Interest of new alkylsulfonylhydrazide-type compound in the treatment of alcohol use disorders

Article abstract of DOI:10.1007/s00213-018-4917-5

Rationale: Recent preclinical research suggested that histone deacetylase inhibitors (HDACIs) and specifically class I HDAC selective inhibitors might be useful to treat alcohol use disorders (AUDs). Objective: The objective of this study was to find a new inhibitor of the HDAC-1 isoenzyme and to test its efficacy in an animal model of AUDs. Methods: In the present study, we prepared new derivatives bearing sulfonylhydrazide-type zinc-binding group (ZBG) and evaluated these compounds in vitro on HDAC-1 isoenzyme. The most promising compound was tested on ethanol operant self-administration and relapse in rats. Results: We showed that the alkylsulfonylhydrazide-type compound (ASH) reduced by more than 55% the total amount of ethanol consumed after one intracerebroventricular microinjection, while no effect was observed on motivation of the animals to consume ethanol. In addition, one ASH injection in the central amygdala reduced relapse. Conclusions: Our study demonstrated that a new compound designed to target HDAC-1 is effective in reducing ethanol intake and relapse in rats and further confirm the interest of pursuing research to study the exact mechanism by which such inhibitor may be useful to treat AUDs.

Full text of DOI:10.1007/s00213-018-4917-5

Psychopharmacology
https://doi.org/10.1007/s00213-018-4917-5
ORIGINAL INVESTIGATION
Interest of new alkylsulfonylhydrazide-type compound
in the treatment of alcohol use disorders
Jérôme Jeanblanc1,2 & Erika Bourguet2,3 & Diana Sketriené1,2 & Céline Gonzalez1,2 & Gautier Moroy4
Rémi Legastelois1,2 & Mathieu Létévé2,3 & Aurélie Trussardi-Régnier2,5 & Mickaël Naassila1,2
&
Received: 19 October 2017 /Accepted: 18 April 2018
#
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Rationale Recent preclinical research suggested that histone deacetylase inhibitors (HDACIs) and specifically class I HDAC
selective inhibitors might be useful to treat alcohol use disorders (AUDs).
Objective The objective of this study was to find a new inhibitor of the HDAC-1 isoenzyme and to test its efficacy in an animal
model of AUDs.
Methods In the present study, we prepared new derivatives bearing sulfonylhydrazide-type zinc-binding group (ZBG) and
evaluated these compounds in vitro on HDAC-1 isoenzyme. The most promising compound was tested on ethanol operant
self-administration and relapse in rats.
Results We showed that the alkylsulfonylhydrazide-type compound (ASH) reduced by more than 55% the total amount of
ethanol consumed after one intracerebroventricular microinjection, while no effect was observed on motivation of the animals
to consume ethanol. In addition, one ASH injection in the central amygdala reduced relapse.
Conclusions Our study demonstrated that a new compound designed to target HDAC-1 is effective in reducing ethanol intake and
relapse in rats and further confirm the interest of pursuing research to study the exact mechanism by which such inhibitor may be
useful to treat AUDs.
.
.
.
.
.
Keywords Alkylsulfonylhydrazide Histone deacetylase Zinc-binding group Inhibition Ethanol self-administration Rats
Abbreviations
FDA
HAT
Food Drug Administration
Histone acetyltransferase
ASH
AUD
aCSF
CeA
Alkylsulfonylhydrazide
Alcohol use disorder
Artificial cerebrospinal fluid
Central nucleus of amygdala
HDAC Histone deacetylase
HDACI Histone deacetylase inhibitor
i.c.v.
Intracerebroventricular
Jérôme Jeanblanc and Erika Bourguet contributed equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00213-018-4917-5) contains supplementary
material, which is available to authorized users.
3
* Erika Bourguet
Institut de Chimie Moléculaire de Reims, CNRS UMR 7312,
Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay,
51096 Reims Cedex, France
erika.bourguet@univ–reims.fr
* Mickaël Naassila
mickael.naassila@u–picardie.fr
4
5
Molécules Thérapeutiques In Silico (MTi), INSERM UMR-S 973,
Université Paris Diderot, Sorbonne Paris Cité, 35 rue Hélène Brion,
75013 Paris, France
1
Groupe de Recherche sur l’Alcool et les Pharmacodépendances
(GRAP), INSERM U1247, Université de Picardie Jules Verne,
C.U.R.S. (Centre Universitaire de Recherche en Santé), Chemin du
Thil, 80000 Amiens, France
Unité Derm-I-C, EA n°7319, Faculté de Médecine de Reims,
Université de Reims Champagne-Ardenne, 1 rue du Maréchal Juin,
51096 Reims Cedex, France
2
Structure Fédérative de Recherche-Champagne Ardenne Picardie
Santé (SFR-CAP Santé), Amiens, France

Psychopharmacology
PBS
PFA
SAHA
TSA
ZBG
Phosphate-buffered saline
Paraformaldehyde
Suberoylanilide hydroxamic acid
Trichostatin A
treated with SAHA and the effect is still present only 1 day
post-treatment. Warnault et al. (2013) also demonstrated that
all the HDACIs are selective of ethanol since they did not
observe any effect on saccharin intake. Sodium butyrate has
been shown to be very effective in reducing ethanol intake in a
model of ethanol intake escalation. Simon-O’Brien et al.
(2015) have demonstrated that sodium butyrate can either pre-
vent ethanol intake escalation or reduce intake once rats dis-
play escalation of ethanol intake. The same authors have also
demonstrated that sodium butyrate is very effective in reduc-
ing ethanol intake in dependent rats that have been exposed to
chronic and intermittent ethanol vapor. The effect of sodium
butyrate on ethanol dependent animals was mimicked by MS-
275 (10 mg/kg once a day, via the intraperitoneal way) since it
decreased the operant ethanol self-administration after the sec-
ond injection in ethanol-dependent rats (Simon-O’Brien et al.
