Investigation of the Role of Chirality in the Interaction with σ Receptors and Effect on Binge Eating Episode of a Potent σ1 Antagonist Analogue of Spipethiane

Article abstract of DOI:10.1021/acschemneuro.9b00261

The enantiomers of the potent σ₁ receptor antagonist (±)-1 were synthesized and evaluated for their affinity at σ₁, σ₂ receptors and dopamine transporter (DAT). Analogously to (±)-1, both of the enantiomers showed very high affinity for the σ₁ receptor and unprecedented selectivity over both the σ₂ receptor and DAT. The lack of enantioselectivity between (+)-1 and (-)-1 indicated that the center of chirality in the 2-position of the benzothiochromane nucleus does not play a crucial role in the interaction with any of the studied targets. Docking studies confirmed that the configuration of the enantiomers has only marginal effects on the molecular interactions with the σ₁ receptor. In in vivo studies in a female rat model of binge eating, (±)-1 dose-dependently decreased the binge eating episode elicited by a history of intermittent food restriction and stress, confirming and strengthening the important role played by the σ₁ receptor in bingeing-related eating disorders.

Full text of DOI:10.1021/acschemneuro.9b00261

Letter
Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
pubs.acs.org/chemneuro
Investigation of the Role of Chirality in the Interaction with σ
Receptors and Effect on Binge Eating Episode of a Potent σ1
Antagonist Analogue of Spipethiane
Fabio Del Bello,† Maria Vittoria Micioni Di Bonaventura, Alessandro Bonifazi,†,‡
§
⊥
⊥
‡
§
Bernhard Wu
̈
n∥sch, Dirk Schepmann, JoLynn B. Giancola, Emanuela Micioni Di Bonaventura,
Giulio Vistoli, Gianfabio Giorgioni,† Wilma Quaglia,* Alessandro Piergentili,
,† †
and Carlo Cifani§
†
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy
School of Pharmacy, Pharmacology Unit, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy
Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse −
§
‡
Intramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States
⊥
Institut fur Pharmazeutische und Medizinische Chemie, Universitat Munster, Corrensstraße 48, 48149 Munster, Germany
̈ ̈ ̈
̈
Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milano, Italy
∥
*
S Supporting Information
ABSTRACT: The enantiomers of the potent σ receptor
1
antagonist (±)-1 were synthesized and evaluated for their
affinity at σ , σ receptors and dopamine transporter (DAT).
1
2
Analogously to (±)-1, both of the enantiomers showed very
high affinity for the σ receptor and unprecedented selectivity
1
over both the σ2 receptor and DAT. The lack of
enantioselectivity between (+)-1 and (−)-1 indicated that
the center of chirality in the 2-position of the benzothiochro-
mane nucleus does not play a crucial role in the interaction
with any of the studied targets. Docking studies confirmed
that the configuration of the enantiomers has only marginal
effects on the molecular interactions with the σ receptor. In
1
in vivo studies in a female rat model of binge eating, (±)-1
dose-dependently decreased the binge eating episode elicited by a history of intermittent food restriction and stress, confirming
and strengthening the important role played by the σ receptor in bingeing-related eating disorders.
1
KEYWORDS: Selective σ antagonists, chirality, docking studies, binge eating episode, palatable food
1
igma (σ) receptors are transmembrane proteins implicated
found in the mitochondria associated membrane of the
endoplasmic reticulum (ER) and in the nuclear and plasma
membranes. It has been reported to aid the inositol
S
in several cellular functions and are among the most
1
9
enigmatic proteins from a biological point of view. At present,
they are considered to be a receptor family different from both
the opioid receptor family and the phencyclidine site of the
triphosphate receptor in the ER membrane in a chaperone-
2+
like mode, modulating proper Ca -signaling. Moreover, it
2
NMDA receptor, and comprise σ and σ receptor subtypes,
1
2
regulates glutamatergic, dopaminergic, and cholinergic neuro-
which are widespread expressed in the central nervous system
+
+
transmission as well as the opening of some cation (K , Na ,
3
(
CNS) and in several peripheral tissues. They are also
2+
3
and Ca ) channels.
localized in the immune and endocrine systems and are
Due to their wide localization throughout the human body
and their involvement in many physiological functions, the σ1
receptors are considered highly appealing targets for the
treatment of several diseases. In particular, centrally active σ1
receptor agonists might be useful in depression, cognition,
3
overexpressed in several tumor cell lines.
