Methods for the treatment of alcohol intoxication and dependence

Alcohol-related disorders are treated by the administration of adenosine antagonists and adenosine agonists to a host. Adenosine antagonists are used to inhibit both acute intoxication and chronic dependence by administering prior to alcohol consumption. The symptoms associated with alcohol withdrawal syndrome may be treated by administering adenosine agonists which reduce the physiological dependence on alcohol during the withdrawal period.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
Alcohol intoxication and dependence are serious health problems worldwide. 
Acute alcohol intoxication can seriously impair an individual's motor 
function, causing temporary incapacity which exposes the individual and 
others to potential accidents. Long term alcohol dependence can cause 
mental and physical disabilities which are detrimental to the individual 
and society. The problem is exacerbated by physical dependence on the 
alcohol which results in "alcohol withdrawal syndrome" as the individual 
ceases alcohol consumption. Alcohol withdrawal syndrome is characterized 
by tremors, weakness, sweating, hyperflexia, and, in the worst case, 
delirium tremens. 
For these reasons, it would be highly desirable to provide methods to 
prevent both acute intoxication and long term alcohol dependence as well 
as for reversing established dependence. It would be particularly 
desirable if methods could be found which could facilitate withdrawal from 
long term alcohol dependence by reducing or eliminating the symptoms of 
alcohol withdrawal syndrome. 
2. Description of the Background Art 
Ethanol-induced changes in cAMP signal transduction appear to play a role 
in the acute and chronic effects of ethanol. See, for example, Nagy et al. 
(1988) Proc. Natl. Acad. Sci. USA 85:6973-6976; Gordon et al. (1986) Proc. 
Natl. Acad. Sci. USA 83:2105-2108; Valverius et al. (1987) Mol. Pharmac. 
32:217-227; and Charness et al. (1988) Biochem. Biophys. Res. Comm. 
155:138-143. Ethanol acutely increases receptor-stimulated cAMP levels in 
NG108-15 neuroblastoma.times.glioma hybrids (Gordon et al. (1986) supra.). 
In contrast, chronic exposure to ethanol causes a decrease in 
receptor-dependent cAMP levels (Gordon et al. (1986) supra. and Charness 
et al. (1988) supra.). This reduction appears to be significant in chronic 
alcoholism since cells from alcoholics exhibit decreases in both adenosine 
receptor-stimulated and PGE.sub.1 receptor-stimulated cAMP levels (Nagy et 
al. (1988) supra.; and Diamond et al. (1987) Proc. Natl. Acad. Sci USA 
84:1413-1416. 
Dar et al. have investigated the effects of certain adenosine antagonists 
(including caffeine, theophylline, and isobutylmethylxanthine on acute and 
chronic alcohol intoxication. Dar et al. (1985) Life Sciences 
33:P1363-1374, set forth that theophylline ameliorated certain symptoms of 
intoxication while having no effect on others, depending on the time 
between theophylline administration and ethanol injection. Dipyridamole 
(which inhibits cellular adenosine uptake) was demonstrated to have a 
potentiating effect on certain symptoms of ethanol intoxication. Dar and 
Wooles (1986) Life Sciences 39:1429-1437, describe the effect of 
administration of caffeine, isobutylmethylxanthine, and theophylline over 
a 10-day period to mice. The intoxicating effect of ethanol was increased 
in mice fed isobutylmethylxanthine and caffeine, while theophylline 
appeared to have no effect. Dar et al. (1987) Psychopharmacology 91:1-4 
demonstrates significant potentiation of ethanol-induced ataxia (loss of 
motor control) in mice pretreated with caffeine, theophylline, and 
isobutylmethylxanthine. 
SUMMARY OF THE INVENTION 
Methods for the treatment of alcohol intoxication and withdrawal are 
provided. Alcohol intoxication is inhibited by the administration of 
adenosine antagonists to a host prior to consumption of ethanol. Useful 
antagonists include those specific for the A.sub.1 and/or A.sub.2 
adenosine receptors and, preferably, those which are not transported into 
the cells and do not inhibit adenosine uptake. In individuals dependent on 
alcohol or susceptible to such dependence, long term administration of 
such adenosine antagonists may reverse or inhibit physiological 
dependence. In addition, the symptoms of alcohol withdrawal syndrome may 
be reduced by administering adenosine or adenosine agonists to the host. 