2015). In addition, the effect of intracerebroventricular (i.c.v.)
injections of MS-275 was also tested in a model of voluntary
heavy drinking in which rats self-administered excessive eth-
anol intake for 2 months (Jeanblanc et al. 2015). MS-275
decreased operant ethanol self-administration (75% decrease)
after the second i.c.v. injection (one injection per day) at the
dose of 500 μM, the motivation to consume ethanol (25%
decrease) and relapse after a period of abstinence (50% de-
crease). Altogether, the results demonstrated that both non-
selective HDACIs and more selective inhibitors targeting the
class I HDACs are effective in reducing ethanol intake and
relapse in rats displaying excessive ethanol intake and/or eth-
anol dependence. Attention has now turned to more potent
and selective compounds that may prove to be more effective.
We previously demonstrated that HDAC-1 mRNA levels
are increased after either acute or repeated ethanol injections
in the striatum (Legastelois et al. 2013). Interestingly, HDAC-
1 is predominantly localized in the nucleus and is part of stable
transcriptional complexes that are recruited to gene promoters
by DNA-binding proteins thus suggesting gene-specific rather
than global and unspecific transcriptional regulation
(Kazantsev and Thompson 2008). Results obtained with
MS-275, which has been described as more selective inhibitor
of HDAC-1 (Khan et al. 2008 but see Glaser et al. 2004), on
ethanol-related behaviors are encouraging in terms of
targeting specifically HDAC-1 to treat AUD, but this mole-
cule may suffer from its poor brain penetration (Hooker et al.
2010). Despite, its poor brain penetration, we demonstrated its
efficacy to reduce ethanol intake after one daily peripheral
injection for 2 days (Simon-O’Brien et al. 2015). In this con-
text, it seems of great interest to develop new molecules
targeting specifically HDAC-1 in order to potentially achieve
higher efficacy (potential effect after only one administration
and less side effects).
Zinc-binding group
Introduction
Alcohol use disorder (AUD) is a chronic disorder character-
ized by the loss of control over ethanol intake, compulsive use
of ethanol, and development of a negative emotional state
during ethanol withdrawal. A large body of evidence has
shown that epigenetic mechanisms and especially those in-
volving histone acetylation/deacetylation may play a role in
the development of AUD (Botia et al. 2012; Jangra et al. 2016;
Barbier et al. 2017; Berkel and Pandey 2017; Pandey et al.
2017). New strategies for the treatment of AUDs are a press-
ing need, and it has been suggested that targeting histone
deacetylase (HDAC) may have great potential (Jangra et al.
2016; Bourguet et al. 2018; Warnault et al. 2013; Legastelois
et al. 2017).
Histone acetyltransferases (HATs) and HDACs regulate
gene transcription by acetylation-deacetylation of histones,
respectively (Mottet and Castronovo 2008). Up to date, 18
human isoforms of HDAC have been identified and are listed
in four classes based on their sequence homology with the
yeast (Saccharomyces cerevisiae) transcriptional regulator
RPD3, their location, and their molecular weight. All classes
of HDACs contain a zinc ion in their active site except for
class III, whose activity is regulated by NAD+. Members of
the classical HDAC family are differentiated into two phylo-
genetic classes, called class I and class II. The HDACs of class
I (HDAC-1, HDAC-2, HDAC-3, and HDAC-8) are mostly
located in the nucleus, whereas HDACs from class II
(HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-9, and
HDAC-10) sharing homologies with the yeast Hda1 protein
can shuttle between the cytoplasm and the nucleus. Class III
HDACs have been identified to belong to the Sir2 family,
requiring NAD+ co-factor for deacetylation, while HDAC-11
is the only isoenzyme of the class IV (de Ruijter et al. 2003).
Preclinical studies demonstrated that HDAC inhibitors
(HDACIs) could be useful tools to treat AUDs. Non-specific
HDACIs such as sodium butyrate, TSA, and the FDA-
approved SAHA (Fig. 1a) have been shown to reduce ethanol
intake and ethanol-withdrawal-induced anxiety (Sakharkar
et al. 2012; Pandey et al. 2008; Warnault et al. 2013). In mice
and rats, SAHA (50–100 mg/kg) inhibited ethanol intake and
the motivation of rats to seek ethanol (Warnault et al. 2013).
The same authors showed that both TSA (0.1–0.4 mg/kg) and
the more selective HDAC class I inhibitor MS-275 (5–20
mg/kg) are also effective in reducing ethanol intake in mice.
The reduction of ethanol intake is lasting as long as mice are
All current HDACIs are characterized by a common
pharmacophore with three key-elements for inhibitor-
enzyme interactions: a metal-binding domain that interacts

Psychopharmacology
Fig. 1 a Pharmacophore of different HDACIs. b Structures of molecules
envisioned as HDACIs. c Reagents and conditions: (a) Cbz-beta-Ala-OH,
HBTU, 2,4,6-collidine, CH2Cl2, RT, 18 h, 48%; (b) TFA, CH2Cl2, RT, 1 h
100%; (c) Me-SO2-Cl, Et3N, THF, RT, 2 h, 50%. d detailed binding mode
of the compound 4 with HDAC-1. HDAC-1 is in white cartoon represen-
tation. Compound 4 and residues implicated in the interaction are in stick
representation. The carbon atoms of 4 are colored in magenta, and those
of HDAC-1 are in green. The Zn atom is in gray sphere
with the active site, a linker domain, and a surface recognition
group interacting with the residues on the rim of the active site
(Fig. 1a) (Chavan and Somani 2010). In 2003, Auge et al.
(2003) introduced a sulfonylhydrazide group (R–SO2–NH–
NH–CO–), as new zinc-binding group (ZBG) replacing the
hydroxamate function and docking experiments of
sulfonylhydrazide type inhibitors evidenced a NH and CO
chelation with the Zn2+ ion similarly to hydroxamate ZBG.