While the σ receptor has been cloned from various tissues
1
4
−6
of different species,
the σ subtype has only recently been
2
cloned and identified as transmembrane protein 97
7
(
TMEM97). The crystal structures of the human σ receptor
1
in complex with the potent σ ligands 4-IBP and PD144418
1
provided the first detailed description of the σ architecture,
Received: May 2, 2019
Accepted: July 12, 2019
Published: July 12, 2019
1
revealing a triangular trimer with only one transmembrane
8
domain in each protomer. The σ receptor is predominantly
1
©
XXXX American Chemical Society
A
DOI: 10.1021/acschemneuro.9b00261
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
Figure 1. Chemical structures of BD-1063, spipethiane, and (±)-1.
a
Scheme 1
a
23
(
a) Pyrrolidine, MeOH; (b) (+)-O,O′-di-p-toluoyl-D-tartaric acid, crystallization from EtOH; (c) (−)-O,O′-di-p-toluoyl-L-tartaric acid,
crystallization from EtOH; (d) 2 N NaOH.
1
0
delirium, akathisia, and other hyperkinetic movement as well
as in neurodegenerative diseases.
Moreover, the affinities of (±)-1 and its enantiomers were
also evaluated at dopamine transporters (DAT), considering
that DAT have been demonstrated to be involved in binge
11,12
σ1 receptor antagonists
have been demonstrated to be efficacious in neuropathic pain,
as well as in visceral, orofacial, and inflammatory pain.
Moreover, σ1 antagonists can be used as antiaddictive
compounds, blocking several effects induced by psychostimu-
While a collection of evidence has
demonstrated the role of the σ receptor in drug abuse, just
one study reports that it is involved in compulsive-like eating.
Moreover, very recently a correlation between σ receptor and
food reinforced operant responding has been discussed.
Binge eating is a central trait of many eating disorders, such
as bulimia nervosa and binge eating disorder (BED), consisting
of compulsive, non-homeostatic consumption of abnormal
amounts of highly palatable food within a few hours, as
described by the DSM-V. BED is among the most frequent
eating disorders, in which subjects assume an unusually large
quantity of food, feeling unable to stop eating. Although it
affects about 2−5% of adults and is more frequent in women
than in men, effective drugs are limited, leading to the
impetus for innovative treatments. The study by Cottone et
13
24
eating disorders and that several σ1 ligands also bind
22,25
DAT.
1
4,15
16,17
lant
and ethanol.
RESULTS AND DISCUSSION
■
Racemic compound (±)-1 was prepared as reported in ref 23
1
18
(
Scheme 1). The enantiomers (+)-1 and (−)-1 were separated
1
by fractional crystallization of the salts of the racemic
19
compound (±)-1 with (+)-O,O′-di-p-toluoyl-D- and
(
−)-O,O′-di-p-toluoyl-L-tartaric acid, respectively (Scheme 1).