Administration of adenosine or adenosine agonists will reduce 
physiological dependence on ethanol during the withdrawal period.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The following definitions are set forth to illustrate and define the 
meaning and scope of the various terms used to describe the present 
invention. 
As used herein, the term "host" means a vertebrate host subject to alcohol 
intoxication and dependence, particularly referring to human hosts. Those 
individuals dependent on alcohol or susceptible to alcoholism may be 
identified by biologic tests described in detail in copending application 
Ser. No. 07/161,628, filed on Feb. 29, 1988, the disclosure of which is 
incorporated herein by reference. Briefly, basal and receptor-stimulated 
cAMP levels are measured in freshly isolated lymphocytes, where a 
reduction in the concentration compared to the expected normal value is 
diagnostic of alcoholism. Alternatively, an increase in adenosine 
receptor-stimulated cAMP levels measured in cultured cells not exposed to 
ethanol compared to the expected normal value is indicative of a 
predisposition toward alcoholism. Also, decreased adenosine 
receptor-stimulated cAMP levels in cultured cells from alcoholics exposed 
to relatively low concentrations of ethanol limited time periods where no 
decrease in adenosine receptor-stimulated cAMP occurs when normal cultured 
cells are exposed to ethanol (under the same conditions) is diagnostic of 
alcoholism susceptibility. 
"Adenosine antagonist" refers to an active agent having adenosine receptor 
blocking activity. Two distinct adenosine receptor classes exist, 
designated A.sub.1 receptors (including subclasses which inhibit adenylate 
cyclase activity when activated) and A.sub.2 receptors which stimulate 
adenylate cyclase activity when activated. Adenosine antagonists are 
capable of combining with either or both of these receptors but are 
incapable of stimulating the normal activity which occurs when adenosine 
binds to such receptors. As a result, stimulation upon subsequent exposure 
to adenosine or adenosine agonists will be blocked or inhibited. 
Adenosine antagonists suitable for use in the methods of the present 
invention include those specific or preferential for either or both the 
A.sub.1 and the A.sub.2 receptor. Exemplary adenosine antagonists include 
PD115,199 (an A.sub.2 antagonist available from Parke-Davis); 
3-(3-hydroxyphenyl)-5H-thiazolo[2,3b]-guinazoline (an A.sub.2 antagonist); 
1,3-diethyl-8-phenylxanthine and other substituted phenylxanthines 
(A.sub.1 antagonists). Preferred adenosine antagonists according to the 
present invention are A.sub.2 specific, not taken up by the cells to which 
they bind, and do not inhibit adenosine uptake. Cellular uptake may be 
measured by techniques described in Aranow and Ullman (1986) J. Biol. Che. 
261:2014-2019. 
PD 115,199 has the following formula: 
##STR1## 
PD 115,199 is described in Chemical Abstracts 106:149316x (1987). 
As used herein, the term "adenosine agonist" means an active agent capable 
of combining with either the A.sub.1 or A.sub.2 receptor and capable of 
stimulating the associated receptor activity. The term adenosine agonist 
will also include partial adenosine agonists which are capable of 
partially stimulating adenosine receptor activity, i.e., providing a 
lesser activity than would be obtained with a like concentration of 
adenosine. 
Adenosine agonists suitable for use in the methods of the present invention 
include those specific or preferential for either or both the A.sub.1 and 
the A.sub.2 receptor. Exemplary adenosine agonists suitable for use in the 
methods of the present invention specific for the A.sub.2 receptor include 
CGS21680 (Ciba-Geigy) and 2-phenylaminoadenosine (CV1808). Exemplary 
adenosine agonists specific for the A.sub.1 receptor include 
N,6-cyclohexyl-adenosine and N,6-cyclopentyladenosine. 
Adenosine agonist CGS 21680C has the following formula: 
##STR2## 
and is described in Chemical Abstracts 112:30385c (1989). 
