Neuroscience Letters 735 (2020) 135177

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Research article

The antagonistic activity profile of naloxone in μ-opioid receptor agonist- T

induced psychological dependence
Atsushi Nakamuraa, Kana Yasufukub, Shinji Shimadaa, Hiroyuki Aritomia, Youko Furuea,
Hiroki Chibaa, Mami Muramotoa, Kenji Takaseb, Katsumi Koikeb, Tomoko Matsumotoc,
Tomoka Shimadac, Ryosuke Watarid, Takanobu Matsuzakid, Toshiyuki Asakib,
Toshiyuki Kanemasab, Masahide Fujitab,*
a
Research Area for Pharmacological Evaluation, Shionogi TechnoAdvance Research Co., Ltd, 1-1, 3-chome, Futaba-cho, Toyonaka, 561-0825, Osaka, Japan
b
Laboratory for Drug Discovery and Disease Research, Shionogi & Co., Ltd., 1-1, 3-chome, Futaba-cho, Toyonaka, 561-0825, Osaka, Japan
c
Research Area for Candidate Selection, Shionogi TechnoAdvance Research Co., Ltd, 1-1, 3-chome, Futaba-cho, Toyonaka, 561-0825, Osaka, Japan
d
Laboratory for Drug Discovery and Development, Shionogi & Co., Ltd., 1-1, 3-chome, Futaba-cho, Toyonaka, 561-0825, Osaka, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Naloxone is a μ-opioid receptor antagonist that has been used to prevent overdose-related respiratory depression
Competitive antagonist and deaths by the illicit use of opioids. Naloxone can also deter the abuse potential of opioids, but little has been
Conditioned place preference reported regarding its antagonistic activity profile against opioid-induced psychological dependence. This study
Dopamine release aimed to confirm the antagonistic activity profile of naloxone against several μ-opioid receptor agonists and
μ-Opioid receptor agonist
investigate whether naloxone could affect the psychological dependence induced by widely used μ-opioid re-
Naloxone
ceptor agonist, oxycodone. In the Guanosine-5’-o-(3-thio) triphosphate (GTPγS) binding assay, naloxone
(30−30,000 nM) inhibited the GTPγS binding induced by oxycodone, hydrocodone, morphine, and fentanyl. It
elicited parallel rightward shifts in the concentration-response curves, indicating that naloxone possessed a
competitive antagonistic activity profile against these μ-opioid receptor agonists. In the conditioned place
preference test, oxycodone (0.01−1 mg/kg, i.v.) produced dose-dependent increases in place preference. The
increased place preference induced by oxycodone (1 mg/kg) was significantly attenuated by co-administration of
naloxone at a dose of 0.5 mg/kg but not 0.01 mg/kg. Naloxone (0.5 mg/kg, i.v.) also blocked oxycodone (1 mg/
kg)-induced dopamine release in nucleus accumbens; however, at a lower dose (0.01 mg/kg), it did not affect the
intrinsic dopamine release by oxycodone. These results indicate that the psychological dependence of oxycodone
could be antagonized by naloxone, depending on the dose. This characterization might lead to a better under-
standing of the competitive antagonistic activity profile of naloxone for μ-opioid receptor in the brain.

1. Introduction intravenous (i.v.) administration is most effective in many fatal cases

Clinically, several μ-opioid receptor agonists such as oxycodone,
hydrocodone, morphine, and fentanyl have been used to manage
moderate to severe pain. The clinical use of opioids is often accom-
panied by many side effects including emesis, constipation, and seda-
tion [1]; the increased rate of overdose-related respiratory depression
and deaths caused by the illicit use of μ-opioid receptor agonists is one
of the most serious issues that not only impacts the public health ne-
gatively but also increases economic burden [2]. Naloxone is an an-
tagonist with a high binding affinity for the μ-opioid receptor [3] and
has been used to prevent such fatal incidents. Several routes adminis-
tration of naloxone have been developed for clinical use, but its
because of rapid onset of action that can rescue overdose-related re-
spiratory depression [4].
