European Journal of Pharmacology, 109 (1985) 321-325 321 Elsevier

SUPPRESSION BY PROGABIDE OF ETHANOL WITHDRAWAL SYNDROME IN RATS

FABIO FADDA, ENRICA MOSCA, ROLANDO MELONI and GIAN LUIGI GESSA *

Institutes of Physiology and Pharmacology *, University of Cagliari, Italy

Received 6 June 1984, revised MS received 20 November 1984, accepted 4 December 1984

F. FADDA, E. MOSCA, R. MELONI and G.L. GESSA, Suppression by progabide of ethanol withdrawal syndrome in rats, European J. Pharmacol. 109 (1985) 321-325.

Progabide, a specific and clinically used GABA receptor agonist, was tested for its ability to suppress ethanol withdrawal syndrome. Male rats were rendered physically dependent on ethanol by feeding for 12 days on a liquid diet in which ethanol isocalorically replaced dextrose. Progabide (100-400 mg/kg i.p.), administered 8 h after ethanol was withdrawn, produced a dose-related inhibition of both tremors and audiogenically induced seizures. A single dose of 400 mg/kg of progabide completely suppressed all ethanol withdrawal reactions. Seizures were more sensitive to the drug than tremors. The results support the view that a decrease in GABA transmission plays a role in ethanol withdrawal symptoms and suggest that progabide may be tested as a possible treatment of ethanol withdrawal syndrome in man.

Progabide Ethanol withdrawal GABA

1. Introduction

The prolonged consumption of large quantities of ethanol results in the development of tolerance and physical dependence in both man (Victor and Adams, 1953; Mendelson, 1964; Gross et al., 1974) and laboratory animals (Freund, 1971; Majchro- wicz, 1975; Goldstein and Pal, 1971). Rats are frequently used in the study of withdrawal from chronic ethanol treatment (Lester and Freed, 1973). During ethanol withdrawal, rats show tremors, spasticity, spontaneous convulsions and general CNS hyperexcitability (Majchrowicz, 1975), a symptomatology similar to that seen in man (Vic- tor and Adams, 1953). Moreover, seizures can be evoked in rats by intense auditory stimulus several hours after the interruption of chronic ethanol treatment when blood ethanol concentration has declined (Hunter et al., 1973). This condition of hyperexcitability, tremor and susceptibility to audiogenic seizures in rats has been used as an animal model of alcohol withdrawal syndrome.

* To whom all correspondence should be addressed.

0014-2999/85/$03.30 1985 Elsevier Science Publishers B.V.

It has been suggested that the development of withdrawal hyperexcitability might be due to a reduced GABAergic function (Ticku, 1980; Ticku and Burch, 1980; Volicer, 1980; Hunt, 1983). In fact, drugs which increase the availability of GABA or directly activate GABA receptors suppress some withdrawal signs in both animals and humans (Seller and Kalant, 1976; Goldstein, 1972; Noble et al., 1976; Cooper et al., 1979; Frye et al., 1983). However, there is no agreement on changes in GABA receptor binding in ethanol dependence. In fact, Ticku (1980) and Ticku and Butch (1980) reported that GABA affinity for its receptors is reduced in brain tissue from chronic ethanol- treated animals and during ethanol withdrawal, while no change was observed by Hemmingsen et al. (1982) in GABA receptors in ethanol depen- dence. These results prompted us to study the effect of a new GABA receptor agonist, progabide (4- ([(4-chlorophenyl)(5- fhioro-2-hydrophenyl)- methylene]amino}butanamide) (SL 76002), on audiogenic seizures and tremors, which are the most reliable signs of ethanol withdrawal syn- drome in animals. Progabide has a broad spectrum

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of anticonvulsant properties (Worms et al., 1982), is much less toxic than other GABA receptor stimulants, such as isoguvacine or muscimol, en- ters the brain to a much greater extent than the latter compounds (Baraldi et al., 1979; Chase and Tamminga, 1979), and its neuropharmacological profile has been more extensively studied than that of other GABA agonists which penetrate the blood brain barrier, such as THIP and kojic amine (Krogsgaard-Larsen et al., 1979; Yarbrough et al., 1979).

