Expression pattern of synaptic vesicle protein 2 (SV2) isoforms in patients with temporal lobe epilepsy and hippocampal sclerosis.

AIMS: Synaptic vesicle proteins 2 (SV2) are neuronal vesicles membrane glycoproteins that appear as important targets in the treatment of partial and generalized epilepsies. Therefore, we analysed the expression of SV2 isoforms in the hippocampus of patients with temporal lobe epilepsy (TLE). METHODS: SV2A, SV2B and SV2C immunostaining and QuantiGene branched DNA assay were performed on biopsies from 31 consecutive TLE patients with mesial temporal sclerosis (MTS) and compared with 10 autopsy controls. SV2 expression was further compared with Timm's staining, and synaptophysin, Zinc transporter 3 (ZnT3), dynorphin, vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) expression. RESULTS: In TLE patients, SV2A and SV2B expression was decreased in areas of synaptic loss. SV2C, which is weakly expressed or absent in the hippocampus of controls, was overexpressed in 10/11 cases with classical MTS1A and mossy fibre sprouting but not in cases with other types of MTS. SV2C staining was located in the inner molecular layer of the dentate gyrus and colocalized with dynorphin, ZnT3 and VGLUT1, suggesting selective expression in presynaptic glutamatergic Zn(2+) -rich terminals of abnormal sprouting fibres. SV2 expression patterns correlated with histological subtypes of MTS, but not with clinical features or therapeutic regimens in this patient cohort. CONCLUSION: In classical MTS1A, the expression of SV2 isoforms is altered with a marked decrease of SV2A and SV2B paralleling synaptic loss and a selective increase of SV2C in sprouting mossy fibres. These findings suggest a different physiology of sprouting synapses and the possibility to target them with SV2C-specific strategies.

Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A.

BACKGROUND AND PURPOSE: Screening of 12,000 compounds for binding affinity to the synaptic vesicle protein 2A (SV2A), identified a high-affinity pyrrolidone derivative, brivaracetam (ucb 34714). This study examined its pharmacological profile in various in vitro and in vivo models of seizures and epilepsy, to evaluate its potential as a new antiepileptic drug. EXPERIMENTAL APPROACH: The effects of brivaracetam and levetiracetam on epileptiform activity and seizure expression were examined in rat hippocampal slices, corneally kindled mice, audiogenic seizure-susceptible mice, maximal electroshock and pentylenetetrazol seizures in mice, hippocampal-kindled rats, amygdala-kindled rats and genetic absence epilepsy rats. KEY RESULTS: Brivaracetam and levetiracetam reduced epileptiform responses in rat hippocampal slices, brivaracetam being most potent. Brivaracetam also differed from levetiracetam by its ability to protect against seizures in normal mice induced by a maximal electroshock or maximal dose of pentylenetetrazol. In corneally kindled mice and hippocampal-kindled rats, brivaracetam induced potent protection against secondarily generalized motor seizures and showed anti-kindling properties superior to levetiracetam. In amygdala-kindled rats, brivaracetam induced a significant suppression in motor-seizure severity and, contrary to levetiracetam, reduced the after-discharge at a higher dose. Audiogenic seizure-susceptible mice were protected more potently against the expression of clonic convulsions by brivaracetam than by levetiracetam. Brivaracetam induced a more complete suppression of spontaneous spike-and-wave discharges in genetic absence epilepsy rats than levetiracetam. CONCLUSIONS AND IMPLICATIONS: Brivaracetam has higher potency and efficacy than levetiracetam as an anti-seizure and anti-epileptogenic agent in various experimental models of epilepsy, and a wide therapeutic index.

Antiepileptic drug discovery: lessons from the past and future challenges.

