Oleoyl alanine (HU595): a stable monomethylated oleoyl glycine interferes with acute naloxone precipitated morphine withdrawal in male rats

Article abstract of DOI:10.1007/s00213-020-05570-4

Rationale: Oleoyl glycine, a little studied fatty acid amide similar in structure to anandamide, interferes with nicotine addiction in mice and acute naloxone-precipitated morphine withdrawal (MWD) in rats. Because endogenous oleoyl glycine is subject to rapid enzymatic deactivation, we evaluated the potential of more stable analogs to interfere with opiate withdrawal. Objectives: The potential of monomethylated oleoyl glycine (oleoyl alanine, HU595) to interfere with somatic and aversive effects of acute naloxone-precipitated MWD, its duration, and mechanism of action was assessed in male Sprague Dawley rats. The potential of dimethylated oleoyl glycine (HU596) to interfere with the aversive effects of naloxone-precipitated MWD was also investigated. Results: Oleoyl alanine (HU595) interfered with somatic and aversive effects produced by naloxone-precipitated MWD at equivalent doses (1 and 5?mg/kg, i.p.) as we have reported for oleoyl glycine; however, oleoyl alanine produced a longer lasting (60?min) interference, yet did not produce rewarding or aversive effects on its own and did not modify locomotor activity. HU596 was not effective. The interference with aversive effects of naloxone-precipitated MWD by oleoyl alanine was prevented by both a PPARα antagonist and a CB₁ receptor antagonist. Accordingly, the compound was found to inhibit FAAH and activate PPARα in vitro. Finally, oleoyl alanine also reduced acute naloxone-precipitated MWD anhedonia, as measured by decreased saccharin preference. Conclusions: Oleoyl alanine (also an endogenous fatty acid) may be a more stable and effective treatment for opiate withdrawal than oleoyl glycine.

Full text of DOI:10.1007/s00213-020-05570-4

Psychopharmacology
https://doi.org/10.1007/s00213-020-05570-4
ORIGINAL INVESTIGATION
Oleoyl alanine (HU595): a stable monomethylated oleoyl glycine
interferes with acute naloxone precipitated morphine withdrawal
in male rats
1
2
1
1
1
1
Samantha M. Ayoub & Reem Smoum & Mathew Farag & Harkirat Atwal & Stephen A. Collins & Erin M. Rock
&
1
3
3
4
1
Cheryl L. Limebeer & Fabiana Piscitelli & Fabio Arturo Iannotti & Aron H. Lichtman & Francesco Leri
&
3
,5
2
1
Vincenzo Di Marzo & Raphael Mechoulam & Linda A. Parker
Received: 8 January 2020 /Accepted: 25 May 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Rationale Oleoyl glycine, a little studied fatty acid amide similar in structure to anandamide, interferes with nicotine addiction in
mice and acute naloxone-precipitated morphine withdrawal (MWD) in rats. Because endogenous oleoyl glycine is subject to
rapid enzymatic deactivation, we evaluated the potential of more stable analogs to interfere with opiate withdrawal.
Objectives The potential of monomethylated oleoyl glycine (oleoyl alanine, HU595) to interfere with somatic and aversive
effects of acute naloxone-precipitated MWD, its duration, and mechanism of action was assessed in male Sprague Dawley rats.
The potential of dimethylated oleoyl glycine (HU596) to interfere with the aversive effects of naloxone-precipitated MWD was
also investigated.
Results Oleoyl alanine (HU595) interfered with somatic and aversive effects produced by naloxone-precipitated MWD at equivalent
doses (1 and 5 mg/kg, i.p.) as we have reported for oleoyl glycine; however, oleoyl alanine produced a longer lasting (60 min)
interference, yet did not produce rewarding or aversive effects on its own and did not modify locomotor activity. HU596 was not
effective. The interference with aversive effects of naloxone-precipitated MWD by oleoyl alanine was prevented by both a PPARα
antagonist and a CB receptor antagonist. Accordingly, the compound was found to inhibit FAAH and activate PPARα in vitro.
1
Finally, oleoyl alanine also reduced acute naloxone-precipitated MWD anhedonia, as measured by decreased saccharin preference.
Conclusions Oleoyl alanine (also an endogenous fatty acid) may be a more stable and effective treatment for opiate withdrawal
than oleoyl glycine.
.
.
.
.
Keywords Oleoyl alanine Naloxone-precipitated morphine withdrawal Conditioned place aversion Saccharin preference
.
.
.
.
.
.
Anhedonia Somatic withdrawal Nausea Oleoyl glycine Rat PPARα CB
1
Introduction
*
Linda A. Parker
parkerl@uoguelph.ca
Oleoyl glycine has recently been identified as an endog-
enous fatty acid amide released in the insular cortex of
mice exposed to traumatic brain injury (Donvito et al.
1
2
3
4
Department of Psychology and Collaborative Neuroscience
Program, University of Guelph, Guelph, ON N1G 2W1, Canada
2
019). The insular cortex is a region implicated in nic-
otine addiction in humans (Naqvi et al. 2007) and rats
Abdolahi et al. 2010). When systemically administered,
Institute of Drug Research, Medical Faculty, Hebrew University of
Jerusalem, Jerusalem, Israel
(
Institute of Biomolecular Chemistry, Endocannabinoid Research
Group, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples, Italy
oleoyl glycine altered withdrawal responses in nicotine-
dependent mice as well as the rewarding effects of nic-
otine, as assessed in the conditioned place preference
Department of Pharmacology and Toxicology, Medical College of
Virginia Campus, Virginia Commonwealth University,
Richmond, VA, USA
(
CPP) paradigm (Donvito et al. 2019). The insular cor-
tex is also essential for the sensation of nausea in
humans (Penfield and Faulk 1955; Napadow et al.
2013; Sclocco et al. 2016) and rats (Contreras et al.
5
Canada Excellence Research Chair on the Microbiome/
Endocannabinoid Axis in Metabolomic Health, Université Laval,
Quebec City, Canada

