The potential rewarding and reinforcing effects of the substituted benzofurans 2-EAPB and 5-EAPB in rodents

Article abstract of DOI:10.1016/j.ejphar.2020.173527

Accounts regarding the use of novel psychoactive substances continue to escalate annually. These include reports on substituted benzofurans (SBs), such as 1-(1-benzofuran-2-yl)-N-ethylpropan-2-amine (2-EAPB) and 1-(1-benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB). Reports on the deaths and adverse consequences from the use of SBs warrant the investigation of their mechanism, possibly predicting the effects of similar compounds. Accordingly, we investigated the possible rewarding and reinforcing effects of 2-EAPB and 5-EAPB through conditioned place preference (CPP), self-administration, and locomotor sensitization tests. We also determined the possible influence of 2-EAPB and 5-EAPB administration on dopamine- and plasticity-related proteins in the nucleus accumbens and ventral tegmental area. 2-EAPB and 5-EAPB induced CPP at different doses and were self-administered by rats. Only 5-EAPB induced locomotor sensitization in mice. 2-EAPB and 5-EAPB did not alter the expressions of dopamine D1 and D2 receptors in the nucleus accumbens, nor changed tyrosine hydroxylase and dopamine transporter expressions in the ventral tegmental area. Both 2-EAPB and 5-EAPB enhanced deltaFosB, but not transcription factor cyclic AMP-response-element binding protein and brain-derived neurotrophic factor in the nucleus accumbens. Hence, the potential rewarding and reinforcing effects on rodents induced by 2-EAPB and 5-EAPB may possibly be associated with alterations in other neurotransmitter systems (besides mesolimbic) and/or neuro-plastic modifications.

Full text of DOI:10.1016/j.ejphar.2020.173527

European Journal of Pharmacology 885 (2020) 173527
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
The potential rewarding and reinforcing effects of the substituted
benzofurans 2-EAPB and 5-EAPB in rodents
Leandro Val Saysona, Raly James Perez Custodioa, Darlene Mae Ortiza, Hyun Jun Leea,
,
,d,*
Mikyung Kimb, Youngdo Jeongc, Yong Sup Leec, Hee Jin Kima **, Jae Hoon Cheonga
a Uimyung Research Institute for Neuroscience, Department of Pharmacy, Sahmyook University, 815 Hwarang-ro, Nowon-gu, Seoul, 01795, Republic of Korea
b Department of Chemistry & Life Science, Sahmyook University, 815 Hwarang-ro, Nowon-gu, Seoul, 01795, Republic of Korea
c Medicinal Chemistry Laboratory, Department of Pharmacy & Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, 26
Kyungheedae-ro, Seoul, 02447, Republic of Korea
d School of Pharmacy, Jeonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do, 54896, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Accounts regarding the use of novel psychoactive substances continue to escalate annually. These include reports
on substituted benzofurans (SBs), such as 1-(1-benzofuran-2-yl)-N-ethylpropan-2-amine (2-EAPB) and 1-(1-
benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB). Reports on the deaths and adverse consequences from the
use of SBs warrant the investigation of their mechanism, possibly predicting the effects of similar compounds.
Accordingly, we investigated the possible rewarding and reinforcing effects of 2-EAPB and 5-EAPB through
conditioned place preference (CPP), self-administration, and locomotor sensitization tests. We also determined
the possible influence of 2-EAPB and 5-EAPB administration on dopamine- and plasticity-related proteins in the
nucleus accumbens and ventral tegmental area. 2-EAPB and 5-EAPB induced CPP at different doses and were self-
administered by rats. Only 5-EAPB induced locomotor sensitization in mice. 2-EAPB and 5-EAPB did not alter the
expressions of dopamine D1 and D2 receptors in the nucleus accumbens, nor changed tyrosine hydroxylase and
dopamine transporter expressions in the ventral tegmental area. Both 2-EAPB and 5-EAPB enhanced deltaFosB,
but not transcription factor cyclic AMP-response-element binding protein and brain-derived neurotrophic factor
in the nucleus accumbens. Hence, the potential rewarding and reinforcing effects on rodents induced by 2-EAPB
and 5-EAPB may possibly be associated with alterations in other neurotransmitter systems (besides mesolimbic)
and/or neuro-plastic modifications.
2-EAPB
5-EAPB
Conditioned place preference
Self-administration
Locomotor sensitization
Abuse potential
1. Introduction
substituted benzofurans (SBs) (Dolan et al., 2017; Eshleman et al.,
2019). Considered as the third most prominent group of novel psycho-
Globally, novel psychoactive substances constantly emerge from
clandestine markets in order to circumvent the regulations prohibiting
their use and distribution (Beltgens, 2017; Johnson et al., 2013; Smith
and Garlich, 2013). As a result, the number of cases for the identification
of these substances by drug monitoring agencies increases annually
(Addiction, 2014; Welter-Luedeke and Maurer, 2016; UNODC, 2014). A
contributing problem is the constant introduction of modifications to the
structure of recognized illicit drugs (Kikura-Hanajiri et al., 2014;
Weaver et al., 2015). This creates new compounds that may possibly be
susceptible for recreational use; some of such compounds include
active substances that recently emerged in the past decade (Roque Bravo
et al., 2019), these empathogens (heightens emotional state) were re-
ported to function mainly on the serotonergic system (Eshleman et al.,
2019), yet still generating downstream effects on dopaminergic proteins
(Rickli et al., 2015). Their actions on these neurotransmitter networks
enable addictive effects, as evidenced by previous studies (Adamowicz
et al., 2014; Cha et al., 2016; Dawson et al., 2014; Fuwa et al., 2016),
making several of these SBs illegal in some countries (Fuwa et al., 2016).
Among these recent SBs are 1-(1-benzofuran-2-yl)-N-ethylpropan-2--
amine (2-EAPB) and 1-(1-benzofuran-5-yl)-N-ethylpropan-2-amine
* Corresponding author. Uimyung Research Institute for Neuroscience, Department of Pharmacy, Sahmyook University, 815 Hwarang-ro, Nowon-gu, Seoul, 01795,
Republic of Korea.