As used herein, the term "treatment" means any administration of the 
adenosine antagonists or agonists for mediating the effect of short term 
alcohol exposure or long term alcohol dependence in a vertebrate host, 
particularly a human, and includes: 
(i) inhibiting the symptoms of acute alcohol intoxication; 
(ii) lessening or inhibiting the long term effects of alcohol intoxication, 
including both acute and chronic intoxication; and 
(iii) relieving the symptoms of alcohol withdrawal. 
As used herein, "alcohol" means ethanol. 
Administration of the adenosine antagonists and agonists for use in the 
method of this invention can be via any of the accepted modes of 
administration. These methods include, but are not limited to, oral, 
parenteral, transdermal, intraarticular and otherwise systemic 
administration. Oral administration is preferred. The compounds are 
administered in a therapeutically effective amount either alone or in 
combination with a suitable pharmaceutically acceptable carrier or 
excipient. 
Depending on the intended mode of administration, the adenosine antagonist 
or agonist of choice may be incorporated in any pharmaceutically 
acceptable dosage form, such as, for example, tablets, transdermal 
patches, pills, capsules, powders, liquids, suspensions, emulsions, 
aerosols or the like, preferably in unit dosage forms suitable for single 
administration of precise dosages, or sustained release dosage forms for 
continuous controlled administration. Preferably the dosage form will 
include a pharmaceutically acceptable excipient and, in addition, may 
contain other medicinal agents, pharmaceutical agents, carriers, 
adjuvants, etc. 
For solid dosage forms, non-toxic carriers include but are not limited to, 
for example, pharmaceutical grades of mannitol, lactose, starch, magnesium 
stearate, sodium saccharin, the polyalkylene glycols, talcum, cellulose, 
glucose, sucrose and magnesium carbonate. Liquid pharmaceutically 
administrable dosage forms can, for example, comprise a solution or 
suspension of an active adenosine agent and optional pharmaceutical 
adjuvants in a carrier, such as, for example, water, saline aqueous 
dextrose, glycerol, ethanol and the like, to thereby form a solution or 
suspension. If desired, the pharmaceutical composition to be administered 
may also contain minor amounts of non-toxic auxiliary substances such as 
wetting or emulsifying agents, pH buffering agents and the like. Typical 
examples of such auxiliary agents are sodium acetate, sorbitan 
monolaurate, triethanolamine, sodium acetate, triethanolamine oleate, etc. 
Actual methods of preparing such dosage forms are known, or will be 
apparent, to those skilled in the art; for example, see Remington's 
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 16th 
Edition, 1980. The composition of the formulation to be administered will, 
in any event, contain a quantity of the active adenosine agent in an 
amount effective for treatment. 
Parenteral administration is generally characterized by injection, either 
subcutaneously, intramuscularly or intravenously. Injectables can be 
prepared in conventional forms, either as liquid solutions or suspension, 
solid forms suitable for solution or suspension in liquid prior to 
injection, or as emulsions. Suitable excipients are, for example, water, 
saline, dextrose, glycerol, ethanol and the like. In addition, if desired, 
the injectable pharmaceutical compositions to be administered may also 
contain minor amounts of non-toxic auxiliary substances such as wetting or 
emulsifying agents, pH buffering agents and the like. 
The amount of active adenosine antagonist or agonist administered will, of 
course, be dependent on the subject being treated, the severity and nature 
of the affliction, the manner of administration, the potency and 
pharmacodynamics of the particular agent and the judgement of the 
prescribing physician. However, the therapeutically effective dosage for 
use in this invention will generally be in the range from about 0.01 
.mu.g/kg (body weight) to 5 mg/kg. 
In treating acute alcohol intoxication, adenosine antagonists will be 
administered to the host prior to ethanol consumption, usually from about 
10 to 60 minutes prior to such consumption, more usually from about 10 to 
30 minutes before such consumption. The dosage administered will generally 
be within the amounts outlined above. By administering the adenosine 
antagonist prior to ethanol consumption, the acute intoxication effect of 
the alcohol in the host can be reduced or substantially inhibited. 
In treating chronic alcohol dependence, the adenosine antagonists will be 
administered over a prolonged period, typically for at least one week, 
more usually being from about 2 to 26 weeks, and frequently the treatment 
period will be indefinte. The administration will usually be at least 
daily, more usually being several times a day, although slow release 
dosage forms may reduce the frequency of administration. 