Psychopharmacology
2
007; Limebeer et al. 2018; Tuerke et al. 2012), as well
i.p.) in preventing naloxone-induced MWD CPA; it also
assessed the potential of a lower dose of oleoyl alanine
(1 mg/kg i.p.) and a longer duration of action (120 min) to
suppress CPA. Experiment 3 ensured that oleoyl alanine
(HU595) did not produce CPA or CPP on its own.
Experiment 4 evaluated the potential of dimethylated oleoyl
glycine (HU596) to interfere with naloxone-precipitated
MWD CPA. Having determined that HU595 (oleoyl ala-
nine), but not HU596, interfered with MWD CPA, experi-
as morphine withdrawal (MWD) in rats (Li et al. 2009;
Li et al. 2013; Wills et al. 2016). Petrie et al. (2019)
recently reported that systemic oleoyl glycine prevented
acute naloxone-precipitated MWD-induced conditioned
place aversion (CPA). Rock et al. (2020) extended this
finding by demonstrating that oleoyl glycine also inter-
fered with somatic effects of naloxone-precipitated
MWD, including the reaction of conditioned gaping
which is a measure of nausea in rats (Grill & Norgren
ment 5 evaluated the mechanism of action (CB receptor and
1
1
978; Parker 2014; McDonald et al. 1997).
Oleoyl glycine is a fatty acid amide closely related to
PPARα) of the effect. Experiment 6 evaluated the potential
of oleoyl alanine to interfere with decreased saccharin pref-
erence following exposure to acute naloxone-precipitated
MWD, a commonly used measure of anhedonia in rodents
(Scheggi et al. 2018). Finally, experiment 7 employed the
in vitro techniques of competitive activity-based protein
anandamide. Anandamide is hydrolyzed rapidly by ami-
dases in the body. In order to stabilize anandamide, Abadji
et al. (1994) synthesized methanandamide, in which a
methyl group was attached to the carbon atom next to
the nitrogen atom of anandamide. Methanandamide pos-
sesses a remarkable stability to fatty acid amide hydrolase
(
(
FAAH) and has a higher affinity for the cannabinoid 1
CB ) receptor than anandamide. In order to stabilize
1
oleoyl glycine, the Jerusalem group synthesized the same
type of derivative, namely monomethylated oleoyl glycine
(
oleoyl alanine; HU595) as well as dimethylated oleoyl
glycine (HU596), in which one or two methyl groups,
respectively, were attached to the carbon atom next to
the nitrogen atom. Like oleoyl glycine, oleoyl alanine is
found, together with other N-acyl alanines and N-acyl ami-
no acids, in both invertebrate and mammalian tissues, in-
cluding the mouse brain (Tortoriello et al. 2013; Leishman
et al. 2016).
We have previously demonstrated that oleoyl glycine pre-
vents the somatic withdrawal responses elicited by acute
naloxone-precipitated MWD which include abdominal con-
tractions, lying flattened on belly, diarrhea, mouthing move-
ments, and the nausea-like conditioned gaping reactions in
the TR test by a peroxisome proliferator-activated receptor
alpha (PPARα) and a cannabinoid 1 (CB ) receptor mecha-
1
nism of action (Rock et al. 2020). This finding is consistent
with the in vitro action of oleoyl glycine as a PPARα ago-
nist and a fatty acid amide hydrolase (FAAH) inhibitor
(
5
Donvito et al. 2019). Furthermore, oleoyl glycine (1 and
mg/kg, i.p.) prevented the aversive effects of naloxone-
precipitated MWD in a CPA test (Petrie et al. 2019). Here,
we compared the effectiveness of HU595 (monomethylated
oleoyl glycine; oleoyl alanine) and HU596 (dimethylated
oleoyl glycine) with that of oleoyl glycine in interfering with
somatic and affective effects of acute naloxone-precipitated
MWD in rats. In experiment 1, we evaluated the potential of
oleoyl alanine (HU595; 5 mg/kg, i.p.) to interfere with so-
matic withdrawal symptoms at the same dose that oleoyl
glycine was effective (Rock et al. 2020). Experiment 2 com-
pared the duration of action (10 and 60 min) of oleoyl ala-
nine (5 mg/kg, i.p.) with that of oleoyl glycine (5 mg/kg,
Fig. 1 Structure of oleoyl glycine, monomethyl oleoyl glycine (oleoyl
alanine, HU595), and dimethyl oleoyl glycine (HU596)

