Enhancing endogenous adenosine A2A receptor signaling induces slow-wave sleep without affecting body temperature and cardiovascular function

Article abstract of DOI:10.1016/j.neuropharm.2018.10.022

Insomnia is one of the most common sleep problems with an estimated prevalence of 10%–15% in the general population. Although adenosine A_2A receptor (A_2AR) agonists strongly induce sleep, their cardiovascular effects preclude their use in treating sleep disorders. Enhancing endogenous A_2AR signaling, however, may be an alternative strategy for treating insomnia, because adenosine levels in the brain accumulate during wakefulness. In the present study, we found that 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, denoted A_2AR positive allosteric modulator (PAM)-1, enhanced adenosine signaling at the A_2AR and induced slow wave sleep (SWS) without affecting body temperature in wild-type male mice after intraperitoneal administration, whereas the SWS-inducing effect of this benzoic acid derivative was abolished in A_2AR KO mice. In contrast to the A_2AR agonist CGS 21680, the A_2AR PAM-1 did not affect blood pressure or heart rate. These findings indicate that enhancing A_2AR signaling promotes SWS without cardiovascular effects. Therefore, small molecules that allosterically modulate A_2ARs could help people with insomnia to fall asleep.

Full text of DOI:10.1016/j.neuropharm.2018.10.022

Accepted Manuscript
Enhancing endogenous adenosine A receptor signaling induces slow-wave sleep
2A
without affecting body temperature and cardiovascular function
Mustafa Korkutata, Tsuyoshi Saitoh, Yoan Cherasse, Shuji Ioka, Feng Duo, Rujie
Qin, Nobuyuki Murakoshi, Shinya Fujii, Xuzhao Zhou, Fumihiro Sugiyama, Jiang-Fan
Chen, Hidetoshi Kumagai, Hiroshi Nagase, Michael Lazarus
PII:
S0028-3908(18)30807-4
10.1016/j.neuropharm.2018.10.022
NP 7393
DOI:
Reference:
To appear in: Neuropharmacology
Received Date: 7 May 2018
Revised Date: 10 October 2018
Accepted Date: 14 October 2018
Please cite this article as: Korkutata, M., Saitoh, T., Cherasse, Y., Ioka, S., Duo, F., Qin, R., Murakoshi,
N., Fujii, S., Zhou, X., Sugiyama, F., Chen, J.-F., Kumagai, H., Nagase, H., Lazarus, M., Enhancing
endogenous adenosine A receptor signaling induces slow-wave sleep without affecting body
2A
temperature and cardiovascular function, Neuropharmacology (2018), doi: https://doi.org/10.1016/
j.neuropharm.2018.10.022.
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Enhancing endogenous adenosine A2A receptor signaling induces slow-wave sleep
without affecting body temperature and cardiovascular function
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Mustafa Korkutataa, b, Tsuyoshi Saitoha, Yoan Cherassea, Shuji Iokaa, Feng Duoc,
Rujie Qinc, Nobuyuki Murakoshic, Shinya Fujiia, Xuzhao Zhoua, Fumihiro Sugiyamad,
Jiang-Fan Chene, Hidetoshi Kumagaif, Hiroshi Nagasea, and Michael Lazarusa, *
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aInternational Institute for Integrative Sleep Medicine (WPI-IIIS), University of
Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
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bPh.D. Program in Human Biology, School of Integrative and Global Majors,
University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan
cCardiovascular Division, Faculty of Medicine, Graduate School of Comprehensive
Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
dLaboratory of Animal Resource Center, Faculty of Medicine, University of Tsukuba,
Tsukuba, Ibaraki 305-8575, Japan
eDepartment of Neurology, Boston University School of Medicine, Boston, MA
02118, USA
fDepartment of Cardiovascular Medicine, Graduate School of Medicine, The
University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan

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*Corresponding Author: Dr. Michael Lazarus, Principal Investigator and Associate
Professor, International Institute for Integrative Sleep Medicine, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan, Phone/fax: 81-29-853-
3681, Email: lazarus.michael.ka@u.tsukuba.ac.jp

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Abstract
Insomnia is one of the most common sleep problems with an estimated prevalence of
10% to 15% in the general population. Although adenosine A2A receptor (A2AR)
agonists strongly induce sleep, their cardiovascular effects preclude their use in
treating sleep disorders. Enhancing endogenous A2AR signaling, however, may be an
alternative strategy for treating insomnia, because adenosine levels in the brain
accumulate during wakefulness. In the present study, we found that 3,4-difluoro-2-
((2-fluoro-4-iodophenyl)amino)benzoic acid, denoted A2AR positive allosteric
modulator (PAM)-1, enhanced adenosine signaling at the A2AR and induced slow
wave sleep (SWS) without affecting body temperature in wild-type male mice after
intraperitoneal administration, whereas the SWS-inducing effect of this benzoic acid
derivative was abolished in A2AR KO mice. In contrast to the A2AR agonist CGS
21680, the A2AR PAM-1 did not affect blood pressure or heart rate. These findings
indicate that enhancing A2AR signaling promotes SWS without cardiovascular effects.
Therefore, small molecules that allosterically modulate A2ARs could help people with
insomnia to fall asleep.
Keywords
Adenosine A2A receptor, allosteric modulator, insomnia, slow-wave-sleep, body
temperature, cardiovascular function
Abbreviations
CHO, Chinese hamster ovary; EEG, electroencephalography; EMG,
electromyography; ECG, electrocardiography; PAM, positive allosteric modulator;
REM, rapid eye movement; SWS, slow-wave sleep.