Historically, most antiepileptic drugs (AEDs) have been discovered either by serendipity, or the screening of compounds using acute seizure models. However, an increasing understanding of the molecular mechanisms underlying epileptogenesis has led to more rational approaches to drug discovery, which have focused on either enhancing inhibitory gamma-amino butyric acid (GABA)-ergic, or antagonizing excitatory glutamatergic, neurotransmission. Unfortunately, AEDs generated using such strategies have poor efficacy and safety profiles, as they interfere with normal cell processes, while ignoring the complex underlying pathophysiology of epilepsy. Recently, however, the use of new epilepsy models has led to the discovery of levetiracetam, an AED with a truly unique mechanism of action, devoid of anticonvulsant activity in normal animals, but with potent seizure suppression in genetic and kindled chronic epilepsy models, and an unusually high safety margin. The recent identification of brivaracetam and seletracetam, which optimize this unique mechanism of action, may further improve the medical management of epilepsy. The experience with levetiracetam, brivaracetam and seletracetam reveals that new experimental epilepsy models can detect AEDs possessing a unique mechanism of action and thereby target the future challenge of providing clinicians novel additions to the current armamentarium of AEDs.

The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents.

1. In this study in vitro and in vivo approaches were combined in order to investigate if the anti-epileptic mechanism(s) of action of levetiracetam (LEV; Keppra) may involve modulation of inhibitory neurotransmission. 2. GABA- and glycine-gated currents were studied in vitro using whole-cell patch-clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound-susceptible mice. The effect of LEV was compared with reference anti-epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. 3. LEV contrasted the reference AEDs by an absence of any direct effect on glycine-gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA-gated currents. A similar action on GABA-elicited currents was observed with the reference AEDs, except ethosuximide. 4. These minor direct effects contrasted with a potent ability of LEV (EC(50)=1 - 10 microM) to reverse the inhibitory effects of the negative allosteric modulators zinc and beta-carbolines on both GABA(A) and glycine receptor-mediated responses. 5. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of beta-carbolines on GABA-gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine-gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. 6. LEV (17 mg kg(-1)) produced a potent suppression of sound-induced clonic convulsions in mice. This protective effect was significantly abolished by co-administration of the beta-carboline FG 7142, from a dose of 5 mg kg(-1). In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg(-1)) was without any effect on the protection afforded by LEV. 7. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti-epileptic mechanism(s) of action of LEV.

Levetiracetam: the preclinical profile of a new class of antiepileptic drugs?

Levetiracetam is a new antiepileptic drug (AED) devoid of anticonvulsant activity in the two classic screening models for AEDs, the maximal electroshock and pentylenetetrazol seizure tests in both mice and rats. This contrasts a potent seizure suppression in genetic and kindled mice and rats and against chemoconvulsants inducing partial seizures in rats. The highly selective action in "epileptic" animals distinguishes levetiracetam from classic and other new AEDs that have nearly equipotent effects in normal and "epileptic" animals. Levetiracetam induces minor behavioral alterations in normal and in kindled mice and rats. This results in an unusually high safety margin in animal models reflecting both partial and primary generalized epilepsy. Furthermore, experiments in the kindling model suggest that levetiracetam may possess antiepileptogenic properties due to a potent ability to prevent the development of kindling in mice and rats at doses devoid of adverse effects. Electrophysiologic recordings from different experimental models suggest that levetiracetam exerts a selective action against abnormal patterns of neuronal activity, which probably explains its selective protection in epileptic animals and its unique tolerability. This effect appears to derive from one or more novel mechanisms of action that do not involve a conventional interaction with traditional drug targets implicated in the modulation of inhibitory and excitatory neurotransmission. Instead, ligand-binding assays have disclosed a brain-specific binding site for levetiracetam. These studies reveal a unique preclinical profile of levetiracetam, distinct from that of all known AEDs, suggesting that levetiracetam could represent the first agent in a new class of AEDs.

Can gap-junction blockade preferentially inhibit neuronal hypersynchrony vs. excitability?