Psychopharmacology
profiling (ABPP) to determine if HU595 and HU596 bind
with FAAH and luciferase assay to determine if HU595 and
HU596 bind with PPARα.
alanine (HU595), and HU596 were first dissolved in ethanol,
Tween 80 was then added to the solution, and the ethanol was
evaporated off with a nitrogen stream; after which, the saline
was added. The final vehicle (VEH) consisted of 1:9 (Tween/
saline). Oleoyl glycine was prepared as a 5 mg/ml solution;
oleoyl alanine (HU595) and HU596 (see Fig. 1 for molecular
structure of each) were prepared as 1 mg/ml and 5 mg/ml
solutions; all drugs were administered i.p. at a volume of
Materials and methods
Animals
1
ml/kg. Lithium chloride (LiCl; Sigma Aldrich) was prepared
The 209 subjects were male Sprague Dawley rats (200 to
in a 0.15 M solution with sterile water and was administered
i.p. at a volume of 20 ml/kg (127.2 mg/kg dose).
Synthesis of monomethyl oleoyl glycine (oleoyl ala-
nine; HU595)
2
50 g on arrival in the laboratory) purchased from Charles
River Labs, St. Constant, Quebec. Animals were pair-housed
in an opaque Plexiglas cage while receiving food and water ad
libitum, except in experiment 6 in which animals were single-
housed with food and water ad libitum. They were exposed to
a 12/12 h reverse light/dark cycle where the lights turn on at
Oxalyl chloride (2.0 M solution in methylene chlo-
ride, 3.5 ml, 7 mmol) was added dropwise under nitro-
gen atmosphere to a solution of oleic acid (1 g,
3.54 mmol) and N,N-dimethylformamide (266 μl,
3.64 mmol) in dry methylene chloride (10 ml). The
reaction mixture was stirred for 1 h and then the solvent
was evaporated under a nitrogen flow. The crude mate-
rial in methylene chloride (10 ml) was added to a solu-
tion of alanine (945 mg, 10.62 mmol) and 2 N potassi-
um hydroxide in an ice bath. Then, the reaction mixture
was stirred for 1 h, water (10 ml) was added, and the
mixture was acidified to pH 3 with 1 N HCl. The prod-
uct was extracted with ether (3 × 50 ml) and dried
7
p.m. All experiments were conducted during the rats’ dark
cycle. The colony room housing all of the rats was kept at
1 °C. All animal procedures were approved by the Animal
2
Care Committee of the University of Guelph and adhere to the
guidelines of the Canadian Council of Animal Care.
Drugs
Morphine and naloxone (Ontario Veterinary College (OVC)
pharmacy) were prepared with saline at a concentration of 20
and 1 mg/ml, respectively, before injecting subcutaneously
(
MgSO ), and solvent was evaporated under reduced
4
(
s.c.) at a volume of 1 ml/kg. Oleoyl glycine (prepared by
the Jerusalem group as previously described (Donvito et al.
019)), HU595 (oleoyl alanine; monomethylated oleoyl gly-
pressure. The crude material was chromatographed on
silica gel (eluting with chloroform:methanol) to yield
white powder. Melting point 42 °C; NMR (CDCl3,
ppm) 6.11–6.01 (d, 1H), 5.35–5.32 (m, 2H), 4.62–4.51
2
cine), and HU596 (dimethylated oleoyl glycine) were dis-
solved in a vehicle mixture of ethanol, Tween 80, and physi-
ological saline in a 1:1:18 ratio. When oleoyl glycine, oleoyl
(
m, 1H), 2.43 (bs, 1H), 2.25–2.20 (t, 2H), 2.01–1.97

Psychopharmacology
(
1
m, 4H), 1.66–1.60 (m, 2H), 1.46–1.44 (d, 3H), 1.30–
.26 (m, 20H), 0.89–0.85 (t, 3H(; LC-MS (-H) 352 m/z.
Synthesis of dimethyl oleoyl glycine (HU596)
investigation of conditioned gaping reactions in experiment
1, the rats were placed in taste reactivity (TR; Grill and
Norgren 1978) chambers with their cannula attached to an
Oxalyl chloride (2.0 M solution in methylene chloride,
.5 ml, 7 mmol) was added dropwise under nitrogen atmo-
infusion pump (Model KDS100, KD Scientific, Holliston,
MA, USA) for fluid delivery. The TR chamber was in a dark
room next to a 25-W light source. The TR chambers used
were made of clear Plexiglas (22.5 × 26 × 20 cm) and placed
on a table with a clear glass top. A mirror beneath the chamber
at a 45° angle facilitated viewing of the ventral surface of the
rat to observe orofacial responses. A Sony video camera
(Handycam, Henry’s Camera; Waterloo, ON) with a
FireWire connection to a computer was focused on the mirror
and used to record the rats from the mirror beneath the cham-
ber. The videos were later scored using The Observer. For
experiments 2–5, a place conditioning apparatus with remov-
able floors was used as described by Wills et al. (2016). The
conditioning apparatus was a rectangular box (60 × 25 ×
25 cm) made of black Plexiglas and a wire mesh lid. During
conditioning, removable metal floors characterized by either a
hole surface (1 cm in diameter spaced 1 cm apart from each
other) or a grid surface (1/2 cm horizontal bars spaced 1 cm
apart) were placed upon a black rubber mat on top of the black
Plexiglas surface. The different floors serve as contextual cues
that differentiate the treatment floor from the VEH floor.
During the test and pretest trials, black metal floors split into
two equal halves (half hole and half grid surface) were placed
into the conditioning boxes. The tactile stimulus properties of
the two floor halves were identical to their matching floor
counterparts used in conditioning. EthoVision software
(Noldus, Inc., Netherlands) was used to automatically capture
the movement of the rat among the floors, which was collected
by a video camera attached to the ceiling. For experiment 6,
assessment of saccharin preference was conducted in the
3
sphere to a solution of oleic acid (1 g, 3.54 mmol) and N,N-
dimethylformamide (266 μl, 3.64 mmol) in dry methylene
chloride (10 ml). The reaction mixture was stirred for 1 h
and then the solvent was evaporated under a nitrogen flow.
The crude material in methylene chloride (10 ml) was added
to a solution of 2-aminoisobutyric acid (1.1 g, 10.62 mmol)
and 2 N potassium hydroxide in an ice bath. Then, the reaction
mixture was stirred for 1 h, water (10 ml) was added, and the
mixture was acidified to pH 3 with 1 N HCl. The product was
extracted with ether (3 × 50 ml) and dried (MgSO ), and sol-
4
vent was evaporated under reduced pressure. The crude ma-
terial was chromatographed on silica gel (eluting with
chloroform:methanol) to yield a yellowish powder. NMR
(
CDCl3, ppm) 5.42 (m, 2H), 2.18–2.13 (m, 6H), 1.56 (s,
H), 1.53 (m, 2H), 1.31–1.29 (m, 20H), 0.88 (t, 3H(; LC-
6
MS (-H) 366 m/z.
Apparatus
In experiment 1, for investigation of the acute naloxone-
precipitated MWD somatic reactions, the observation cham-
bers consisted of 4 black Plexiglas boxes (22.5 × 26 × 20 cm)
with an opaque lid, sitting on top of a clear glass-topped table.
A closed-circuit Panasonic WV-CP484 video camera was
placed below each of the chambers to video record somatic
withdrawal behaviors that were FireWired to a computer for
later scoring using “The Observer” event recording software
(
Noldus Information Technology Inc., Leesburg, VA). For