Some environmental and drug-related factors contribute to over-
dose-related respiratory depression and deaths, but the potential for
abuse associated with opioid misuse and addiction is considered to be
the leading cause [5]. Opioid abusers often use i.v. administration route
to attain a larger magnitude of euphoria and many abuse-deterrent
approaches including formulating agonist/ antagonist combinations
[6], adding aversive agents [7], modifying the delivery system [8], and
developing prodrugs [9] have been challenged. Of these approaches, a
recent study on the human potential for abuse raised the possibility that
i.v. co-administration of oxycodone and naloxone (1:0.5 ratio) could

⁎
Corresponding author at: Neuroscience Laboratory for Drug Discovery and Disease Research Laboratory, Shionogi & Co., Ltd., Osaka, Japan.
E-mail address: masahide.fujita@shionogi.co.jp (M. Fujita).

https://doi.org/10.1016/j.neulet.2020.135177
Received 18 May 2020; Received in revised form 11 June 2020; Accepted 15 June 2020
Available online 20 June 2020
0304-3940/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Nakamura, et al.
produce lesser euphoria than that of oxycodone [10]. This is a rational
pharmacological challenge, but little has been reported about the an-
tagonist activity profile of naloxone and its appropriate ratio to be used
in preventing opioid abuse.
For many decades, the conditioned place preference paradigm has
been used to evaluate the psychological dependence related to opioid
abuse in rodents [11]. A dose of μ-opioid receptor agonists causes place
preference by activating the mesolimbic dopamine system, which is
projected from the ventral tegmental area to the nucleus accumbens
(NAC) [12]. Thus, the concentration of dopamine released in NAC is
considered to be one of the critical parameters in the brain to estimate
the psychological dependence induced by μ-opioid receptor agonists.
This study aimed to investigate the ability of naloxone to prevent μ-
opioid receptor agonist- induced psychological dependence. Of these
agonists, oxycodone was consumed the most globally from 2000 to
2014 [13], and was thus selected for this study. Here, we have revealed
that naloxone can inhibit oxycodone-induced increases in place pre-
ference and dopamine release, indicating the antagonism of naloxone
against oxycodone-induced psychological dependence.
2. Materials and methods
2.1. Drugs
Naloxone hydrochloride (naloxone) was obtained from Tocris
Bioscience (Bristol, UK). Oxycodone hydrochloride (oxycodone) and
morphine hydrochloride (morphine) were obtained from Shionogi &
Co., Ltd. (Osaka, Japan). Hydrocodone bitartrate (hydrocodone) and
fentanyl citrate (fentanyl) were obtained from Mallinckrodt Inc.
(Hobart, NY, USA). All drugs were dissolved in dimethyl sulfoxide
(Nacalai Tesque, Inc., Kyoto, Japan) and 0.9 % physiological saline
(Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) for in vitro
and in vivo experiments, respectively. For in vivo experiments, drugs
were administered intravenously at a volume of 2 mL/kg. In the an-
tagonist study, 1–50 % of naloxone to oxycodone was used as a dose
ratio (1:0.01, 1:0.1, and 1:0.5 of oxycodone and naloxone). In addition,
the molecular weight of oxycodone hydrochloride (351.82 g/mol) and
naloxone hydrochloride (363.84 g/mol) was used to calculate a molar
ratio of active pharmaceutical ingredients (1:0.0097, 1:0.097, and
1:0.48 of oxycodone and naloxone).
2.2. Animals
This study used 160 male Sprague Dawley rats weighing 180−240 g
(Charles River Laboratories Japan, Inc., Kanagawa, Japan). The rats
were housed in a room maintained at 23 °C ± 1 °C under a 12-h light/
dark cycle with ad libitum access to food and water. Each rat was used
only once. All procedures for the animal experiments were approved by
the Animal Care and Use Committee of Shionogi Research Laboratories
(Osaka, Japan) and were performed according to the guidelines of the
Association for Assessment and Accreditation of Laboratory Animal
Care International (AAALAC) guidelines.
2.3. [35S]-GTPγS binding assay
The antagonism of naloxone against recombinant human μ-opioid
receptor (PerkinElmer Life and Analytical Sciences, Inc., Kanagawa,
Japan) was measured by a functional [35S]-GTPγS assay, as previously
described [3]. In this assay, [35S]-GTPγS bindings of μ-opioid receptor
agonists, oxycodone, hydrocodone, morphine and fentanyl, were de-
termined in absence and presence of naloxone.