The enantiomeric excess, determined by HPLC using
DAICEL - Chiralpak AD-H (4.6 mm × 250 mm) as the
chiral stationary phase and n-hexane/2-propanol 90/10 v/v as
the mobile phase, was found to be >99% for (+)-1 and >97%
20
1
for (−)-1 (see the Supporting Information). H NMR
spectroscopy studies confirmed that the enantiomeric purity
is >97% (detection limit) for both the enantiomers. Indeed,
2
1
1
the H NMR spectrum of the racemic compound (±)-1 in the
presence of the chiral solvating reagent (S)-(+)-2,2,2-trifluoro-
1
8
al. demonstrated that the σ antagonist BD-1063 (Figure 1)
σ pK = 8.05, σ /σ selectivity ratio = 71) reduced binge-
1-(9-anthryl)-ethanol shows two pairs of doublets at 3.50 ppm
1
22
(
for the PhCH N protons, whereas only two doublets were
1
i
1
2
2
like eating in a dose-dependent manner and blocked the
increased eating rate in palatable rats. This result suggests that
the σ1 receptors might be implicated in neurobiological
adaptations underlying compulsive-like eating.
We have reported a series of spipethiane analogues showing
observed for (+)-1 and (−)-1 at 3.51 and 3.49 ppm,
respectively.
The affinities of (±)-1 and its enantiomers (+)-1 and (−)-1
18
3
were determined at the σ receptor, using [ H]-(+)-pentazo-
1
3
cine as the radioligand, and at the σ receptor, using [ H]-di-o-
2
high affinity for the σ receptor and selectivity over the σ
tolylguanidine as the radioligand in the presence of an excess of
1
2
subtype. Among these ligands, compound (±)-1 (Figure 1) (σ1
(+)-pentazocine to ensure occupancy of σ receptors. The
1
pK = 10.28) behaved as a very potent σ receptor antagonist
experiments were performed on guinea pig brain and rat liver
i
1
23
and showed the outstanding σ /σ selectivity ratio of 29 510.
membrane preparations for the σ1 and σ2 receptors,
1
2
26,27
Since compound (±)-1 shows both a markedly higher σ1
respectively, according to previously reported procedures.
affinity and σ /σ selectivity with respect to BD-1063, to better
The DAT assay was performed with rat striatal membranes
1
2
3
clarify the role played by the σ receptor in compulsive-like
using the radioligand [ H]-WIN35,428, analogously to
1
28,29
eating disorders, it was evaluated in a female rat model of binge
eating.
Given that the biological profile of ligands might
quantitatively and qualitatively be influenced by stereo-
chemistry, the presence of a center of chirality in (±)-1
prompted us to preliminarily separate the two enantiomers
previously reported procedures.
The affinity values, expressed as pK , are reported in Table 1
i
22
together with those of BD-1063 for useful comparison.
The data reported in Table 1 reveal that, similarly to the
racemic compound (±)-1, both enantiomers (+)-1 and (−)-1
show very high affinity for the σ receptor and impressive σ /σ
1
1
2
(
+)-1 and (−)-1 and evaluate their affinities for the σ and σ
selectivity. Moreover, (±)-1 and its enantiomers display
submicromolar DAT affinity higher than 1 μM, and
1
2
receptors.