Psychopharmacology
home cage in the colony room. Bottles with sipper tubes were
inserted into the home cage with the saccharin-containing bot-
tle placed on the right side of the cage across all rats.
preferences. EthoVision software tracked the movement of
rats from a video camera in the ceiling throughout the trial
to determine how much time was spent on each floor. There
were no significant differences in time spent on the hole or the
grid floor in any experiment. Floors and conditioning boxes
were washed between each trial.
Surgery
In experiment 1, all 16 rats, under isoflurane anesthesia, were
surgically implanted with an intraoral cannula according to the
procedure described by Limebeer et al. (2010).
One day following the pretest, the rats received two 3-day
conditioning trial cycles in order to produce naloxone-
precipitated MWD-induced CPA (as described by Petrie
et al. 2019). On day 1 of each cycle, the floor opposite to the
assigned drug floor was paired with s.c. saline injection. The
rats were injected i.p. with the VEH and 10 min later were
injected i.p. with saline. Ten minutes after a saline injection,
the rats were placed into the conditioning box with the
assigned saline-paired floor for 20 min. On day 2 of each
cycle, the rats received a high dose of morphine (20 mg/kg,
s.c.), 24 h after the saline conditioning trial the previous day.
After the injection, rats were placed in an empty Plexiglas
cage and monitored for signs of respiratory distress and stim-
ulated when necessary until they recovered and were returned
to the home cage. On day 3 of the cycle, 24 h post morphine
injections, the rats were injected i.p. with VEH, 5 mg/kg
oleoyl glycine, or 5 mg/kg HU595 (oleoyl alanine), 10 or
60 min prior to receiving an injection of naloxone (1 mg/kg,
s.c.). Ten minutes later, they were placed into the conditioning
box with the assigned naloxone-paired floor for 20 min. Four
days later, all rats underwent a second 3-day conditioning
cycle. Five days following the last naloxone trial, a 10-min
drug-free test trial was given. The test trial consisted of the
same procedures as the pretest trial, but the rats were given s.c.
saline injection 10 min prior to the test. During the test trial,
EthoVision tracked the amount of time the rats spent on each
floor surface. The groups (n = 12/group) were VEH–10 min,
VEH–60 min, oleoyl glycine–10 min, oleoyl glycine–60 min,
HU595–10 min, and HU595–60 min.
Procedures
Experiment 1: Potential of oleoyl alanine (HU595) to prevent
acute naloxone-precipitated MWD somatic effects in rats
Following 3 days of recovery from intraoral surgery, the rats
received an adaptation trial in which they were placed in the
TR chamber with their cannula attached to an infusion pump
(
Model KDS100, KD Scientific, Holliston, MA, USA) for
fluid delivery. Water was infused into their intraoral cannula
for 2 min at the rate of 1 ml/min. The conditioning procedure
began 2 h following the adaptation trial. Each rat received two
conditioning trial cycles separated by 48 h during which sac-
charin was paired with acute naloxone-precipitated MWD and
somatic reactions were monitored. On the first trial of each
cycle, all rats were injected with morphine (20 mg/kg, s.c.)
and were placed in an empty Plexiglas cage in the colony
room. They were monitored for signs of respiratory distress
and returned to their home cage once fully ambulatory. On the
second day of each cycle, 24 h after the morphine injection,
the rats were injected i.p. (1 ml/kg) with VEH (n = 8) or
HU595 (5 mg/kg; n = 8) and 20 min later (see Rock
et al. 2020) were placed in the TR chamber and intraorally
infused with 0.1% saccharin solution for 2 min at the rate of
1
ml/min. Immediately after the saccharin infusion, the rats
were injected with naloxone (1 mg/kg, s.c.) and were then
placed in the somatic withdrawal observation chamber for
To further investigate the duration of effectiveness of
oleoyl alanine, an additional group was administered
5 mg/kg i.p., HU595 (n = 12), 120 min prior to receiving nal-
oxone over each of the two conditioning trials. Finally, be-
cause oleoyl glycine interfered with MWD-induced CPA at a
lower dose of 1 mg/kg, i.p. (Petrie et al. 2019), an additional
group (n = 12) was administered a dose of 1 mg/kg, i.p., of
HU595 10 min prior to naloxone on both conditioning trials.
3
0 min. On the second naloxone-precipitated MWD trial, the
rats were videotaped for somatic reactions (mouthing move-
ments, abdominal contractions, lying flattened on belly) and
instances of diarrhea were recorded (yes or no). Body weight
was measured 2 h post naloxone to determine percentage body
weight loss. Seventy-two hours following the final MWD tri-
al, the rats were given a 2-min drug-free TR test during which
they were intraorally infused with saccharin at the rate of 1 ml/
min and their orofacial reactions were recorded.
Experiment 3: Potential of oleoyl alanine (HU595) to produce
CPA or CPP on its own
Experiment 2: Potential of oleoyl glycine and oleoyl alanine
In order to determine if oleoyl alanine produced CPP or CPA
on its own, rats (n = 12) received two conditioning trial cycles
(as in experiments 2 and 3) with HU595 and VEH beginning
the day following a 10-min pretest. On each conditioning trial
cycle, they received 1 ml/kg i.p. injections of HU595
(5 mg/kg) or VEH (24 h apart; with VEH and HU595 trials
(
HU595) to prevent acute naloxone-precipitated
MWD-induced CPA at 10 min and 60 min prior to naloxone
The rats (n = 96) were placed in the CPA testing apparatus for
a 10-min drug-free pretest trial to measure baseline floor