In treating the symptoms of alcohol withdrawal, adenosine agonists will be 
administered to the host after the consumption of alcohol has ceased and 
for so long as the host is subject to the symptoms. Typically, the 
treatment will last for a period of from 3 to 30 days, more typically from 
about 7 to 21 days, although in some cases the treatment may last 
indefinitely. The dosage of the agonist will generally be within the 
amounts outlined above. The administration will be at least daily, more 
usually being multiple times during the day. Again, slow release dosage 
forms may find use. The following examples are offered by way of 
illustration, not by way of limitation. 
EXPERIMENTAL 
NG108-15 cells were grown in defined media as described (Gordon et al. 
(1986) supra.) to a final density of 12-20.times.10.sup.6 cells per flask. 
Cells were incubated at 37.degree. C. for 10 min in 9 ml of assay medium 
containing 100 mM ethanol with or without 1 U/ml adenosine deaminase (ADA) 
or 10.sup.31 5 M isobutylmethylxanthine (a concentration which blocks 
adenosine A.sub.2 receptors but does not inhibit phosphodiesterase 
activity) and acetylated cAMP levels determined (Gordon et al. (1986) 
supra.). The effects of ethanol on cAMP production are illustrated in FIG. 
1. Bars represent means .+-.SEM, n=4-9. The asterisk (*) indicates a 
significant difference from cells not treated with ethanol (p&lt;0.002, 
student's t-test). cAMP levels in the absence of ethanol were 18.4.+-.2.4 
pmoles/10.sup.6 cells (n=16). 
Ethanol-induced decreases in receptor-dependent cAMP levels appear to be 
due to heterologous desensitization of receptors coupled to the 
stimulatory guanine nucleotide regulatory protein, G.sub.s. This 
desensitization is caused by a decrease in messenger RNA for the 
.alpha.-subunit protein (Mochly-Rosen (1988) Nature 333:848-850). Since 
heterologous desensitization in other systems is preceded by initial 
increases in intracellular cAMP (Sibley et al. (1985) Nature 317:124-129), 
cAMP levels should be increased by treatment with ethanol. When NG108-15 
cells were incubated with 200 mM ethanol for 10 min in the absence of 
added agonist, a 60% increase in intracellular cAMP concentration was 
observed (FIG. 1). Since ethanol does not directly stimulate adenylate 
cyclase activity but only receptor-dependent cAMP production (Rabin et al. 
(1983) J. Pharmac. Exptl. Thera. 227:551-556), the possibility that 
ethanol increases the extracellular concentration of a stimulatory agonist 
was considered. Neural cells (Green (1980) Supramol. Structure 
13:175-182), lymphocytes (Newby et al. (1981) Biochem. J. 200:399-403; 
Fredholm et al. (1978) Biochem. Pharmac. 27:2675-2682), and other cell 
types (Purines: Pharmacology and Physiological Roles, Ed. T. W. Stone, 
Macmillan Press, Ltd., London, 1985) release adenosine, and adenosine has 
been implicated in the effects of ethanol (Proctor et al. (1984) Science 
224:519-521; Dar et al. (1983) Life Sci. 33:1363-1374). Therefore, 
adenosine concentrations in the medium of control and ethanol-treated 
cells were measured using high pressure liquid chromatography (HPLC). The 
results are shown in FIG. 2. 
Representative chromatograms of control (A) and ethanol-treated cells (B) 
are shown in FIG. 2. NG108-15 cells (5.times.10.sup.6 cells/well) were 
incubated with 2 ml of PBS in the presence or absence of 200 mM ethanol 
for 10 min. Fluorescent derivatives of adenosine were prepared (Green 
(1980) supra.) and injected onto a reverse-phase HPLC column equilibrated 
with 1.2 mM KPO.sub.4, pH 5 and eluted with a 0-60% methanol gradient. 
Peak areas were compared with those of known amounts of 1,N.sup.6 
-ethenoadenosine. A significant increase in the concentration of 
extracellular adenosine was found when NG108-15 cells were incubated with 
200 mM ethanol. Within 10 min, adenosine concentrations reached 37.+-.1.2 
nM/5.times.10.sup.6 cells in ethanol-treated cells while control cultures 
had 18.2.+-.3.7 nM adenosine (n=4, p&lt;0.005). 
