Epilepsy Research, 13 (1992) 4348 0920-1211/92/SO5.00 0 1992 Elsevier Science Publishers B.V. All rights reserved EPIRES 00507
Effects of loreclezole on epileptic activity and on EEG and behaviour in rats with absence seizures
N. Ates, E.L.J.M. van Luijtelaar, W.H.I.M. Drinkenburg, J.M.H. Vossen and A.M.L. Coenen Department
(Received 17 February 1992; revision received 12 May 1992; accepted 18 May 1992) Key words: Loreclezole; Spike-wave discharges; EEG, WAG/Rij rats; Absence epilepsy; Antiepileptic drugs
The antiepileptic profile of loreclezole, a new putative antiepileptic compound, has been determined in rats of the WAG/Rij strain, a genetic model of generalized absence epilepsy. In addition, the effects of 0, 5, 10 and 20 mg/kg loreclezole on the spectral content of the background EEG and on spontaneous behaviour of rats were investigated. Both the number of spike-wave discharges and their total duration dose-dependently decreased following administration of loreclezole. Furthermore, the behaviour of the animals was not markedly influenced and significant changes in the background EEG were not noticed after administration. These data suggest that the broad-spectrum antiepileptic loreclezole can be a valuable new drug in the treatment of absence epilepsy.
Some antiepileptic drugs are preferentially active against generalized tonic-clonic convulsions (e.g., diphenylhydantoin and carbamazepine), while some specifically prevent generalized absence seizures (e.g., ethosuximide and trimethadione). On the other hand, some show an efficacy against both types of epilepsy (clonazepam, phenobarbital and valproate). These latter compounds are called broad-spectrum antiepileptic drugs. Promising new compounds are currently undergoing preclinical and clinical evaluation. Loreclezole (R 72063) is an example of a new antiepileptic drug undergoing development. This triazole deriCorrespondence to: Dr. N. Ates, Department of Psychology, University of Nijmegen, P.O. Box 9104, 6500 HE Nijmegen, Netherlands.
vate blocks tonic-clonic seizures induced by pentylenetetrazol and allylglycine in a variety of animal models. Loreclezole fails, however, to have activity against maximal electroshock seizures16. The antiepileptic profile of loreclezole suggests that it may interact in a unique manner with the benzodiazepine receptor complex or signal transduction mechanism12. In animal studies, plasma concentrations of about 1 mg/l have been found to be effective and maximum blood levels are reached l-2 h after oral administration16. Until now, however, the effects of loreclezole were mainly evaluated in behavioural models, while few EEG-controlled studies were undertaken. Genetic rat models are the pertinent choice for human absence epilepsy and the WAG/Rij strain is such a model*. All members of this inbred strain show the electrophysiological and behavioural manifestations of generalized absence
epilepsy4,‘4v15. They spontaneously exhibit spikewave discharges (SWD), probably of thalamic origin”‘. The pharmacological profile of SWD resembles that of human absence epilepsy”. In the present EEG-controlled experiment, it was investigated whether loreclezole has an effect on generalized absence epilepsy of the WAG/Rij strain. In order to determine whether loreclezole also has side effects, spontaneous behaviour and background EEG were additionally measured and analysed. Materials and metbods Thirty-two male and female WAG/Rij rats, 6 months old and weighing 24&320 g, were used as experimental subjects. The animals were individually housed and had ad lib access to food and water. A 12-h light-dark cycle with white lights on at 20.00 h was maintained throughout the experiment. The experiment took place in the dark phase of the 24-h day. EEG electrodes (Plastics One, MS 333/2-A) were implanted under complete anaesthesia (Nembutal, 60 mg/kg i.p.) in the frontal and in the parietal region of the cortex with coordinates A 2.0, L 3.5 and A -6.0, L 4.0, respectively, with skull surface flat and bregma zero zero. An earth electrode was placed in the cerebellum. The animals were allowed to recover for at least 10 days before the experiments were started. Experiments were performed in freely moving animals. Loreclezole was synthesized at Janssen Pharmaceutica (Beerse, Belgium). It was dissolved in water and administered in suspension at dosages of 5, 10 and 20 mg/kg in a volume of 1 ml. Additionally, a 0 mg/kg control group was used to control for loreclezole treatment by injecting WAG/Rij rat with vehicle saline. Each group consisted of 8 subjects. The rats were adapted to the experimental and recording conditions for 18 h and a baseline EEG was then recorded for 1 h, starting at 10 h. Then, animals were intraperitoneally injected with loreclezole or solvent and, subsequently, the EEG was recorded for 4 consecutive hours. The EEG signal was amplified and filtered by an Elema-Schiinander polygraph and only frequencies between 1 and 70 Hz were allowed to pass. The