Psychopharmacology
in counterbalanced order) 20 min (Petrie et al. 2019) prior to
placement in the conditioning box lined with the grid or hole
floor (counterbalanced) for 20 min, while their locomotion
was tracked by EthoVision. Three days after the final condi-
tioning day, the rats received a 10-min drug-free as described
previously.
final saline or naloxone injection where all rats received two
bottles (saccharin (0.1%) and reverse osmosis water) in their
home cage and consumption of both liquids was measured at
the same intervals used during pretest. As in the pretest, the
amounts consumed were converted to saccharin PRs.
Experiment 7: Effect of oleoyl alanine (HU595) and HU596
on the rat serine hydrolase proteome for FAAH activity
and on luciferase assay for PPARα activity
Experiment 4: Potential of dimethyloleoyl glycine (HU596)
to interfere with MWD CPA
The rats (n = 32) were treated as in experiment 2, except that
they were pretreated with VEH (n = 12), 1 mg/kg HU596 (n =
Competitive activity-based protein profiling (ABPP) assay of
activity at FAAH For preparation of proteomes, rat brains were
dounced homogenized in an isotonic buffer consisting of
8
), or 5 mg/kg HU596 (n = 12) 10 min prior to naloxone.
20 mM HEPES, 2 mM DTT, 0.25 M sucrose, and 1 mM
Experiment 5: Mechanism of action of oleoyl alanine (HU595)
interference with acute naloxone-precipitated MWD-induced
CPA
MgCl , pH 7.2. Lysed proteomes were then subjected to a
2
low-speed spin (1400×g, 5 min) to remove debris, and ultra-
centrifugation (100,000×g, 45 min) to separate membrane and
cytosolic fractions. The supernatant was removed and saved
as the soluble proteome, while the pellet was washed and
resuspended in isotonic resuspension solution (20 mM
HEPES, 2 mM DTT) by pipetting and saved as the membrane
proteome. Rat brain membranes were diluted to 1 mg/ml prior
to use (Chang, et al. 2012). Proteomes (50 μl) were preincu-
bated with either DMSO or 10–30 μM concentrations of
oleoyl glycine (OG), HU595, and HU596 at 37 °C. After
20 min, FP-Rh (1.0 μl, 50 μM in DMSO, a kind gift from
Ben Cravatt) was added and the mixture was incubated for
another 30 min at 37 °C. Reactions were quenched with SDS
loading buffer (12.5 μl, 5×) and run on SDS-PAGE (sodium
dodecyl sulfate polyacrylamide gel electrophoresis; Chang
et al., 2013). Following gel imaging, serine hydrolase activity
was determined by measuring fluorescent intensity of gel
band corresponding to FAAH using ImageJ 1.43u software.
The 33 rats were treated as in experiment 2 (with only the 10 min
pretreatment time) except that, on the naloxone trials, they also
received an injection of VEH, MK886 (1 mg/kg, i.p.; PPARα
antagonist), or AM251 (1 mg/kg, i.p.; CB receptor antagonist)
1
20 min prior to an injection of HU595 (5 mg/kg, i.p.). Ten
minutes following pretreatment with HU595, the rats were
injected with naloxone (1 mg/kg, s.c.) and 10 min later were
placed in the MWD chamber. The groups were VEH-HU595
(n = 10), MK886-HU595 (n = 11), and AM251-HU595 (n = 12).
Petrie et al. (2019) previously demonstrated that at the doses
employed here neither AM251 nor MK886 modified the strength
of CPA produced by acute naloxone-precipitated MWD.
Experiment 6: Effect of oleoyl alanine (HU595) on saccharin
preference following acute naloxone-precipitated MWD
In order to familiarize the rats with saccharin, the rats (n = 80)
were pre-exposed to two bottles (saccharin (0.1%) and reverse
osmosis water) in their home cage and consumption of both
liquids was measured at 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. The
cumulative amount of saccharin and water consumed at each
of the six intervals was converted into a cumulative saccharin
preference ratio (PR) (cumulative saccharin consumed/(cumu-
lative saccharin + cumulative water consumed)). Using pretest
PR scores, rats were assigned to one of 4 groups (N = 20/
group): saline–saline, saline–naloxone, morphine–saline, or
morphine–naloxone. Seventy-two hours following the pretest,
the rats received two cycles of acute naloxone-precipitated
MWD. On day 1, rats were injected s.c. with saline or
Luciferase assay of PPARα activity Cell culture, reagents, and
transfections
Fibroblast-like (COS-7) cells were propagated in
Dulbecco’s modified Eagle medium (DMEM; Cat. no.
11965092, Life Technology, Milan, Italy) supplemented
with 10% FBS, and 1% penicillin and streptomycin (Cat.
no. 15070063, Life Technology, Milan, Italy) in a humidi-
fied atmosphere of 95% air/5% CO at 37 °C. For cell
2
transfection, cells were seeded onto 24-well plastic plates
2
at 2 × 103 cells/cm density. After plating, the cells were
transfected on the next day with (a) pM1-hPPARα-Gal4,
(b) TK-MH100x4-Luc containing the UAS enhancer ele-
ments, and (c) Renilla luciferase (pRL, cat. E2231;
Promega, Milan, Italy). The combination of plasmids was
transfected into the cells by use of Lipofectamine LTX (Life
Technology, Milan, Italy) following the manufacturer’s in-
struction. After 24 h, the cells were treated with vehicle
(DMSO 0.003%) and HU595 and HU596 (up to 50 μM).
2
0 mg/kg morphine and were monitored in empty cages for
signs of respiratory distress. Twenty four hours later, the rats
in each group were pretreated with an i.p. injection of VEH
(n = 10) or 5 mg/kg HU595 (n = 10) 10 min prior to receiving
a s.c. injection of saline or naloxone. Over the next 48 h, this
cycle was repeated once more, except 10 min following the