** Corresponding author.
E-mail addresses: hjkim@syu.ac.kr (H.J. Kim), cheongjh@syu.ac.kr (J.H. Cheong).
https://doi.org/10.1016/j.ejphar.2020.173527
Received 31 March 2020; Received in revised form 25 August 2020; Accepted 28 August 2020
Available online 29 August 2020
0014-2999/© 2020 Elsevier B.V. All rights reserved.

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
(5-EAPB) (Fuwa et al., 2016; King, 2014; Uchiyama et al., 2014).
Both 2-EAPB and 5-EAPB contain 2-ethylaminopropyl (EAP); how-
ever, the EAP (outlined in red) in 2-EAPB is located on R2 (Fig. 1C),
whereas in 5-EAPB, it is on R5 (Fig. 1D). Both compounds are also
structurally similar to methamphetamine (Fig. 1B) except for the pres-
ence of a furan ring (outlined in blue) and an additional methyl on the
amine site (red arrow). Another SB similar to these compounds is 5-APB
(an Ecstasy analog). Initially developed as a monoamine transport in-
hibitor (Dawson et al., 2014; Rickli et al., 2015), 5-APB was found to
elicit conditioned place preference (CPP) (Cha et al., 2016). Other
similar SBs are 6-MAPB, 5-MAPB, and 6-APB that have all been reported
for recreational purposes due to their empathogenic and
psycho-stimulating properties (Shimshoni et al., 2017). Several deaths
from the chronic use of 6-APB and 5-APB have also been reported
(Adamowicz et al., 2014; Nugteren-van Lonkhuyzen et al., 2015), with
one death from 5-EAPB (Deville et al., 2019). These accounts strongly
indicate the dangers and adverse consequences arising from their use,
thus necessitating investigations on the neuronal mechanisms by which
these drugs affect behavior. Indeed, such investigations are crucial for
2-EAPB and 5-EAPB, for which substantial relevant information is
generally lacking (Deville et al., 2019; Uchiyama et al., 2014). Extensive
studies on their pharmacodynamics could also generate significant in-
formation that would be applicable in predicting the likely effects of
similarly structured compounds.
2. Materials and methods
2.1. Animals
All animals used in the study were purchased from Hanlim Animal
Laboratory Co. (Hwasung, Korea) and were housed in a temperature-
and humidity-controlled room (temperature: 22 ± 2 ◦C, relative hu-
midity: 55 ± 5%) with a 12/12 h light/dark (07:00–19:00 light) cycle.
Different cohorts of animals were used for each test. Only male subjects
were used to avoid the confounding effects of hormonal changes in fe-
males (Fattore et al., 2008). Adolescent rodents were used since this
stage of development was previously shown to increase novelty-seeking
and exploratory behavior, and thus sensitivity to abuse studies
(Zakharova et al., 2009). Sprague-Dawley rats (6 weeks old) were used
in the CPP paradigm, SA test, and western blotting. For the SA test, rats
were caged individually; for other tests, 4–6 rats were housed per cage.
For locomotor sensitization test, five C57BL/6J mice (6 weeks old) were
housed per cage. Animals were habituated in the animal room for 5 days
prior to experiments. They had access to food and water, except when
rats underwent lever training and the actual SA sessions. Animal use in
this study was in accordance with the Principles of Laboratory Animal
Care (NIH Publication No. 85-23, 1985 revision) and the Animal Care
and Use Guidelines of Sahmyook University (SYUIACUC 2019-001).
In this study, we explored the potential rewarding and reinforcing
effects of 2-EAPB and 5-EAPB. In particular, we employed the CPP
paradigm and the self-administration (SA) test in rats for any evidence of
addictive effects. Furthermore, we also investigated the effects of these
drugs on mice locomotor activity, since increased locomotor activity and
sensitization are common characteristics of most addictive psychosti-
mulants (Modi et al., 2006; Shimosato and Ohkuma, 2000). Since the
neurochemistry of addiction is well-established to involve the meso-
limbic dopamine system, we also examined the influence of 2-EAPB and
5-EAPB on various dopamine-related proteins, specifically dopamine D1
receptor, dopamine D2 receptor, tyrosine hydroxylase, and dopamine
transporter. We also examined neuroplasticity-related proteins associ-
ated with addiction, namely phosphorylated (p-) cyclic
AMP-response-element binding protein (CREB), deltaFosB, and
brain-derived neurotrophic factor (BDNF). Additionally, all experiments
were simultaneously duplicated using methamphetamine.
2.2. Drugs
2-EAPB hydrochloride was synthesized in five steps from benzofuran
(Taniguchi et al., 2010). Briefly, benzofuran was treated with phos-
phorous oxychloride and N,N-dimethylformamide. The resulting 2-for-
mylbenzofuran was condensed with nitroethane and then reduced
with LiAlH4 to give 1-(benzofuran-2-yl)propan-2-amine. This compound
was acetylated using acetyl chloride and then reduced by LiAlH4 to give
1-(benzofuran-2-yl)-N-ethylpropan-2-amine. The resulting amine was
treated with hydrochloride to give 2-EAPB as a hydrochloride salt. Its
structure was confirmed by the following spectroscopic analyses.
H-NMR (400 MHz, D2O) δ 7.57 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz,
1H), 7.28 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 6.70 (s, 1H), 3.68
(q, J = 6.5 Hz, 1H), 3.21-3.05 (m, 4H), 1.28 (d, J = 6.5 Hz, 3H), 1.22 (t,
J = 7.3 Hz, 3H); 13C NMR (100 MHz, D2O) δ 153.84, 146.32, 130.34,
127.86, 125.58, 122.04, 111.56, 106.43, 55.16, 40.05, 38.68, 15.11,
Fig. 1. Chemical structures of (A) a general substituted benzofuran, (B) methamphetamine (METH), (C) 2-EAPB, and (D) 5-EAPB. The furan ring is outlined in blue;
the EAP group is outlined in red; an additional methyl on the amine site is indicated by the red arrow. (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article.)