Adenosine stimulates the production of cAMP via the A.sub.2 adenosine 
receptor, which is positively coupled to adenylate cyclase (Daly in: Adv. 
Cyclic Nucleotides and Protein Phosphorylation (eds. D. M. F. Cooper and 
K. B. Seamon) Vol. 19, pp. 29-46 (Raven Press, N.Y., 1985). The increase 
in intracellular cAMP levels produced by acute ethanol (FIG. 1) could 
therefore be due to activation of adenosine A.sub.2 receptors by the 
extracellular adenosine accumulated in the presence of ethanol. If this 
were the case, then degradation of extracellular adenosine should prevent 
stimulation of cAMP production by ethanol. Adenosine deaminase (ADA) was 
used to deaminate adenosine to inosine, a nucleoside with low affinity for 
the adenosine receptor. When NG108-15 cells were incubated with ADA, 
stimulation of cAMP production by ethanol was completely abolished (FIG. 
1). Moreover, treatment of the cells with an adenosine receptor 
antagonist, isobutylmethylxanthine (IBMX), also completely blocked 
ethanol-induced increases in cAMP levels (FIG. 1). These data suggest that 
acute exposure to ethanol causes an increase in extracellular adenosine. 
Accumulated adenosine then binds to the A.sub.2 receptor to stimulate cAMP 
production. 
In contrast to the acute stimulation of cAMP levels by ethanol, chronic 
exposure to ethanol causes a decrease in or desensitization of adenosine 
receptor and PGE.sub.1 receptor-dependent cAMP production (Gordon et al. 
(1986) supra.; Mochly-Rosen et al. (1988) supra.). If adenosine were 
responsible for ethanol-induced heterologous desensitization, ADA should 
prevent this response. The following experiment was performed to test this 
theory. 
NG108-15 cells were maintained in 100 mM ethanol for 48 hr with and without 
1 U/ml ADA. Adenosine receptor- and PGE.sub.1 receptor-stimulated cAMP 
production were then determined in the absence of ethanol as previously 
described (Gordon et al. (1986) supra.). In FIG. 3, desensitization is 
expressed as the percent decrease in ethanol-treated cells compared to 
cells never exposed to ethanol. ADA was active throughout the incubation 
period (data not shown). Asterisks indicate a significant difference from 
cells not treated with ethanol (*p&lt;0.025, **p&lt;0.001, student's t-test). 
FIG. 3 shows that when NG108-15 cells are co-incubated for 48 hours with 
ethanol and 1 U/ml ADA, a concentration sufficient to block the acute 
increase in cAMP (FIG. 1), chronic ethanol-induced desensitization of 
adenosine receptor-stimulated cAMP levels is substantially blocked and 
desensitization of the PGE.sub.1 receptor is prevented completely. 
These results indicate that accumulation of extracellular adenosine is 
required for the heterologous desensitization produced by chronic exposure 
to ethanol, as well as for the acute effects of ethanol on cAMP levels. In 
other preparations, adenosine can cause homologous and heterologous 
desensitization of receptors (Kenimer et al. (1981) Mol. Pharmac. 
20:585-591; Newman et al. (1983) Biochem. Pharmac. 32:137-140); 
desensitization is dependent on the concentration of agonist and time of 
exposure (Kenimer et al. (1981) supra.). Consistent with these results, 
Green (1980) supra., has found in C1300 neuroblastoma cells, that 10-20 nM 
of endogenously released adenosine is sufficient to desensitize adenosine 
receptor-dependent cAMP production during an overnight culture. 
If accumulation of extracellular adenosine is required for ethanol-induced 
heterologous desensitization, then cells which do not release adenosine 
should not desensitize after chronic exposure to ethanol. Adenosine uptake 
and release are mediated via a single bidirectional transporter which is 
inactive in the nucleoside transport-deficient mutants 80-2A6 and 160-D4 
of the S49 lymphoma cell line. Transport-deficient clones fail to 
transport adenosine and virtually all other nucleosides. S49 wild type 
(WT) and adenosine transport mutants (80-2A6 and 160-D4) were grown in 
defined media (11, without phytohemagglutinin) with 1 U/ml ADA for at 
least 2 weeks. They were then grown in the presence or absence of 100 mM 
ethanol with or without 1.5 U/ml ADA for 48 hours. Accumulation of 
adenosine over 5 min was determined by incubating 1.0.times.10.sup.7 
cells/ml in sterilized PBS in the absence or presence of 200 mM ethanol. 
