Novel acylethanolamide derivatives that modulate body weight through enhancement of hypothalamic pro-opiomelanocortin (POMC) and/or decreased neuropeptide y (NPY)

Article abstract of DOI:10.1021/jm300484d

Newly synthesized acylethanolamide derivatives oleoyl-l-valinolamide (1), oleoyl-d-valinolamide (2), elaidoyl-l-valinolamide (3), elaidoyl-d-valinolamide (4) stearoyl-l-valinolamide (5), and palmitoyl-l-valinolamide (6) were investigated in mice as antiob

Full text of DOI:10.1021/jm300484d

Article
pubs.acs.org/jmc
Novel Acylethanolamide Derivatives That Modulate Body Weight
through Enhancement of Hypothalamic Pro-Opiomelanocortin
(POMC) and/or Decreased Neuropeptide Y (NPY)
Yosefa Avraham,*,†,⊥ Jehoshua Katzhendler,‡ Lia Vorobeiv,† Shira Merchavia,† Chana Listman,†
Eithan Kunkes,† Fida’ Harfoush,§ Sawsan Salameh,§ Aviva F. Ezra,‡ Nikolaos C. Grigoriadis,∥
Elliot M. Berry,† and Yousef Najajreh*,§,⊥
†Department of Human Nutrition and Metabolism, Braun School of Public Health, Faculty of Medicine, Hebrew University of
Jerusalem, Jerusalem, Israel
‡Institute of Drug Research, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, 91120 Jerusalem, Israel
§Anticancer Drugs Research Lab, Faculty of Pharmacy, Al-Quds University, Abu-Dies, P.O. Box 20002, Jerusalem, Palestinian
Authority
∥Department of Neurology, AHEPA University Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece
ABSTRACT: Newly synthesized acylethanolamide derivatives oleo-
yl-L-valinolamide (1), oleoyl-D-valinolamide (2), elaidoyl-L-valinola-
mide (3), elaidoyl-D-valinolamide (4) stearoyl-L-valinolamide (5), and
palmitoyl-L-valinolamide (6) were investigated in mice as antiobesity
compounds. Compounds 1, 2, 5, 6 significantly decreased body
weight by 6.57% following eight injections of 1 mg/kg ip during 39
days, while 3 and 4 showed no such activity. Receptor binding
indicated that no compound activated CB1, CB2, PPARα, or TRPV1
receptors. Hypothalamic RT-PCR showed that mRNA expression of
the anorexigenic genes POMC and CART was up-regulated by 1, 2, 5
and 1, 2, respectively, while that of the orexigenic genes NPY and
CaMKK2 was down-regulated by the respective compounds 1, 5, 6
and 1, 2, 5. Oleoyl-L-valinolamide enhances anorectic pathways and
lead to decreased glucose levels, enhanced locomotor activity, and
improved cognition. Effects of oleoyl-L-valinolamide on weight were dose-dependent, and it could be given orally. 1, 2, 4, 5
down-regulated FAAH mRNA expression.
Several neuronal centers in the lateral hypothalamic nuclei are
considered to be the “hunger” center, while ventromedial nuclei
serve as the “satiety” center. In addition, the paraventricular and
arcuate hypothalamic nuclei (ARC) are sites where multiple
hormones, released from the gut and adipose tissue, regulate
food intake and energy expenditure. Two distinct types of
neurons in ARC are important for the control of food intake.