Epileptic activity involves a hypersynchronous firing of large populations of brain neurons, some of which are hyperexcitable. This study explored to what extent gap-junction blockade inhibits neuronal synchronization vs. neuronal excitability. We investigated the effects of the gap-junction blockers (GJBs) 1-heptanol, 1-octanol and carbenoxolone vs. the loop diuretic furosemide on spontaneous and evoked epileptiform field potentials, induced in CA3 area of rat hippocampal slices by a 'high K(+)-low Ca(2+)' perfusion fluid. This milieu induced frequent (>30 min(-1)) spontaneous bursts, led single fimbrial stimuli to evoke repetitive population spikes (PSs), and increased PS amplitudes. Both furosemide and the three GJBs gradually reduced spontaneous field bursting, or even stopped it within one hour. The anti-bursting activity of carbenoxolone showed dose-response dependence in the concentration range 50-400 microM. 1-heptanol and 1-octanol markedly and similarly depressed all the epileptiform markers of the evoked responses, whereas carbenoxolone did not reduce the number of repetitive PSs evoked by single stimuli. By its minor effect on evoked responses, carbenoxolone resembled furosemide, rather than its congeners GJBs. These results favor the possibility that selective gap-junction blockade might antagonize epileptic synchronization, without depressing neuronal excitability.

Levetiracetam does not modulate neuronal voltage-gated Na+ and T-type Ca2+ currents.

This study investigated whether the mechanism of action of levetiracetam (LEV) is related to effects on neuronal voltage-gated Na+ or T-type Ca2+currents. Rat neocortical neurones in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na+ current. Additionally, visually identified pyramidal neurones in the CA1 area of rat hippocampal slices were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study low-voltage-gated (T-type) Ca2+ current. LEV (10 microM-1 mM) did not modify the Na+ current amplitude and did not change (200 microM) the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the Na+ current. Likewise, LEV (32-100 microM) did not modify the amplitude and did not change the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the T-type Ca2+current. In conclusion, neuronal voltage-gated Na+ channels do not appear directly involved in the antiepileptic mechanism of action of LEV, and LEV was devoid of effect on the low-voltage-gated (T-type) Ca2+ current in hippocampal neurones.

Neuroprotective properties of the novel antiepileptic drug levetiracetam in the rat middle cerebral artery occlusion model of focal cerebral ischemia.

Levetiracetam (LEV) is a new antiepileptic drug with a promising preclinical profile involving both anticonvulsant and antiepileptogenic effects in kindling models. The latter stimulated the present study to compare its neuroprotective properties with the potent and selective, non-competitive NMDA antagonist, MK-801, in the rat middle cerebral artery occlusion model. Twenty-four hours after a transient occlusion of 90 minutes the animals were sacrificed and infarct volume and lesion distribution were determined from stained coronal sections. LEV was administered by intraperitoneal (i.p.) bolus injections of 5.5, 11, 22 and 44 mg x kg(-1), 30 minutes before occlusion followed by a continuous 24 hour i.p. infusion of 1.25, 2.6, 5.1 and 10.2 mg x kg(-1) per hour, respectively. LEV administration did not alter body temperature but reduced the infarct volume by 33% (P< 0.05) at the highest dose tested. An i.p. bolus injection of 0.04, 0.12 and 0.4 mg x kg(-1) of MK-801 followed by continuous i.p. infusion of 0.036, 0.108 and 0.36 mg x kg(-1) per hour, reduced the infarct volume by 49, 51 and 74% (P< 0.05), respectively. However, only the highest dose of MK-801 induced a significant reduction in the infarct volume (P< 0.05) and this was associated with hypothermia. These results suggest that LEV possesses neuroprotective properties which may be relevant for its antiepileptogenic action.

Pharmacological evaluation of a diarylmethylene-piperidine derivative: a new potent atypical antipsychotic?

A new diaryl-methylene piperidine derivative, 2, displayed an atypical antipsychotic profile both in vitro and in vivo. The main pharmacological characteristics of this compound appears to reside in a more potent antagonism of the 5-HT2 serotonergic receptor than of the D2 dopaminergic receptor. This confirms that molecules displaying a D2/5-HT2 binding ratio < 1 possess clozapine-like antipsychotic activity.

Levetiracetam inhibits the high-voltage-activated Ca(2+) current in pyramidal neurones of rat hippocampal slices.