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1. Introduction
Insomnia is one of the most common sleep problems with an estimated prevalence of
10% to 15% in the general population and 30% to 60% in the older population (Roth,
2007). Moreover, insomnia frequently co-occurs with a wide range of psychiatric
disorders, including depression and anorexia (de Zambotti et al., 2017; Seow et al.,
2018). The most widely prescribed agents for the treatment of insomnia are
benzodiazepines and non-benzodiazepines, which are central nervous system
depressants that enhance signaling of the inhibitory neurotransmitter γ-aminobutyric
acid (Wafford and Ebert, 2008). These medications, however, are plagued by a wide
range of adverse effects, including muscle relaxation, rebound insomnia, changes in
appetite, next-day sedation, cognitive impairment, amnesic effects, and development
of drug tolerance and dependence (Aragona, 2000; Vgontzas et al., 1995). Orexin
receptor antagonists were also recently developed and approved for treating insomnia
(Cox et al., 2010). The major issues of these drugs are next-morning sleepiness with
possible muscle weakness, strange dreams, sleep-walking, and other nighttime
behaviors or suicidal ideation (Jacobson et al., 2014). Moreover, because orexin
receptor antagonists mostly work by preventing arousal from sleep, they are generally
ineffectual in people who have problems falling asleep. A highly selective adenosine
A2A receptor (A2AR) agonist, CGS 21680, produces profound increases in sleep after
infusion into the subarachnoid space underlying the ventral surface region of the
rostral basal forebrain in rats, the lateral ventricle of mice, or the lateral preoptic area
of rats (Satoh et al., 1999; Scammell et al., 2001; Urade et al., 2003; Methippara et al.,
2005). Administration of an A2AR agonist is not considered to have clinical potential
for the treatment of sleep disorders, however, due to its adverse cardiovascular effects,
which include hypotension and tachycardia (de Lera Ruiz et al., 2014). A positive

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allosteric modulator (PAM) may evoke selective physiologic A2AR responses because,
in contrast to an A2AR agonist, its actions are limited to when and where adenosine is
released. Adenosine levels in the brain progressively increase during wakefulness
(Porkka-Heiskanen et al., 1997), and therefore allosteric modulation of A2ARs to
promote the somnogenic effects of the increased adenosine may be an alternative
strategy for treating insomnia.
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In the present study, we identified a small lipophilic monocarboxylate (3,4-difluoro-2-
((2-fluoro-4-iodophenyl)amino)benzoic acid), denoted A2AR PAM-1, that induces
slow-wave sleep (SWS), the major part of sleep characterized by slow and high-
voltage brain waves, by enhancing A2AR signaling without affecting body
temperature, blood pressure, or heart function in mice.
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2. Material and methods
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2.1. Reagents
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Adenosine (Nacalai Tesque, Kyoto, Japan), CGS 21680 (Sigma-Aldrich, St. Louis,
MO), Cremophor® EL (Sigma-Aldrich), DMSO (Nacalai Tesque), DMEM (Nacalai
Tesque), FBS (Nichirei Biosciences, Tokyo, Japan), HBSS (Gibco, Waltham, MA),
hygromycin B (Wako, Tokyo, Japan), ketamine hydrochloride (Ketalar, Daiichi
Sankyo, Tokyo, Japan), nonessential amino acids (NEAA; Nacalai Tesque),
penicillin/streptomycin (Wako), pentobarbital (Somnopentyl, Kyoritsu Seiyaku,
Tokyo, Japan), puromycin (InvivoGen, San Diego, CA), saline (Otsuka, Tokyo,
Japan), Ultrance cAMP-kit (PerkinElmer, Waltham, MA), xylazine hydrochloride
(Celactal, Bayer, Tokyo, Japan), ZM241385 (Tocris Bioscience, Bristol, UK), 3-
isobutyl-1-methylxanthine (IBMX; Tocris Bioscience) and HEPES (Gibco).
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2.2. Animals
Male mouse lines on a C57BL/6 background, including wild-type and A2AR KO
(Chen et al., 1999) mice, which were maintained at the International Institute of
Integrative Sleep Medicine and weighing 21-27 g (10-15 weeks old), were used in the
experiments. The animals were housed in an insulated and soundproof recording
chamber that was maintained at an ambient temperature of 23 ± 0.5°C with a relative
humidity of 50 ± 5% and an automatically controlled 12 h light/12 h dark cycle (light
on at 8:00, illumination intensity ≈ 100 lux). All animals had free access to food and
water. This study was performed in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the US National Institutes of
Health (2011). Experimental protocols were in compliance with relevant Japanese and
institutional laws and guidelines and approved by the University of Tsukuba animal
ethics committee (protocol #14-322). Every effort was made to minimize the number
of animals used as well as any pain and discomfort experienced by the animals.
2.3. Mouse A2AR-expressing Chinese hamster ovary cells
The flag epitope-tagged open reading frame of A2AR was amplified by PCR from
mouse brain total RNA. The resultant amplicon was cloned into a pMXs-IRES-Puro
retroviral vector (Kitamura et al., 2003). The plasmid was then transfected into the
retrovirus packaging cell line Plat-E (Morita et al., 2000). The supernatant of
transfected Plat-E cells was recovered after 24 h and applied to Chinese hamster
ovary (CHO) cells strongly expressing the ecotropic receptor for the retrovirus
(Montminy et al., 1990). Mouse A2AR-expressing CHO (mA2AR-CHO) cells were
selected in DMEM supplemented with 5% FBS and 1% NEAA by treatment with
hygromycin B (250 µg·ml-1) and puromycin (10 µg·ml-1). The mA2AR-CHO cells