(1) Pro-opiomelanocortin (POMC) neurons activated by
anorexigenic hormones release α-melanocyte-stimulating hor-
mone (α-MSH) acting on the satiety center. (2) Neurons
activated by orexigenic peptides such as ghrelin releasing
neuropeptide Y (NPY) and agouti-related peptide (AgRP)
activate the hunger center. The ARC integrates neural (mostly
vagal) and humoral inputs such as enteropeptides including
orexigenic (ghrelin and orexins) and anorexigenic peptides
(cholecystokinin, polypeptide YY, glucagon-like peptide 1,
oxyntomodulin, leptin, and others) that have a physiological
role in regulating appetite and satiety. Peripheral and central
INTRODUCTION
■
The prevalence of obesity is increasing worldwide and has
serious public health consequences leading to increased
mortality and morbidity resulting from type 2 diabetes,
hypertension, stroke, cardiovascular, respiratory problems,
gallbladder disease, osteoarthritis, and sleep apnea, as well as
certain cancers. Diet and exercise remain the main cornerstones
of obesity management. However, since these options are the
most difficult over the long-term, many obese patients would
benefit from drug treatment to reduce body weight and prevent
complications.1−12 The only currently available drug on the
market, orlistat,13 has significant gastrointestinal side effects
that limit its use. The recently approved new drug Qnexa
consists of phentermine, which may cause dry mouth,
constipation, insomnia, and palpitations, and topirimate,
which may cause birth defects, dizziness, paresthesia,
disturbances in attention, headache, dysgeusia, alopecia, and
hypokalemia.14 Other drugs such as sibutramine13,14 and
rimonabant13,14 have been removed from the market.15
The control of body weight concerns the control of adipose
Received: April 5, 2012
Published: February 5, 2013
tissue mass through the key role of the hypothalamus.7−10
© 2013 American Chemical Society
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modulators of feeding behavior act through specific receptors in
the afferent (mostly vagal) nerves and hypothalamic neurons
implicated in adiposity signaling and regulation of food
intake.16 Leptin was found to correlate with body fat mass
and affects both food consumption and energy expendi-
ture17−21 (Table 1 and Figure 2). Other compounds involved
TRPV1 expression in the peripheral vagal sensory nerves is
involved in the central control of food intake.31,32
The endocannabinoids anandamide and 2-AG are endoge-
nous activators of TRPV1.32 Another factor in hypothalamic
intracellular signaling is the enzyme CaMKK2, a member of the
serine/threonine-specific protein kinase family. Hypothalamic
CaMKK2 functions as an AMPK kinase to phosphorylate and
activate AMPK in the ARC and is implicated in ghrelin-induced
pathways that regulate NPY and AgRP in NPY neurons.
Inhibition of CaMKK2 in mice reduces appetite and promotes
weight loss.33,34
5-HT1A receptors are involved in 5-HT reuptake by the
presynaptic neurons from the synaptic cleft. 5-HT (serotonin)
release is stimulated by food intake and causes satiety.35,36 The
5-HT1A receptor has been shown to interact with brain-derived
neurotrophic factor (BDNF), which may play a major role in
regulation of mood and anxiety.36
As part of our efforts to search for novel fatty acid
aminoethanolamide derivatives that have new biological
activities, we have prepared several different conjugates. Our
hypothesis is that in contrast to the endogenous acylethano-
lamides which are regulated by defined endogenic signals
(anabolic/catabolic pathways) the nonendogenous acylethano-
lamides will probably display long-term effects or will function
on a different anorexigenic target. Thus, it seems likely that the
antiobesity effects and/or pathways of endogenous oleoyletha-
nolamide and that of the nonendogeous oleoylvalinolamide will
be different. Support for this premise came from the
experiment of Astarita et al.38 demonstrating that compared
to oleoylethanolamide which was entirely hydrolyzed by mouse
liver homogenate during 60 min period at 30 °C, its substituted
analogue oleoylalaninolamide was not affected at all under the
same experimental conditions.
Table 1. Compounds Inhibiting Food Intake
CRH/urocortin
5HT
α-MSH
POMC
leptin
CaMKK2 inhibitors CCK peptide
dopamine
CB1/CB2
PPARα (NR1C1)
GPR119, GPR55
agonists37
TRPV1
antagonists37
agonists37
agonists37
histamine
GABA
NEα1 agonist
CART
include the endocannabinoids which affect food consumption
and weight, behavior, and cognitive function including brain
rewarding processes.22−28 Orally administered endocannabi-
noids can affect food intake and digestive processes via their
effects on peristalsis29 and catecholamines.26,27Anandamide
(AEA) and the other N-acylethanolamines, e.g., oleoylethano-
lamide (OEA), palmitoylethanolamide (PEA), and linoleoyle-
thanolamide (LEA) (Figure 1), are formed by several
One of our aims was to assess the antiobesity effect of
branched lipophilic groups residing on ethanolamine at α
position to the amine. To test this, acyl-L- and D-ethanolamide
derivatives were synthesized, purified, and characterized. Four
fatty acids were selected to study the effects of saturation, cis
and trans configurations C18:1,cis-9 and C18:1,trans-9, and
chain lengths C18:0 and C16:0. The following acylaminoetha-
nolamides were synthesized and purified: oleoyl-L-valinolamide
(1), oleoyl-D-valinolamide (2), elaidoyl-L-valinolamide (3),
elaidoyl-D-valinolamide (4), stearoyl-L-valinolamide (5), and
palmitoyl-L-valinolamide (6). These compounds were tested for
their effects on food consumption, body weight, activity,
cognitive function, neurological score, glucose levels, liver
enzymes, histopathology, and survival.
