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

Elsevier

321

S U P P R E S S I O N BY PROGABIDE OF E T H A N O L WITHDRAWAL S Y N D R O M E 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 received20 November1984, accepted4 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withdrawn, produced a dose-related inhibition of both tremors and audiogenically induced seizures.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; Majchrowicz, 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 (Victor 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 correspondenceshould be addressed.
0014-2999/85/$03.30 1985 ElsevierSciencePublishers 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 ethanoltreated animals and during ethanol withdrawal,
while no change was observed by Hemmingsen et
al. (1982) in GABA receptors in ethanol dependence. 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 syndrome in animals. Progabide has a broad spectrum

322

of anticonvulsant properties (Worms et al., 1982),

is much less toxic than other GABA receptor
stimulants, such as isoguvacine or muscimol, enters 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 T H I P and kojic amine
(Krogsgaard-Larsen et al., 1979; Yarbrough et al.,
1979).
The present results show that progabide suppresses 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 dextrose 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 concentration 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 / k g 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 audiogenically 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

T.

I, ~l-t

Sal

Ethanol

(g/kg)

Fig. 1. Effect of ethanol on withdrawal tremors in ethanol-dependent rats (see Methods). Ethanol was withdrawn at midnight 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 represent the S.E.M. * P < 0.05, ** P < 0.01, with respect to control.

extension and intensity, by lifting the rats vertically 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 development of tonic-clonic seizures.
Different groups of withdrawn rats were treated
intraperitoneally with ethanol (20% solution in

m-t

8O

60

40_

I11
~

2CL

tt

I
i
Sal

I'"
3

Ethanol (g/kg)

Fig. 2. Effect of ethanol on audiogenic seizures in ethanol-dependent rats (see Methods). Ethanol was withdrawn at midnight 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.

323

3. Results

it

S a I.

50

100

200

300

400

SL 7 6 0 0 2 ( r r ~ j / k g )

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.

80

~.

40

2o_

tl

Sal

50

100

200

300

[..
400

S L ?6002~ngfkg )

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.

The intraperitoneal administration of ethanol,

1-3 g / k g 30 min before the test, produced a doserelated inhibition of both tremors and seizures
(figs. 1, 2). The dose of 3 g / k g completely suppressed tremors and audiogenically induced
seizures. Moreover, unlike in control animals,
ethanol failed to increase motor activity or produce ataxia in these rats.
t/rogabide (100-400 mg/kg) produced a doserelated reduction in tremors and frequency of
audiogenic seizures in withdrawn rats (figs. 3, 4).
Thirty min following the highest dose of progabide, 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 excessive 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 developed (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

324

treatment in the management of ethanol withdrawal syndrome in man.

From a more theoretical point of view, the
finding that a GABA-mimetic agent, such as progabide, 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 receptor 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. Indeed, the same authors reported that the benzodiazepine, 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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