Extracellular adenosine concentrations were determined as in FIG. 2. 
Accumulation of adenosine over 24 hours in the absence or presence of 100 
mM ethanol was determined as described by Green (1980) supra.. Assays for 
receptor-stimulated cAMP levels were carried out as in FIG. 3. Basal 
levels of cAMP were 2.41.+-.0.28 pmoles/10.sup.6 cells for all cell types 
and did not change with chronic exposure to ethanol. cAMP levels of 
ethanol-treated cells are expressed as a percentage of cAMP in cells never 
exposed to ethanol. Values represent means .+-.SEM. Number of 
determinations in indicated in parentheses. The asterisk (*) indicates a 
significant difference from cells not exposed to ethanol (p&lt;0.001). The 
results are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
ADENOSINE TRANSPORT IS REQUIRED FOR ETHANOL-INDUCED 
HETEROLOGOUS DESENSITIZATION IN S49 CELLS 
cAMP LEVELS 
EXTRACELLULAR ADENOSINE 100 mM EtOH, 48 hr. 
(nM/10.sup.7 cells) 100 uM PIA 
1 uM PGE.sub.1 
Cell Type 
No EtOH EtOH (% of control) 
__________________________________________________________________________ 
Wild Type 
5 min 
4.4 .+-. 0.9 (5) 
200 nM 
17.6 .+-. 3.6 (5) 
65 .+-. 7* (27) 
36 .+-. 6* (6) 
24 hr. 
21.9 .+-. 0.3 (2) 
100 nM 
56.7 .+-. 10.2 (2) 
Wild Type + 
ND ND 118 .+-. 31 (4) 
96 .+-. 17 (5) 
Chronic ADA 
80-2A6 5 min 
0 (2) 200 mM 
0 (2) 115 .+-. 11 (6) 
82 .+-. 7 (4) 
24 hr. 
0 (3) 100 mM 
0 (3) 
160-D4 ND ND 94 .+-. 9 (4) 
98 .+-. 7 (4) 
__________________________________________________________________________ 
Incubation of S49 wild type cells with maximally-effective concentrations 
of PIA or PGE.sub.1 increased cAMP levels 1.92.+-.0.41-fold (n=18) and 
13.5.+-.3.5-fold (n=6) over basal, respectively. Similar results were 
obtained in the adenosine transport deficient cells (data not shown), 
indicating that coupling of adenosine receptor and PGE.sub.1 receptor to 
adenylate cyclase is normal in the mutant cells. 
S49 wild type cells showed a significant increase in the concentration of 
extracellular adenosine when exposed to ethanol for 5 min or 24 h (Table 
1) but the 80-2A6 mutant cell line failed to accumulate extracellular 
adenosine under these conditions (Table 1). As expected, when S49 wild 
type cells were treated with 100 mM ethanol for 48 hours, adenosine 
receptor and PGE.sub.1 receptor-stimulated cAMP levels were reduced to 
65.+-.7 and 36.+-.6% of control, respectively (Table 1). As in NG108-15 
cells, addition of ADA to S49 wild type cells prevented ethanol-induced 
heterologous desensitization (Table 1). By contrast, there was no 
desensitization of adenosine receptor- or PGE.sub.1 receptor-stimulated 
cAMP levels when the adenosine transport-deficient cells were exposed to 
100 mM ethanol for 48 hours. (Table 1). Thus, the adenosine transporter is 
required for ethanol-induced heterologous desensitization of 
receptor-dependent cAMP production in S49 cells. Although the adenosine 
transporter is a non-specific nucleoside carrier, other nucleosides have 
very low affinities for the adenosine receptor and do not stimulate cAMP 
production (Daly (1985) supra.). In addition, exposure of S49 wild type 
cells to inosine for 48 hr. did not desensitize the adenosine receptor 
(data now shown). Taken together these results indicate that accumulation 
of extracellular adenosine mediates ethanol-induced heterologous 
desensitization. 