Figure 1. Structures of the natural endogenous N-acylethanolamides.
enzymatic pathways from their precursors which are the N-
acylated ethanolamine phospholipids. Endogenous OEA and
PEA are thought to inhibit food intake by acting on receptors in
the intestine. OEA, PEA, and possibly also LEA may be
involved in regulation of food intake by selective prolongation
of feeding latency and postmeal intervals. These N-acylethanol-
amines are formed locally in the intestine, where they can
activate PPARα located in proximity to their site of synthesis.
Prolonged intake of dietary fat (45% energy) may promote
overconsumption of food by decreasing the endogenous levels
of OEA, PEA, and LEA in the intestine.30
Of the endogenous N-acylethanolamides, AEA and OEA
have been extensively studied.30 Pharmacologically, most of the
biological functions of AEA are mediated by the two
cannabinoid receptors CB1 and CB2.28 Interestingly, there is
emerging evidence for the participation of TRPV1 and
capsaicin signaling in the regulation of feeding and body
weight. Dietary administration of capsaicin and other chemi-
cally related “vanilloid” compounds can reduce food intake and
increase energy expenditure in animals and humans.31,32
Acutely administered capsaicin can reduce food intake, an
effect that may be related to altered satiety or alternatively via
visceral fat and anorexia. Furthermore, activation of TRPV1 by
capsaicin affects lipid metabolism, prevents adipogenesis in
stimulated preadipocytes, and prevents obesity in male mice.
RESULTS
■
Chemistry. According to Scheme 1 the synthesis of
compounds 1−6 was performed to produce tens to hundreds
of milligrams of pure products. The coupling reaction of the
appropriate fatty acid and oleoyl-L-valinolamide proceeded at
standard EDC/HOBt coupling conditions with DMF as a
solvent at room temperature for 24 h. Following solvent
evaporation the resultant mixtures were applied to silica column
chromatography (dichloromethane/ethyl acetate, 1:1, v/v).
The products obtained ranged from a viscous oily state in the
case of oleoyl-L-valinolamide (1) to solid in the case of stearoyl-
L-valinolamide (6) in yields of 65−90%.
The synthesis of compounds 1−6 was implemented
according to several distinct protocols including use of DIC,
DCC, and EDC as coupling reagents. In addition the reaction
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Figure 2. Endogenous anorexigenic/orexigenic signaling pathways. The frame on the left defines the anorexigenic pathways discussed in this paper.
was assisted by microwave in solution and on solid support
employing DIC or EDC as coupling agents. All the three
methods employing DIC, DCC, and EDC in solution resulted
in yields of 60−90%; however, the workup of EDC is of some
advantage, since it is water-soluble and can be extract into the
aqueous medium following the washing of the organic phase
with acidic water.
The reaction with DCC/NHS in pyridine at room
temperature resulted in poor yields in the range of 23−61%.
However, at elevated temperatures of 50−60 °C the products
were obtained at reasonable yields of 60−85%. The reaction
with DIC/HOBt was carried out in DMF at room temperature
and produced good yields. Since the purity of the resulting
products by the EDC method is relatively high even prior to
applying them for further purification on silica columns, this
method is our method of choice in the current presentation.