2.4. Conditioned place preference test
A conditioned place preference study was conducted using a shuttle
box (1460 mm × 450 mm × 600 mm, w × l × h; KN-80; Natsume
2
Neuroscience Letters 735 (2020) 135177
Seisakusyo Co., Ltd., Tokyo, Japan) that was made of acrylic resin
board and divided into two equal-sized compartments [14]. One com-
partment was white with a textured floor and the other was black with
a smooth floor to create equally preference compartments. The place
conditioning paradigm consisted of three phases (the pre-conditioning
test, drug conditioning and the post-conditioning test). In the pre-
conditioning test, the rats that had not been treated with either drugs or
saline were then placed on the platform. The time spent in each com-
partment during a 900 s session was recorded. Conditioning sessions (4
days for drugs, 4 days for saline) were conducted once daily for 8 days.
Immediately after i.v. administration of oxycodone and naloxone, these
rats were placed in the compartment opposite that in which they had
spent the most time in the preconditioning test for 60 min. On alter-
native days, these animals received saline and were placed in the other
compartment for 60 min. On the day after the final conditioning ses-
sion, a postconditioning test that was identical to the preconditioning
test was performed.
2.5. In vivo microdialysis study and quantification of dopamine
The rat in vivo microdialysis study and quantification of dopamine
in NAC were performed. Before implantation of a cannula, the rats were
anesthetized with inhaled isoflurane and/or subcutaneous administra-
tion of three types of anesthesia mixture (15 mg/kg alphaxanm,
0.15 mg/kg medetomidine, and 2.5 mg/kg butorphanol) for surgery. A
guide cannula (AG-8; Eicom, Kyoto, Japan) was implanted into the NAC
in the anesthetized rats (from the bregma: anterior, +4.0 mm; lateral,
−0.8 mm; ventral, −6.8 mm; angle of 16 degrees) according to the
atlas of Paxinos and Watson and fixed to the skull with cranioplastic
cement. Two to five days after surgery, microdialysis probes (A-I-8-02,
2-mm membrane length; Eicom) were slowly inserted into the NAC
through guide cannulas under anesthesia, and the rats were placed in
experimental cages. The probes were perfused continuously (2 mL/min)
with artificial cerebrospinal fluid: 0.9 mM MgCl2, 147.0 mM NaCl,
4.0 mM KCl, and 1.2 mM CaCl2. Outflow fractions were collected every
20 min. After 3 baseline fractions were collected from the rat NAC, rats
were given drugs or saline. Dialysis samples were collected for 60 min
after treatment and analyzed by high-performance liquid chromato-
graphy (Eicom) with electrochemical detection (Eicom). Dopamine was
separated by column chromatography, and identified and quantified by
the use of standards, as described previously [15].
2.6. Measurement of plasma and brain concentrations
The concentrations of oxycodone and naloxone in the plasma and
brain were measured by liquid chromatography with tandem mass
spectrometry method. The rats were given with i.v. administration of
oxycodone or naloxone. Blood samples and whole brain without cere-
bellum samples were collected from an indwelling arterial cannula at 5,
15, and 60 min after drug administration, and transferred into the ice-
cold polypropylene tubes. The blood samples were centrifuged at 3000
× g for 10 min at 4 °C. The brain samples were homogenized in saline
solution. The obtained plasma samples and brain samples were stored
frozen at −80 °C until the assay. The concentrations in the plasma and
brain samples were determined according to the similar method de-
scribed previously [16].
2.7. Statistical analysis
The data are expressed as mean ± standard error of the mean
(SEM). GraphPad Prism 6.0 software (San Diego, CA, USA) was used to
perform the statistical analyses. The statistical significance of differ-
ences among groups was assessed by a one- or two-way analysis of
variance (ANOVA), followed by Dunnett’s multiple comparison test. A
probability value (p) of < 0.05 was considered to be statistically sig-
nificant. The pharmacokinetic parameters of oxycodone and naloxone
A. Nakamura, et al.
Fig. 1. Naloxone antagonism against oxycodone, hydrocodone, morphine, and fentanyl at human μ-opioid receptors as indicated by the [35S]-GTPγS binding assay.
Concentration-response curves of oxycodone (A), hydrocodone (B), morphine (C), and fentanyl (D) with different concentrations of naloxone in the binding of [35S]-
GTPγS to membranes containing the human μ-opioid receptor. Data are presented as the mean and SEM for 3 independent experiments.
were calculated using noncompartmental analysis module in Phoenix
WinNonlin software (version 8.1; Certara L.P., Princeton, NJ) based on
a non-compartment model analysis with uniform weighting.