Psychopharmacology
Luciferase assay
SEM) somatic MWD reactions and conditioned gaping in the
TR test for the groups pretreated with VEH or HU595 prior to
naloxone. Independent t tests revealed that the group pretreated
with oleoyl alanine displayed significantly fewer MWD-elicited
mouthing movements, abdominal contractions, and bouts of ly-
ing on belly following the second naloxone injection and
displayed significantly fewer conditioned gaping reactions dur-
ing the drug-free TR test than did the group pretreated with VEH
After 18 h of treatment, the cells were harvested and
processed for the luciferase and Renilla luciferase
Promega) using the Dual-Luciferase Reporter Assay
(
System (Promega, cat. E1910) and detected using the
GloMax Luminometer (Promega).
(ps < 0.01). However, the groups did not significantly differ in
Data analysis
the instances of diarrhea or in percentage body weight loss.
For experiment 1, somatic opioid withdrawal behaviors demon-
strated by VEH and oleoyl alanine groups were each entered into
separate independent sample t tests. For experiment 2, separate
Experiment 2: Potential of oleoyl glycine and oleoyl
alanine (HU595) to prevent acute naloxone-
precipitated MWD at 10 min and 60 min prior to
naloxone
2
× 2 mixed-factors ANOVAs were performed for each pretreat-
ment group (VEH, OlGly, HU595) at each time (10 or 60 min) as
a between-subjects factor and floor (grid or hole) as a within-
subjects factor. Also, the data for each of the two additional
groups were entered into a paired t test for each floor to determine
if oleoyl alanine prevented the MWD-induced CPA at a dose of
At a dose of 5 mg/kg, i.p., HU595 (oleoyl alanine) produced
longer lasting prevention of acute naloxone-precipitated MWD
than did oleoyl glycine. Figure 2 presents the mean seconds spent
on the saline-paired floor and on the MWD-paired floor for each
of the pretreatment groups. Among the vehicle-pretreated groups
(upper section of Fig. 2), the analysis revealed only significant
effects of time (F(1,22) = 5.3; p < 0.031), and floor (F(1,22) = 13.9;
p < 0.001), with the rats at both times demonstrating an aversion
to the MWD-paired floor. Among the oleoyl glycine pretreated
groups (middle section of Fig. 2), the analysis revealed a signifi-
1
mg/kg and at a delay of 120 min. For experiment 3, the amount
of time spent on the VEH- and HU595-paired floors was entered
into a paired sample t test during the test of place preference. The
distance traveled on the VEH and oleoyl alanine (HU595) con-
ditioning trials was entered into a 2 × 2 repeated measures
ANOVA with drug (HU595 or VEH) and conditioning trials
(1 and 2) as within-subjects factors. For experiment 4, a 3 × 2
cant time effect (F(1,22) = 18.1; p < 0.001), a floor effect (F(1,22) =
mixed-factors ANOVA was performed with the between-
subjects factor of pretreatment (VEH 1 mg/kg or 5 mg/kg
HU596) and the within-subjects factor of floor (grid or hole).
For experiment 5, time spent on each floor was entered into a
17.4; p < 0.001), and a time × floor interaction (F(1,22) = 7.0; p =
0.015); subsequent paired t tests revealed that oleoyl glycine
prevented the MWD-induced CPA when given 10 min before
naloxone, but not when given 60 min before naloxone. Among
the HU595 (oleoyl alanine) pretreated groups (bottom section of
Fig. 2), the analysis revealed no significant effects; that is, the rats
did not display an aversion to the MWD-paired floor when ad-
ministered HU595 at either 10 min or 60 min before naloxone.
However, when administered 120 min prior to naloxone (data not
displayed), oleoyl alanine no longer interfered with the MWD
CPA; the rats spent significantly (t (11) 2.6; p = 0.026) less time
on the MWD-paired floor (Μ = 193.5 ± 29.6 s) than on the saline-
paired floor (M = 355.5 ± 34.1). As was evident with oleoyl gly-
cine (Petrie et al., 2019) at a dose of 1 mg/kg, oleoyl alanine
interfered with the MWD (data not displayed); the time spent
on the MWD-paired floor (M = 264.3 ± 18.3 s) did not signifi-
cantly differ from the time spent on the saline-paired floor (M =
305.2 ± 20.2 s; t (11) = 1.1).
3
× 2 mixed-factors ANOVA with pretreatment (VEH, 1 mg/kg
MK886, 1 mg/kg AM251) as a between-subjects factor and floor
grid or hole) as a within-subjects factor. For experiment 6, the
(
PR scores during the test were entered into a 2 (pretreatment,
VEH or HU595) × 4 (group SS, SN, MS, MN) × 6 (time 2 h, 4 h,
6
h, 8 h, 12 h, and 24 h) mixed-factors ANOVA. In experiment 7,
the differences among treatments for the luciferase assay were
evaluated by t tests.
Results
Experiment 1: Potential of oleoyl alanine (HU595) to
interfere with somatic effects of MWD and condi-
tioned gaping in rats
Experiment 3: Potential of oleoyl alanine to produce
CPA or CPP
Oleoyl alanine (HU595) interfered with the naloxone-
precipitated somatic MWD effects of abdominal contractions,
lying on belly, and mouthing movements, but not diarrhea or
body weight loss. Also, oleoyl alanine, like oleoyl glycine, inter-
fered with MWD-induced conditioned gaping reactions in rats,
reflective of conditioned nausea. Table 1 presents the mean (±
Oleoyl alanine produced neither CPA nor CPP following two
pairings with a distinctive floor (data not shown). A paired t
test revealed that the rats did not significantly differ in their
preference for the oleoyl alanine-paired floor (mean = 278 ±