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were subsequently maintained in DMEM supplemented with 5% FBS, 1% NEAA,
1% penicillin/streptomycin, and 250 µg·ml-1 hygromycin B at 37°C in an atmosphere
of 5% CO2.
2.4. cAMP assay
Activation of A2ARs was quantified by cyclic adenosine monophosphate (cAMP)
accumulation in CHO cells expressing mouse A2ARs. CHO cells were suspended in
HBSS containing 1 M HEPES and 0.25 M IBMX in 384-well micro-plates (2×103
cells/well), and incubated with adenosine and A2AR PAM-1 at the indicated
concentrations for 30 min at 25°C. The detection mixture containing the Eu-cAMP
tracer and ULight-anti-cAMP antibody was added and incubated for 1 h at 25°C. A
micro-plate reader (ARVO X5, Perkin Elmer; excitation: 340 nm; emission: 665 nm)
was used to measure the Förster resonance energy transfer (FRET) signal. All
experiments were performed according to the manufacturer’s instructions (LANCE
Ultra cAMP Kit, PerkinElmer). The cAMP levels are based on the dynamic range
(“linear portion”) of the cAMP standard curve and normalized to the baseline or
adenosine treated group.
2.5. Stereotaxic surgery for the placement of EEG/EMG electrodes
Mice were anesthetized with pentobarbital [50 mg·kg-1, intraperitoneal (i.p.)] and then
placed in a stereotaxic apparatus. Electroencephalogram (EEG) and electromyogram
(EMG) electrodes for polysomnographic recordings were chronically implanted in the
mice (Oishi et al., 2016). The implant comprised two stainless steel screws (1 mm in
diameter) inserted through the skull above the cortex (anteroposterior, +1.0 mm; left-
right, -1.5 mm from bregma or lambda) according to the atlas of Paxinos and Franklin

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(Paxinos and Franklin, 2004) that served as the EEG electrodes. Two insulated,
stainless steel Teflon-coated wires were placed bilaterally into both trapezius muscles
and served as the EMG electrodes. All electrodes were attached to a micro connector
and fixed to the skull with dental cement.
2.6. Pharmacologic treatment and infusion cannula implantation
For control data, mice were injected with saline or vehicle (10 ml·kg-1 body weight,
i.p.) at 22:00 or 21:30, respectively. A2AR PAM-1 was dissolved in saline
immediately before use and administered intraperitoneally at 22:00 on the
experimental day at a dose of 30, 60, or 75 mg·kg-1. ZM241385 (15 mg·kg-1, i.p.) was
dissolved in vehicle (5% DMSO, 5% Cremophor® EL in saline) and injected into
C57BL/6J mice at 21:30. Mice were randomly assigned to groups that received
control or drug injections.
For intracerebroventricular (i.c.v.) infusion of A2AR PAM-1, a stainless-steel cannula
was inserted into mice during surgery 0.5 mm anterior and 1.6 mm lateral to bregma
to a depth of 1.6 mm below the dura at an angle of 20°, thus placing the cannula into
the lateral ventricle. To ensure correct placement of cannula, a plastic tube filled with
saline was attached to the infusion cannula; a drop in the meniscus indicated that the
cannula tip was in the ventricle. During the experiments, the mice were infused
continuously using an infusion pump with artificial cerebrospinal fluid into the lateral
ventricle of the brain at a speed of 1 µl·h-1. Sleep-wakefulness states were monitored
for a period of 36 h after infusion of each compound. Saline infusion recordings were
obtained in each animal for 36 h, beginning at 20:00, which served as the control for
the same animal. In the next experiment, A2AR PAM-1 (200 nmol·h-1) was infused
into the lateral ventricle of the mouse brain for 12 h (20:00 to 8:00).

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2.7. Vigilance state assessment based on EEG/EMG polygraphic recordings
Ten days after surgery, the mice were individually housed in transparent barrels in an
insulated soundproof recording chamber and connected to the EEG-EMG recording
cables for 3 to 5 days of habituation before starting the polygraphic recordings. To
evaluate the spontaneous sleep-wake cycle, each animal was recorded for 24 h
beginning at 20:00, the onset of the dark period. The animals then entered the
pharmacologic phase of the study in which sleep-wakefulness parameters were
recorded for 36 h. The data collected during the first 24 h also served as baseline
comparison data for the second experimental day. Cortical EEG/EMG recordings
were amplified, filtered (EEG 0.5-30 Hz; EMG 20-200 Hz), and digitized at a
sampling rate of 128 Hz, and then recording using data acquisition software
SleepSign® (Kissei Comtec, Matsumoto, Japan). The vigilance states were classified
offline in 10-s epochs into three stages, i.e., wakefulness, rapid eye movement (REM)
sleep, and SWS by SleepSign® (ver 3.4) according to standard criteria (Oishi et al.,
2016). As a final step, defined vigilance stages were examined visually, and corrected
when necessary.
2.8. Blood pressure and heart rate measurement
The blood pressure of the mice was measured using the tail-cuff method with a BP-
98A blood pressure device (Softron, Tokyo, Japan). The same time period (13:00 –
16:00) was selected for testing the blood pressure of each mouse (9-12 weeks old) to
avoid normal daily variations in blood pressure. Five consecutive days were used to
habituate the mice to the device. To optimize cardiovascular circulation, mice were
wrapped in a cotton sheet and, except for the tail, maintained at 37°C within a

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cylinder heater. A programmable sensor with an inflatable balloon attached to a tail
cuff was used to monitor tail pulse waves and measure blood pressure when the pulse
waves were stable and rhythmic. Blood pressure measurement was read and recorded
by the software. After five consecutive training days, mice were randomly assigned to
one of three groups and injected with saline (10 ml·kg-1, i.p.), A2AR PAM-1 (75
mg·kg-1, i.p.) or CGS 21680 (1 mg·kg-1, i.p.). Blood pressure was measured at 30 min,
1 h 30 min, and 2 h 30 min after injection (at each time-point, 20 readings for each
mouse were collected). After testing, the mice were gently picked up by the tail and
gently returned to their cages.
The heart rate of the mice was measured by telemetry. Mice were anesthetized with
ketamine hydrochloride (80 mg·kg-1, i.p.) and xylazine hydrochloride (8 mg·kg-1, i.p.)
and a PhysioTel F20-ETA mouse telemetry transmitter (Data Science International, St.
Paul, MN) was placed in the midline of the mouse back and fixed with surgical
sutures. The negative (white) electrode was placed in the trapezius muscle, while the
positive (red) electrode was sutured to a muscle in the back opposite the xiphoid
process. Each mouse was singly housed in a cage after surgery with a distance of at
least 1 m between cages to avoid interference between telemetry transmitters. After 7
days of recovery, the mice were randomly assigned to one of three groups and
injected with saline (10 ml·kg-1, i.p.), A2AR PAM-1 (75 mg·kg-1, i.p.), or CGS 21680
(1 mg·kg-1, i.p.). The transmitted cardiovascular signal was analyzed for 2 h after the
injections using Data Science International software.
2.9. Heart rhythm measurement
The cardiac rhythm of mice was measured by electrocardiography (ECG). Mice were
anesthetized with ketamine hydrochloride (80 mg·kg-1, i.p.) and xylazine