In the present work, we sought to evaluate whether
sulfonylhydrazide ZBG-bearing derivatives may be consid-
ered as a novel class of HDACIs that may target HDAC-1
based on our structure-activity relationships and molecular
docking experiments. To test this hypothesis,
alkylsulfonylhydrazide chelators were introduced (Fig. 1b).
H e r e, ou r p r i m a r y a i m w a s t o t e s t o u r n ew
alkylsulfonylhydrazide-type compound (ASH), the benzyl
3-(2-(methylsulfonyl)hydrazinyl)-3-oxopropylcarbamate
compound, targeting HDAC-1, on ethanol operant self-
administration and relapse in rats. We also wanted to test
whether the injection of the molecule directly in the central
amygdala (CeA) could reduce relapse after an extinction of
the behavior. The role of the CeA in the control of emotional
behavior, anxiety, and relapse for drugs of abuse is well de-
scribed in the literature (Roberto et al. 2012; Gilpin et al.
2015). For example, Pandey and colleagues (Pandey et al.
2008) demonstrated that the anxiolytic effect of acute expo-
sure to ethanol is associated to an increase in the expression of
Arc and BDNF within the central but not the basolateral
amygdala. On the opposite, during withdrawal, when anxiety
increases, expression of both genes is decreased and
microinfusion of BDNF into CeA is able to attenuate
anxiety-like behaviors during withdrawal. Moreover, several
reports and reviews already implicated epigenetic mecha-
nisms occurring within the CeA during alcohol exposure, con-
sumption, and withdrawal (Moonat et al. 2013; Simon-
O’Brien et al. 2015; Pandey et al. 2017). Thus, in order to
evaluate the effect of a new HDACI on relapse after extinc-
tion, CeAwas an obvious neurobiological substrate candidate
to test our compound.
Materials and methods
Chemistry
All the reagents and the experiments carried out for chemistry
and the synthesis of the compounds are detailed in supplemen-
tary informations.

Psychopharmacology
HDAC assays
but no programmed events occurred. The effort required to ob-
tain the reward was progressively increased (from one press to
three consecutive presses to obtain the reward), and then the
duration of the sessions were progressively decreased (from
1 h to 15 min). Animals were trained for at least 5 weeks before
the beginning of the experimental manipulations. During the
self-administration sessions, number of lever presses and number
of ethanol deliveries were recorded using PackWin software
(Bioseb, Vitrolles, France). This paradigm allows us to observe
ethanol intake reaching a level near 1 g/kg/15 min which are
above the intoxication levels.
All details regarding HDAC assays are detailed in supplemen-
tary informations.
Docking
All details regarding docking studies are detailed in supple-
mentary informations.
Binge drinking behavior in the operant
self-administration paradigm
Sucrose self-administration
Animals
Ten naïve rats were submitted to a procedure of self-
administration of a 4% sucrose solution in operant chambers as
described for the ethanol self-administration group. After two
overnight sessions under a fixed ratio one schedule (one press
on the active lever leads to a delivery of 0.1 mL of a 4% sucrose
solution), rats underwent daily sessions of 1 h for 3–4 days under
a FR3 schedule, 30 min and then 15 min for at least 2 weeks
before the surgery aiming at the implantation of guide cannula
targeting the lateral ventricle. Four rats had to be excluded from
the study due to a lack of acquisition of the operant task.
Male Long-Evans rats (320–345 g at the beginning of the exper-
iment) were obtained from Charles River (L’Arbresle, France).
Animals were individually housed under a light/dark cycle of
12 h (lights on at 7:00 am) with food and water available ad
libitum. Experiments were carried out in accordance with the
guidelines for Care and Use of Laboratory Animals (National
Institutes of Health) and the European community regulations
for animal use in research (CEE no. 86/609) and were approved
by the local research ethics committee (CREMEAP; no. 260912-
10). The total number of animals used in this study was 40 and
we had to exclude a total of 10 rats (misplacement of the cannula
or no acquisition of the operant task).
Surgery
Stereotaxic surgery was used to implant each rat with one cannula
(i.c.v. group) or two cannulae (central nucleus of amygdala, CeA
group). Rats were continuously anesthetised with isoflurane during
the surgery. Four holes were drilled for screws, one or two other
holes were drilled for the placement of cannulae (26GA, 12 mm,
Phymep). The coordinates for the lateral ventricle were −0.8 mm
posterior to bregma, + 1.4 mm lateral to the medial suture, and −
3 mm from the skull surface. The coordinates for the CeA were −
1.80 mm from bregma, 4.0 mm laterally from median suture, and
−6.0 mm from skull surface. The cannulae were fixed with dental
cement. The injectors used for the microinfusions extended 1 mm
below the tip of the cannula for the i.c.v. group and 2 mm for the
CeA group. Subject weights were monitored daily after the surgery
for five consecutive days to ensure recovery. One week after recov-
ery, subjects returned to self-administration training and were habit-
uated to the microinjection procedure with two sham injections. The
experimental microinjections began upon acquisition of stable
responding for ethanol.
Reagents
ASH compound was dissolved to a concentration of 125, 250,
and 500 μM in an artificial cerebrospinal fluid (aCSF) solution
(CMA Microdialysis, Solna, Sweden). Ethanol 96% was pur-
chased from VWR (Fontenay-sous-Bois, France). Phosphate-
buffered saline (PBS) was made with NaCl 137 mM, KCl
2.7 mM, Na2HPO4 10 mM, and KH2PO4 1.8 mM purchased
from Sigma-Aldrich (Saint-Quentin Fallaviers, France).
Paraformaldehyde (Sigma-Aldrich, Saint-Quentin Fallaviers,
France) was dissolved in PBS at 4%. Sodium pentobarbital
sodium (54.7 mg/100 mL) and buprenorphine (Buprecare) were
purchased from Centravet (Dinan, France).
