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Effects of 3-hydroxy,3-ethyl,3-phenylpropionamide (HEPP) on rat models of generalized and focal epilepsy

Sim6n Brailowskya, Teresa Montiela, Elizabeth HernAndeza, Christian Marescauxb and Marguerite Vergnesb olnstituto de Fisiologfa Celular, U.N.A.M., Mexico 04510 D. F. (Mexico) and bL. N. B.C., Centre de Neurochimie, C.N.R.S.-I.N.S.E.R.M., Stwbourg67084(France)

(Received 10 September 1991; revision received 20 January 1992; accepted 22 January 1992) Key words: GABA withdrawal syndrome; Absences; Spike-and-wave; Antiwnvulsants;

EEG

The GABA withdrawal syndrome (GWS) is a new model of focal epilepsy in which paroxysmal activity is induced through the interruption of a chronic, intracortical infusion of GABA. Preliminary studies have shown extraordinary resistance of this epileptogenic activity to classic antiwnvulsants including diazepam, the most effective agent for treating status epilepticus. However, GWS can be inhibited by GABA itself. The rat with petit mal-like seizures is a genetic model of generalized non-convulsive epilepsy (GNCE), with behavioral characteristics and electrical (spike-and-wave discharges) signs resembling absences. Moreover, GABAmimetics aggravate this type of seizure. Rats with GWS induced by cessation of a localized GABA infusion (50&d/h for 24 h), and the rat model of GNCE, were treated with HEPP, a new anticonvulsant agent. In the case of GWS, the drug produced a significant decrease of focal spike activity in animals which started discharging at low frequencies while in rats with higher frequency discharge, HEPP was without effect. HEPP administered on the second day of the GWS in naive rats had no effect. In rats with GNCE, doses of 50 and 100 mgikg i.p. blocked the spike-and-wave discharges. The higher dose produced sedation in this absence seizures model. Although the mechanism of action of HEPP is still unknown, its unique antiepileptic profile deserves further studies.

INTRODUCTION The search for new anticonvulsant agents and of animal models with epileptic syndromes to test these drugs is of key importance to the neuropharmacology of epilepsy. Carvajal et a1.4 synthesized 5-hydroxy-5-ethyl-5 Correspondence to: Dr. Sim6n Brailowsky, Departamento de Neurociencias, Instituto de Fisiologfa Celular, U.N.A.M., Apdo. Postal 70-600, Mexiw 04510 D.F., Mexico.

phenyl-butyramide (HEPB) while investigating drugs capable of enhancing GABAergic neurotransmission. Although this compound did not significantly increase cerebral y-aminobutyric acid (GABA) levels4, it showed anticonvulsant effects against electroshock seizures in rats and mice, and against convulsions induced by bicuculline and by glutamate decarboxylase inhibitors. The drug also reduced the duration and spread of afterdischarges produced by hippocampal electrical stimulation in cats’. Interestingly, HEPB potentiated

0920-1211/92/$05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved

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convulsions induced by strychnine, a glycine receptor antagonist’. More recently, Carvajal’s group has synthesized the lower homologues of HEPB: 3-hydroxy-3ethyl-3-phenyl-propionamide (HEPP) and 2-hydroxy-2-ethyl-2-phenyl-acetamide (HEPA). Of these two compounds HEPP displays the lowest toxicity and the highest protective index against seizures induced by electroshock, pentetrazol, 4aminopyridine, bicuculhne and thiosemicarbazide’. The present work reports effects of HEPP on two rat models of epilepsy: the GABA withdrawal syndrome (GWS) and the genetic model of absence seizures. The GWS is a new model of focal epileptogenesis induced by the cessation of localized, intracortical infusions of GABA”“. Paroxysmal activity arises at the GABA infusion site and lasts from hours to days, depending on infusion time and dose. The distinct electro-clinical profile of GWS consists of spikes, polyspikes and spike-and-wave discharges (SWD) which may be associated with limb myoclonus 2. The GWS has been found both in baboons3 and in rats. Preliminary pharmacological studies show that this focal activity is quite resistant to both ‘classic’ (i.e., barbiturates, phenytoin, carbamazepine, valproate, progabide, benzodiazepines) and new anticonvulsant agents (i.e., NMDA receptor antagonists, such as amino-phosphonoheptanoate (APH), ketamine or MK-801). This reduced sensitivity of the GWS to anticonvulsant drugs is time-dependent. Thus, APH or benzodiazepines have no effect on the first day of epileptic activity, but are effective on the second day of the syndrome. The Wistar rat with spontaneous SWD is an experimental model of generalized non-convulsive epilepsy (GNCE). Vergnes et al.” developed a colony of rats that fulfil the criteria for animal models of absence seizures: (1) electro-clinical similarities with human petit ma1 seizures; (2) arrested movement initiation and reduced responsiveness during SWD; (3) increased occurrence of SWD with decreased arousal and SWD blockade by arousal or movement; (4) characteristic developmental profile; and (5) a pharmacological responsiveness resembling human absences (i.e., antiepileptic ef-