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hydrochloride (8 mg·kg-1, i.p.) and fixed with needles on a styrofoam platform. Mice
were then gently pushed into a position where the two front paws and the left rear
paw are in contact with 25-gauge needles that served as ECG electrodes. For
intracardiac electrography, the throat of the mice was opened and the internal jugular
vein was isolated to insert a catheter along the course of the vein to the right atrium.
Electrographic signals were 5.000-10.000-fold amplified and filtered (0.5-250 Hz)
with an AC-601G system (Nihon Kohden, Tokyo, Japan). The same time period
(10:00 – 12:00) was selected for testing the heart rhythm to avoid normal daily
variations in the cardiac rhythm. Mice were randomly assigned to groups that
received A2AR PAM-1 (75 mg·kg-1, i.p.) or CGS 21680 (1 mg·kg-1, i.p.) injections.
After recording the baseline for 1-2 minutes, mice were injected with drugs and
recording continued for 30 minutes. The data were analyzed using LabChart Pro
software (ADInstruments, Dunedin, New Zealand).
2.10. Body temperature measurement
The core body temperature of the mice was measured using Thermochron iButtons
(KN Laboratories, Osaka, Japan). iButtons were programmed to monitor core body
temperature every 5 min for 14 consecutive days beginning at the end of the recovery
period. The mice were anesthetized with pentobarbital (50 mg·kg-1, i.p.). The skin of
the abdomen was shaved and cleaned with 70% ethanol and a longitudinal, 2-cm
incision was made along the midline. One iButton cleaned with 70% ethanol was
placed in the peritoneal cavity and the incision was closed with nylon sutures. The
mice were housed individually in cages after surgery and experiments were conducted
after a 10-day recovery period. iButtons were removed from the animals after cervical

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dislocation under anesthesia and RhManager software (KN Laboratories, Osaka,
Japan) was used to collect the recorded data from the iButtons.
2.11. Synthesis of A2AR PAM-1
A solution of 2,3,4-fluorobenzoic acid (1.35 g, 7.68 mmol), 2-fluoro-4-iodoaniline
(1.91 g, 8.06 mmol), and lithium amide (0.702 g, 30.6 mmol) in tetrahydrofuran (10.5
mL) was reacted using a standard method (Cai et al., 2008) to give 3,4-difluoro-2-((2-
fluoro-4-iodophenyl)amino)benzoic acid (A2AR PAM-1, 2.99 g, 99%) as a brown
solid (Figure S1); IR (KBr) 3311, 1673, 1602, 1520, 1500, 1444, 1273, 768 cm-1; 1H
NMR (400 MHz CD3OD) δ = 7.89 (1 H, ddd, J = 2.3, 6.0, 9.2 Hz), 7.48 (1 H, dd, J =
1.8, 10.5 Hz), 7.41 (1 H, ddd, J =1.4, 1.8, 8.5 Hz), 6.91 (1 H, ddd, J = 7.3, 9.4, 9.4
Hz), 6.75 (1 H, ddd, J = 5.6, 8.5, 8.5 Hz); 13C NMR (100 MHz acetone-d6) δ = 169.9,
155.7 (dd, JC,F = 252.1, 4.8 Hz), 155.6 (d, JC,F = 252.1 Hz), 143.6 (dd, JC,F = 247.8,
14.9 Hz), 137.4 (dd, JC,F = 7.7, 2.9 Hz), 135.0 (d, JC,F = 3.8 Hz), 131.9(d, JC,F = 11.5
Hz), 129.8 (dd, JC,F = 9.6, 3.8 Hz), 125.8 (d, JC,F = 21.0 Hz), 123.8 (d, JC,F = 5.8 Hz),
116.4, 110.1 (d, JC,F = 18.2 Hz), 84.7 (d, JC,F = 6.7 Hz); HRMS-ESI: m/z [M-H]- calcd
for C13H6F3INO2, 391.9395; measured, 391.9414.
2.12. Formation of the sodium salt of A2AR PAM-1
Aqueous sodium hydroxide (100 µM, 754 µL) was added to a stirred solution of A2AR
PAM-1 (0.266 g, 75.4 mmol) in ethanol (20.0 mL) at 0°C. The mixture was stirred for
45 min at room temperature and then concentrated in vacuo and freeze-dried. The
residue was dissolved in water and filtered. The filtrate was freeze-dried to obtain the
sodium salt of A2AR PAM-1 (0.265 g, 89%) as a gray solid (m.p. 290−291°C; Anal.