2

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
10.60; HR-MS calculated for C13H18NO [M+H]+ 204.1383, found
204.1387.
response levers (4.5 cm long), a stimulus-light source situated above the
left lever, and a centrally located house light (2.5 W, 24 V) on top of the
chamber. Downward pressure of 25 g on a lever resulted in an auto-
mated response. Adjacent to the operant chamber was a mechanically
operated syringe pump that delivered solutions (0.01 mL/s) through
Teflon tubes that were attached to the I.V. catheter of the rats. This was
connected to a swivel system that allowed the free movement of rats.
Built-in Graphic State Notation software (Coulborn Instruments)
allowed automatic control over the experimental parameters and data
collection.
5-EAPB hydrochloride was synthesized in four steps from
benzofuran-5-carbaldehyde according to the procedure similar to that
used for the synthesis of 2-EAPB hydrochloride (Taniguchi et al., 2010).
Briefly, benzofuran-5-carbaldehyde was condensed with nitroethane
and then reduced with LiAlH4 to give 1-(benzofuran-5-yl)
propan-2-amine; this compound was acetylated and then reduced by
LiAlH4 to give 1-(benzofuran-5-yl)-N-ethylpropan-2-amine. The result-
ing amine was treated with hydrochloride to give 5-EAPB as a hydro-
chloride salt. Its structure was confirmed by the following spectroscopic
analyses. 1H NMR (400 MHz, D2O) δ 7.73 (1H, d, J = 2.0 Hz), 7.52 (1H,
s), 7.51 (1H, d, J = 8.4 Hz), 7.19 (1H, dd, J = 8.4, 1.7 Hz), 6.83 (1H, d, J
= 2.0 Hz), 3.54 (1H, m), 3.17-3.10 (2H, m), 3.10-2.86 (2H, m),
1.22-1.18 (6H, m). 13C NMR (100 MHz, D2O) δ 153.84, 146.32, 130.34,
127.86, 125.58, 122.04, 111.56, 106.43, 55.16, 40.05, 38.68, 15.11
10.60. HRMS calculated for C13H18NO [M+H]+ 204.1383, found
204.1389.
2.4.2. Procedure
The entire protocol was based on our previous studies (Custodio
et al., 2017; Custodio et al., 2019). Training for the drug-paired lever
pressing was conducted for three consecutive days (30 min/session, 2
sessions/day) for a contingent food-pellet reward on a continuous
schedule of reinforcement. Rats acquiring greater than 80 pellets on the
last session of training were selected and prepared for surgery. Standard
operating techniques were carried out as previously described (Custodio
et al., 2019). The rats were left for 5 days to recover from surgery. After
the recovery period, rats were given a consumable daily diet of pellets
(approximately 20 g) and were subjected to a 2-h daily SA session under
a fixed-ratio (FR) 1 schedule for 7 consecutive days, and a FR2 schedule
for 3 days. During the SA test, both levers (right/left) were available to
the rats. Pressing the left lever (active lever) resulted in a delivery of 0.1
mL of 2-EAPB (0.03, 0.1, or 0.3 mg/kg/infusion), 5-EAPB (0.03, 0.1, or
0.3 mg/kg/infusion), methamphetamine (0.1 mg/kg/infusion) or saline
(n = 7). Simultaneously, the house light was switched off, and the
stimulus light was illuminated and remained lit for 20 s after the end of
the infusion (the time-out period). Lever presses during periods of
“time-out” were recorded but had no effect. Right-lever presses (inactive
lever) were recorded but not reinforced.
Methamphetamine was purchased from Sigma-Aldrich (St. Louis,
Missouri, U.S.). All the drugs were dissolved in 0.9% sterile saline and
administered intraperitoneally (i.p., for CPP, locomotor sensitization,
treatment for western blotting) or intravenously (for SA). All dosages of
drugs (2-EAPB, 5-EAPB, and methamphetamine) used in the present
study were based on previous publications that evaluated the rewarding
and reinforcing properties of amphetamine derivatives (Cain et al.,
2008; Custodio et al., 2017; Marona-Lewicka et al., 1996).
2.3. Conditioned place preference test
2.3.1. Apparatus
The CPP apparatus consisted of two compartments measuring 47 ×
47 × 47 cm3. Each compartment provided distinct visual and tactile
cues, one had black walls with smooth flooring, while the other
compartment had black walls with white dots and rough black flooring.
The compartments could be separated from each other by a detachable
guillotine door. Illumination throughout the experiment was maintained
at 12 lux. An automated system (Ethovison, Noldus, Netherlands) was
utilized for the recording and analyses of animal movements.
2.5. Locomotor sensitization
2.5.1. Apparatus
The locomotor activity of the mice was measured in a square, black-
Plexiglas container with an open-field arena (42 × 42 × 42 cm3). A
computer system (Ethovison, Noldus, Netherlands) was utilized to re-
cord the total distance moved (cm) of each mouse.
2.3.2. Procedure
The protocol used was similar to that used in our previous studies
(Custodio et al., 2020; Custodio et al., 2019), with some modifications.
The test consisted of three phases: (1) habituation [3 days] and
pre-conditioning [1 day], (2) conditioning [6 days], and (3)
post-conditioning [1 day]. During the habituation phase, rats (n = 8)
were given access to both compartments for 15 min on three consecutive
days. The pre-conditioning phase began the following day during which
the time spent in each compartment was recorded for 15 min. Rats were
assigned to groups based on the pre-conditioning phase, specifically,
their non-preferred side was designated as the drug-paired compart-
ment. During the conditioning phase, the guillotine doors were closed.
Rats received an i.p. injection of 2-EAPB (1, 3, and 10 mg/kg), 5-EAPB
(0.3, 1, 3, and 10 mg/kg), methamphetamine (1 mg/kg), or saline, and
were randomly placed in one of the compartments for 30 min. On
alternate days, rats received saline injections and were confined in the
compartment other than the drug-paired compartment. Immediately
following the last conditioning day, the post-conditioning phase began
wherein rats were drug-free and allowed to access both compartments as
during the pre-conditioning phase.