The effect of the new antiepileptic drug levetiracetam (LEV; KEPPRA) on the neuronal high-voltage-activated (HVA) Ca(2+) current was investigated on pyramidal neurones, visually identified in the CA1 area of rat hippocampal slices. Nystatin-perforated patch clamp recordings were made under experimental conditions designed to study HVA Ca(2+) currents. The HVA current, activated by steadily increasing voltage-ramps, was reversibly eliminated by Cd(2+) and depressed by either nimodipine, or omega-Conotoxin GVIA. After 30 min perfusion of the slices with LEV 32 microM, the current decayed to 55+/-9% (mean+/-SEM; n=9) of the initial value, which is significantly (P<0.05, two-tailed t-test) lower than the rundown to 84+/-10% in a control group (n=10) of neurones. The limited, but significant depression of the neuronal HVA Ca(2+) current, produced by LEV at a clinically relevant concentration, might contribute to the antiepileptic action of the drug.

Failure of GPI compounds to display neurotrophic activity in vitro and in vivo.

The aim of this study was to evaluate the neurotrophic and neuroprotective properties of a series of immunophilin ligands and to assess the potential involvement of FK506 Binding Protein 12 kDa (FKBP12) rotamase inhibition in this activity. Both FK506 and rapamycin induced a potent inhibition of the FKBP12 rotamase activity (pIC(50) values of 7.3 and 7.4, respectively) but only a modest inhibition was observed with 1-(3,3-dimethyl-2-oxo-pentanoyl)-pyrrolidine-2-carboxylic acid S-3-pyridin-3-yl-propyl ester (GPI 1046) (5.8), its N-oxide (5.4) and thioester (6.3) analogues. Compared to nerve growth factor, all these immunophilin ligands only induced marginal increases in neurite outgrowth of rat dissociated newborn dorsal root ganglia cells. Furthermore, systemic administration of GPI 1046 and its N-oxide and thioester analogues failed to prevent striatal dopamine depletion induced by acute or chronic i.p. treatment with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP). These results suggest that inhibition of FKBP12 rotamase activity is not predictive for neurotrophic and neuroprotective properties of immunophilin ligands and question their therapeutic utility in neurodegenerative diseases like Parkinson's disease.

Inhibition of neuronal hypersynchrony in vitro differentiates levetiracetam from classical antiepileptic drugs.

Field potentials were recorded from rat hippocampal slices in order to compare the electrophysiological action of the new antiepileptic drug (AED), levetiracetam (LEV), with that of the classical AEDs, valproate, clonazepam and carbamazepine, on epileptiform responses induced by a 'high K(+)-low Ca(2+)' perfusion fluid. Increasing [K(+)] from 3 to 7.5 mM and decreasing [Ca(2+)] from 2.4 to 0.5 mM, in the bathing fluid, produced population spikes (PSs) of increasing amplitudes in the CA3 area of the slices, repetitive PSs evoked by single stimuli, and spontaneous bursts. Clinically relevant concentrations of LEV, 32 and 100 micro M, consistently reduced the second (PS(2)) and third (PS(3)) population spikes, and the number (N) of repetitive PSs per evoked response. Levetiracetam 32 micro M also opposed the increase in amplitude of the first PS (Delta PS(1)). Neither valproate 1 mM, nor clonazepam 1 micro M, nor carbamazepine 50 micro M, produced any decrease in Delta PS(1)and in PS(2), but all decreased N. These results show that LEV contrasts to reference AEDs by its ability to antagonize neuronal (hyper)synchronization, in the highly seizure-prone CA3 area of rat hippocampal slices.

Absence of negative impact of levetiracetam on cognitive function and memory in normal and amygdala-kindled rats.

The effect of the new antiepileptic drug (AED) levetiracetam (LEV, Keppra) on cognitive function was studied in normal and amygdala-kindled rats by using the Morris water maze test. In addition, we investigated the effect of LEV on long-term potentiation (LTP) in rat hippocampal slices. Sodium valproate (VPA) was used as comparator in all studies. Clonazepam (CZP) and carbamazepine (CBZ) were used in normal rats. The results indicated that doses of LEV known to suppress motor seizures did not alter cognitive performance. In contrast, similar doses of the classic AEDs all decreased learning performance of the rats. Likewise, VPA did alter LTP but LEV was inactive. Amygdala-kindled rats were more sensitive than normal rats to the effects of VPA. These results suggest that LEV may be devoid of negative impact on cognition in epileptic patients.