B
DOI: 10.1021/acschemneuro.9b00261
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
Table 1. Affinity Values, Expressed as pK of (± )-1, Its
Specifically, the two calculated, optimized complexes appear
to be stabilized by the same interaction pattern, which can be
summarized as follows: (1) the ammonium head of the ligand
stabilizes a clear salt bridge with Glu172 reinforced by the H-
bond with Tyr103, which in turn also approaches the
thiochroman-4-one moiety; (2) the thiochroman-4-one system
contacts a set of apolar side-chains (i.e., Met 93, Leu95,
Leu105, Ile178, Leu182), and its carbonyl group elicits a H-
bond with Thr181); (3) the phenyl ring is inserted in a
subpocket completely lined by aromatic residues such as
Trp89, Phe107, Phe133, His154, and Trp164. Clear similarity
between the two generated complexes is further confirmed by
the similar values obtained for some representative scoring
functions such as the primary ChemPLP function (−123.49
kcal/mol (S) vs −124.17 kcal/mol (R)), the electrostatic-
based APBS score (−10.55 kcal/mol (S) vs −10.39 kcal/mol
(R)), and the empirical XScore (−10.99 kcal/mol (S) vs
i
Enantiomers (+)-1 and (−)-1 and BD-1063, at σ and σ
1
2
a
Receptors and DAT
pKi
compd
σ1
σ2
6.53
6.87
6.39
6.20
DAT
σ1/σ
σ1/DAT
2
(
(
(
±)-1
+)-1
−)-1
10.85
10.92
10.66
8.05
5.80
5.77
5.67
20 893
11 220
18 621
71
112 202
141 253
97 724
891
b
b
b
BD-1063
5.10
a
Equilibrium dissociation constants (K ) were derived from IC
i
50
30
values using the Cheng−Prusoff equation. The IC50 values were
determined by displacement of [ H]-(+)-pentazocine and [ H]-DTG
3
3
in the presence of 500 nM (+)-pentazocine from the σ and σ
1
2
3
receptors, respectively, and by displacement of [ H]WIN35,428 from
DAT. Each experiment was performed in triplicate. pK and pIC
i
50
values are means from three to five experiments, which agreed within
b
±
20%. Data from ref 22.
−
11.03 kcal/mol (R)). Overall, the presented docking results
are in consensus with the observed lack of stereoselectivity
between the two studied enantiomers, which are indeed able to
stabilize almost identical interaction patterns with the σ1
receptor.
consequently an uncommon selectivity for the σ receptor even
over these transporters. This result is noteworthy, considering
1
that most potent σ ligands also bind DAT with high
1
2
2,25
affinity.
Significant differences in the affinities toward the
Though the enantiomers (+)-1 and (−)-1 show similar
σ , σ receptors and DAT between the enantiomers (+)-1 and
1
2
affinities at σ , σ and DAT, they might manifest a different
1
2
(−)-1 were not observed, indicating that the configuration of
pharmacokinetic profile. The possible role of the configuration
in determining the metabolic fate of the considered
enantiomers was analyzed in silico by predicting the metabolic
reactions they can undergo by a similarity-based approach
involving the MetaQSAR database as described in ref 31. In
detail and considering the molecules within MetaQSAR
showing a Tanimoto similarity greater than 0.5, the following
metabolic reactions can be figured out: (1) oxygenation of
sulfide to sulfoxide and to sulfone; (2) hydroxylation on the
carbon atom in alpha to both heteroatom and unsaturated
systems; (3) hydroxylation on the phenyl ring (preferentially in
para position); (4) reduction of the carbonyl group to alcohol.
Among these putative reactions, (1) and (4) involve sites of
metabolism close to the chiral center. In particular, the S-
oxidation can show a certain degree of substrate stereo-
selectivity especially when catalyzed by FMO enzyme, whose
cavity seems to be more sensitive to the substrate configuration
compared to CYP450 enzymes as reported in previous
the stereocenter in 2-position of the benzothiochromane
system of (±)-1 does not play a crucial role in the interaction
with these molecular targets. To rationalize the lack of
stereoselectivity between the enantiomers (+)-1 and (−)-1 at
the σ receptor, docking studies on the crystal structure of the
1
8
human σ receptor were performed. In Figure 2, the best
1
32
studies. Focusing attention on metabolic reactions occurring
close to the chiral center, one may suppose that differences in
the metabolism of the two enantiomers might be eventually
due to the S-oxidation reactions rather than the reduction of
the carbonyl group considering that the involved aldehyde
dehydrogenases are well-known for their product rather than
substrate stereoselectivity.
Compared to BD-1063, the affinity values of (±)-1, (+)-1,
and (−)-1 are significantly higher at the σ receptor and only
1
slightly higher at the σ receptor and DAT. Consequently,
2
marked increases in σ /σ and σ /DAT selectivity ratios are
1
2
1
observed for (±)-1, (+)-1, and (−)-1 with respect to BD-1063.