2.5.2. Procedure
This test was based on our previous protocol (Custodio et al., 2017;
Custodio et al., 2020), with few modifications. The behavioral assay
consisted of four phases: habituation, drug treatment (T1-T7), drug
abstinence (A1-A7), and drug challenge (Ch). For the first two days,
mice were habituated to the apparatus for 30 min. On the third day (T0),
locomotor activity was recorded and used as a baseline parameter.
Thereafter, 2-EAPB (1, 3, and 10 mg/kg), 5-EAPB (1, 3, and 10 mg/kg),
methamphetamine (1 mg/kg), or saline was administered to the mice (n
= 8) for 7 days. Mice were then challenged with the same dose and drug
after 7 days of abstinence. Locomotor activity was evaluated for 30 min
immediately following the first, third, and seventh day of both drug or
saline treatment and abstinence, as well as on the challenge day.
2.6. Western blotting
The procedure for obtaining protein, gel preparation, and blot
analysis was based on our previous studies (Custodio et al., 2019; Kim
et al., 2019), with some modifications. Rats (n = 6) were treated with
2-EAPB (10 mg/kg), 5-EAPB (1 mg/kg), methamphetamine (1 mg/kg),
or saline using the treatment schedule employed in CPP in order to
associate possible modified protein levels with the probable induction of
CPP behavior. Treatment was done in the CPP experiment room, but the
test was not conducted. The rats were killed by decapitation 24 h after
the last drug administration and used for protein extraction. Brains were
2.4. Self-administration test
2.4.1. Apparatus
Standard operant chambers (Coulborn Instruments, Allentown, PA,
USA) were kept inside sound-attenuating boxes with built-in ventilation
fans. Each operant chamber included a pellet dispenser, left and right
3

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
rapidly and carefully removed and placed in ice-cold saline to prevent
damage to the brain. The nucleus accumbens and ventral tegmental area
were sliced into sections using a rat-brain matrix. The regions were then
isolated from the slices and immediately frozen at ꢀ 80 ◦C until further
Tukey’s post-test indicated that rats treated with 2-EAPB (10 mg/kg), 5-
EAPB (1 mg/kg), and methamphetamine had significantly higher CPP
scores compared to the saline-treated group.
use. Tissues were lysed with 500 μL homogenization buffer (RIPA buffer
3.2. 2-EAPB and 5-EAPB are self-administered by rats
[Biosesang Inc., Seongnam, Korea], cOmplete™ ULTRA protease in-
hibitor cocktail tablets [05892791001; Sigma-Aldrich], and Phos-
STOP™ phosphatase inhibitor cocktail tablets [04906845001,
A two-way ANOVA in Fig. 3A exhibits significant differences be-
tween treatment groups [F (3, 24) = 38.4, P < 0.001], SA days [F (6,
144) = 21.5, P < 0.001], and an interaction between treatment and days
[F (18, 144) = 3.48, P < 0.001] under FR1, and also significant differ-
ences between treatment groups [F (3, 24) = 102, P < 0.001] and an
interaction between treatment and SA days [F (6, 48) = 13.0, P < 0.001]
under FR2. Tukey’s post-test showed significantly increased lever
pressing in rats that self-administered 2-EAPB (0.1 and 0.3 mg/kg/
infusion). Two-way ANOVA of Fig. 3B shows significant differences
between treatment groups [F (3, 24) = 120, P < 0.001], SA days [F
(4.35, 104) = 4.29, P < 0.01], and an interaction of the two [F (18, 144)
= 4.06, P < 0.001] under FR1, and also between treatment groups [F (3,
24) = 120, P < 0.001], SA days [F (2.00, 48.0) = 19.8, P < 0.001], and
an interaction of treatment and days [F (6, 48) = 3.78, P < 0.01] under
FR2. Tukey’s post-test showed significantly increased lever pressing in
rats that self-administered 5-EAPB at all doses. Fig. 3C two-way ANOVA
displays a significant difference only between treatment groups [F (1,
12) = 442, P < 0.001] under FR1, and between treatment groups [F (1,
12) = 880, P < 0.001] under FR2. Bonferroni’s post-test exhibited
significantly increased lever pressing in rats that self-administered
methamphetamine. Two-way ANOVA of Fig. 3D displays a significant
difference between treatments [F (3, 24) = 27.9, P < 0.001], SA days [F
(3.97, 95.3) = 13.5, P < 0.001], and an interaction between the two [F
(18, 144) = 2.33, P < 0.01] under FR1, and significant differences be-
tween treatments [F (3, 24) = 18.4, P < 0.001] and an interaction be-
tween treatment and SA days [F (6, 48) = 3.73, P < 0.01] under FR2.