Chronic electrode implantation entails epileptiform field potentials in rat hippocampal slices, similarly to amygdala kindling.

Evoked field potentials were recorded in the CA3, CA1 and dentate gyrus (DG) of hippocampal slices from amygdala kindled, non-stimulated amygdala electrode-implanted, and non-implanted age-matched rats to evaluate the consequences on hippocampal neuronal networks of kindling stimulation versus electrode implantation. No overt modification of field potentials was detected in either the CA1 or the DG areas. In contrast, a very significant increase in the occurrence of repetitive population spikes evoked by single stimuli was detected in the CA3 area in slices from both amygdala kindled and non-stimulated amygdala implanted rats. The epileptiform pattern of CA3 field potentials was at least as well expressed in implanted non-stimulated, as in kindled rats, suggesting that electrode implantation has a major contribution to this marker of epileptogenesis.

Evidence for a unique profile of levetiracetam in rodent models of seizures and epilepsy.

The protective and adverse effect potentials of levetiracetam ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide) in rodent models of seizures and epilepsy were compared with the profile of several currently prescribed and newly developed antiepileptic drugs. Levetiracetam was devoid of anticonvulsant activity in the acute maximal electroshock seizure test and in the maximal pentylenetetrazol seizure test in mice (up to 540 mg/kg, i.p.) but exhibited potent protection against generalised epileptic seizures in electrically and pentylenetetrazol-kindled mice (ED50 values = 7 and 36 mg/kg, respectively, i.p.). This differs markedly from established and most new antiepileptic drugs which induce significant protection in both the acute seizure tests and the kindling models. Furthermore, levetiracetam was devoid of anticonvulsant activity in several maximal chemoconvulsive seizure tests although an interesting exception was the potent protection observed against secondarily generalised activity from focal seizures induced by pilocarpine in mice (ED50 value = 7 mg/kg, i.p.), pilocarpine and kainic acid in rats (minimum active dose = 17 and 54 mg/kg, respectively, i.p.). The protection afforded by levetiracetam on the threshold for methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)-induced seizures persisted after chronic administration (17-170 mg/kg, i.p., twice daily/14 days) and levetiracetam did not lower the seizure threshold for the proconvulsant action of the inverse benzodiazepine receptor agonist, N-methyl-beta-carboline-3-carboxamide (FG 7142). The main metabolite of levetiracetam (ucb L057; (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid) was found to be inactive in sound-sensitive mice after acute administration of doses up to 548 mg/kg, i.p. Levetiracetam induced only minor behavioural alterations in both normal and amygdala-kindled rats (54-1700 mg/kg, i.p.) resulting in an unusually high safety margin between rotarod impairment and seizure suppression of 148 in corneally kindled mice and 235 in Genetic Absence Epilepsy Rats from Strasbourg. In comparison, existing antiepileptic drugs have ratios between 2 and 17 in the corneally kindled mouse model. These studies reveal a unique profile of levetiracetam in rodent models. Characteristics are a general lack of anticonvulsant activity against maximal, acute seizures and selective protection with a very high safety margin in genetic and kindled animals and against chemoconvulsants producing partial epileptic seizures. This activity differs markedly from that of the established and newly introduced antiepileptic drugs and appears to derive from the parent compound since its major metabolite was inactive in all models studied. Together these results therefore suggest that levetiracetam may offer an effective, broad-spectrum treatment of epileptic seizures in patients, with a minimum of adverse effects.

Validation of corneally kindled mice: a sensitive screening model for partial epilepsy in man.

