network excitability in the rat neocortex

Neonatal seizures are associated with a high likelihood of adverse neurological outcomes, including mental retardation, behavioral disorders, and epilepsy. Early seizures typically involve the neocortex, and post‐neonatal epilepsy is often of neocortical origin. However, our understanding of the consequences of neonatal seizures for neocortical function is limited. In the present study, we show that neonatal seizures induced by flurothyl result in markedly enhanced susceptibility of the neocortex to seizure‐like activity. This change occurs in young rats studied weeks after the last induced seizure and in adult rats studied months after the initial seizures. Neonatal seizures resulted in reductions in the amplitude of spontaneous inhibitory postsynaptic currents and the frequency of miniature inhibitory postsynaptic currents, and significant increases in the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in pyramidal cells of layer 2/3 of the somatosensory cortex. The selective N‐methyl‐d ‐aspartate (NMDA) receptor antagonist d ‐2‐amino‐5‐phosphonovalerate eliminated the differences in amplitude and frequency of sEPSCs and mEPSCs in the control and flurothyl groups, suggesting that NMDA receptors contribute significantly to the enhanced excitability seen in slices from rats that experienced recurrent neonatal seizures. Taken together, our results suggest that recurrent seizures in infancy result in a persistent enhancement of neocortical excitability.     

Estradiol-induced changes in the activity of hippocampal neurons in network culture are suppressed by co-incubation with gabapentin

The ovarian steroid hormone estradiol, in addition to its function in the maintenance and regulation of reproductive capacity, can alter neuronal excitability. Estradiol is proconvulsant, increases neuronal excitability and decreases the threshold for seizure activity. Over one-third to one-half of women with epilepsy experience catamenial seizures, which are seizures influenced by cyclical hormone changes. These hormone-sensitive seizures respond to the anti-epileptic drug gabapentin, which is a structural analogue of the inhibitory amino acid neurotransmitter GABA. We studied the effects of 17-beta-estradiol alone and estradiol co-incubated with gabapentin on neuronal activity in network cultures of rat hippocampal neurons using a fluorescent calcium binding dye fluo-3 AM, FM 1-43 labeling of synaptic vesicles and electrophysiological recordings. Significant changes in the neuronal network activity were observed in the estradiol-treated neuronal cultures; the reactivity of the neurons to KCl depolarization induced intracellular calcium changes, and FM 1-43 destaining was increased as was the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSC). All these excitatory effects of estradiol were nullified by co-incubating the neurons with a combination of estradiol and gabapentin. This suggests that gabapentin can indeed affect the estradiol-induced changes in neuronal network hyperexcitability by influencing the neuronal calcium levels, exocytosis and synaptic activity. Our findings could provide an understanding of the cellular basis of hormone-sensitive seizure control by gabapentin.     

Effect of zonisamide on molecular regulation of glutamate and GABA transporter proteins during epileptogenesis in rats with hippocampal seizures

Epileptiform discharges and behavioral seizures may be the consequences of excess excitation associated with the neurotransmitter glutamate, or from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove amino acids from the synaptic cleft. Excitation initiated by the synaptic release of glutamate is attenuated by the action of glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino-acid carrier-1 (EAAC-1). GABA is removed from synaptic regions by the action of the transporters proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3). In this experiment, albino rats with chronic, spontaneous recurrent seizures induced by the amygdalar injection of FeCl3 were treated for 14 days with zonisamide (ZNS) (40 mg/kg, i.p.). Control animals underwent saline injection into the same amygdalar regions. Treatment control for both groups of intracerebrally injected animals was i.p. injection of equal volumes of saline. Western blotting was used to measure the quantity of glutamate and GABA transporters in hippocampus and frontal cortex. ZNS caused increase in the quantity of EAAC-1 protein in hippocampus and cortex and down regulation of the GABA transporter GAT-1. These changes occurred in both experimental and ZNS treated control animals. These data show that the molecular effect of ZNS, with up-regulation of EAAC-1 and decreased production of GABA transporters, should result in increased tissue and synaptic concentrations of GABA. Although many antiepileptic drugs have effects on ion channels when measured in vitro our study suggests that additional mechanisms of action may be operant. Molecular effects on regulation of transporter proteins may aid in understanding epileptogenesis and inform investigators about future design and development of drugs to treat epilepsy.