Tukey’s post-test showed significantly greater number of infusions ac-
quired by rats that self-administered 2-EAPB at all doses. Fig. 3E two-
way ANOVA exhibits significant differences between treatments [F (3,
24) = 36.6, P < 0.001], SA days [F (4.55, 109) = 5.35, P < 0.001], and
an interaction between treatment and days [F (18, 144) = 1.74, P <
0.05] under FR1, and significant differences between treatments [F (3,
24) = 27.6, P < 0.001] and SA days [F (1.90, 45.7) = 4.69, P < 0.05]
only under FR2. Tukey’s post-test presented significantly greater num-
ber of infusions acquired by rats that self-administered 5-EAPB at all
doses. Two-way ANOVA of Fig. 3F exhibits significant differences be-
tween treatments only [F (1, 12) = 152, P < 0.001] under FR1, but with
significant differences between treatments [F (1, 12) = 443, P < 0.001]
and SA days [F (1.98, 23.8) = 4.13, P < 0.05] under FR2. Bonferroni’s
post-test revealed significantly greater number of infusions acquired by
rats that self-administered methamphetamine. One-way ANOVA of
Sigma-Aldrich]). Tissue extracts were centrifuged at 16000 g at 4 ◦C
◦
for 20 min. Samples were heated at 95 C for 5 min 20
μg of protein
lysates were then loaded onto 10% sodium-dodecyl sulfate/polyacry-
lamide gel electrophoresis (SDS/PAGE) gels, separated, and transferred
to nitrocellulose membranes. Blots were blocked with 5% bovine serum
albumin in Tris-buffered saline in 0.1% Tween-20 (TBST) solution for 1
h and incubated with specific primary antibodies (Mouse Monoclonal
Anti-Dopamine D1 receptor Antibody [MA1-46024 Invitrogen, (Salyer
et al., 2011); Rabbit Polyclonal Anti-Dopamine D2 receptor Antibody
[D2R-201AP FabGennix International, Inc., (Rentesi et al., 2013)];
Rabbit Polyclonal Anti-Tyrosine hydroxylase Antibody [AB152;
Sigma-Aldrich, (Kawahata et al., 2009)]; Rabbit Monoclonal
Anti-Dopamine transporter [ab184451 Abcam]; Rabbit Monoclonal
Anti-CREB Antibody [#9197S Cell Signaling Technology]; Rabbit
Monoclonal Anti-Phospho-CREB Antibody [#9198S Cell Signaling
Technology]; Rabbit Monoclonal Anti-FosB Antibody [ab184938
Abcam]; Rabbit Monoclonal Anti-BDNF Antibody [ab108319 Abcam];
Mouse Monoclonal Anti-Beta-actin (β-actin) Antibody [A5441
Sigma-Aldrich]) overnight at 4 ◦C. The next day, blots were washed
three
times in TBST
and incubated
with horseradish
peroxidase-conjugated anti-rabbit (1:3000) or anti-mouse secondary
antibodies (1:5000) for 1 h. After three washes with TBST, the blots were
visualized using enhanced chemiluminescence (Clarity Western ECL;
Bio-Rad Laboratories, Hercules, CA, USA) and ChemiDoc Imaging Sys-
tem (Image Lab software, version 6.0; Bio-Rad). Values for
phosphorylation-independent protein levels were normalized to β-actin.
Phosphorylated
proteins
were
normalized
to
their
phosphorylation-independent form. Fold change was determined by
normalizing to the values of the saline group.
2.7. Data analysis
All values were expressed as mean ± standard error of the mean (S.E.
M.). Results from CPP, western blotting, and mean number of infusions
in SA were analyzed by one-way analysis of variance (ANOVA), followed
by Tukey’s post-test. Data from the active-lever responses and the
number of infusions during SA test under the FR schedule of reinforce-
ment were analyzed using two-way repeated measures ANOVA with
treatments as the between-subject factor and SA days as the within-
subject factor (note that data from each FR schedule were analyzed
separately). Tukey’s or Bonferroni’s post-test was utilized for further
comparison. Comparison of the mean number of infusions between sa-
line and methamphetamine was analyzed using unpaired t-test. Data
from locomotor sensitization were analyzed using a two-way repeated
measures ANOVA with treatments as between-subject factor and days as
within-subject factor, with Tukey’s or Bonferroni’s test for post-hoc
analyses. Values of P less than 0.05 was the criterion for statistical sig-
nificance. All statistical analyses were performed using GraphPad Prism
software v. 8.01 (San Diego, CA, USA).
3. Results
3.1. 2-EAPB and 5-EAPB induce CPP in rats at different doses
Fig. 2. The effects of 2-EAPB and 5-EAPB on rat place preference. Rats were
conditioned with 2-EAPB (1, 3, and 10 mg/kg) and 5-EAPB (0.3, 1, 3, and 10
mg/kg), METH (1 mg/kg), or saline (SAL). 2-EAPB and 5-EAPB induced CPP in
rats, suggesting their rewarding effects similar to METH. Data are presented as
means ± S.E.M. n = 8. ***P < 0.001 significantly different from the SAL group
(One-way ANOVA, Tukey’s post-test).
Fig. 2 illustrates the CPP scores of rats treated with 2-EAPB EAPB (1,
3, and 10 mg/kg), 5-EAPB (0.3, 1, 3, and 10 mg/kg), methamphetamine
(1 mg/kg) or saline. One-way ANOVA indicated a significant difference
among the treatment groups [F (8, 63) = 6.01, P < 0.001]. Furthermore,
4

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
Fig. 3. The effects of 2-EAPB and 5-EAPB on rat SA (A, B, C): The total active-lever responses; (D, E, F): total number of infusions; (G, H, I): mean number of infusions
for each treatment group during the 2-h, 10-day SA experiment in rats. 2-EAPB and 5-EAPB were modestly self-administered by rats, obtaining a higher number of
active lever responses and infusions as compared with the SAL group, indicating its reinforcing effects. However, 2-EAPB and 5-EAPB have weaker reinforcing effects
as compared to METH. Values are mean ± S.E.M. n = 7. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the SAL group (active lever responses and number of
infusions [Two-way RM-ANOVA, Tukey’s or Bonferonni’s post-test]; mean number of infusions [One-way ANOVA, Tukey’s post-test; Unpaired t-test (METH)]).
Fig. 3G shows a significant difference between treatment groups [F (3,
36) = 13.9, P < 0.001], with a Tukey’s post-hoc analysis showing an
increased mean number of infusions of 2-EAPB at all doses. In Fig. 3H,
one-way ANOVA shows a significant difference between treatment
groups [F (3, 36) = 40.0, P < 0.001]; a Tukey’s post-test exhibited an
increased mean number of infusions of 5-EAPB at all doses. Unpaired t-
test of Fig. 3I shows a significantly higher mean number of infusions
acquired by rats that self-administered methamphetamine [t = 35.1, df
= 18, P < 0.001] compared to saline.