Epileptogenesis induced by electrical kindling of rats appears to be superior to the acute maximal electroshock seizure (MES) test in normal animals in predicting the efficacy and adverse effect potential of drugs in patients with partial epilepsy. Unfortunately, inclusion of such kindling models in primary screening is hampered by the laborious and expensive procedure of stimulation and recording with implanted brain electrodes. Furthermore the size of the rats excludes their use in initial testing where compound availability is often limited for the 'first batch synthesis'. The present study demonstrates that chronic electrical stimulation with corneal electrodes in mice can rapidly yield large numbers of kindled animals with a seizure phenomenology reflecting partial seizures in man. A pharmacological characterisation showed that corneally kindled mice can be used repeatedly for several drug experiments with reproducible results. The seizure protection and adverse effect potential obtained with proven antiepileptic drugs were similar to the effects observed in amygdala kindled rats and their corresponding clinical profile in partial epilepsy. Protection was obtained with vigabatrin and levetiracetam in this new model despite their lack of anticonvulsant activity in the acute MES test. Furthermore, in agreement with clinical findings with NMDA antagonists, MK-801 revealed more severe adverse effects in corneally kindled mice than in normal animals. These results suggest that corneal kindling of mice represents a sensitive and valid screening model for the identification of new therapies for partial epilepsy in man.

Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure.

To explore further alterations in skeletal muscle in chronic heart failure (CHF), we examined myosin heavy chain (MHC) isoforms from biopsies of the vastus lateralis in nine male patients with class II-III (CHF) (left ventricular ejection fraction (LVEF) 26 +/- 11%, peak oxygen consumption (peak VO2) 12.6 +/- 2 mL.kg-1.min-1) and nine age-matched sedentary normal males (NL). The relative content of MHC isoforms I, IIa, and IIx was determined by gel electrophoresis as follows: The normal sedentary group (NL) had a higher percent of MHC type I when compared with the patients (NL 48.4 +/- 7% vs CHF patients 24 +/- 21.6%, P < 0.05, no difference between MCH IIa (NL 45.1 +/- 10.5% vs CHF 56.0 +/- 12.5%), and CHF patients had a higher relative content of MHC type IIx than did the normal group (NL 6.5 +/- 9.6% vs CHF 20.0 +/- 12.9%, P < 0.05. Three of nine patients had no detectable MHC type I. In patients relative expression of MHC type I (%) was related to peak VO2 (r = 0.70, P < 0.05). Our results indicate that major alterations in MHC isoform expression are present in skeletal muscle in CHF. These alterations parallel previously reported changes in fiber typing that may affect contractile function i skeletal muscle and possibly exercise performance. The absence of MHC type I in some CHF patients suggests that skeletal muscle changes in this disorder are not solely a result of deconditioning, buy may reflect a specific skeletal muscle myopathy in this disorder.

Anticonvulsant profile of the imidazoquinazolines NNC 14-0185 and NNC 14-0189 in rats and mice.

The anticonvulsant effects of NNC 14-0185 (3-(3-cyclopropyl-5-isoxazolyl)-6-fluoro-5-morpholino-imidazo[1,5- a] quinazoline) and NNC 14-0189 (3-(5-cyclopropyl-1,2, 4-oxadiazol-3-yl)-7-fluoro-5-(4-methyl-1-piperazinyl)-imidazo[1,5- a] quinazoline) in mice and rats were evaluated and compared with those of diazepam, clonazepam and the novel beta-carboline, abecarnil. Following i.p. administration, NNC 14-0185 and NNC 14-0189 prevented audiogenic seizures in DBA/2 mice and the clonic convulsions induced in mice by pentylenetetrazole, DMCM (methyl 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), 3-mercaptopropionic acid and a low dose of bicuculline. NNC 14-0185 and NNC 14-0189 prevented seizures induced by pentylenetetrazole in rats and were also effective anticonvulsants in amygdala-kindled rats. In general, the anticonvulsant potencies of NNC 14-0185 and NNC 14-0189 were comparable to those of the reference benzodiazepines. However, like abecarnil, they were not effective against the seizures induced in mice by maximal electroshock and a high dose of bicuculline. The anticonvulsant effects of NNC 14-0185 and NNC 14-0189 against pentylenetetrazole-induced seizures were apparent within 5 min of i.p. injection and persisted for at least 2 h. These effects appeared to be mediated by benzodiazepine receptors since they were inhibited by concurrent administration of flumazenil. Both NNC 14-0185 and NNC 14-0189 showed greater separation between their anticonvulsant and muscle relaxant effects (measured as impaired rotarod performance) than did diazepam. In this respect, their therapeutic windows were similar (NNC 14-0185) to or better (NNC 14-0189) than that of abecarnil. Tolerance did not develop to the anticonvulsant effects of NNC 14-0185 and NNC 14-0189 over a 4-day test. In comparison, the anticonvulsant effects of diazepam and abecarnil were attenuated by repeated drug administration. Thus, NNC 14-0185 and NNC 14-0189 have a promising anticonvulsant and side-effect profile in comparison with diazepam, clonazepam and abecarnil. The potential use of these compounds in the treatment of epilepsy should be explored further.