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Calcd for C13H6NO2·Na·1.5H2O: C, 35.32; H, 2.05; N, 3.17. Measured: C, 35.34; H,
1.91; N, 3.14). The sodium salt of A2AR PAM-1 was used for all in-vivo experiments.
2.13. Statistical analysis
Statistical analyses were carried out using Systat Software (SigmaPlot). All results are
presented as mean ± standard error of the mean (SEM). Two-tailed Student’s t-tests
were used for statistical comparisons between two groups (Fig. 1A, B, D, E, Fig. 2C,
D, F, Fig. 3B, D, Fig. 4B, Fig. 5A, B, Fig. S2A, B, Fig. S4B, Fig. S5A-C, and Fig.
S6A-C). For t-tests, the normality of each dataset was established using the
Kolmogorov–Smirnov test. Two-way repeated-measures analysis of variance
(ANOVA) followed by the Tukey test were used for dose-response effects on the
amounts of the SWS, REM sleep, and wakefulness (Fig. 2B, Fig. 3A, C, Fig. 4A, Fig.
S3A and Fig. S5A) (Chrivia et al., 1993). In all of the cases, P <0.05 was considered
significant (significance levels are indicated in figures as *: P<0.05, **: P <0.01 or
***: P <0.001).
3. Results
3.1. Screening of small-molecule compounds for allosteric A2AR modulation
We established CHO cells that express mouse A2ARs (Figure S2) using a retrovirus-
mediated gene transfer method (Kitamura et al., 2003). We used these mA2AR-CHO
cells to screen 1173 small-molecule compounds for their allosteric effects at A2ARs.
The compounds were synthesized in Dr. Hiroshi Nagase’s laboratory at the University
of Tsukuba. A2AR activity in CHO cells was determined by measuring cAMP
produced after adding adenosine and small-molecule compounds using a fluorescence
resonance energy transfer immunoassay. Because a one-compound-one-well approach

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may be wasteful research conduct due to a likely small number of active compounds
in our library, we tested initially 391 mixtures containing three compounds each in
triplicates. We selected mixtures that significantly enhanced the effects of adenosine
at the A2ARs (P<0.01, unpaired t-test) for individual compound testing and found that
eight of the mixtures showed an effect according to this criterion (Mixture 124:
t(4)=27.9, P<0.0001, Mixture 181: t(4)=31.5, P<0.0001, Mixture 194: t(4)=30.9,
P<0.0001, Mixture 211: t(4)=9.6, P=0.0006, Mixture 274: t(4)=11, P=0.0003, Mixture
319: t(4)=6.81, P=0.0024, Mixture 332: t(4)=8.62, P=0.0009, Mixture 346: t(4)=4.71
P=0.0091, unpaired t-test; Figure 1A). Further individual testing of compounds in the
eight mixtures revealed that only compound 371 (3,4-difluoro-2-((2-fluoro-4-
iodophenyl)amino)benzoic acid) in mixture 124 enhanced adenosine-induced A2AR
activation (t(4)=9.14, P=0.0007, unpaired t-test; Figure 1B). A cell culture bioassay
revealed that cAMP levels were not altered by treating A2AR-expressing or native
CHO cells with compound 371 in the absence of adenosine or by treating native CHO
with adenosine and compound 371 (Figure 1C), suggesting that compound 371 is
likely a positive allosteric modulator for A2ARs, and we therefore named this
compound A2AR PAM-1. Co-treatment of A2AR-expressing CHO cells with 150 nM
adenosine and various concentrations of A2AR PAM-1 (i.e., 25, 50, and 100 µM)
amplified adenosine A2AR-evoked cAMP accumulation in a dose-dependent manner
by 42% ± 1.4%, 46% ± 1.1%, and 50% ± 1.0%, respectively (25 µM A2AR PAM-1:
t(4)=4.47, P=0.011, 50 µM A2AR PAM-1: t(4)=7.21, P=0.0019, 50 µM A2AR PAM-1
vs. 25 µM A2AR PAM-1: t(4)=4.71, P=0.0092, 100 µM A2AR PAM-1: t(4)=9 P=0.0008,
100 µM A2AR PAM-1 vs. 25 µM A2AR PAM-1: t(4)=8.08, P=0.0012, 100 µM A2AR
PAM-1 vs. 50 µM A2AR PAM-1: t(4)=3.65, P=0.021, unpaired t-test; Figure 1D).
Similarly, co-treatment of A2AR-expressing CHO cells with 100 µM A2AR PAM-1

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and 50, 100, or 150 nM adenosine increased A2AR activity in the CHO cells in a dose-
dependent manner by 55% ± 0.4%, 66% ± 1.5%, and 72% ± 1.7%, whereas 100 µM
A2AR PAM-1 did not significantly enhance the cellular activity of A2AR-expressing
CHO cells treated with 250 nM adenosine (50 nM Adenosine: t(4)=14.9, P=0.0001, 50
nM Adenosine vs. 100 nM Adenosine: t(4)=7.04, P=0.0021, 50 nM Adenosine vs. 150
nM Adenosine: t(3)=12.40, P=0.0011, 50 nM Adenosine vs. 250 nM Adenosine:
t(4)=11.79, P=0.00029, 100 nM Adenosine: t(4)=6.18, P=0.034, 150 nM Adenosine:
t(3)=4.98, P=0.015, unpaired t-test; Figure 1E).
3.2. Intraperitoneal administration of A2AR PAM-1 induces SWS without affecting
body temperature in mice
We then tested the effect of intraperitoneal administration of A2AR PAM-1 on the
sleep/wake behavior of wild-type mice. We analyzed EEG and EMG recordings made
after saline or A2AR PAM-1 injections during the dark period at 22:00, when mice
usually spend most of their time awake. Although baseline sleep and wake of mice 24
h prior to treatment was not significantly different between the saline and A2AR PAM-
1 groups during the dark period (Figure S3), A2AR PAM-1 dose-dependently
increased SWS after the injections for the following 8 h (SWS: F(1,106)=13.97, P=0.033,
two way repeated measures ANOVA-Tukey test, 30 mg·kg-1 A2AR PAM-1 vs. 60
mg·kg-1 A2AR PAM-1: t(7)=4.36, P=0.0032, 30 mg·kg-1 A2AR PAM-1 vs. 75 mg·kg-1
A2AR PAM-1: t(6)=5.45, P=0.0015, unpaired t-test; Figure 2A, B, D). The total
amount of SWS was increased by 60.8 ± 11.4 min for 8 h with the highest dose of
A2AR PAM-1 (i.e., 75 mg·kg-1) compared with saline treatment, whereas wakefulness
was decreased by 59.2 ± 12.8 min (SWS: t(7)=4.27, P=0.0036, Wake: t(7)=4.33,
P=0.0034, unpaired t-test; Figure 2C). Intraperitoneal injection of A2AR PAM-1 did