Ethanol self-administration
Naïve rats were exposed to a 20% intermittent access paradigm
with access to two bottles: one containing tap water and the other
a 20% ethanol solution, every other day in the homecage. This
procedure induces escalation of ethanol consumption in several
strains of rats. After 4 weeks of 20% intermittent access, rats
were trained to self-administer ethanol. The self-administration
chambers contained two levers: an active lever for which presses
resulted in delivery of a 0.1 mL of fluid reward (a 20% ethanol
solution) and an inactive lever, for which presses were counted,
Microinfusions
The intracerebral route of administration (i.c.v. and intra CeA)
was preferred because of the synthetized quantity available, to
avoid potential peripheral side effects and because we have pre-
viously shown that MS-275 has the same behavioral effect when
injected either via the i.c.v. or the i.p. route (Simon-O’Brien et al.

Psychopharmacology
2015). ASH compound (0, 150, 250, 500, or 1000 μM) was
microinfused i.c.v. and intra-CeA 3 h before the beginning of
the self-administration sessions (as we previously done in
Jeanblanc et al. 2015). For the test of ASH on relapse, only the
dose of 500 μM was microinfused. Microinfusions were per-
formed with a Harvard pump 11 Plus Advanced (Phymep,
France) and 25-mL Hamilton syringes (1702 N, Bonaduz,
Switzerland). Injectors were built using stainless steel tubing
(outer diameter 0.229 mm, inner diameter 0.127 mm,
Phymep). ASH compound was microinfused at a speed of
1 μL/min for the i.c.v. group (2 μL total) and at 0.5 μL/min for
the CeA group (0.3 μL). All the injections were performed fol-
lowing a Latin square design.
comparisons, data were analyzed by a Student’s t test.
Significance for all tests was set at p < 0.05.
Results
Chemistry
The coupling was carried out between tert-butylcarbazate and
Cbz-beta-alanine with HBTU as coupling agent in the pres-
ence of 2,4,6-collidine. The product 2 was obtained with a
yield of 48%. Deprotection of the Boc protective group was
performed with TFA and the compound was obtained in quan-
titative yield. Then, the reaction with methanesulfonyl chlo-
ride in THF with Et3N allowed us to obtain compounds 4
(alkylsulfonylhydrazide-type compound (ASH) for benzyl
3-(2-(methylsulfonyl)hydrazinyl)-3-oxopropylcarbamate) in
50% yield (Fig. 1c).
Progressive ratio
To evaluate the effect of ASH compound on the motivation to
consume alcohol, we performed a progressive ratio test. In this
test, the delivery of each reward requires an increased number
of active lever presses for still the same volume of 0.1 mL of
the 20% ethanol solution. The schedule of increase is as fol-
lows: 3, 4, 5, 7, 9, 12, 15, 17, 20, 22, 25, 28, 30, 33, and 35.
The breaking point, i.e., the maximum effort rats are willing to
make to obtain a single reward reflects the level of motivation
to consume the drug. ASH compound (500 μM) was i.c.v.
microinfused 3 h before the session.
HDAC assays
The inhibitory activity of compound 4 (ASH) towards HDACs
from a total nuclear extract using a HDAC colorimetric assay kit
(Active Motif, Inc., Belgium) was performed. Half maximal in-
hibitory concentration (IC50) of ASH was estimated to 8 mM. The
ASH compound decreased the emitted fluorescence displaying
more than 51% of HDAC-1 inhibitory activity.
Relapse after extinction
Docking
After several weeks of self-administration, rats were trained to
extinguish their pressing behavior during sessions in which
both levers were present, but the presses on any of them led
to no delivery of ethanol. After 1 week, the number of active
lever presses decreased, and for each animal, the threshold
determining the extinction was set to 10% of the baseline for
three consecutive days. The relapse test is based on the re-
acquisition paradigm in which ethanol is available again (un-
der a FR3 schedule) after a free prime of 0.1 mL. ASH com-
pound was microinfused into the CeA 3 h prior to the test.
Based on the blind docking experiments, compound 4 has the
best affinity for HDAC-1 in the vicinity of the catalytic site.
Further docking experiments with a better accurately were there-
fore focused on the catalytic site to identify the most likely bind-
ing mode between HDAC-1 and compound 4. The analysis of
docking results shows that the lowest energy models are stabilized
by the chelation of Zn atom by the sulfonyl group (Fig. 1d) and by
insertion of the methyl group of the sulfonylhydrazide moiety into
a hydrophobic pocket formed by Met 30, Gly 138, Leu 139, Phe
150, Gln 260, Gly 300, Gly 301, and Tyr 303. Two hydrogen
bonds are observable, the first one between the imidazole ring of
His 178 and the oxygen atom of the carbamate function and the
second one between the carbonyl group of Gly 149 and the NH
group close to the carbonyl of the sulfonylhydrazide moiety.
Moreover, the binding mode is stabilized by a π-stacking interac-
tion between the phenyl group of 4 and the aromatic ring of Tyr
204 sidechain.
Histology
Rats were deeply anesthetized with pentobarbital and were sub-
mitted to a transcardiac perfusion with 50 mL of PBS and 50 mL
of PFA 4%. Brains were stored in a sucrose 20%/PFA 4% solu-
tion for 48 h. Brains were then cut in slices of 100 μm of thickness
for histological verification of the cannulae placements.
Statistical analysis
Histology
Data were analyzed by one-way ANOVA with repeated mea-
sures followed by a Tukey post hoc test when indicated by
significant effects of treatments or interactions. For simple
No rats were excluded from the i.c.v. study due to misplacement
of the cannulae, while two rats had to be excluded from the
analysis of the intra-CeA study. Schematic representation of the

Psychopharmacology
placements of the different cannula is shown in Supplementary
Fig. 1 (a) for the i.c.v. group and (b) for the intra-CeA group.