ANOVA shows a significant difference between treatment groups [F
(4, 35) = 9.18, P < 0.001], treatment days [F (7, 245) = 21.1, P <
0.001], and an interaction between the two [F (28, 245) = 5.91, P <
0.001]. Tukey’s post-test showed that only methamphetamine enhanced
locomotor activity on the first, third, and seventh days of treatment
compared to saline. In Fig. 4B, a two-way ANOVA reveals a significant
difference between treatment groups [F (4, 35) = 10.4, P < 0.001],
treatment days [F (7, 245) = 33.5, P < 0.001], and an interaction be-
tween the two [F (28, 245) = 7.64, P < 0.001]. A Tukey’s post-hoc
analysis showed that 5-EAPB (3 and 10 mg/kg) and methamphet-
amine enhanced mice locomotor activity on the first, third, and seventh
days of treatment and on the challenge day compared to saline. Fig. 4C
displays a two-way ANOVA indicating a significant difference between
treatments [F (4, 35) = 19.2, P < 0.001], between days [F (1, 35) = 69.4,
P < 0.001], and an interaction of treatment and days [F (4, 35) = 5.57, P
= 0.001], with Bonferroni’s post-test showing only methamphetamine
treatment having a significant difference between the first day of
3.3. 5-EAPB but not 2-EAPB induces locomotor sensitization in mice
Fig. 4 exhibits the locomotor response of mice on the first, third, and
seventh days of treatment following 2-EAPB and 5-EAPB (1, 3, 10 mg/
kg), methamphetamine (1 mg/kg) or saline administration for 7 days,
and the comparison between the distance moved of mice between the
first day of treatment and the challenge day. In Fig. 4A, a two-way
5

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
Fig. 4. Effects of 2-EAPB and 5-EAPB on mice locomotor sensitization. (A, B): The locomotor activity of mice (distance moved, cm) before drug treatment (T0), on
the first (T1), third (T3), and seventh (T7) days of treatment, on the first (A1), third (A3), and seventh (A7) days of drug abstinence, and challenge day (Ch). Only 5-
EAPB and METH increased the locomotor activity of mice supported by the significant difference in their distance moved compared to SAL. Values are mean ± S.E.M.
n = 8. *P < 0.05, **P < 0.01, and ***P < 0.001 significantly different from the SAL group (Two-way ANOVA, Tukey’s post-test). (C, D): Comparison of locomotor
activity during the first day of drug treatment versus drug challenge. Only 5-EAPB and METH induced locomotor sensitization in mice confirmed by the significant
difference in their distance moved between T1 and Ch. Values are mean ± S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 significantly different from T1 (Two-way
ANOVA, Bonferroni’s post-test).
treatment and the challenge day. Fig. 4D shows the results of a two-way
ANOVA indicating significant differences between treatment groups [F
(4, 35) = 17.8, P < 0.001], between days [F (1, 35) = 81.0, P < 0.001],
and an interaction of the two [F (4, 35) = 5.47, P = 0.002]. Bonferroni’s
post-hoc analysis showed that treatment with 5-EAPB (at all doses) and
methamphetamine produced a significantly higher distance moved be-
tween the first day of treatment and the challenge day.
expression in the nucleus accumbens of rats treated with 2-EAPB (10
mg/kg), 5-EAPB (1 mg/kg), methamphetamine (1 mg/kg), or saline. A
one-way ANOVA in Fig. 6C exhibited a significant difference among
groups [F (3, 20) = 4.84, P < 0.05]. Tukey’s post-hoc analysis revealed
increased deltaFosB expressions after 2-EAPB and 5-EAPB administra-
tion. No significant changes in p-CREB or BDNF expressions were
exhibited between treatment groups in Fig. 6B [F (3, 20) = 1.38, P <
0.277], and 6D [F (3, 20) = 0.688, P < 0.570].
3.4. 5-EAPB and 2-EAPB showed no alterations in dopamine-related
proteins in the nucleus accumbens and ventral tegmental area of rats
4. Discussion
Fig. 5 illustrates the effects on dopamine D1 receptor and dopamine
D2 receptor expression in the nucleus accumbens, and tyrosine hy-
droxylase and dopamine transporter expression in the ventral tegmental
area of rats treated with 2-EAPB (10 mg/kg), 5-EAPB (1 mg/kg),
methamphetamine (1 mg/kg), or saline. In Fig.s 5B, 5C, and 5F, a one-
way ANOVA showed no significant differences in dopamine D1 receptor
[F (3, 20) = 2.25, P < 0.114], dopamine D2 receptor [F (3, 20) = 2.23, P
< 0.116], and dopamine transporter [F (3, 20) = 1.83, P < 0.174] ex-
pressions in rat brain after the administration of 2-EAPB or 5-EAPB. A
one-way ANOVA of Fig. 5E exhibited a significant difference among
groups [F (3, 20) = 10.6, P < 0.001], with a Tukey’s post-hoc analysis
revealing a significant increase in tyrosine hydroxylase expression after
methamphetamine administration only.
Treatment with specific doses of 2-EAPB (10 mg/kg) and 5-EAPB (1
mg/kg), along with METH (1 mg/kg), generated place preference in rats.
These results are similar to the previously reported data for CPP induced
by the SB 5-APB (1 and 2 mg/kg) (Cha et al., 2016). This suggests that
2-EAPB and 5-EAPB may possess potential rewarding effects similar to
methamphetamine (Pandy et al., 2018). Interestingly, 2-EAPB required
a higher dose for CPP induction than 5-EAPB. This observation might
suggest a greater tendency for 5-EAPB abuse given its ability to elicit a
reward-like effect at a lower dose, which is comparable to metham-
phetamine, than 2-EAPB. The two compounds were also modestly
self-administered by rats at certain doses, as shown by the higher
active-lever responses and number of infusions across the SA days (FR1
and FR2 schedules) compared to saline. This indicates that they may also
possibly possess potential reinforcing properties, although at lower
˜
3.5. 2-EAPB and 5-EAPB alter deltaFosB expression in the nucleus
accumbens
strengths compared to methamphetamine (dela Pena et al., 2013).