Psychopharmacology
Table 1 Acute naloxone-
precipitated MWD behaviors in
MWD behavior
VEH pretreatment
HU595 pretreatment
Statistical significance
experiment 1 (from Rock et al.,
Mouthing movements (f)
Abdominal contractions (f)
Lying on belly (s)
57.2 (± 6.6)
13.1 (± 2.4)
722.3 (± 120.4)
0.5 (± 0.2)
30.8 (± 7.4)
5.0 (± 0.8)
102.0 (± 57.9)
0.8 (± 0.2)
2.4 (± 0.4)
3.5 (± 0.9)
t (14) = 2.6; p = 0.025
t (14) = 3.2; p = 0.006
t (14) = 4.6; p < 0.001
t (14) = 1.0; ns
2
019). Data presented = mean (±
SEM); f, frequency; s, seconds
Diarrhea (yes/no)
%
Body weight loss
2.7 (± 0.5)
t (14) = 0.5; ns
Conditioned gaping in TR test (f)
7.8 (± 2.9)
t (14) = 3.2; p = 0.007
3
8.0 s) and the VEH-paired floor (mean = 281 ± 41.8 s).
18.46; p < 0.001), group × time (F(15,360) = 11.98; p < 0.001),
and pretreatment × group × time (F(15,360) = 5.07; p < 0.001).
To evaluate the triple interaction, separate single-factor
ANOVAs for the group factor were conducted for each pre-
treatment condition at each interval. These revealed a signifi-
cant effect at 2 h (F(3,36) = 13.0; p < 0.001), 4 h (F(3,36) = 8.9;
p < 0.001), and 6 h (F(3,36) = 3.3; p = 0.03) among the VEH
pretreated groups, but not among the HU595 pretreated
groups. Subsequent Bonferroni post hoc comparison tests re-
vealed that among the VEH pretreated groups, group MN
displayed significantly suppressed saccharin PRs than all oth-
er groups after 2 and 4 h of drinking (ps < 0.01).
Oleoyl alanine did not modify the activity level during the
conditioning trials; the analysis of the distance traveled on
the oleoyl alanine conditioning trials and the vehicle condi-
tioning trials revealed no significant effects.
Experiment 4: Potential of dimethyl oleoyl glycine
(
HU596) to interfere with an acute naloxone-
precipitated MWD CPA
Dimethylated oleoyl glycine (HU596) was ineffective at doses
of 1 or 5 mg/kg in preventing MWD CPA. Figure 3 presents
the mean (± SEM) seconds spent on the saline floor and the
MWD floor in experiment 4. The analysis revealed only a
statistically significant effect of floor (F(1,29) = 36.9;
p < 0.001). All groups displayed MWD-induced CPA.
Experiment 7: Effect of oleoyl alanine (HU595) and
HU596 on the rat serine hydrolase proteome for FAAH
and on luciferase assay for PPARα
Experiment 5: Mechanism of action of oleoyl alanine
The top section of Fig. 6 a and b shows the effect of
two concentrations of oleoyl alanine and HU596 on a
rat brain serine hydrolase proteome, including fatty acid
amide hydrolase, in comparison with OlGly. Of the
three compounds, only oleoyl glycine, in agreement
with our previous report (Donvito et al., 2019), and
oleoyl alanine inhibited the binding of the serine hydro-
lase probe to FAAH (by about 40% at 10 μM), without
affecting the binding to the other serine hydrolases that
can be identified with this method. As shown in Fig. 6
b, the effect of oleoyl alanine at 10 μM was comparable
with that of oleoyl glycine, whereas HU596 did not
show significant inhibition at either concentration tested.
The bottom section of Fig. 6 c and d shows the effect of
three concentrations of oleoyl alanine and HU596 on PPARα
activity in a luciferase assay, in comparison with the standard
PPARα agonist, fenofibrate. Both compounds revealed
PPARα activity at a 50 uM concentration, but HU596 was
more potent and efficacious than oleoyl alanine, with a signif-
icant effect being observed at a 30 μM concentration.
(HU595) interference with acute naloxone-induced
MWD CPA
Oleoyl alanine interfered with the MWD CPA, but pre-
treatment with either MK886 or AM251 prevented this
interference as seen in Fig. 4. The analysis revealed a
significant effect of floor (F(1,30) = 52.2; p < 0.001), and
a significant interaction of pretreatment group by floor
(
F(2,30) = 4.6; p = 0.018). Subsequent paired t tests re-
vealed that groups MK886-HU595 and AM251-HU595
spent less time on the MWD-paired floor than on the
saline-paired floor (ps < 0.01), but group VEH-HU595
did not show significant CPA.
Experiment 6: Effect of oleoyl alanine (HU595) on
saccharin preference following acute naloxone-
precipitated MWD
Acute naloxone-precipitated morphine withdrawal suppressed
saccharin preference for the first 4 h of the test among VEH
pretreated rats, but not among HU595 pretreated rats. Figure 5
presents the mean cumulative saccharin PR across the 24 h of
testing for rats pretreated with VEH or HU595. The 2 × 4 × 6
mixed-factors ANOVA revealed significant effects of time
Discussion
Oleoyl glycine is a brain lipid capable of reducing nicotine
self-administration and morphine withdrawal signs acting via
(F(5,360) = 44.44; p < 0.001), pretreatment × time (F(5,360) =

Psychopharmacology
Fig. 2 Mean (± SEM) seconds
spent on the saline-paired floor
and the MWD-paired floor for
each pretreatment group (VEH,
oleoyl glycine, and HU595
(
oleoyl alanine) at each pretreat-
ment time in experiment 2.
**p < 0.001, *p < 0.05 differ-
*
ence between floor preference
distinct molecular targets (Donvito et al., 2019; Petrie et al.,
potentially more stable to hydrolysis, analogs of oleoyl gly-
cine on the effects of morphine withdrawal. Oleoyl alanine
(HU595) interfered with acute naloxone-precipitated opiate
withdrawal when assessed by the somatic and affective effects
of MWD (experiments 1, 2, and 6) in male Sprague Dawley
2
019; Rock et al., 2019). Like other N-acyl-glycines, this en-
dogenous molecule is subject to inactivation by enzymatic
hydrolysis (Huang et al., 2001; Donvito et al., 2019). We have
investigated here the capability of two methylated, and hence