ASH compound decreases ethanol self-administration
We tested the effect of ASH compound on operant ethanol
self-administration in an original model of voluntary binge
drinking in rats. We used a specific behavioral paradigm to
achieve high voluntary ethanol intake to mimic binge drink-
ing. In this model, rats drink more than 0.8 g of pure ethanol
per kilogram of body weight in only 15 min and display clear
signs of intoxications such as sedation and ataxia at the end of
the 15-min session. Intracerebroventricular injections of ASH
compound were performed 3 h prior to the self-administration
sessions as we have already performed in our previous studies
with MS-275 (Jeanblanc et al. 2015). This delay was chosen
because it allows changes in gene expression to occur. Each
rat (n = 12) received a microinfusion of each ASH compound
concentration with a 1-week period of washout between two
microinfusions. As shown in Fig. 2a, we observed a dose-
dependent decrease in the number of active lever presses to
obtain the delivery of alcohol. A one-way ANOVA with re-
peated measures indicated a main effect of the factor treat-
ment (F(4, 44) = 5.24, p = 0.002). The post hoc test (Tukey)
conducted on the data revealed significant differences be-
tween the group aCSF and the dose of 500 μM (p < 0.05).
No other doses were significantly different from aCSF (all
p’s > 0.05). Significant differences were observed between
the group 125 μM and both 250 and 500 μM groups. The
total amount of alcohol consumed during these 15-min ses-
sions was analyzed using a one-way ANOVA with repeated
measures (Fig. 2b). This analysis indicated a significant effect
of the factor treatment (F(4, 44) = 5.46, p < 0.001). The post
hoc test revealed a significant difference between the aCSF
group and the 500 μM group (p < 0.05). No other group was
significantly different from the aCSF group (all p’s > 0.05).
Significant differences were observed between the group
125 μM and both 250 and 500 μM groups. The test also
revealed a significant difference between the 500 and the
1000 μM groups. Further analysis of the pattern of drinking
for the aCSF group and the 500 μM group indicates that there
is no difference between both groups as regards with the
latency to obtain the first reward (p > 0.05, Fig. 2c). On the
opposite, the latency to the delivery of the last reward is
significantly shorter for the group 500 μM as compared to
the aCSF group (p < 0.05, Fig. 2d). In addition, and as a
consequence, the duration of the consummatory episode is
shorter for the 500 μM group (p < 0.05, Fig. 2e). The analysis
of drinking pattern after the 500 μM dose of ASH compound
microinfusion was also conducted using the cumulative de-
liveries. For this analysis, only rats showing at least one de-
livery during the session were included (n = 10). As shown in
Fig. 2f, the initiation of the drinking episode is similar in both
Fig. 2 The ASH compound reduces alcohol self-administration. a The
number of active lever presses is dose-dependently reduced after the i.c.v.
microinfusion of the ASH compound. Results are expressed as mean
SEM of active lever presses. n = 12, *p < 0.05, **p < 0.01. b The total
amount of alcohol consumed during the 15-min session is dose-
dependently reduced after the i.c.v. microinfusion of the ASH compound.
Results are expressed as mean SEM of pure ethanol consumed in g/kg/
15 min. n = 12, *p < 0.05, **p < 0.01. The most efficient dose of ASH
compound (500 μM) does not alter the latency for the first press but leads
to a premature end d and a reduced duration of the drinking episode. e
Results are expressed as mean SEM in minutes. n = 11, *p < 0.05. One
rat was removed from this analysis because it did not obtain any reward
over the 15-min session. f The ASH compound modifies the pattern of
drinking over the 15-min session. g Number of active lever presses for 4%
sucrose. Results are expressed as mean SEM of cumulative active lever
presses. n = 12, *p < 0.05
groups (aCSF and ASH compound at 500 μM). It is only
after few deliveries that ASH compound blocked the increase
in ethanol consumption along the self-administration session.
The two-way repeated measures ANOVA conducted on the
data revealed a main effect of the factor treatment (F(1,36) =
5.11, p = 0.050), of the factor time (F(4,36) = 12.40, p <
0.001), and indicated an interaction between both factors
(F(4,36) = 2.79, p < 0.05). The post hoc test indicated a sig-
nificant difference between groups aCSF and ASH 500 μM
for the time points 6–9, 9–12, and 12–15 min (all p’s < 0.05).
ASH compound (250 and 500 μM) was i.c.v. microinfused in
rats trained to self-administer 4% sucrose and show no effect

Psychopharmacology
on the level of sucrose self-administration (Fig. 2g). A one-
way ANOVA with repeated measures analysis revealed no
effect of the factor treatment (F(2,10) = 1,88, p > 0.05).
Rats underwent to a regular 15-min session of ethanol self-
administration the day after the microinfusion (Fig. 3a), and
the results indicate that there is no more differences between
the groups (F(4, 44) = 0.16, p > 0.05).
and humans (López-Moreno et al. 2015). A large body of ev-
idence suggests that epigenetic regulation of gene expression,
including histone acetylation and deacetylation, contributes to
the development of ethanol reward-related behaviors linked to
ethanol addiction (Pandey et al. 2008; Bourguet et al. 2018;
Pandey et al. 2017). Recent studies have also suggested that
targeting a specific HDAC class and isoenzyme such as
HDAC-1 (Jeanblanc et al. 2015; Bourguet et al. 2018) or
HDAC-2 (Sakharkar et al. 2014) can reduce ethanol-related
behaviors. Numerous studies that have investigated the role of
HDAC in ethanol-related behaviors used non-specific
HDACIs, and thus further studies are needed in order to deci-
pher which HDAC isoenzyme may play a role in particular
components of ethanol addiction. Finding new epigenetic drugs
targeting specific HDAC isoenzyme could be useful in order to
avoid toxic side effects that are currently seen with available
HDACIs. Because MS-275, suggested as more selective inhib-
itor of HDAC-1, has been shown to have promising potential to
treat ethanol-related behaviors but may suffer from its poor
brain penetration capacity, there is an interest to further develop
new compounds targeting HDAC-1. In the present study, using
docking and enzyme activity tests, we identified an
alkylsulfonylhydrazide-type compound as a good inhibitor of
HDAC-1. We found that administration of ASH via the i.c.v.
route, but not directly in the CeA, decreased ethanol operant
self-administration. As shown with other HDACIs, the effect of
ASH lasted only the first day of administration. Moreover,
when injected directly in the CeA, ASH decreased resumption
of ethanol self-administration after abstinence.