Therefore, due to their interesting selectivity profiles, (±)-1
and its enantiomers would allow us to confirm and strengthen
Figure 2. Superposition of the calculated binding poses of (S)-1
(blue-colored ligand) and (R)-1 (red-colored ligand).
the hypothesis that the σ receptor system plays an important
1
role in compulsive-like eating disorder. Since racemic
binding poses of the two enantiomers (+)-1 and (−)-1 at the
receptor are compared. The observation that the
enantiomers reveal an almost identical arrangement supports
that the configuration has only marginal effects on the
interactions.
compound (±)-1 shows σ affinity and selectivity profiles
1
σ
indistinguishable to those of both enantiomers, it was selected
for the in vivo study, using a well-characterized animal model
of binge eating. The episode of binge eating for palatable food
was induced by combining stress with three consecutive cycles
1
C
DOI: 10.1021/acschemneuro.9b00261
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
Figure 3. Systemic injections of compound (±)-1 decreased the binge eating episode in rats with cycles of intermittent food restrictions and stress.
Mean ± SEM (n = 6−8 for each group, as shown for each bar) palatable food intake (kcal/kg) in the first 15 min (left) and the total 2 h (right) test
#
#
session. p < 0.01 vs the other vehicles groups; **p < 0.01 vs food restricted and stressed vehicle rats. At 15 min after palatable food access and at
the end of the test (2 h time point), three-way ANOVA showed a significant interaction among the three factors (food restriction, stress, and
treatment) [15 min: F(3,90) = 3.16, p < 0.05] [2 h: F(3,90) = 2.8, p < 0.05].
3
3
of food restriction/refeeding in female rats, which were used
in consideration of the high prevalence of BED in adolescent
mechanism of action remained unclear. The ability of the σ
1
14
antagonists to reduce the motivational effect of cocaine,
21
15
16,17
and young adult females.
methamphetamine, and alcohol
makes it conceivable
34
As previously reported by us, the body weight of rats was
reduced in the 4 days of food restriction (in restricted rats),
but during refeeding period the rats regained their body weight
to values of controls (nonrestricted rats) the last day of each
cycle (values not displayed). At 15 min after palatable food
access and at the end of the test (2 h time point), three-way
ANOVA showed a significant interaction among the three
factors (food restriction, stress, and treatment) [15 min: F(3,90)
that the blockade of the σ1 receptor may control the
motivation for palatable food consumption under particular
conditions, such as food restrictions and acute stress in our
binge eating rat model, modulating the dopaminergic system.
METHODS
■
Chemistry. General. Instruments used for melting point
determination, IR, NMR and mass spectra, elemental analyses and
optical rotation are given in references.
=
3.16, p < 0.05] [2 h: F(3,90) = 2.8, p < 0.05]. As shown in
35,36
Figure 3, posthoc comparisons indicated that the palatable
food intake of the vehicle restricted and stressed group was
significantly higher than that in the other vehicle groups
Resolution of (2-(1-Benzylpiperidin-4-yl)thiochroman-4-one)
(±)-1 by Fractional Crystallization. A solution of (+)-O,O’-di-p-
toluoyl-D-tartaric acid (5.73 g, 14.8 mmol) in ethyl acetate (EtOAc)
23
(
nonrestricted and nonstressed; nonrestricted and stressed;
(30 mL) was added to a solution of racemic compound (±)-1 (5 g,
4.8 mmol) in EtOAc (60 mL) and the mixture was left at r.t. for 24
h. The white solid was crystallized five times from EtOH (2.24 g): mp
1
restricted and nonstressed) (15 min: p < 0.01; 2 h: p < 0.05).