EEG was subsequently stored in digitized form on a magneto-optical disk (DATA Instruments, ATCODAS). SWD were visually scored according to criteria elaborated earlieri4. The number of SWD and the time accumulated by summing the durations of individual episodes of SWD were determined for each animal. These values were calculated for the baseline hour and for the 4 post-injection hours. Additionally, the spectral content of the interictal EEG phases was determined during representative periods of passive wakefulness in the first hour following loreclezole or vehicle administration. The minimum time period that was analysed was 25 s. These epochs were digitized with a rate of 512 samples per second. The power spectrum was calculated by means of a Fast Fourier Transformation (FFT) with a bin density of 1 Hz and normalized. Z-scores are then obtained with mean = 0 and variance = 1, to compensate for interindi~dual differences in amplification’. The contents of the following EEG frequency bands were determined: the delta band (l-4 Hz), the theta band (610 Hz), the sigma band (11-14 Hz), the beta 1 band (15-30 Hz) and the beta 2 band ( > 30 Hz). Spontaneous behaviour of the rats was observed and the duration of following behavioural categories was quantified for 30 min starting 30 min after injection: ‘exploratory’ behaviour (walking, rearing, sniffing, digging), ‘automatic’ behaviour (grooming, eating, drinking) and ‘passive’ behaviour (sitting, lying and standing still)3. Behavioural observations were done through a window from an adjacent room. The data were recorded and analysed with a PC registration software package’. The overall effects induced by the compound with respect to epileptic activity, behaviour and Zscores of EEG spectral analysis, were statistically analysed by means of a one-factor (doses) ANOVA, while post-hoc pairwise comparisons with Tukey’s protected t-test were used to test differences between groups. Results Effects of loreclezole on epileptic activity are presented in Figs. 1 and 2. During the 4-h post-injection period, the number of SWD was si~i~cantly
5 n ” BAsFLml?. HOUR
REGISTRATION TIME ~5
Fig. 1. Effects of loreclezole on the mean and standard deviation of the number of SWD during a baseline hour and 4 post-drug hours (n = 8). Statistical analyses were performed by means of ANOVA and post-hoc pairwise comparison test. l different from 0 mg/kg at P x0.05. **different from 0 mg/kg at P ~0.01.
REGISTRATION TIME 10
Fig. 2. Effects of loreclezole on the mean and standard deviation of the total duration of SWD during a baseline hour and 4 post-drug hours (n = 8). Statistical analyses were performed by means of ANOVA and post-hoc pairwise comparison test. ** different from 0 mg/ kg at P ~0.01.
The mean Z-scores of power density of the five frequency bands of the interictal EEG of the saline group were: delta = 2.41, theta = 2.27, sigma = 0.78, beta 1 = 0.13 and beta 2 = -0.48. The administration of loreclezole did not induce any significant changes EEG.
of the duration during solvent,
on the mean and standard
of passive and explorative
the first post-drug 5, 10 or 20 mg/kg
i.p. * different
of the background
Loreclezole exerted an effect in two categories of spontaneous behaviour, namely, passive and exploratory behaviour (F(3,32) = 2.99, P < 0.05 and F(3,32) = 3.63, P-=0.05) (Fig. 3). Loreclezole did not influence the behaviour after 5 mg/kg. However, 10 mg/kg loreclezole induced an increase in the duration of exploratory behaviour and a decrease in the duration of passive behaviour, compared to both saline and 20 mg/kg. The group receiving 20 mg/kg showed a significant increase in the duration of passive behaviour compared to the saline and 10 mg/kg groups.
Fig. 3. Effects of loreclezole
a single injection from 0 mg/kg
of at P
~0.05.** different from 0 mg/kg at P