Repetitive transcranial magnetic stimulations of the rat. Effect of acute and chronic stimulations on pentylenetetrazole-induced clonic seizures.

Repetitive transcranial magnetic stimulation (RTMS) has been reported to induce epileptic seizures in normal control and in epileptic patients. Therefore we characterized the effect of acute or chronic stimulations with RTMS on the induction of pentylenetetrazole (PTZ)-induced clonic seizures in the rat. Male Wistar rats were stimulated with a 13-cm coil with a stimulus frequency of 50 Hz. The motor threshold (Tm) was determined by a single transcranial stimulus. Acute stimulation was performed with a stimulus intensity of 0.9 x Tm and 1.5 x Tm using a duration of the train of stimuli of five seconds. Chronic stimulation was performed with a duration of the train of stimuli of one and five seconds using a stimulus intensity of 1.8 x Tm, every day for 30 days. Time to onset of PTZ-induced clonic seizure was determined after the acute stimulation or the last stimulation in chronic RTMS. In the groups of rats receiving acute RTMS (0.9 and 1.5 x Tm) no seizures developed. No differences were observed in time to onset of clonic seizures after PTZ injection compared to control rats. In the group of rats receiving chronic RTMS some rats showed facial contractions, chewing or head movements during or immediately after the stimulations. None of the rats developed tonic or clonic seizures in relation to RTMS. Time to onset of PTZ clonic seizures was reduced in both groups receiving RTMS with a stimulus duration of one (P < 0.01) and five (P < 0.05) seconds compared to control rats. The results from this study suggest that acute suprathreshold stimulation with a stimulus frequency of 50 Hz does not affect the induction of clonic PTZ seizures, whereas chronic (daily) stimulations have a facilitatory effect. This indicates that chronic stimulation with RTMS may induce a kindling process in the rat. Chronic RTMS stimulation may therefore represent an interesting alternative non-invasively kindling model to chemical and electrical stimulations.

Anticonvulsant activity of novel derivatives of 2- and 3-piperidinecarboxylic acid in mice and rats.

The relative ability of derivatives of 2-piperidinecarboxylic acid (2-PC; pipecolic acid) and 3-piperidinecarboxylic acid (3-PC; nipecotic acid) to block maximal electroshock (MES)-induced seizures, elevate the threshold for electroshock-induced seizures and be neurotoxic in mice was investigated. Protective index (PI) values, based on the MES test and rotorod performance, ranged from 1.3 to 4.5 for 2-PC benzylamides and from < 1 to > 7.2 for 3-PC derivatives. PI values based on elevation of threshold for electroshock-induced seizures and rotorod performance ranged from > 1.6 to > 20 for both types of derivatives. Since preliminary data indicated that benzylamide derivatives of 2-PC displace [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine (TCP) binding to the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor in the micromolar range and such low affinity uncompetitive antagonists of the NMDA receptor-associated ionophore have been shown to be effective anticonvulsants with low neurological toxicity, the 2-PC derivatives were evaluated in rat brain homogenates for binding affinity to the PCP site. Although all compounds inhibited [3H]TCP binding, a clear correlation between pharmacological activity and binding affinity was not apparent. Select compounds demonstrated minimal ability to protect against pentylenetetrazol-, 4-aminopyridine- and NMDA-induced seizures in mice. Corneal and amygdala kindled rats exhibited different sensitivities to both valproic acid and the nonsubstituted 2-PC benzylamide, suggesting a difference in these two models. Enantiomers of the alpha-methyl substituted benzylamide of 2-PC showed some ability to reduce seizure severity in amygdala kindled rats.