Moreover, posthoc comparisons showed a significant
reduction in palatable food consumption in the binge eating
group (restricted and stressed rats) treated with (±)-1, at 3
and 7 mg/kg at 15 min and 2 h time points in a dose−response
relationship. These doses did not reduce palatable food intake
in the other three groups. Compound (±)-1 at the dose of 1
mg/kg was ineffective in all groups of rats. Therefore, we found
that systemic injections of (±)-1 selectively blocked the
episode of binge eating without a general inhibition of food
intake, observed for serotonergic drugs, such as fluoxetine or
sibutramine in the same animal model. Considering the
unprecedented selectivity of (±)-1 for σ over σ and DAT,
these results confirm and strengthen the important role played
by the σ receptor in the pharmacotherapy of bingeing-related
eating disorders. Despite several works revealing the potential
effect of σ receptor antagonists to treat drug abuse, the precise
2
0
1
85−186 °C, [α] = +153.8 (c 1, CH OH). The salt was dissolved
D
3
in water (50 mL) and the solution was cooled to 0 °C, made basic
with 2 N NaOH and extracted with EtOAc (3 × 30 mL). Removal of
20
dried solvent afforded (+)-1 (1.18 g): mp 80−82 °C, [α]
=
D
1
+
144.66 (c 1, CHCl ), H NMR (CDCl ): δ 1.38−2.02 (m, 7H),
3
3
2.80−3.12 (m, 4H), 3.35 (m, 1H), 3.50 (dd, 2H), 7.10−8.11 (m,
9H). The free base was converted into the oxalate salt, that was
2
0
D
recrystallized from MeOH: mp 218−220 °C, [α]
CH OH). ESI/MS: m/z 338.2 [M + H] . Anal. Calcd for
= +137.2 (c 1,
+
3
C H NOS.C H O : C, 64.62; H, 5.89; N, 3.28, S, 7.50. Found: C,
2
1
23
2
2
4
33
64.36; H, 6.23; N, 3.34, S, 7.42.
A solution of the amine recovered from the mother liquor (1.7 g,
1
2
5
(
.04 mmol) in EtOAc (20 mL) was treated with a solution of
−)-O,O’-di-p-toluoyl-L-tartaric acid (1.95 g, 5.04 mmol) in EtOAc
(10 mL) and left at r.t. for 24 h. The white solid was crystallized four
1
2
0
D
times from EtOH (1.74 g): mp 182−183 °C, [α] = −148.54 (c 1,
CH OH). The salt was dissolved in water (20 mL), and the solution
1
3
D
DOI: 10.1021/acschemneuro.9b00261
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
was cooled to 0 °C, made basic with 2 N NaOH and extracted with
EtOAc (3 × 10 mL). Removal of dried solvent afforded (−)-1 (0.9 g):
ORCID
Fabio Del Bello: 0000-0001-6538-6029
Maria Vittoria Micioni Di Bonaventura: 0000-0002-8044-1206
Alessandro Bonifazi: 0000-0002-7306-0114
2
0
1
mp 81−82 °C, [α]
= −143.57 (c 1, CHCl ). The H NMR
D
3
23
spectrum was identical to those of (±)-1 and the enantiomer (+)-1.
The free base was converted into the oxalate salt, that was
recrystallized from MeOH: mp 219−220 °C, [α] = −137.0 (c 1,
CH OH). ESI/MS: m/z 338.2 [M + H] . Anal. Calcd for
2
0
Bernhard Wunsch: 0000-0002-9030-8417
̈
D
+
3
Giulio Vistoli: 0000-0002-3939-5172
Gianfabio Giorgioni: 0000-0002-9576-6580
Wilma Quaglia: 0000-0002-7708-0200
Alessandro Piergentili: 0000-0001-6135-6826
Carlo Cifani: 0000-0001-6180-828X
C H NOS.C H O : C, 64.62; H, 5.89; N, 3.28, S, 7.50. Found: C,
21
23
2
2
4
6
4.38; H, 5.89; N, 3.34, S, 7.42.
Receptor Binding Studies. The experimental details of σ and σ
1
2
26−29
receptors and DAT are given in references.