Microwave assisted method in solution and on solid support
(DIC/HOBt/DMF) at 85 °C was not of any advantage and
displayed significantly modest yields. Some of the reasons for
low yields in the preceding method are due to (1) the
probability of the valinol to react also through the free alcohol
residue to result an ester and (2) the ability of the valinol to
eliminate water in the presence of excess of HOBt or during
treatment with NaOH. Elimination of water was confirmed in
several cases by mass spectrometry. .
NMR Assignment. A detailed NMR study of compounds 1,
2, 3, 4, including, 1H NMR, 13C NMR, 1H−13C HSQC
(heteronuclear single quantum correlation), HETCOR, and
1
1H−1H COSY measurements, resulted in H and 13C chemical
shift assignments, as listed in Table 2.
The two methyl groups 4a and 5a of the valinol moiety that
are coupled to the methine proton 3a (1H−1H COSY pattern)
displayed two distinct doublets at 0.93 and 0.96 ppm that in the
HETCOR correlated with the 13C signals at 18.97 and 19.64
ppm, respectively. Although HETCOR is significantly less
sensitive than the HSQC experiment, it provides higher
resolution in the carbon dimension that allowed us to
distinguish between the two methyl groups 4a and 5a. The
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methyl groups are not magnetically equivalent because of
rotational restriction around the (CH3)2CH−CH bond.
NOESY experiments reveal interactions between H-7a and
H-2, H-7a and H-(4a and/or 5a). Therefore, it is inferred that
the OH proton of 7a is hydrogen-bonded to the carbonyl group
of C-1 to form a seven-member ring arrangement that reduces
conformational freedom in the isopropyl residue. The steric
rigidity of this part is not due to an amphipathic structure
where the lipophilic fatty acid chain and the isopropyl group are
oriented to one side, since (a) there is no indication in the
NOESY that there are interactions between H-(4a,5a or 3a)
and H-(2−18) and (b) the two doublets of 4a and 5a are also
seen in the nonconjugated valinol at δH 0.84 and 0.86 and in
the depsi valine (HO-Val-OH) at 0.91 and 1.04 ppm. The
existence of the H(7a)−H(4a,5a) interaction may indicate that
one of the methyl groups of 4a and 5a is oriented toward the
C(O)···H−O part of the seven-member ring system and
probably impedes the free rotation of the isopropyl group.
Scheme 1. Synthesis of Compounds 1−6, N-
a
Acylvalinolamides
1
The H chemical shifts of 1a and 2a were deduced from the
edited phase sensitive HSQC and HECTOR, making it possible
to unambiguously assign the CH2 (1a) resonance and
distinguish it from the resonance of CH(2a).
The chemical shift of the OH proton 7a is in the range 5.62−
5.68 ppm, and since it is engaged in a hydrogen bond with the
carbonyl, it does not exchange and is expected to appear as
either a triplet or double doublet. Actually it appears as a broad
doublet, implying an unresolved double doublet signal
generated by two nonequivalent protons. Since H-1a and H-
2a partially overlap, we have carefully examined the NMR
spectrum of L-valinol and found that the two protons of the
CH2−OH appeared as two double doublets at 3.56 and 3.24
ppm coupled to H-7a and H-2a. Two distinct peaks related to
H-1a were also observed in HSQC spectra, indicating that in
addition to 4a and 5a, the methylene group (1a) of the valinol
is also conformationally constrained.
a
Reagents and conditions: (a) in 25 mL of dry DMF, fatty acid 1.0
equiv, L-valinol or D-valinol 1.4 equiv, EDC·HCl 1.2 equiv, HOBt 1.0
equiv; (b) rt, 24 h.