ASH compound does not alter the motivation
to consume ethanol
Motivation to consume was evaluated during a progressive ratio
protocol in which the effort to obtain a delivery of ethanol in-
creases progressively after each delivery. We observed (Fig. 3b)
that the microinfusion of the dose of 500 μM has no effect
(Student’s t test, p > 0.05) on the value of the breaking point.
ASH compound microinfused within the CeA does not
alter ethanol consumption but reduces relapse
after extinction
The dose of 500 μM was microinfused 3 h prior to the self-
administration sessions and had no effect on the total amount
of ethanol consumed (Fig. 4a, p > 0.05). Rats were then sub-
mitted to seven sessions of extinction (Fig. 4b) and re-exposed
to ethanol during a re-acquisition session. We found that the
aCSF group relapsed moderately to about 50% of their previ-
ous baseline of ethanol consumption (0.53 g/kg/15 min vs.
0.91 g/kg/15 min). The ASH compound group relapsed sig-
nificantly less than the aCSF group with an average of 0.22 g/
kg/15 min (p < 0.05, Fig. 4c).
After the identification of the ASH compound as a prefer-
entially inhibitor of HDAC-1 isoform, we evaluated its effica-
cy on ethanol-related behaviors. We used the operant self-
administration procedure in an animal model of binge-like
consumption, i.e., intake of large quantity of ethanol during
a short period of time. First, we tested the dose-response effect
of the ASH compound on operant self-administration when
microinfused 3 h prior to the sessions via the i.c.v. route, and
Discussion
Acute and chronic ethanol intake has been shown to change
HDAC gene expression within the peripheral blood in both rats
Fig. 3 a The ASH compound has no more effect on alcohol self-
administration 27 h after its i.c.v. microinfusion. Results are expressed
as mean SEM of pure ethanol consumed in g/kg/15 min. n = 12. b The
ASH compound, at the dose of 500 μM i.c.v. administrated, does not
modify the motivation to consume alcohol. Results are expressed as mean
SEM of the breaking point meaning the maximum effort a rat is willing
to make to obtain one single reward (0.1 mL of 20% ethanol). n = 8

Psychopharmacology
Fig. 4 a Intra-CeA microinfusion of ASH compound (500 μM) does not
alter alcohol self-administration. Results are expressed as mean SEM of
pure ethanol consumed in g/kg/15 min. n = 12. b Rats extinguished their
pressing behavior after seven consecutive days of extinction sessions in
which the levers were available, but no alcohol was delivered at any time.
The extinction criterion was set to three consecutive sessions with a level
of consumption below 10% of the baseline level when alcohol is
available. Results are expressed as mean SEM of pure ethanol
consumed in g/kg/15 min. n = 12. c ASH compound microinfused into
the CeA 3 h prior to the test reduces the relapse after a period of
abstinence (extinction). Results are expressed as mean SEM of pure
ethanol consumed in g/kg/15 min. n = 10, *p < 0.05
we observed a U shape curve with the maximum efficacy (−
55% of ethanol consumed) at the dose of 500 μM, while an
absence of effect was also observed at the highest dose
(1000 μM). All the different HDACIs so far identified are
not specific to only one HDAC isoform but have different
affinity for several isoforms. It cannot be ruled out that the
absence of effect at the highest dose of our ASH compound is
due to its action through other HDAC isoforms than the
HDAC-1. By analyzing the pattern of drinking, by the mean
of the latencies to the first reward, the delay to obtain the last
reward and the profile of the cumulative ethanol deliveries, we
can conclude that the ASH compound does not alter the initi-
ation of the consumption episode but reduces its total duration
by inducing its premature termination. Unlike MS-275 that
postponed the initiation of the drinking episode (Jeanblanc
et al. 2015), ASH compound produces its effect only after a
certain amount of ethanol consumed. In addition, we found
that the ASH compound has no effect on the motivation to
consume ethanol in the progressive ratio schedule. All togeth-
er, these results suggest that the ASH compound does not
affect the motivation to consume ethanol and that either there
is an aversive effect due to the ASH compound in the presence
of ethanol which makes the rats stop pressing to obtain more
ethanol or there is a substitutive effect of our ASH compound
since ethanol exposure leads to a hyperacetylation of various
proteins including histones (Shepard and Tuma 2009).
Interestingly, the overall effect of our ASH compound is
similar to the one observed with the MS-275 which is de-
scribed as a HDAC-1, HDAC-3, HDAC-8, and HDAC-9 on
the reduction of alcohol self-administration (Jeanblanc et al.
2015). Here, with the ASH compound, the effect occurs after
the first administration, whereas with the MS-275, two con-
secutive daily microinfusions were necessary to significantly
decrease ethanol self-administration. However, the mecha-
nisms leading to the reduction of ethanol consumption of
these two drugs seem different. Indeed, MS-275 reduces eth-
anol consumption by a reduction of the motivation to consume
ethanol, whereas the ASH compound has no effect on the
motivation but stop the drinking episode after a certain
amount of consumed ethanol. These differences could be
due to a difference in the inhibitory spectrum towards
HDACs isoforms. In addition, we found that our ASH com-
pound has no effect on sucrose self-administration suggesting
that it does not have any locomotor effects. Moreover, this
absence of effect may suggest a specific effect to alcohol or
drug of abuse and not a general effect on all the rewarding
substances.