3. Results
3.1. The antagonistic activity profile of naloxone against several μ-opioid
receptor agonists
At first, the antagonistic activity profile of naloxone against several
μ-opioid receptor agonists was examined by a functional GTPγS assay.
Naloxone (30−30,000 nM) inhibited the opioid-induced GTPγS
binding and elicited parallel rightward shifts in the concentration-re-
sponse curves of oxycodone (Fig. 1A), hydrocodone (Fig. 1B), morphine
(Fig. 1C), and fentanyl (Fig. 1D).
3.2. Effect of naloxone on oxycodone-induced place preference and
dopamine release in NAC
Next, we used oxycodone as the most commonly consumed μ-opioid
receptor agonist and investigated naloxone antagonism on psycholo-
gical dependence in rats. Oxycodone (0.01−1 mg/kg) produced a dose-
dependent increase in the preference for drug-associated place times;
oxycodone at 0.3 mg/kg and 1 mg/kg increased the place preference
significantly as compared with that of the vehicle (Fig. 2A; p = 0.0001
vehicle vs oxycodone 0.3 mg/kg, p = 0.0028 vehicle vs oxycodone 1
mg/kg). Based on these results, we used 1 mg/kg of oxycodone as an
adequate evaluation dose for further experiments. In the conditioned
place preference study, the 1:0.01 combination (1 mg/kg oxycodone +
3
Neuroscience Letters 735 (2020) 135177
0.01 mg/kg naloxone) did not alter place preferences compared with
that of oxycodone alone, but the 1:0.5 combination (1 mg/kg oxyco-
done + 0.5 mg/kg naloxone) significantly reduced the place preference
(Fig. 2B; p = 0.0178 oxycodone 1 mg/kg vs oxycodone 1 mg/kg +
naloxone 0.5 mg/kg). To understand the underlying mechanism in-
volved in changes in psychological dependence, we also evaluated the
effect of naloxone on dopamine release in NAC. Oxycodone (1 mg/kg)
significantly increased the dopamine release as compared with that of
the vehicle, which was dose-dependently reduced by the co-adminis-
tration of naloxone (Fig. 2C; two-way ANOVA, p < 0.0001, vehicle vs
oxycodone 1 mg/kg; Dunnett's post-hoc test, p < 0.0001, vehicle vs
oxycodone, 1 mg/kg at 6–60 min post-administration). The 1:0.01
combination (1 mg/kg oxycodone + 0.01 mg/kg naloxone) did not
produce any changes as compared with that in oxycodone alone,
whereas the 1:0.1 mixture (1 mg/kg oxycodone + 0.1 mg/kg naloxone)
and 1:0.5 combination (1 mg/kg oxycodone + 0.5 mg/kg naloxone)
reduced the dopamine release significantly (two-way ANOVA,
p = 0.0029, oxycodone 1 mg/kg vs oxycodone 1 mg/kg + naloxone
0.1 mg/kg; Dunnett's post-hoc test, p < 0.0001, vehicle vs oxycodone 1
mg/kg, at 6–48 min post-administration; two-way ANOVA, p < 0.0001,
oxycodone 1 mg/kg vs oxycodone 1 mg/kg + naloxone 0.5 mg/kg;
Dunnett's post-hoc test, p < 0.0001, vehicle vs oxycodone 1 mg/kg at
6–60 min post-administration).
3.3. The plasma and brain concentrations of co-administered oxycodone
and naloxone
When measuring the plasma concentration after co-administration
with oxycodone (1 mg/kg) and naloxone (0.01−0.5 mg/kg), the
A. Nakamura, et al.
highest plasma concentrations of oxycodone and naloxone were ob-
served at 5 min post-administration as compared with those at 15 min
and 60 min, respectively. Afterward, the concentrations gradually de-
creased (Fig. 3A and B). The ratio of the plasma and brain concentra-
tions of oxycodone to naloxone did not change among the cotreatment
groups (Fig. 3C). The rat serum protein binding rates of oxycodone and
naloxone were 18.9 % and 61.0 %, respectively.