The present results show that progabide sup- presses tremors and audiogenic seizures of ethanol withdrawal in rats.

2. Materials and methods

Male Sprague-Dawley rats, weighing 150-200 g, were used. The animals were housed 1 per cage, under an artificial light-dark cycle (light on from 7 : 00 to 19 : 00) and at a constant temperature of 22C. The rats were rendered ethanol-dependent by the assumption of a nutritionally complete liquid ethanol diet (50 ml/day), in which ethanol isocalorically replaced dextrose. A balanced dex- trose diet (Piccioni, Brescia, Italy), derived from values shown to adequately maintain rats and permit their growth, was used. Fresh diet was presented daily at 11:00 in 100 ml graduated bottles for a period of 2 weeks. After 2 days of the dextrose diet, the rats had continuous access for 12 days to the ethanol diet. The ethanol concentra- tion was 6% (w/v) for the first 2 days, 7% for the next 5 days and 8% for the last 5 days. During the last period, the rats ingested 16-18 g/kg of ethanol daily.

The withdrawal period began 6-7 h after the ethanol diet was removed (at midnight). The symptoms observed were hyperactivity, tremors, tail rigidity, irritability, spasticity and spontaneous convulsions of the clonic-tonic type. All these signs were recorded. However, only tremors and audio- genically induced seizures were scored in the evaluation of the drug effect in suppressing withdrawal syndrome. These signs were evaluated 8.5 h after the alcohol diet was removed. Tremors were rated on a scale of 1 to 3 on the basis of their

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Fig. 1. Effect of ethanol on withdrawal tremors in ethanol-de- pendent rats (see Methods). Ethanol was withdrawn at mid- night and 8 h later saline or ethanol was administered i.p. Thirty min after treatment, tremor scores were assessed. At least 6 rats were used for each treatment. Vertical bars repre- sent the S.E.M. * P < 0.05, ** P < 0.01, with respect to control.

extension and intensity, by lifting the rats verti- cally by the tail, according to the method of Frye and Breese (1981). Immediately after the tremor test, susceptibility to audiogenic seizures was evaluated. The rats, placed in a cylindrical box (40 40 cm), were individually exposed to a sound generated by an electric bell (100 dB) for 1 min. The bell sound produced a running episode which preceded the loss of righting reflex and the devel- opment of tonic-clonic seizures.

Different groups of withdrawn rats were treated intraperitoneally with ethanol (20% solution in

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Fig. 2. Effect of ethanol on audiogenic seizures in ethanol-de- pendent rats (see Methods). Ethanol was withdrawn at mid- night and 8 h later saline or ethanol was administered i.p Thirty min after treatment, susceptibility to audiogenic seizure,, was assessed. * P < 0.05, ** P < 0.01, with respect to control.

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Fig. 3. Effect of progabide on withdrawal tremors in ethanol-dependent rats (see Methods). Ethanol was withdrawn at midnight and 8 h later saline or progabide was administered i.p. Thirty min after treatment, tremor scores were assessed. At least eight rats were used for each treatment. Vertical bars represent the S.E.M. * P < 0.05, ** P < 0.01, with respect to

control.

H20), different doses of progabide (a gift from Synthelabo, LERS, Paris) (suspended in saline containing 1% Tween 80 and injected immediately after preparation) or the same volume of Tween 80 solution (5 ml/kg).

For tremors, the statistical significance of the results was analyzed using the Wilcoxon test and for audiogenic seizures, the chi-square analysis was used.

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[.. Fig. 4. Effect of progabide on audiogenic seizures in ethanol-dependent rats (see Methods). Ethanol was withdrawn at midnight and 8 h later saline or progabide was administered i.p. Thirty min after treatment, susceptibility to audiogenic seizures was assessed. * P < 0.05,** P < 0.01, with respect to control.