Remarkably, 5-EAPB induced greater SA responses at lower doses than
2-EAPB, probably implying a stronger inclination for self-administering
5-EAPB. Given that agents with increased serotonin-releasing efficacy
Fig. 6 demonstrates the effects on p-CREB, deltaFosB, and BDNF
6

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
Fig. 5. Effects of 2-EAPB (10 mg/kg), 5-EAPB (1 mg/kg), METH (1 mg/kg), or SAL on dopamine-related protein levels. (A): Representative blots of the target
proteins in the nucleus accumbens. Protein levels of (B) dopamine D1 receptor (DRD1) and (C) dopamine D2 receptor (DRD2). (D): Representative blots of the target
proteins in the ventral tegmental area. Protein levels of (E) tyrosine hydroxylase (TH) and (F) dopamine transporter (DAT). Only METH significantly increased TH
expression. Values are mean ± S.E.M. n = 6. ***P < 0.001 significantly different from the SAL group (One-way ANOVA, Tukey’s post-test).
Fig. 6. Effects of 2-EAPB (10 mg/kg), 5-EAPB (1 mg/kg), METH (1 mg/kg), or SAL on plasticity-related protein levels. (A) Representative blots of the target proteins
in the nucleus accumbens. Protein levels of (B) p-CREB, (C) deltaFosB, and (D) BDNF. Both 2-EAPB and 5-EAPB significantly increased deltaFosB levels in the nucleus
accumbens. Values are mean ± S.E.M. n = 6. *P < 0.05 significantly different from the SAL group (One-way ANOVA, Tukey’s post-test).
7

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
relative to dopamine (such as SBs) normally decrease reinforcement
(Gomila et al., 2017; Rickli et al., 2015), it may seem peculiar how
2-EAPB and 5-EAPB still induced SA responses, although lower than
methamphetamine. 5-EAPB possesses relatively higher affinity for the
dopamine D1 and D2 receptors are determined (through competitive
receptor antagonism) or actual dopamine levels in the mesolimbic sys-
tem are characterized. Further studies are underway for the determi-
nation of other potential neuromodulators that may be implicated in the
rewarding effects of 2-EAPB and 5-EAPB.
dopamine transporter (IC50 = 4.9 μM) than other SBs (Rickli et al.,
2015); hence, it may be plausible that the capability of the two com-
pounds to induce SA might be associated with a combination of dopa-
minergic and serotonergic mediation. Further investigations are needed
to elucidate this, particularly since there are no binding-affinity data
available for 2-EAPB to our knowledge. In addition, only 5-EAPB (1, 3,
and 10 mg/kg) and methamphetamine (1 mg/kg) were capable of
inducing locomotor sensitization in mice, highlighting yet another
pharmacological difference between the two SBs. Their different effects
on mouse locomotor activity may again be attributed to the relatively
higher dopamine transporter affinity of 5-EAPB because other SBs such
Neuronal adaptations have been exhibited during the expression of
addictive behaviors after chronic exposure to abused drugs (Russo et al.,
2010). These changes are generally accompanied by modifications in
gene expression, as evidenced by the varying expression levels of spe-
cific transcriptional regulators in the brain after repeated drug exposure.
One of these transcription factors is CREB that is normally activated by
the cAMP pathway. Altered p-CREB has been shown in the nucleus
accumbens after repeated exposure to addictive drugs (McClung and
Nestler, 2003). Another transcription factor, deltaFosB (Fos family), was
previously identified to accumulate in the nucleus accumbens after
repeated treatment of abused substances (McClung and Nestler, 2003).
Protein expression results exhibited significantly increased deltaFosB
levels in the nucleus accumbens of rats after 2-EAPB and 5-EAPB
treatments. These are consistent with previous reports of deltaFosB in-
duction by morphine and cocaine (McClung and Nestler, 2003;
Zachariou et al., 2006). The overall effect of enhanced deltaFosB in-
creases medium spiny-neuron density in the nucleus accumbens,
thereby leading to sensitized behavioral responses to drugs of abuse
(Russo et al., 2010). This suggests that deltaFosB induction in the nu-
cleus accumbens may have also contributed to the development of the
potential rewarding effects by 2-EAPB and 5-EAPB. However, none of
the drugs (including methamphetamine) were able to modify p-CREB
expression, unlike in previous reports of abused substances (Pluzarev
and Pandey, 2004). The lack of significance in p-CREB/CREB ratio after
methamphetamine treatment might be because its effect on p-CREB
expression is a significant reduction only after a long period of with-
drawal following continuous exposure (McDaid et al., 2006). The lack of
significant changes in deltaFosB expression after methamphetamine
administration may not be due to prolonged euthanizing time after last
treatment, but probably due to intermittent treatment frequency, as
studies that do report increased deltaFosB expression exposed their
subjects to methamphetamine continuously over several days (Custodio
et al., 2019; Wen et al., 2020). Furthermore, one of the gene targets of
CREB is BDNF, a neurotrophic factor that contributes to neuronal
growth and synaptic differentiation, and has been well-implicated in
drug-reward mechanisms (Li and Wolf, 2015). In contrast to previous
reports (Koo et al., 2012; Ren et al., 2015), 2-EAPB, 5-EAPB, and
methamphetamine treatments did not alter BDNF expression in the
nucleus accumbens, despite their induction of potential rewarding ef-
fects. However, these results are also consistent with a previous study
showing the normalization of increased BDNF 24 h after cocaine SA
(Graham et al., 2007). Since the brain extraction in our study was per-
formed 24 h after the last drug administration, we may not have
detected the possible transient increase in BDNF. Overall, the modifi-
cation in the expression of deltaFosB may possibly suggest a facilitating
role in the induction of the potential rewarding effects of 2-EAPB and
5-EAPB.
as 5-APB (IC50 = 6.1 μM (Rickli et al., 2015)) and 6-APDB (IC50 = 33 μM
(Rickli et al., 2015)) also induced dose-dependent horizontal stimulation
at similar doses (Roque Bravo et al., 2019). Supplementary experiments
to determine the dopamine transporter affinity of 2-EAPB may be
necessary to elucidate its lack of stimulant effects. It is of note that
5-EAPB elicited sensitization at the CPP-inducing dose (1 mg/kg) and at
higher doses (3 and 10 mg/kg), similar to other addictive drugs (Jing
et al., 2014; Shimosato and Ohkuma, 2000). Overall, our behavioral
assessments moderately suggest that the alterations engendered by
2-EAPB are comparable to the effects of some cannabinoids (Tampus
et al., 2015) and amphetamine derivatives (Custodio et al., 2017),
whereas the elicited changes by 5-EAPB are similar to the effects of
opioid-analgesic treatments in adolescent C57BL/6 mice (Niikura et al.,
2013). The variation between the behaviors induced by the two com-
pounds may possibly stem from their conformational and potential
binding-affinity differences, since structure-activity relationships influ-
ence the addictive (and other) effects of drugs (Glennon and Dukat,
2016; Wiley et al., 2016). Taken together, our results suggest that
2-EAPB and 5-EAPB, although with varying potencies, may hold a sig-
nificant likelihood for abuse due to their potential rewarding and rein-
forcing effects.