4. Discussion
There have been many non-clinical reports regarding the pharma-
cological effects of naloxone against the μ-opioid receptor agonists such
as DAMGO and morphine [17,18]. However, the antagonistic activity
profile of naloxone for clinically used μ-opioid receptor agonists has not
been revealed. In this study, naloxone dose-dependently ameliorated
the increased GTPγS activation by several μ-opioid receptor agonists
including oxycodone, fentanyl, and hydrocodone. A previous study
reported that the Schild slope of naloxone is 1.0 based on Schild re-
gression analysis [3], indicating that naloxone is a competitive an-
tagonist against these μ-opioid receptor agonists.
A previous report indicates that oxycodone (0.5 mg/kg, i.v.)-in-
duced dopamine release is abolished by naloxone (3 mg/kg, i.v.) in the
nucleus accumbens [19], suggesting that oxycodone increases the do-
pamine release via μ-opioid receptor activation. Another research group
4
Neuroscience Letters 735 (2020) 135177
Fig. 2. Effects of naloxone on oxycodone-in-
duced place preference and dopamine release
in the NAC.
Rats were placed and conditioned in either
compartment for 60 min after intravenous ad-
ministration of oxycodone (0.03–1 mg/kg) (A),
or co-administration of oxycodone (1 mg/kg)
and naloxone (0.01 or 0.5 mg/kg) (B). The
preference for drug paired place was calculated
as the mean difference in times spent in the
compartment between the post-conditioning
test and the pre-conditioning test in each group
[14]. Data are presented as the mean and SEM
for 10–21 rats (A, B). **p < 0.01 or ***p <
0.001 vs. the vehicle-treated group, and *p <
0.05 vs. the vehicle-treated group with oxyco-
done (one-way ANOVA, Dunnett's post hoc
test). For in vivo microdialysis experiments, the
rats were intravenously administered vehicle,
oxycodone, or naloxone at time 0 (C). Dopa-
mine release (%) was calculated as the per-
centages of the corresponding baseline levels,
and data are represented as the mean and SEM
for 7 or 8 rats. F(4, 60) = 14.85, ###p < 0.001
vs oxycodone (1 mg/kg) (two-way ANOVA,
Dunnett's post-hoc test).
reported that a dopamine D3 antagonist suppresses oxycodone (3 mg/
kg, i.p.)-induced place preference [20]. The relationship between oxy-
codone-induced dopamine release and place preference might be par-
tially predictable, but there is little evidence regarding the effect of
naloxone in the oxycodone-induced place preference, and comparable
experimental protocols including administration route and dose have
not been used to fully clarify this. The present study shows that oxy-
codone-induced place preference could be dose-dependently inhibited
by naloxone. Administering a combination of oxycodone and naloxone
at a dose ratio of 1:0.5 (molar ratio of 1:0.48), but not 1:0.01 (molar
ratio of 1:0.0097), did not produce any preference for drug-associated
place times. This finding is partly consistent with the results on drug
discrimination paradigms in mice [21]. In the microdialysis study, we
revealed that co-administration of naloxone (0.01−0.5 mg/kg) reduced
the oxycodone-induced dopamine release in NAC dose-dependently,
and a combination dose ratio of 1:0.5, but not 1:0.1 and 1:0.01, of
oxycodone and naloxone did not cause the dopamine release. These
results show that 50 % administration dose ratio of naloxone to oxy-
codone sufficiently reduces the intrinsic oxycodone-induced psycholo-
gical dependence, and a ratio of lower than 10 % may not be effective.
A previously reported clinical study revealed a similar antagonism by
50 % dose ratio of naloxone to oxycodone in opioid-experienced drug
users [10]. In another study with partial μ-opioid receptor agonist bu-
prenorphine, 25 % dose ratio of naloxone also reduced the abuse
A. Nakamura, et al.
liability of buprenorphine [22], suggesting that an appropriate ratio of
naloxone could antagonize μ-opioid receptor agonist-induced psycho-
logical dependence. This is the first report showing a mechanism-based
antagonistic activity profile of naloxone in psychological dependence.