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3. Results

The intraperitoneal administration of ethanol, 1-3 g/kg 30 min before the test, produced a dose- related inhibition of both tremors and seizures (figs. 1, 2). The dose of 3 g/kg completely sup- pressed tremors and audiogenically induced seizures. Moreover, unlike in control animals, ethanol failed to increase motor activity or pro- duce ataxia in these rats.

t/rogabide (100-400 mg/kg) produced a dose- related reduction in tremors and frequency of audiogenic seizures in withdrawn rats (figs. 3, 4).

Thirty min following the highest dose of pro- gabide, withdrawn rats showed none of the withdrawal signs, such as spasticity, tail stiffness, etc., and appeared completely normal. Moreover, seizure susceptibility was suppressed at this time.

4. Discussion

Due to the high rate of morbidity and mortality in severe ethanol withdrawal in man (Salum et al., 1972; Thompson et al., 1975), a drug effective in the treatment of this syndrome is an important issue.

Drugs used in the treatment of this condition are chloral hydrate, paraldehyde, barbiturates (Isbell et al., 1955; Olbriel, 1979; Shaw, 1980; Kramp and Rafaelson, 1978), which produce ex- cessive CNS depression at doses needed to control ethanol withdrawal (Greenblatt and Greenblatt, 1972), and benzodiazepines, chlordiazepoxide and diazepam. The latter, even at the high doses used in withdrawal syndrome, reduce but do not fully suppress ethanol withdrawal tremors in human alcoholics and have been shown to fail to control hallucinations and confusion once they have devel- oped (Kramp and Rafaelson, 1978; Woo and Greenblatt, 1979; Aaronson et al., 1982).

Progabide is a relatively non-toxic GABA agonist. It has been used in the treatment of various forms of epilepsy in humans (Loiseau et al., 1981). The doses giving protection against ethanol withdrawal syndrome in rats are far below the LDs0 of the compound (Worms et al., 1982). Therefore, progabide may be tested as a possible

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treatment in the management of ethanol with- drawal syndrome in man.

From a more theoretical point of view, the finding that a GABA-mimetic agent, such as pro- gabide, counteracts ethanol withdrawal supports the view that a decrease in GABA transmission plays a role in this condition. Indeed, there is a great deal of evidence suggesting that acute and chronic ethanol modifies GABAergic activity in brain (Ticku, 1980; Ticku and Burch, 1980; Volicer, 1980; Hunt, 1983; Mereu and Gessa, 1984), and that progabide and its immediate metabolite formed in the brain, SL 75102, are specific and pure agonists for GABA receptors (Worms et al., 1982).

However, while there is general agreement that seizures in ethanol withdrawal are sensitive to direct and indirect GABA agonists (Cooper et al., 1979; Frye and Breese, 1981), it has been recently reported by Frye et al. (1983) that withdrawal tremors are completely resistant to GABA recep- tor stimulants.

Contrary to this conclusion, our results indicate that progabide also protects against withdrawal tremors, although less effectively than against seizures. It is possible that tremors are only partly dependent on a deficit in GABA transmission.

However, the negative results of Frye et al. (1983) with muscimol and ~,-vinyl-GABA might be due to the inability of intracisternally injected GABA mimetics to reach the target area in concentrations sufficient to inhibit tremors. In- deed, the same authors reported that the benzodi- azepine, chlordiazepoxide, reverses both seizures and tremors. Benzodiazepines are considered to act by potentiating GABA transmission (Guidotti et al., 1979). This picture is further complicated by the finding that tremors in cats were resistant to diazepam (Aaronson et al., 1982).

In conclusion, our results question the view that withdrawal tremors are completely resistant to GABA receptor stimulants and are independent of GABAergic control (Frye et al., 1983) and indicate that further studies are required to fully assess the role of GABA in the control of the different signs of ethanol withdrawal syndrome.

Acknowledgements

The present study was supported by C.N.R. grant 83.02669.56 and a grant from the Italian Ministry of Public Instruction. We thank our technician Mr. P. Suffiotti.

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