One of the major issues in the treatment of patients with
AUDs is the maintenance of the abstinence period and thus to
avoid the occurrence of relapse. The withdrawal-induced anx-
iety is often described as a main reason for the relapse (Becker
2008; Simioni et al. 2012), and several studies have implicated

Psychopharmacology
epigenetic mechanisms in a specific brain structure, namely
the central amygdala, as a key factor for relapse (Silberman
et al. 2009; Gilpin et al. 2015). We tested here the efficacy of
the ASH compound on both ethanol self-administration and
on relapse after a period of abstinence. Interestingly, the
microinfusion directly into the CeA has no effect on the total
amount of ethanol consumed in a regular self-administration
session. This results suggest either that epigenetic modifica-
tions induced by ASH within the CeA has no role in the
control of ethanol consumption or that the final quantity of
ASH is high for a small structure such as CeA and thus that we
are on the ascending arm of the BU shape^ curve observed in
Fig. 2a, b (the total amount of ASH compound is about 315 ng
when i.c.v. microinfused, while it is of about 45 ng for the intra
CeA microinfusion).
On the other hand, after a week of abstinence conducted in
a behavioral extinction paradigm, we performed a relapse test
using a re-acquisition protocol in which one delivery of etha-
nol is given Bfor free^ before the animals can again get as
much ethanol as they want by pressing the active lever three
times during the 15-min session. Control animals relapse to
half of their pre-extinction baseline level, whereas animals
treated with ASH only relapse at 20% of their pre-extinction
baseline level.
Thus, we can conclude that the ASH compound has a rapid
but transient effect on ethanol consumption after a single ad-
ministration. This decrease in ethanol consumption is not me-
diated by a decrease in the motivation to consume ethanol but
appears after a certain amount of ethanol consumed as if there
was a BSTOP^ signal leading to the early termination of the
drinking episode. Although the overall effect on ethanol use
appears to be identical, the underlying mechanisms are cer-
tainly different as the ASH compound and the MS-275 does
not have the same effects on the motivation to consume etha-
nol and the consumption profile. It is possible that, due to the
difference of the two compounds regarding HDAC specificity,
they do not target the same molecular mechanisms. The ques-
tion of specificity supports the interest of pursuing research in
the synthesis of new ultra-specific compounds of each of the
different HDAC isoforms in order to really succeed from a
purely fundamental point of view to identify the role of each
HDAC in addictive behaviors and on the other hand, from a
clinical point of view, choosing the treatment to the patients
according to their addictive symptomatology (therapeutic ob-
jective oriented either towards the reduction of the consump-
tion, or the maintenance of abstinence, or the reduction in
craving, or the decreased in motivation to consume drugs,
etc.). Moreover, we show that the administration of an
HDAC-1 inhibitor directly into the CeA nucleus makes it
possible to reduce relapse.
after a single injection needs to be further investigated and
especially regarding the genes whose expression is change
and that may be involved in gating ethanol intake. In the same
vein, the effect of the HDAC inhibitor does not last 1 day after
treatment, and thus, it could be very interesting to explore the
effect of a chronic treatment. The mechanistic knowledge will
help develop new therapeutic interventions targeting specific
components of ethanol addiction.
Conclusion
In conclusion, we report for the first time the interest of new
alkylsulfonylhydrazide compound in the treatment of AUDs.
In the animal model of voluntary operant ethanol binge drink-
ing, only one i.c.v. microinjection of the ASH compound sig-
nificantly inhibited ethanol intake, whereas two consecutive
injections were necessary with other HDACIs (Jeanblanc et al.
2015; Simon-O’Brien et al. 2015). Our ASH compound is not
only able to reduce ethanol self-administration but also it sig-
nificantly reduced relapse when directly injected into the CeA.
In addition, we demonstrate that the mechanism of action of
our ASH is different to the MS-275 one despite the chemical
similarity suggesting a difference in the molecular targets. On
the basis of these results, the design of next generation of
alkylsulfonylhydrazide will be engaged to unravel the exact
mechanism of action of this type of molecules.
Acknowledgements Financial support by SFR CAP-Santé, Université de
Reims Champagne-Ardenne, Université de Picardie, Conseil Régional de
Picardie, CNRS, Ministry of Higher Education and Research (MESR), is
gratefully acknowledged.
Compliance with ethical standards
Experiments were carried out in accordance with the guidelines for Care
and Use of Laboratory Animals (National Institutes of Health) and the
European community regulations for animal use in research (CEE no. 86/
609) and were approved by the local research ethics committee
(CREMEAP; no. 260912-10).
References
Auge F, Hornebeck W, Decarme M, Laronze JY (2003) Improved
gelatinase a selectivity by novel zinc binding groups containing
galardin derivatives. Bioorg Med Chem Lett 13:1783–1786
Barbier E, Johnstone AL, Khomtchouk BB, Tapocik JD, Pitcairn C,
Rehman F, Augier E, Borich A, Schank JR, Rienas CA, Van
Booven DJ, Sun H, Natt D, Wahlestedt C, Heilig M (2017)
Dependence-induced increase of alcohol self-administration and
compulsive drinking mediated by the histone methyltransferase
PRDM2. Mol Psychiatry 22:1746–1758
Becker HC (2008) Alcohol dependence, withdrawal, and relapse.