Computational Methods. Docking simulations were based on
the σ receptor resolved structure in complex with PD144418 (PDB
1
Author Contributions
ID: 5HK1). The protein and ligand structures were prepared by
37
F.D.B. and M.V.M.D.B. contributed equally. F.D.B., G.G.,
W.Q., and A.P. designed the novel compounds and planned
the procedures for their synthesis. They developed the
chemical synthesis, characterized the novel compounds,
wrote the relative chemical experimental parts and drafted
the main text of the manuscript. A.B., B.W., and D.S. and
performed binding experiments at σ and σ receptors. A.B.
and J.B.G. performed binding experiments at DAT. G.V.
carried out docking experiments. M.V.M.D.B., E.M.D.B., and
C.C. performed in vivo assays and drafted the relative
discussion. All authors critically discussed and approved the
final version of the manuscript.
applying standard protocols as previously described. Docking
38
simulations were performed by using PLANTS and focusing the
search within a 12 Å radius sphere around the bound ligand. For each
enantiomer, 10 poses were generated and scored by the ChemPLP
scoring function with the speed equal to 1. The obtained best poses
were then minimized by fixing all atoms outside a 10 Å radius sphere
around the bound ligands.
1
2
In Vivo Studies. Material and Methods. Subjects, Diet
Composition, and Binge Eating Experimental Procedure. One
hundred and forty-four female Sprague−Dawley rats (Charles River,
Italy) were used. The palatable food was prepared by mixing Nutella
(
Ferrero, Italy) chocolate cream (52%), ground food pellets (4RF18)
(
33%), and water (15%). The binge eating procedure was described
in detail in our previous studies.
33,39−42
Funding
Drug. Compound (±)-1 was synthesized according to previously
23
The work was supported by the Italian Ministry of University
and Research under Grant PRIN- 2015KP7T2Y to C.C., by
grant from the University of Camerino (Fondo di Ateneo per
la Ricerca 2018), and by the Intramural Research Program of
the National Institute on Drug Abuse (postdoctoral fellowship
to A.B. and postbaccalaureate fellowship to J.B.G.).
reported procedure and dissolved in 5% dimethylsulfoxide in
distilled water. It was administered intraperitoneally (2 mL/kg) at
doses of 1, 3, or 7 mg/kg. On test day, compound (±)-1 or the
respective vehicle was administered 1 h before access to palatable
food.
Experiment 1: Effect of Compound (±)-1 in a Model of Binge
Eating. The purpose of this experiment was to evaluate the effects of
compound (±)-1 on binge eating behavior. One hundred and forty-
four female rats, divided into four groups (n = 36 per group), were
used. They were either exposed or not exposed to three consecutive 8
day cycles of food restriction/refeeding, during which the access to
the palatable food was provided to them for 2 h/day on days 5, 6, 13,
and 14. On day 25, rats were either exposed or not exposed to 15 min
of frustration stress before the 2 h palatable food binge eating test. On
the test day, each group was separated in four subgroups and treated
intraperitoneally with vehicle or (±)-1 compound (1, 3, and 7 mg/
kg), 30 min before the 2 h palatable food access. After testing, the
estrous cycle phase was determined. During the estrous phase, stress-
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
CNS, central nervous system; ER, endoplasmic reticulum;
BED, binge eating disorder; DAT, dopamine transporter;
EtOAc, ethyl acetate.
REFERENCES
■
43,44
induced binge eating was not observed in our model.
8 rats that were in this phase were excluded from the statistical
analysis.
Statistical Analysis. The data are reported as mean ± SEM. The
Therefore,
(
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Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.9b00261.
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AUTHOR INFORMATION
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*
(7) Alon, A., Schmidt, H. R., Wood, M. D., Sahn, J. J., Martin, S. F.,
Corresponding Author
Phone +390737402237. E-mail: wilma.quaglia@unicam.it.
and Kruse, A. C. (2017) Identification of the gene that codes for the
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