coupling constants between the methine and the two doublets
(J = 6.7) was easily measured using two-dimensional
homonuclear J-resolved spectroscopy. It is likely that the two
As is expected, the cis and the trans isomers of the 9-
octadecenoyl part of the molecules are identified by the δc of
Table 2. NMR Data of Compounds 1−4
oleoyl-L-valinolamide
oleoyl-D-valinolamide
elaidoyl-L-valinolamide
(3)
elaidoyl-D-valinolamide
(1)
(2)
(4)
positions 1H (ppm) 13C (ppm) 1H (ppm) 13C (ppm) 1H (ppm) 13C (ppm) 1H (ppm) 13C (ppm)
COSY
7a, 2a
NOESY
1a
3.66
3.72
1.87
0.93
0.96
2.85
5.68
64.32
57.25
29.15
18.97
19.64
3.68
3.73
1.87
0.93
0.96
64.32
57.25
29.14
18.97
19.64
3.67
3.72
1.88
0.94
0.96
64.34
57.26
29.15
18.98
19.64
3.68
3.72
1.87
0.94
0.96
64.41
57.29
29.13
18.98
19.64
7a, 3a, 4a, and/or 5a
2a
3a, 6a, 1a
3a
4a, 5a, 2a
[1a,2a]
4a, 5a
5a, 4a
6a
3a
3a
2a
1a
7a, 3a [1a, 2a]
7a, 3a [1a, 2a]
7a
5.70
5.68
5.62
[1a, 2a], 2, 4a, and/or 5a
1
174.43
37.12
26.01
174.49
37.12
26.01
174.46
37.12
26.02
174.47
37.13
26.02
2
2.21
1.63
2.21
1.63
2.21
1.63
2.22
1.63
3
3, 7a, 4−7
2, 4−7
3
2, 4
4−7
8, 11
9, 10
9, 10
11, 8
12−17
16
3
2.00
5.33
5.33
2
27.31
130.14
129.85
27.36
1.98
5.33
5.33
1.98
27.31
130.14
129.86
27.36
1.95
5.37
5.37
1.95
32.75
130.61
130.32
32.70
1.95
5.37
5.37
1.95
32.76
130.61
130.32
32.70
9, 10, 7, 12
8, 11
9, 10, 4−7, 12−17
8, 11, 7, 12
8, 11
8, 11, 7, 12
9, 10, 7, 12
11, 18
9, 10, 4−7, 12−17
1.25
1.29
0.87
32.04
22.82
14.25
32.04
22.82
14.25
1.25
1.29
0.87
32.04
22.82
14.26
1.25
1.26
0.87
32.05
22.83
14.27
17
1.27
0.87
18
17
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Figure 3. Changes in percentage body weight of mice following treatment with compounds 1−6. Values are mean SEM (%); N = 10 mice per
group. Mean values with a common letter differ: (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001. The arrows indicate days of compounds
administration.
the allyl methylene groups (C-8, C-11), where the carbon
chemical shift of the cis isomer compared to the trans is
relatively upfield. A slight shift in the same direction is ascribed
to the respective double-bonded carbons (9, 10) as is depicted
in Table 2.
Biology. In order to evaluate the effect of compounds 1−6
on body weight in mice, we have initially carried out our
experiments in Sabra mice (an outbreed strain that models
variability as found in human populations). Results on body
weight and food consumption in these mice are presented in
Tables 3 and 4 and Figure 3. The mechanism of action of
compounds 1−6 was inferred from receptor binding assays and
RT-PCR measurements (Tables 5 and 6). The effect of
Figure 5. Activity of treated mice compared to controls. Values are the
compound 1 on body weight through oral administration by
gavage was presented in (Figure 4). Since the decrease in body
mean
SEM (%); N = 10 mice per group. Mean values with a
common letter differ: (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001.
also measured 2-AG levels (Figure 7) following administration
of compound 1. We also examined the dose-dependent activity
of compound 1 in Sabra mice on body weight, motor activity,
cognitive function, and POMC gene expression (Figure 8) and
the impact of compound 1 compared to rimonabant on
cognitive function, neurological score, and ALT levels (Table
8) .
In addition, experiments have been performed in two “gold
standard” mice models for obesity: C57BL/6 mice fed a high
fat diet (40% calories of fat) and the ob/ob mice which are
leptin deficient. In the C57BL/6 mice model we have tested (in
parallel with the Sabra mice) body weight food consumption,
motor activity, and cognitive function (Figure 9). The efficacy
of compound 1 to reduce body weight relative to rimonabant
was tested in both mice models, Sabra and C57BL/6 (Tables 7
and 9). Likewise, the ability to ameliorate the metabolic
syndrome by oleoyl-L-valinolamide was tested in comparison to
rimonabant. In this regard four parameters were compared:
body weight, glucose levels, cholesterol levels, triglycerides
levels, and liver enzymes (Tables 9 and 10). Oleic acid was used
as a control.