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not significantly alter the REM sleep duration during the dark period compared with
saline injection.
Administration of A2AR PAM-1 (75 mg·kg-1, i.p.) to the mice did not significantly
affect the episode numbers of SWS and REM sleep for 8 h in the dark period (Figure
S4A). On the other hand, wake episode numbers lasting 120 to 239 s increased by
307% (t(7)=3.88, P=0.006, unpaired t-test), and wake episode numbers lasting 480 to
959 s and 960 to 1909 s decreased by 47% (t(7)=2.89, P=0.02, unpaired t-test) and
88% (t(7)=4.60, P=0.002, unpaired t-test), respectively, compared with the saline
injection. The mean duration of wake episodes decreased by 38% (t(7)=3.38, P=0.01,
unpaired t-test) compared with saline, but the duration of the SWS and REM sleep
episodes was not significantly different after A2AR PAM-1 (75 mg·kg-1, i.p.)
administration (Figure S4B). A2AR PAM-1 (75 mg·kg-1, i.p.) also did not
significantly affect the number of transitions between SWS, wake, and REM sleep
(Figure S4C).
To assess whether EEG activity was altered by A2AR PAM-1 administration, we
compared the normalized EEG power spectrum of SWS in mice treated with saline or
A2AR PAM-1 (Figure 2E). EEG activity in the frequency range of 0.5–25 Hz during
SWS was indistinguishable between A2AR PAM-1–induced and natural (saline
injection) SWS. These data suggest that A2AR PAM-1 induced physiologic sleep
rather than abnormal sleep.
We also measured the effect of intraperitoneal administration of 75 mg·kg-1 A2AR
PAM-1 or 1 mg·kg-1 of the A2AR agonist CGS 21680 (as positive control) on the body
temperature of the mice during the dark period (Figure 2F). Although CGS 21680
strongly decreased the body temperature for almost 2 h (t(10)=3.68, P=0.0042 at 22:15,
t(10)=10.48, P<0.0001 at 23:15, t(10)=2.33, P=0.041 at 00:05 vs. saline injected group,

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unpaired t-test), A2AR PAM-1 did not affect the body temperature of the mice. These
data suggest that A2AR PAM-1 induces physiologic sleep independent of the body
temperature.
3.3. Sleep-inducing effect of A2AR PAM-1 was suppressed by blocking A2ARs
We further investigated whether A2ARs mediate the sleep-inducing effect of A2AR
PAM-1. First, we pretreated wild-type mice with the selective A2AR antagonist
ZM241385 (15 mg·kg-1, i.p.) or vehicle 30 min before the A2AR PAM-1 injection at
22:00. The dose of ZM241385 was selected based on previous studies (El Yacoubi et
al., 2000; Nakamura et al., 2016). In the presence of ZM241385, A2AR PAM-1
injection produced no significant changes in SWS (Figure 3A), indicating that
ZM241385 completely blocked the A2AR PAM-1–induced SWS. When we calculated
the total amount of SWS for 4 h after the intraperitoneal injection of A2AR PAM-1
(Figure 3B), we found that it did not significantly alter the total amount of SWS after
ZM241385 pretreatment. ZM241385 pretreatment alone also had no significant effect
on SWS compared with vehicle pretreatment (Vehicle + Saline vs. Vehicle + 75
mg·kg-1 A2AR PAM-1: t(8)=4.04, P=0.0037, 15 mg·kg-1 ZM241385 + 75 mg·kg-1
A2AR PAM-1 vs. Vehicle + 75 mg·kg-1 A2AR PAM-1: t(8)=2.63, P=0.029, 15 mg·kg-1
ZM241385 + Saline vs. Vehicle + 75 mg·kg-1 A2AR PAM-1: t(8)=6.10, P=0.00028,
unpaired t-test; Figure 3B).
We then administered 75 mg·kg-1 A2AR PAM-1 (i.p.) into A2AR KO mice and their
wild-type littermates at 22:00. We observed no significant changes in SWS in the
A2AR KO mice compared with saline treatment, whereas SWS was increased by 74.3
± 12.0 min for 6 h in wild-type littermates of A2AR KO mice (F(1,190)=20.83, P=0.003,
two way repeated measures ANOVA-Tukey test, t(14)=5.63, P<0.0001, unpaired t-

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test; Figure 3C, D). Concomitantly, wakefulness was decreased in the wild-type
littermates of A2AR KO mice (F(1,190)=16.14, P=0.005, two way repeated measures
ANOVA-Tukey test, t(14)=5.50, P<0.0001, unpaired t-test), whereas neither REM
sleep in these mice nor wakefulness and REM sleep in the KO mice were affected by
intraperitoneal administration of 75 mg·kg-1 A2AR PAM-1 (Figure S5). Baseline
sleep and wake of the KO mice and their wild-type littermates 24 h prior to treatment
was not different between the saline and A2AR PAM-1 groups during the dark period
(data not shown). These findings suggest that A2AR are necessary for A2AR PAM-1 to
induce SWS.
3.4. Intracerebroventricular administration of A2AR PAM-1 induces SWS in mice
To elucidate whether the sleep-inducing effect of A2AR PAM-1 is mediated via A2ARs
expressed in the brain, we infused A2AR PAM-1 into the lateral ventricle of wild-type
mice at 200 nmol·h-1 during the dark period (20:00 to 8:00) and assessed EEG and
EMG activity. Infusion with A2AR PAM-1 for 12 h increased the time spent in SWS 5
h after the infusion, resulting in a total SWS increase during the dark period of 141.6
± 12.5 min compared with saline infusion (F(1,118)=34.40, P=0.004, two way repeated
measures ANOVA-Tukey test, t(8)=5.67, P=0.00047, unpaired t-test; Figure 4A, B).
Concomitantly, total wakefulness was decreased by 145.5 ± 15.8 min during a 12-h
i.c.v. infusion of A2AR PAM-1 (F(1,118)=43.46, P=0.003, two way repeated measures
ANOVA-Tukey test, t(8)=5.08, P=0.00095, unpaired t-test), whereas REM sleep was
not affected.
Intracerebroventricular infusion of A2AR PAM-1 (200 nmol·h-1) into mice affected
SWS and wake episode numbers during the dark period (Figure S6A). SWS episode
numbers lasting 0 to 29 s, 30 to 59 s, and 60 to 120 s increased by 267% (t(8)=7.56,