Some groups have reported that oxycodone binds selectively to μ-
opioid receptor and oxycodone-induced antinociception is mediated by
the μ-opioid receptor [23–25]. Our previous study also revealed that
the increased GTPγS binding by oxycodone was completely antagonized
by a μ-opioid receptor antagonist β-funaltrexamine [26], indicating that
oxycodone produces pharmacological effects by mainly acting on the μ-
opioid receptor. Another group reported the possible involvement of κ-
opioid receptor in its antinociception [27], but an inadequate dose of
nor-binaltorphimine, a κ-opioid receptor antagonist, might be used for
the pharmacological study. In the present study, oxycodone-induced
place preference and dopamine release were inhibited by naloxone
(0.5 mg/kg) that could antagonize μ-opioid receptor selectively in rats
[28], suggesting that not only the antinociceptive effect but also the
psychological dependence of oxycodone are mediated via the μ-opioid
receptor. In addition, oxycodone exhibits various pharmacological ef-
fects depending on the plasma concentrations [16], but plasma con-
centration of oxycodone (1 mg/kg) that associated with the psycholo-
gical dependence was 28.5 ng/ml in rats, which is almost similar to that
required for the antinociceptive effect (47.6 ng/mL). This also indicates
that naloxone (0.5 mg/kg) might ameliorate the oxycodone (1 mg/kg)-
induced antinociceptive effect.
Pharmacokinetic studies revealed that both oxycodone and na-
loxone were not observed for their pharmacokinetic interaction. When
assuming free concentrations of oxycodone at 5 min (plasma
concentration × Kp brain × unbound fraction ratio), the estimated
concentration (≥ 2 μM) shows about 400 % binding in the GTPγS
functional assay; this suggests that oxycodone at 1 mg/kg could pro-
duce sufficient brain-related pharmacological effects in rats. However,
the estimated free concentrations of naloxone at 0.01 mg/kg and
0.5 mg/kg were calculated to be approximately 10 nM and 300 nM,
respectively. In the GTPγS functional assay, naloxone at 300 nM shifted
5
Neuroscience Letters 735 (2020) 135177
Fig. 3. Plasma and brain concentrations after
co-administration with naloxone and oxyco-
done in rats.
Rats were intravenously administered oxyco-
done (1 mg/kg) and naloxone (0.01, 0.1, or
0.5 mg/kg). Blood samples were collected at 5,
15, and 60 min, and brain samples were col-
lected at 60 min after drug administration. The
time-course of the plasma concentration of
oxycodone (A) and naloxone (B), and the brain
concentrations (C) were measured according to
a previously described method (9). The dis-
tribution ratio between the brain and plasma is
expressed as a Kp brain value. Data are re-
presented as the mean ± SEM for three rats.
the concentration-response curve of oxycodone approximately 100-fold
to the right compared with that of oxycodone alone; however, 10 nM
naloxone did not greatly affect the curve. These findings are in line with
the observed antagonistic response of naloxone following administra-
tion of a 1:0.5, but not a 1:0.01 combination of oxycodone and na-
loxone.
In conclusion, we have found that naloxone exhibits a competitive
antagonistic activity profile for widely prescribed μ-opioid receptor
agonists and inhibits the opioid-induced pharmacological effects in the
brain. Although the respective pharmacological effects observed in this
study do not directly represent the clinical pharmacological effects, our
results provide substantial evidence for the clinical use of naloxone to
manage adverse effects of opioids.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Atsushi Nakamura: Conceptualization, Formal analysis,
Validation, Writing - original draft. Kana Yasufuku:
Conceptualization. Shinji Shimada: Investigation, Methodology.
Hiroyuki Aritomi: Investigation, Methodology. Youko Furue:
Investigation, Methodology, Validation. Hiroki Chiba: Investigation,
Methodology, Validation. Mami Muramoto: Investigation,
Methodology. Kenji Takase: Investigation, Methodology, Validation.
Katsumi Koike: Investigation, Methodology. Tomoko Matsumoto:
Investigation, Methodology, Validation. Tomoka Shimada:
Investigation, Methodology. Ryosuke Watari: Investigation,
Methodology. Takanobu Matsuzaki: Investigation, Methodology,
Validation. Toshiyuki Asaki: Project administration. Toshiyuki
Kanemasa: Project administration. Masahide Fujita: Project admin-
istration, Writing - review & editing.
A. Nakamura, et al.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgments
All authors are employees of Shionogi & Co., Ltd, and Shionogi
TechnoAdvance Research Co., Ltd, the manufacturer of oxycodone and
morphine, and have complete access to the data that supports this
publication. We thank Trent Rogers, PhD, from Edanz Group (https://
en-author-services.edanzgroup.com/) for editing a draft of this manu-
script.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.neulet.2020.135177.
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