Alcohol Res Health 31:348–361
Berkel TD, Pandey SC (2017) Emerging role of epigenetic mechanisms
in alcohol addiction. Alcohol Clin Exp Res 41:666–680
Our current observations raise a number of interesting
questions for future study. The mechanism by which an
HDAC inhibitor can reduce ethanol self-administration 3 h

Psychopharmacology
Botia B, Legastelois R, Alaux-Cantin S, Naassila M (2012) Expression of
ethanol-induced behavioral sensitization is associated with alteration
of chromatin remodeling in mice. PLoS One 7:e47527
Bourguet E, Ozdarska K, Moroy G, Jeanblanc J, Naassila M (2018) Class
I HDAC inhibitors: potential new epigenetic therapeutics for alcohol
use disorder (AUD). J Med Chem 61:1745-1766
Chavan AV, Somani RR (2010) HDAC inhibitors—new generation of
target specific treatment. Mini Rev Med Chem 10:1263–1276
de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB
(2003) Histone deacetylases (HDACs): characterization of the clas-
sical HDAC family. Biochem J 370:737–749
Gilpin NW, Herman MA, Roberto M (2015) The central amygdala as an
integrative hub for anxiety and alcohol use disorders. Biol
Psychiatry 77:859–869
Glaser KB, Li J, Pease LJ, Staver MJ, Marcotte PA, Guo J, Frey RR,
Garland RB, Heyman HR, Wada CK, Vasudevan A, Michaelides
MR, Davidsen SK, Curtin ML (2004) Differential protein acetyla-
tion induced by novel histone deacetylase inhibitors. Biochem
Biophys Res Commun 325:683–690
Hooker JM, Kim SW, Alexoff D, Xu Y, Shea C, Reid A, Volkow N,
Fowler JS (2010) Histone deacetylase inhibitor, MS-275, exhibits
poor brain penetration: PK studies of [C]MS-275 using positron
emission tomography. ACS Chem Neurosci 1:65–73
Jangra A, Sriram CS, Pandey S, Choubey P, Rajput P, Saroha B,
Bezbaruah BK, Lahkar M (2016) Epigenetic modifications, alcohol-
ic brain and potential drug targets. Ann Neurosci 23:246–260
Jeanblanc J, Lemoine S, Jeanblanc V, Alaux-Cantin S, Naassila M (2015)
The class I-specific HDAC inhibitor MS-275 decreases motivation
to consume alcohol and relapse in heavy drinking rats. Int J
Neuropsychopharmacol 18:pyv029
Moonat S, Sakharkar AJ, Zhang H, Tang L, Pandey SC (2013) Aberrant
histone deacetylase2-mediated histone modifications and synaptic
plasticity in the amygdala predisposes to anxiety and alcoholism.
Biol Psychiatry 73:763–773
Pandey SC, Ugale R, Zhang H, Tang L, Prakash A (2008) Brain chro-
matin remodeling: a novel mechanism of alcoholism. J Neurosci 28:
3729–3737
Pandey SC, Kyzar EJ, Zhang H (2017) Epigenetic basis of the dark side
of alcohol addiction. Neuropharmacology 122:74–84
Roberto M, Gilpin NW, Siggins GR (2012) The central amygdala and
alcohol: role of gamma-aminobutyric acid, glutamate, and neuro-
peptides. Cold Spring Harb Perspect Med 2:a012195
Sakharkar AJ, Zhang H, Tang L, Shi G, Pandey SC (2012) Histone
deacetylases (HDAC)-induced histone modifications in the amyg-
dala: a role in rapid tolerance to the anxiolytic effects of ethanol.
Alcohol Clin Exp Res 36:61–71
Sakharkar AJ, Zhang H, Tang L, Baxstrom K, Shi G, Moonat S, Pandey
SC (2014) Effects of histone deacetylase inhibitors on amygdaloid
histone acetylation and neuropeptide Yexpression: a role in anxiety-
like and alcohol-drinking behaviours. Int J Neuropsychopharmacol
17:1207–1220
Shepard BD, Tuma PL (2009) Alcohol-induced protein hyperacetylation:
mechanisms and consequences. World J Gastroenterol 15:1219–1230
Silberman Y, Bajo M, Chappell AM, Christian DT, Cruz M, Diaz MR,
Kash T, Lack AK, Messing RO, Siggins GR, Winder D, Roberto M,
McCool BA, Weiner JL (2009) Neurobiological mechanisms con-
tributing to alcohol-stress-anxiety interactions. Alcohol 43:509–519
Simioni N, Cottencin O, Guardia D, Rolland B (2012) Early relapse in
alcohol dependence may result from late withdrawal symptoms.
Med Hypotheses 79:894–895
Kazantsev AG, Thompson LM (2008) Therapeutic application of histone
deacetylase inhibitors for central nervous system disorders. Nat Rev
Drug Discov 7:854–868
Simon-O’Brien E, Alaux-Cantin S, Warnault V, Buttolo R, Naassila M,
Vilpoux C (2015) The histone deacetylase inhibitor sodium butyrate
decreases excessive ethanol intake in dependent animals. Addict
Biol 20:676–689
Warnault V, Darcq E, Levine A, Barak S, Ron D (2013) Chromatin
remodeling—a novel strategy to control excessive alcohol drinking.
Transl Psychiatry 3:e231
Khan N, Jeffers M, Kumar S, Hackett C, Boldog F, Khramtsov N, Qian
X, Mills E, Berghs SC, Carey N, Finn PW, Collins LS, Tumber A,
Ritchie JW, Jensen PB, Lichenstein HS, Sehested M (2008)
Determination of the class and isoform selectivity of small-
molecule histone deacetylase inhibitors. Biochem J 409:581–589
Legastelois R, Botia B, Naassila M (2013) Blockade of ethanol-induced
behavioral sensitization by sodium butyrate: descriptive analysis of
gene regulations in the striatum. Alcohol Clin Exp Res 37:1143–1153
Legastelois R, Jeanblanc J, Vilpoux C, Bourguet E, Naassila M (2017)
Epigenetic mechanisms and alcohol use disorders: a potential ther-
apeutic target. Biol Aujourdhui 211:83–91
Mottet D, Castronovo V (2008) Histone deacetylases: target enzymes for
cancer therapy. Clin Exp Metastasis 25:183–189
López-Moreno JA, Marcos M, Calleja-Conde J, Echeverry-Alzate V,
Bühler KM, Costa-Alba P, Bernardo E, Laso FJ, de Fonseca FR,
Nadal R, Paz Viveros M, Maldonado R, Giné E (2015) Histone
deacetylase gene expression following binge alcohol consumption
in rats and humans. Alcohol Clin Exp Res 39:1939–1950