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P<0.0001, unpaired t-test), 196% (t(8)=3.47, P=0.008, unpaired t-test), and 154%
(t(8)=2.88, P=0.02, unpaired t-test), respectively, and wake episode numbers lasting 0
to 29 s, 30 to 59 s, and 60 to 120 s also increased by 205% (t(8)=3.97, P =0.004,
unpaired t-test), 177% (t(8)=3.55, P=0.007, unpaired t-test), and 137% (t(8)=2.77,
P=0.02, unpaired t-test), respectively, compared with saline infusion. On the other
hand, episode numbers of REM sleep were not significantly affected by A2AR PAM-1
infusion (200 nmol·h-1, i.c.v.). The mean duration of wake episodes decreased by 72%
(t(8)=3.06, P=0.01, unpaired t-test) compared with the saline-infused group, but mean
episode duration of the SWS and REM sleep did not significantly change after A2AR
PAM-1 (200 nmol·h-1, i.c.v.) administration (Figure S6B). A2AR PAM-1 (200
nmol·h-1, i.c.v.) increased the number of transitions between SWS and wakefulness by
148% (t(8)=4.91, P =0.001, unpaired t-test), and from wakefulness to SWS by 128%
(t(8)=4.26, P=0.002, unpaired t-test) compared with the saline-infused group (Figure
S6C).
Moreover, the EEG activity in the frequency range of 0.5–25 Hz during SWS
episodes was indistinguishable between mice treated with saline or A2AR PAM-1
(Figure 4C). These data suggest that A2AR PAM-1 induces physiologic sleep rather
than abnormal sleep via A2ARs that are likely expressed in the brain.
3.5. Intraperitoneal administration of A2AR PAM-1 does not affect blood pressure or
heart rate
A2AR agonists evoke cardiovascular effects (Hutchison et al., 1989; Kirkup et al.,
1998; Nekooeian and Tabrizchi, 1996). We therefore tested the effect of
intraperitoneal administration of A2AR PAM-1 on blood pressure and heart rate in
wild-type mice. First, we measured blood pressure in mice 30, 90, and 150 min after

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intraperitoneal injection of 75 mg·kg-1 A2AR PAM-1 or 1 mg·kg-1 of the A2AR agonist
CGS 21680 using an electrosphygmomanometer (Figure 5A). The dose of the A2AR
agonist CGS 21680 was selected based on previous studies in mice (Carvalho et al.,
2017; Nakav et al., 2008; Ohta and Sitkovsky, 2001). Compared with saline treatment,
the systolic, and diastolic blood pressures were significantly decreased for up to 90
min after injecting the A2AR agonist CGS 21680 (SBP at 30 min: t(9)=10.55,
P<0.0001, SBP at 90 min: t(9)=7.51, P<0.0001, DBP at 30 min: t(9)=6.60, P<0.0001,
DBP at 90 min: t(9)=5.86, P<0.0001, unpaired t-test) and returned to normal levels
within 150 min after the injection. In contrast, blood pressure was not changed after
intraperitoneal administration of A2AR PAM-1 (75 mg·kg-1) at 30, 90, or 150 min
after treatment. In addition, we measured the heart rate of mice after intraperitoneal
injection of 75 mg·kg-1 A2AR PAM-1 or 1 mg·kg-1 A2AR agonist CGS 21680 using
the telemetry implants (Figure 5B). The heart rate of the mice increased after
intraperitoneal administration of the A2AR agonist CGS 21680 (HR at 60 min:
t(8)=2.34, P=0.047, HR at 75 min: t(8)=2.90, P=0.019, HR at 90 min: t(8)=2.80,
P=0.023, unpaired t-test), whereas the heart rate was not affected by injection of 75
mg·kg-1 A2AR PAM-1. Finally, we monitored the heart rhythm in anesthetized mice
after intraperitoneal administration of 75 mg·kg-1 of A2AR PAM-1 or 1 mg·kg-1 of
A2AR agonist CGS 21680 using intracardiac EGM. We observed sinus arrhythmia in
mice after intraperitoneal administration of A2AR agonist CGS 21680, whereas
injection of 75 mg·kg-1 A2AR PAM-1 did not cause abnormalities of the cardiac
rhythm (Figure 5C).
4. Discussion