Figure 4. Weight curves of oleoyl-L-valinolamide (1) and vehicle
(control) administered on days 6, 18, and 32 by gavage. Values are the
mean
SEM (%); N = 10 mice per group. Mean values with a
common letter differ: (∗) p < 0.05.
weight may be associated with enhanced physical activity, it was
presented in Figure 5. Because of the fact that obesity may be
associated with impaired cognitive function,39 we have
evaluated the effects of these compounds on cognition (Figure
6). Their safety was evaluated by neurological score (Table 8),
and the leading compound 1 was also tested by brain and liver
histopathology. Since previous results indicated that decreased
2-AG levels were associated with a decrease in body weight, we
Effect of Compounds 1−6 on Body Weight. The
compounds were administered to the mice as is described in
trial A.
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Figure 6. Cognitive function as measured during days 8−12 using the eight-arm maze test. (The lower the number the better the performance).
Values are mean SEM; N = 10 mice per group. Mean values with a common letter differ: (∗) p < 0.05.
to CB1, CB2, TRPV1, and PPARα receptors which are
involved in body weight regulation.30These results are
presented in Table 5.
The CB1, CB2 receptor binding assays evaluated the ability
of 10 μM tested compounds to displace the bound antagonist
[3H]SR141716A. The relatively moderate values of 35%, 36%,
and 21% of antagonist replacement in CB1 receptor by 10 μM
of compounds 1, 2, and 6, respectively, indicate that their IC50
values extend at least in the range of 15−50 μM, which is quite
high for functioning as agonists to the CB1 receptor. The same
is inferred from the binding of compound 1 to the CB2
receptor. Because of the low binding affinity of compounds 1−
6 to the receptors TRPV1 and PPARα, it seems likely that the
Figure 7. 2-AG levels in the hypothalamus. Data are expressed as the
anorexigenic signal of compounds 1−6 is not mediated by these
mean SEM (n = 8 for control, n = 10 for oleoyl-L-valinolamide)
Mean values with a common letter differ: (∗∗) p < 0.01.
receptors.
Gene Expression in the Hypothalamus. Mice were
treated as described in trial A. In addition to receptor binding
evaluation, gene expression was measured at the end of the
experiment by real time PCR on the hypothalamus for POMC,
CART, NPY, CAMKK2, 5HT1A, and FAAH genes. The results
are summarized in Table 6.
It is apparent that some compounds elevate the expression of
anorexigenic genes such as the POMC gene (compounds 1, 2,
5) and CART gene (compounds 1, 2, 4), and some elevate the
expression of orexigenic genes such as NPY (compound 3) and
CAMKK2 (compounds 3 and 6).
Each mouse weight was divided by its initial weight. The
percentage of weight reduction of the control group was
subtracted from the percentage of weight reduction of each
group. From Table 3 and Figure 3 it is apparent that
compounds 1, 2, 5, and 6 significantly decreased body weight,
while compounds 3 and 4 were not effective.
Following the above observations, we then considered
whether the decrease in body weight was associated with
decreased food intake.
Effect of Compounds 1−6 on Total Food Consump-
tion. Mice were treated as described in trial A. Food
consumption was measured every day, and the average was
calculated for every week according to the equation presented
in the Experimental Section. Table 4 shows that compounds 1,
2, 5, and 6 significantly decreased food consumption,
compound 3 slightly increased food intake, and compound 4
caused a significant increase in food intake.
Effect of Oral Application of Oleoyl-L-valinolamide
Administered by Gavage. Oral administration (10 mg/kg by
gavage) of oleoyl-L-valinolamide according to trial B resulted in
a significant weight loss of treated mice compared to the
control animals. Figure 4 displays a significant difference (p <
0.05) in weight gain between the control and treated groups
(5−10%) (five mice per cage). This result also indicates that
oleoyl-L-valinolamide is quite stable during its transit time
through the gastrointestinal tract and thus can be administered
orally.