fects of anti-absence drugs and enhancement with GABAergic agents) (see Snead7). Since GWS displays sensitivity to GABA itself (blockade of paroxysmal activity)2, and the rat model of absence seizures shows enhancement of epileptic activity with GABAmimetics”‘, it was of interest to assess the effects of HEPP on these two models of epilepsy. METHODS G WS experiments

Male Wistar rats from the U.N.A.M. breeding colony weighing 250-300 g at surgery were used. Animals were individually housed in plastic cages (37 X 27 X 16 cm) and maintained under controlled temperature and light/dark cycles (1202 h) with food and water ad libitum. Surgical procedures consisted of implantation of epidural screws for EEG recording, a reference electrode over the frontal sinus and stainless steel cannulae (external diameter = 0.5 mm) for both drug infusion and electrocorticography. These cannulae were stereotaxically implanted over the left somatomotor cortex (2 mm posterior to bregma, 2 mm lateral, 1.5 mm depth from bone surface) using the flat skull position2. One week after surgery, a control EEG was obtained to confirm the absence of behavioral or EEG abno~alities. Recordings were obtained with a Grass polygraph (Mod. 78), from homologous areas of the somatomotor region (filters = 0.3-300 Hz). The next day osmotic minipumps (Alza model 2001, delivery rate = 1 yllh) were subcutaneously implanted under halothane anesthesia. The minipumps containing GABA (50 ,@pl, pH = 7.33, purchased from Sigma) or its vehicle, saline (pH = 6.8), were incubated in saline at 37°C for at least 5 h before implantation. For the assessment of minipump function, direct blue (1 mg/5 ml) was added to the solutions. Only those rats with a blue mark at the infusion site were considered for analysis. After 24 h of continuous infusion, the rats were recorded continuously for 30 min, after which the catheters were atraumatically disconnected from the intracerebral cannulae. EEG recording was continued until the appearance of paroxysmal ac-

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tivity. Sixty minutes after GWS began, HEPP was administered i.p. at a dose of 50 mg/kg. The drug was solubilized in warm saline. The same procedure was followed in a separate group of rats but the drug was given 24 h after the beginning of GWS.

Ten days after the termination of paroxysmal activity the rats were deeply anesthetized and prepared for histological analysis (Nissl and glial fibrillary acidic protein (GFAP) staining). Results are expressed as median of frequency discharge and analyzed using the Kruskal-Wallis

EFFECTS OF HEPP ON GWS

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J2oopv I set Fig. 1. EEG effects of HEPP on the GABA withdrawal syndrome (GWS) in the rat. GABA (5O&Uh for 24 h) was infused into the left somatosensory cortex (MCx). The upper half of the figure shows the temporal evolution of the paroxysmal discharges. HEPP was administered i.p. 1 h after the beginning of epileptic activity. The drug induced a decrease in the frequency discharge that lasted for approximately 2 h, with recovery thereafter.

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test, where the comparisons were control vs. HEPP with effect and control vs. HEPP without effect. GNCE experiments

Male Wistar rats from the Strasbourg breeding unit with spontaneous SWD were implanted as described previously’“. HEPP was administered at a 50 mg/kg dose, dissolved in saline, or at 25,50 and 100 mg/kg dose dissolved in a mixture of propylene-glycol (40%)) ethanol (lo%), distilled water (50%) and 2 drops of Tween 80 for a total volume of 10 ml. The mixture was pre-warmed to 55°C and administered i.p. in volumes of 2 ml/kg i.p. Each dose, and its solvent, were given to groups of 8 rats. After 15 min habituation to the recording conditions, the EEG was recorded for 20 min before and 80 min after the injection. Results were expressed in mean duration of electrical seizures per 20-min period and were statistically analyzed using the Mann-Whitney test and the Wilcoxon rank sum test for comparisons between drug and control (dose 0) conditions, and for each dose (SPSSPC + package). Since HEPP was found to be unstable in saline solution, it had to be injected immediately after preparation. A different solvent was therefore used in GNCE experiments. RESULTS G WS experiments