We have noted that in S49 and NG10815 cells, adenosine accumulation is due 
to inhibition of adenosine uptake after acute ethanol exposure. After 
chronic exposure to ethanol, acute ethanol no longer inhibits adenosine 
uptake and consequently no longer causes adenosine accumulation. Uptake of 
[.sup.3 H]-adenosine in freshly isolated lymphocytes from nonalcoholics 
and actively drinking alcoholics was also measured. Lymphocytes were 
incubated with and without 200 mM ethanol for three minutes, and [.sup.3 
H]-adenosine uptake was measured at 10 seconds. The results are 
illustrated in FIG. 4. Values represent means .+-.SEM, n=7 for alcoholics 
and n=9 for nonalcoholics; *p&lt;0.001 compared to uptake in the absence of 
ethanol, and +p&lt;0.001 compared to nonalcoholics. Adenosine uptake into 
freshly-isolated lymphocytes from alcoholics was similar to that observed 
in S49 cells after chronic exposure to alcohol in that it was relatively 
insensitive to acute alcohol; there was less inhibition by ethanol 
compared to uptake in cells from nonalcoholics (FIG. 4). These results 
indicate that the difference in adenosine uptake in lymphocytes from 
alcoholics is due at least in part to the effect of in vivo exposure to 
chronic ethanol on the adenosine transport system. 
Lymphocytes from alcoholics and nonalcoholics were grown in culture for 6 
days, and 100 mM ethanol was added for 24 hours. The cells were washed, 
preincubated for 5 minutes in the absence of ethanol, and then incubated 
in the absence and presence of 100 mM ethanol for 5 minutes. Extracellular 
adenosine was measured by HPLC (n=5-7 for alcoholics and nonalcoholics). 
In these experiments, adenosine concentrations in the absence of ethanol 
did not significantly differ between alcoholics and nonalcoholics 
(207.+-.26 mM/10.sup.7 cells, n=14, including 7 alcoholics and 7 
nonalcoholics). *p&lt;0.05, +p&lt;0.025 compared to cells not exposed to ethanol 
acutely. 
Since extracellular adenosine is required for ethanol-induced heterologous 
desensitization in S49 cells, the effects of ethanol on extracellular 
adenosine accumulation in cultured lymphocytes from nonalcoholics and 
alcoholics was investigated. Exposure of lymphocytes from nonalcoholics to 
100 mM ethanol for 5 minutes increased the concentration of extracellular 
adenosine by 17%. In contrast, ethanol increased extracellular adenosine 
by 37% in lymphocytes from alcoholics (FIG. 5). After chronic exposure to 
100 mM ethanol for 24 hours, rechallenge with ethanol did not increase 
extracellular adenosine in lymphocytes from nonalcoholics. This is similar 
to results in chronically treated S49 cells. However, cultured lymphocytes 
from alcoholics, even after chronic exposure to ethanol, still showed a 
73% increase in extracellular adenosine when rechallenged with 100 mM 
ethanol (FIG. 5). Thus, in addition to a greater increase in extracellular 
adenosine concentration induced by acute ethanol, cells from alcoholics 
continue to accumulate adenosine even after chronic exposure to ethanol. 
This appears to account for their increased sensitivity to ethanol-induced 
heterologous desensitization. These results indicate that administering 
adenosine antagonists to alcoholics would have even greater therapeutic 
effects than on non-alcoholics since alcoholics are releasing much larger 
amounts of adenosine. 
In summary, it has been shown that acute exposure to ethanol increases the 
concentration of extracellular adenosine which then activates adenosine 
A.sub.2 receptors to increase intracellular cAMP levels. Accumulation of 
extracellular adenosine is also required for the development of chronic 
ethanol-induced heterologous desensitization of receptor-stimulated cAMP 
production. Since extracellular adenosine accumulation is greater in 
alcoholics than nonalcoholics, adenosine antagonist therapy should inhibit 
the effects of chronic alcohol abuse. Moreover, adenosine agonists should 
be useful in inhibiting alcohol withdrawal syndrome. 
Although the foregoing invention has been described in detail for purposes 
of clarity of understanding, it will be obvious that certain modifications 
may be practiced within the scope of the appended claims.