Regarding the FAAH enzyme most compounds down-
regulated FAAH gene expression (compounds 1, 2, 4, and 5).
Activity. One of the parameters that may contribute to
weight loss is increased energy expenditure; therefore, we
tested the effects of compounds 1−6 on motor activity. Mice
were treated as in trial A protocol. Motor activity was measured
on day 14 using the open field test. The number of lines
crossed was measured for 3 min for each mouse. Oleoyl-L-
valinolamide (1) and oleoyl-D-valinolamide (2) increased
motor activity (p < 0.001a, p < 0.01b), respectively, while
stearoyl-L-valinolamide (5) and palmitoyl-L-valinolamide (6) (p
< 0.05c, p < 0.05d) decreased it significantly. No change in
motor activity was observed with elaidoyl-L-valinolamide (3)
and elaidoyl-D-valinolamide (4) (Figure 5).
Effect of the Compounds on Cognitive Function. Mice
were treated according to the protocol of trial A, and cognitive
function was evaluated during days 8−12 using the eight-arm
maze test. The scores (AUC) were calculated as follows: (days
2 + 3 + 4 + 5) − 4(day 1). Values are the mean SEM (%); N
Receptor Binding Experiments. Compounds 1−6 were
tested by Ricerca Biosciences LLC for receptor binding activity
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Figure 8. (A) Weight curve (dose−response of compound 1 administered ip). (B) Activity. (C) Cognitive function. (D) POMC gene expression.
Values are the mean SEM (%); N = 10 mice per group. Mean values with a common letter differ: (∗) p < 0.05, (∗∗) p < 0.01.
= 10 mice per group. Mean values with a common letter differ:
(∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001.
while 10 mg/kg extended it only to 30%. Regarding cognitive
activity, it is clear from (Figure 8C) that 1 mg/kg compound 1
improved cognitive function; however, 10 mg/kg did not
change it relative to the controls. It seems that the dose-
response patterns resemble at high dosages a receptor
saturation (or approach to saturation) phenomenon that in
the case of cognitive function may mediate unfavorable signals.
Figure 8D indicates that gene expression of POMC is also
regulated by compound 1 in a dose-dependent manner. 1 and
10 mg/compound 1 amplify the amount of the gene by 50%
and 90%, respectively.
Effect of Oleoyl-L-valinolamide (1) on Female C57BL/6
Mice Fed a High Fat Diet. Mice treatment and oleoyl-L-
valinolamide regimen was as detailed in trial D. From Figure 9
it can be seen that oleoyl-L-valinolamide also affects C57BL/6
mice fed on a high fat diet. Body weight decreased significantly
(Figure 9A) on days 7, 8, 9, 10 (−3.2%, −3.2%, −3.1%, −6.3%,
all at p < 0.05). Food consumption was decreased (Figure 9B)
by 22% (p < 0.05) and 45% (p < 0.001) at week 1 and week 2,
respectively. Activity and cognitive functions were enhanced
(Figure 9C and Figure 9D) .
From Figure 6, it is evident that only oleoyl-L-valinolamide
significantly improved cognitive function (p < 0.05).
Levels of 2-AG in the Hypothalamus. We have shown
previously40 that gradual diet restriction caused a decrease in
brain 2-AG levels while administration of 2-AG elevated food
intake. Thus, it seems likely that 2-AG brain levels are
associated with the state of satiety/hunger. In order to find out
whether this correlation also applied in the case of oleoyl-L-
ethanolamide, we measured 2-AG level in the hypothalamus.
Indeed, Figure 7 demonstrates that after treating mice with
compound 1 (8 × 1 mg/kg for 39 days), the level of 2-AG
decreased significantly in the hypothalamus by 44.5% (p <
0.001) (control (100 7)).
Dose−Response Studies of Weight Gain/Loss, Activ-
ity, Cognitive Function, and POMC Gene Expression
Following Oleoyl-L-valinolamide Administration. The
following studies were according to trial C protocol. At the
end of the experiment (day 16) POMC expression was
evaluated in the hypothalamus using real time PCR. Figure 8
shows dose-dependent effects on weight (8A), activity (8B),
cognitive function (8C), and POMC gene expression (8D).