We also determined the influence of 2-EAPB and 5-EAPB on
dopamine-related proteins in the nucleus accumbens and ventral
tegmental area, brain regions that constitute the mesolimbic dopamine
system. Drugs of abuse have been recognized to modify the expression of
receptors and other proteins in this system, which have all been impli-
cated in the manifestation of addictive-like behaviors in rodents, such as
CPP and SA (Abiero et al., 2019; Botanas et al., 2017; Custodio et al.,
2019). Peculiarly, none of these drugs were able to elicit significant
changes to the expressions of dopamine-related proteins, except for
methamphetamine, which significantly increased tyrosine hydroxylase
in the ventral tegmental area. Increased tyrosine hydroxylase could
correspond to an upregulation in the release of dopamine in the nucleus
accumbens (Abiero et al., 2019), which was also reported to contribute
in facilitating addiction-like behaviors (Custodio et al., 2019). A possible
rationale for the lack of consistent statistical significant changes in ex-
pressions of the other proteins might be due to methodological dissim-
ilarities (e.g. exposure duration, treatment frequency, effective dose,
euthanizing time after last treatment) between our lab and previous
studies (Abiero et al., 2019; Krasnova et al., 2013). Despite these dif-
ferences, our results could still be suggestive of the involvement of other
neurotransmitter systems that might have influenced the development
of drug-seeking behaviors in rodents after 2-EAPB and 5-EAPB exposure
(Lanteri et al., 2008; Pierce and Kumaresan, 2006). In fact, the anti-
convulsant drug pregabalin was capable of eliciting CPP in mice in the
same manner as cocaine, but was unable to alter extracellular dopamine
levels in the nucleus accumbens, suggesting the involvement of addi-
tional receptor mechanisms besides dopamine that could mediate its
rewarding effects (Coutens et al., 2019). Likewise, the participation of
dopaminergic receptors may not be entirely disregarded as a mechanism
for the potential abuse liabilities of these SBs, unless the contribution of
To summarize, 2-EAPB and 5-EAPB induced CPP at different dosages
and were also modestly self-administered by rats, indicating their po-
tential rewarding and reinforcing effects. Only 5-EAPB was able to
induce locomotor sensitization in mice at a dose-dependent manner,
suggesting a possible greater tendency for 5-EAPB drug tolerance and
withdrawal compared to 2-EAPB. The variations in the CPP-inducing
doses and the absence/presence of locomotor alterations could suggest
the involvement of other diverse receptor mechanisms that can influ-
ence behavior. The elicited addictive phenotypes by 2-EAPB and 5-EAPB
treatments may possibly be associated with deltaFosB induction in the
nucleus accumbens, although dopaminergic mediation may not be
completely disregarded. Our investigation has generated preliminary
evidence that 2-EAPB and 5-EAPB possess great potential for abuse;
consequently, we recommend that the legal status of their availability to
8

L.V. Sayson et al.
European Journal of Pharmacology 885 (2020) 173527
Dolan, S.B., Forster, M.J., Gatch, M.B., 2017. Discriminative stimulus and locomotor
effects of para-substituted and benzofuran analogs of amphetamine. Drug Alcohol
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the public should be rapidly rationalized.
Role of funding source
Eshleman, A.J., Nagarajan, S., Wolfrum, K.M., Reed, J.F., Swanson, T.L., Nilsen, A.,
Janowsky, A., 2019. Structure-activity relationships of bath salt components:
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Psychopharmacology 236, 939–952.
This work was supported by the Ministry of Food and Drug Safety
(19182MFDS410) and the National Research Foundation of Korea
(2017R1D1A1A02018695).
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psychoactive benzofurans strongly increase extracellular serotonin level in mouse
corpus striatum. J. Toxicol. Sci. 41, 329–337.
CRediT authorship contribution statement
Glennon, R.A., Dukat, M., 2016. Structure-activity Relationships of Synthetic Cathinones,
Neuropharmacology of New Psychoactive Substances (NPS). Springer, pp. 19–47.
Leandro Val Sayson: Investigation, Data curation, Formal analysis,
Writing - original draft. Raly James Perez Custodio: Investigation,
Formal analysis. Darlene Mae Ortiz: Investigation, Formal analysis.
Hyun Jun Lee: Investigation, Formal analysis. Mikyung Kim: Investi-
gation, Formal analysis. Youngdo Jeong: Investigation, Formal anal-
ysis. Yong Sup Lee: Investigation, Formal analysis. Hee Jin Kim:
Conceptualization, Methodology, Project administration, Validation.
Jae Hoon Cheong: Conceptualization, Methodology, Project adminis-
tration, Validation.
´
Gomila, I., Moranta, C., Quesada, L., Pastor, Y., Dastis, M., Torrents, A., Elorza, M.A.,
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Dynamic BDNF activity in nucleus accumbens with cocaine use increases self-
administration and relapse. Nat. Neurosci. 10, 1029–1037.
Jing, L., Zhang, M., Li, J.-X., Huang, P., Liu, Q., Li, Y.-L., Liang, H., Liang, J.-H., 2014.
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Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
˜
Kim, M., Custodio, R.J., Botanas, C.J., de la Pena, J.B., Sayson, L.V., Abiero, A., Ryoo, Z.
Y., Cheong, J.H., Kim, H.J., 2019. The circadian gene, Per 2, influences
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