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Our observations suggest that enhancing A2AR signaling by intraperitoneal
administration of A2AR PAM-1 induces SWS without cardiovascular effects in mice.
Therefore, A2AR-modulating compounds may provide safe options for the treatment
of insomnia and poor-quality sleep.
Over the past century, several putative hypnogenic substances implicated in the sleep
homeostatic process have been identified, including prostaglandin D2 (Qu et al., 2006),
cytokines (Krueger et al., 1984), anandamide (García-García et al., 2009), urotensin II
peptide (Huitron-Resendiz et al., 2005), and adenosine (Porkka-Heiskanen et al.,
1997). Adenosine represents a state of relative energy deficiency: ATP depletion
positively correlates with an increase in extracellular adenosine levels (Kalinchuk et
al., 2003) and positively associates with sleep (Porkka-Heiskanen et al., 1997).
Adenosine levels in samples collected from several brain areas of cats during
spontaneous sleep-wake cycles by in vivo microdialysis were higher during SWS than
during wakefulness for all probed brain areas (Porkka-Heiskanen et al., 1997). The
observation in animals that adenosine levels are elevated during prolonged
wakefulness may explain why an allosteric modulator could effectively enhance the
sleep-inducing effect of endogenous adenosine in the brain. On the other hand,
adenosine is absent or its concentration is too low in the cardiovascular system under
physiologic conditions to affect blood pressure and heart function after administration
of an allosteric modulator of A2AR.
Medicinal chemistry for A2ARs has been widely developed in recent decades for use
in myocardial perfusion imaging and the treatment of inflammation and neuropathic
pain (de Lera Ruiz et al., 2014). Several A2AR agonists that entered clinical trials
elicited undesirable side effects, however, thus precluding their further development.
On the other hand, allosteric modulators bind at a distinct site other than the natural

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ligand binding site (i.e., the orthosteric site) and exert their effects only in the
presence of the orthosteric ligand (Wenthur et al., 2014). As a consequence, an
allosteric modulator mimics the activity duration of the natural ligand and thus the
pharmacologic response of an allosteric modulator more closely resembles the natural
physiologic activity of the receptor than is possible with a synthetic agonist. Because
efforts to evoke pharmacologic A2AR responses have focused almost exclusively on
the use of orthosteric ligands, however, the possibility that A2AR responses, especially
in the brain, can be fine-tuned using allosteric modulators has received very little
attention (Göblyös and Ijzerman, 2009).
Moreover, it is widely accepted that the basic adenosine scaffold must be maintained
in an A2AR agonist (Fredholm et al., 2011). Thus, the development of adenosine
analogs for treating the central nervous system, including sleep induction for treating
insomnia, is restricted by the poor transport of these drugs through the brain
endothelial cells, which are connected by tight junctions to establish a blood-brain
barrier (BBB) (Pardridge et al., 1994). In contrast, A2AR PAM-1, when administered
intraperitoneally, exhibits a sleep-inducing effect that is likely mediated by A2ARs in
the brain and thus appears to cross the BBB. Small lipophilic monocarboxylates like
A2AR PAM-1 likely pass through the BBB by passive diffusion or via a
monocarboxylate transport system (Tsuji, 2005). Therefore, allosteric modulation of
A2ARs has the potential to cause pharmacologic effects in the central nervous system
after systemic administration, resulting in good quality sleep.
Our study did not investigate how and where the A2AR PAM-1 binds at the receptor to
exert its allosteric effect. Therefore, an important next step will be to examine the
allosteric interactions of A2AR PAM-1 and the receptor using binding assays and
crystal structure analysis. With respect to the latter, a crystal structure of the human

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A2AR bound to a bitopic antagonist revealed a potential allosteric pocket (Sun et al.,
2017) and another study suggested that a sodium ion binding site can be exploited for
allosteric modulation of A2AR (Gutiérrez-de-Terán et al., 2013). Moreover, to solidify
the sleep enhancing effect of the A2AR PAM-1, it may be necessary to test the A2AR
PAM-1 in mice at the time of normal sleep onset, i.e., A2AR PAM-1 administration at
the onset of the light period, or in an animal model of insomnia, for example, a mouse
model mimicking the human first-night effect (Xu et al., 2014).
Due to work schedules and expectations, lifestyle choices, pre-existing medical
conditions, or aging, people are coping with an increasingly wide range of sleep
problems, including difficulties with falling and staying asleep, waking up too early,
and poor-quality ("non-restorative") sleep. Deficiencies in sleep cause significant
social losses due to increased prevalence of mood disorders, lead to decreased
economic productivity, and are linked to traffic and work-related accidents due to
excessive daytime sleepiness (Groeger et al., 2004; Saddichha, 2010; Sutton et al.,
2001). Insufficient sleep is not only by itself a major problem in modern society, but
is also an established risk factor for obesity, diabetes, heart disease, and other lifestyle
diseases (Colten et al., 2006). Moreover, psychiatric illnesses, especially anxiety and
mood disorders, are long recognized to be a frequent cause of insomnia (Okuji et al.,
2002).
5. Conclusions
The findings of our study indicate that enhancing A2AR signaling promotes SWS
without cardiovascular effects. Therefore, small molecules that allosterically
modulate A2ARs could help people with sleep problems to fall asleep and thus also be
a potential treatment for psychiatric disorders.

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Our study was conducted in mice, the most commonly used model organism of
human disease. Results in mice, however, are not particularly reliable for predicting
human study outcomes, mostly due to the limited genetic diversity associated with
common laboratory mice. Therefore, many obstacles remain to be overcome in
generating a novel drug for the treatment of insomnia in humans.
Conflicts of Interests
The authors declare no competing financial interests.
Author contributions
M.K., N.M., Y.C., F.S., and M.L. designed the experiments. M.K., F.D., R.Q., S.F.,
and X.Z. collected and analyzed the data. T.S., S.I., and H.N. synthesized chemical
compounds, J.C. and H.K. contributed mouse or cell lines, M.K., Y.C., and M.L.
wrote the paper. All authors approved the final version of the manuscript.
Funding
This work was supported by the Japan Society for the Promotion of Science [Grant-
in-Aid for Scientific Research B (grant number 17H02215) to M.L.]; the Japan
Science and Technology Agency [CREST grant (grant number JPMJCR1655) to
M.L.]; the Ministry of Education, Culture, Sports, Science, and Technology (MEXT)
of Japan [Grants-in-Aid for Scientific Research on Innovative Areas “Living in
Space” (grant numbers 15H05935, 15K21745, 18H04966) and “WillDynamics”
(grant number 17H06047) to M.L.]; the World Premier International Research Center

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Initiative (WPI) from MEXT (to Y.C., T.S., H.N., and M.L.); and the Naito
Foundation (to M.L.).
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