Only rats (n = 10) which showed focal spikes with cessation of GABA infusion were considered for analysis. Latency in the appearance of epileptic discharges ranged from 27 to 70 min. Two types of response were obtained with HEPP administration (50 mg/kg i.p.) 60 min after the onset of focal paroxysmal activity: either a significant reduction in spike frequency discharge during about 90 min (Fig. 1)) or no antiepileptic effect at all. When animals were grouped into HEPP responders versus non-responders, the latter showed significantly higher spike discharge frequencies during the initial 5 min of the GWS (median = 19.2) than the former (median = 4.2). Nevertheless, 10 min after GWS onset responders (n = 5) reached discharge frequencies similar to

Fig. 2. Time course of the frequency of GWS paroxysmal discharges before and after HEPP administration. The curve starts 5 min after GWS onset and includes the values (median) for all three groups in the first hour (black squares). HEPP (50 mg/kg i.p.) was administered at the arrow. The drug produced a significant (P < 0.05, Kruskal-Wallis test) decrease in frequency discharge in 50% of the rats (group 2, n = 5). In rats which did not respond to the drug (group 3, n = 5), spike frequency discharge was similar to that of untreated animals (‘GWS control’, group 3, n = 9). See text for details.

those of non-responders. A control group (n = 9) with GWS and no treatment showed similar temporal evolution of spike discharges to that of rats where HEPP had no effect (Fig. 2). A different group of rats (n = 5) was treated with HEPP 24 h after the onset of GWS. No effects were observed either in the discharge frequency or in EEG patterns 105 min after drug injection. Histological analysis showed an area of gliosis localized at the cortical infusion site and in the thalamic projection area, similar to one previously reported’. The thalamic degenerative changes were observed in the ventro-lateral group and in the reticular nucleus. Animal variability concerning the thalamic degenerative alterations correlated with differences in cortical location of the infusion cannula but not with drug response. GNCE experiments

As shown in Fig. 3, higher doses of HEPP (50 and 100 mg/kg) significantly decreased the SWD. The effect was observed with saline and with Tween 80 solvents. The Tween 80 solution in-

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Fig. 3. Mean (+ SEM) duration of SWD during five successive 20-min periods (in roman numerals) before (I) and after (II-V) i.p. injection of various doses of HEPP. The doses are given in mgkg, indicating the solvent used: propylene glycol+ethanol+Tween 80 +water on the left, and saline on the right. The drug had significant (‘P < 0.05, **P < 0.005, Wilcoxon test) antiepileptic effects, particularly with the 50 and 100 mgkg doses (n = 8 rats/dose).

duced a discrete anti-absence effect during the first 40 min after its administration (Fig. 3). The antiepileptic action of HEPP was accompanied by sedative effects, more marked with the 100 mg/kg dose, and reversible with sensory stimulation. The 25 mg/kg dose had similar effects to the solvent on its own and was not sedative. DISCUSSION Two possible effects of HEPP on the GWS model of focal epileptogenesis were obtained: either the drug had antiepileptic effects of it did not. The distinctive sign of the population which experienced antiepileptic effects was a low spike frequency discharge (< 5 cps) within the first 5 min of GWS onset, although this frequency reached similar values to the non-responder group (28 vs. 29.1 spikesimin), after 10 min. No further differences were observed between the two groups, until 15 min after HEPP injection, when responders showed a significant reduction in spike discharge. The therapeutic effect was reversible, since spike frequency discharge increased again 2 h after drug injection, to reach values similar to the control. It is interesting to note that the electrographic seizures in GWS are significantly reduced by HEPP,