Psychopharmacology
Fig. 3 Mean (± SEM) seconds
spent on the saline-paired floor
and the MWD-paired floor in ex-
periment 2. Dimethylated oleoyl
glycine (HU596) did not modify
the strength of the CPA.
*
*p < 0.01
rats. As previously reported with OlGly (Donvito et al., 2019;
Petrie et al., 2019), HU595 had no aversive or rewarding ef-
fects of its own using place conditioning (experiment 3).
As was demonstrated by oleoyl glycine (Rock et al., 2019),
oleoyl alanine reduced somatic MWD symptoms and also
reduced the display of conditioned gaping produced by
MWD. Oleoyl glycine and oleoyl alanine were equally effec-
tive in reducing the aversive effects of acute naloxone-
precipitated MWD at doses of 1 and 5 mg/kg, i.p., when
administered 10 min prior to naloxone. However, oleoyl ala-
nine retained its activity for a longer period (60 min, but not
eliminated activity, possibly for reasons discussed below.
Finally, rats experiencing acute naloxone-precipitated MWD
also showed the anhedonic reaction of suppressed saccharin
PRs, but pretreatment with oleoyl alanine prevented this an-
hedonic reaction.
Interference of acute naloxone-precipitated MWD by
oleoyl alanine was reversed by pretreatment with either
a PPARα antagonist or by a CB receptor antagonist, as
1
previously shown with oleoyl glycine (Rock et al.,
2019). These results are consistent with the report that
oleoyl glycine acts in vitro as a PPARα agonist and a
weak FAAH inhibitor. Petrie et al. (2019), however,
reported that the protective effect of oleoyl glycine on
acute naloxone-precipitated MWD-induced CPA was re-
1
20 min) than oleoyl glycine, in agreement with its potentially
higher stability toward FAAH than oleoyl glycine. Hence, we
synthesized an additional molecule, the dimethylated oleoyl
glycine (HU596), which, we assumed, would cause a larger
steric hindrance. However, this chemical change ultimately
versed by pretreatment with a CB antagonist only, sug-
1
gesting that the mechanism of action of oleoyl glycine
Fig. 4 Mean (± SEM) seconds
spent on the saline-paired floor
and the MWD-paired floor in ex-
periment 5 in which rats were
pretreated with MK886 (1 mg/kg,
i.p.) or AM251 (1 mg/kg, i.p.)
prior to treatment with HU595
(
5 mg/kg, i.p.). Asterisks indicate
significance, with **p < 0.01;
*p < 0.001
*

Psychopharmacology
Fig. 5 Mean (± SEM) cumulative
saccharin preference ratios by
group across the two-bottle sac-
charin preference testing period
for both VEH and HU595
pretreated animals. Two asterisks
indicate a significant difference
between groups (p < 0.01)
on the aversive effects of MWD may be secondary to
inhibition of FAAH elevating anandamide and indirectly
is both necessary and sufficient, whereas activation of
PPARα is necessary but not sufficient to exert the in-
terference with naloxone-precipitated MWD. This hy-
pothesis would also explain why HU596 is inactive at
producing this effect despite its potentially enhanced
stability to hydrolysis, whereas it does not contradict
our hypothesis that oleoyl alanine has a more prolonged
duration of action compared with oleoyl glycine due to
its higher stability to hydrolysis. In fact, it is likely that
oleoyl alanine still binds effectively to FAAH but,
activating CB . Here, we found that while oleoyl ala-
1
nine, like oleoyl glycine, was capable of both inhibiting
FAAH and directly activating PPARα at intermediate
micromolar concentrations in vitro, HU596 could only
exert the latter of these effects at the same concentra-
tions. These findings suggest that, at least in the case of
these two compounds, indirect activation of CB1 (via
FAAH inhibition and elevation of anandamide levels)

Psychopharmacology
a
b
(
µM)
OlGly
HU596
HU595
kDa
250-
1
50-
1
00-
7
5-
-
FAAH
5
0-
3
7-
5-
2
c
d
Fig. 6 a and b (top section) In vitro competitive affinity-based proteome
profiling (ABPP) of oleoyl glycine, oleoyl alanine (HU595), and HU596
using the serine hydrolase-directed probe FP-Rh in the membrane frac-
tion of the rat brain proteome. a The selectivity profiles of oleoyl glycine,
HU596, and HU595 (10–30 μM), as judged by competitive ABPP anal-
ysis in the rat brain membrane proteome. b Quantification of % of FAAH
inhibition determined by measuring fluorescent intensity of gel band
using the ImageJ 1.43u software. Data represent the mean ± SD of n =
dependent effect of c oleoyl alanine (HU595) and d HU596 in compar-
ison with fenofibrate, in COS-7 cells transiently transfected with human
PPARα-Gal 4 plasmid. Cells were co-transfected with equimolar
amounts of plasmid encoding for TK-MH100x4-Luc containing the
UAS enhancer elements and Renilla luciferase as transfection efficiency
control and normalize the signal intensity of luciferase. HU595 and
HU596 were dissolved in DMSO (vehicle, 0.003%). Data are expressed
as the mean ± SEM of four independent determinations. *p ≤ 0.05 vs
control group, determined by Student’s t test
2
separate experiments. c and d (bottom section) Luciferase assays in
PPARα-transfected COS-7 cells. Bar graphs show the concentration-
unlike oleoyl glycine, is not hydrolyzed by the enzyme.
Alternatively, PPARα may interfere with naloxone-
precipitated MWD only at a later stage, following
FAAH inhibition, which would explain why the less
enzymatically stable oleoyl glycine is capable of altering
acute MWD in a fashion not mediated by the PPARα
receptor.
The interference with the aversive effects of MWD
by oleoyl alanine by AM251 or MK886 was not the
result of an effect of the pretreatment agent itself, as
Petrie et al. (2019) showed that at the same doses and
under the identical experimental conditions, neither pre-
treatment drug modified the strength of the acute
naloxone-precipitated MWD CPA on their own.
Instead, they blocked either the direct (at PPARα) or
indirect (FAAH inhibition) effects of oleoyl alanine.
The results suggest oleoyl alanine (HU595) may be a
more stable agent to combat the aversive effects of
acute naloxone-precipitated MWD than oleoyl glycine.
At doses of 1 or 5 mg/kg, both compounds prevent
the aversive effects of MWD in a CPA paradigm
(Petrie et al., 2019), but the preventative effect of oleoyl
alanine (HU595) persists over 60 min, unlike that of
oleoyl glycine.
Funding information The research was supported by grants from the
Natural Sciences and Engineering Research Council of Canada (03629)
and PlantExt to LAP and from PlantExt to RM.
Compliance with ethical standards All animal procedures
were approved by the Animal Care Committee of the University of
Guelph and adhered to the guidelines of the Canadian Council of
Animal Care.

Psychopharmacology
Conflict of interest The authors declare that they have no conflict of
interest.
Limebeer CL, Rock EM, Sharkey KA, Parker LA (2018) Nausea-induced
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