since this model of partial epileptogenesis has proved to be extremely resistant to all common anticonvulsants on the first day of GWS appearance. This anticonvulsant effect, however, was not seen when the drug was administered 24 h after GWS onset. The differential sensitivity to anticonvulsants depending on the temporal evolution of the GWS suggests that different physiopathological mechanisms are involved in the genesis of the seizures in this model. GABAergic mechanisms seem to be more important during the first day of paroxysmal activity, since reinitiation of the GABA infusion at the epileptic focus during this time is capable of arresting the abnormal activity*. Excitatory neurotransmission might be involved from the second day on, since antagonists of the NMDA receptor have shown anticonvulsant effects by this time (Menini et al., in preparation). Although the mechanism of action of HEPP is still unknown, the antiepileptic effects revealed by the rat GNCE model might be independent of any GABAergic mechanism, since GABAmimetic agents (muscimol, THIP) produce an enhancement of SWD”; however, benzodiazepines and valproate are effective in this type of seizures. The anticonvulsant effect detected with higher doses in this model must be distinguished from its sedative effects, because spike-and-wave activity is very sensitive to changes in alertness. In summary, HEPP has antiepileptic effects that might be interpreted as partially GABAergic although they probably also involve other neurotransmitter systems. Further pharmacological studies aiming at the synthesis of a more stable and potent analogue of HEPP are now under way. ACKNOWLEDGEMENTS Our thanks to O.C. Snead and to R. Tapia for their comments on the manuscript. We are grateful to Isabel Perez-Montfort for manuscript edition. Supported in part by CONACYT (P228CCOX891625) and DGAPA, UNAM (IN02-10-89).

172 REFERENCES Brailowsky, S., Kunimoto, M., Menini, C., Silva-Barrat, C. and Naquet, R., The GABA-withdrawal syndrome: a new model of focal epileptogenesis, Bruin Res., 442 (1988) 175-179. Brailowsky, S., Kunimoto, M., Silva-Barrat, C., Menini, C. and Naquet, R., Electroencephalographic study of the GABA-withdrawal syndrome in rats, Epilepsia, 31 (1990) 369-377.

Brailowsky, S., Menini, C., Silva-Barrat, C. and Naquet, R., Epileptogenic gamma-aminobutyric acid withdrawal syndrome after chronic, intracortical infusion in baboons, Neurosci. Left., 74 (1987) 75-80.

Carvajal, G., Russek, M., Tapia, R. and Massieu, G., Anticonvulsive action of substances designed as inhibitors of yBiochem. transaminase, aminobutyric-a-ketoghrtaric Pharmacol., 13 (1964) 1059-1069. Meza-Toledo, S.E., Zenteno-Garcia, S.T., MartinezMutioz, D. and Carvajal-Sandoval, G., A new homologous series of anticonvulsants: phenyl-alcohol-amides. Synthesis and pharmacological evaluation, Arzneim. -Forsch., 40

(1990) 1289-1291. 6 Perez de la Mora, M. and Tapia, R., Anticonvulsant effect of 5ethyl,5phenyl,2-pyrrohdinone and its possible relationship to y-aminobutyric acid-dependent inhibitory mechanisms, Biochem. Pharmacol., 22 (1973) 2635-2639. 7 Snead, O.C., y-Hydroxybutyrate model of generalized absence seizures: further characterization and comparison with other absence models, Epilepsia, 29 (1988) 361-36X. 8 Tapia, R., Drucker-Cohn, R.R., Meza-Ruiz, G., Duran, L. and Levi, G., Neurophysiological and neurochemical studies on the action of the anticonvulsant y-hydroxy.yethyl,y-phenyl-butyramide. Epilepsia, 20 (1979) 135-145. 9 Vergnes, M., Marescaux, C., Depauhs, A., Micheletti, G. and Warter, J.M., Spontaneous spike and wave discharges in thalamus and cortex in a rat model of genetic petit mallike seizures, Exp. Neurol., 96 (1987) 127-136. 10 Vergnes, M., Marescaux, C., Micheletti, G., Depaulis, A., Rumbach, L. and Warter, J.M., Enhancement of spike and wave discharges by GABAmimetic drugs in rats with spontaneous petit mal-like epilepsy, Neurosci. Left., 44 (1984) 91-94.

Effects of 3-hydroxy,3-ethyl,3-phenylpropionamide (HEPP) on rat models of generalized and focal epilepsy.

The GABA withdrawal syndrome (GWS) is a new model of focal epilepsy in which paroxysmal activity is induced through the interruption of a chronic, int...
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