Concerning the weight, it is apparent that application of 1 and
10 mg/kg compound 1 reduced weight gain by 4.6% and 6.4%,
respectively, compared to control. The flattening of the dose
response is more pronounced in the case of activity (Figure 8B)
where 1 mg/kg enhanced the motor activity of mice by 20%
Effect of Oleoyl-L-valinolamide (1) on ob/ob Mice
(Trial E). ob/ob mice are obese mice deficient in the leptin
gene. The translated leptin hormone is known to regulate
appetite. Since leptin activates the anorexigenic POMC neurons
in the arcuate nucleus of the hypothalamus (ARC), it was of
interest to find out whether compound 1 is active in LepOB
/
LepOB mice. The results revealed that although oleoyl-L-
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Figure 9. Effect of oleoyl-L-valinolamide (1) on weight (A), food consumption (B), activity (C), and cognitive function (D) on female C57BL/6
mice. Values are the mean SEM (%); N = 10 mice per group. Mean values with a common letter differ: (∗) p < 0.05, (∗∗∗) p < 0.001.
a
Table 3. Effect of Compounds 1−6 on Percentage Body Weight during 39 Days of Experiment
% weight gain (+)/loss (−) at selected days
compd
D-4
D-9
D-14
D-21
D-25
D-30
D-35
D-39
oleoyl-L-valinol amide (1)
oleoyl-D-valinol amide (2)
elaidoyl-L-valinolamide (3)
elaidoyl-D-valinolamide (4)
stearoyl-L-valinolamide (5)
palmitoyl-L-valinolamide (6)
+0.5
+0.4
+3.1
−2.3
+2.1
+1.9
−2.2*
−0.8
−0.1
−1.7
+0.9
−1,8
−3.1
+1.0
−0.3
−1.4
−2.2
−2.4
−1.7
−0.1
−1.0
−1.8
+3.0*
−5.0*
−3.3
0.7
−5.2*
−4.7*
+0.2
−6.2*
−6.0*
1.6
−6.4*
−6.7*
0.0
−1.1
−5.3*
+0.2
1.4
−1.0
−6.0
−6.7
**
***
−5.8*
−7.8
**
**
***
***
−2.1
−4.9
−7.1
−8.5
a
*
**
***
Values are the mean SEM (%); N = 10 mice per group. p < 0.05. p < 0.01.
p < 0.001.
a
Table 4. Percent Food Consumption Vs Control over 6 Weeks
% food consumption related to control
W-4
***
compd
W-2
**
W-3
W-5
W-6
***
***
***
oleoyl-L-valinolamide (1)
oleoyl-D-valinolamide (2)
elaidoyl-L-valinol amide (3)
elaidoyl-D-valinolamide (4)
stearoyl-L-valinolamide (5)
palmitoyl-L-valinolamide (6)
93.8
79.4
98.5
102.9
96.0
97.0
91.2
78.1
90.3
85.9
80.6
83.6
77.3
**
***
**
***
***
***
81.8
108.1
112.0
93.8
104.9*
109.2*
104.5
**
***
108.0
111.4
100.5
97.4
109.1*
**
**
85.7
82.5
**
96.8
85.9
81.4*
a
*
**
***
Values are the mean SEM (%); N = 10 mice per group. p < 0.05. p < 0.01.
p < 0.001.
valinolamide decreased weight in Sabra and C57BL/6 mice, it
had no effect in ob/ob mice even at dosages of 10 mg/kg.
Safety and Brain and Liver Gross Pathology. Neuro-
logical score detected by the NSS method did not show any
changes following administration of the different compounds.
In addition, no toxic effects were observed. All the mice
survived during the experimental periods.
Histology following administration of oleoyl-L-valinolamide
did not show any pathological changes. No major morpho-
logical changes were noticed among the neuronal or glial cells
in brain sections. Moreover, no inflammatory, ischemic,
necrotic, or apoptotic changes were noticed and no
apoptosis/necrosis, inflammation, and centrilobular apoptosis/
necrosis (coagulative) were seen in the liver sections studied. In
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a
Table 5. Screening Assays Performed at 10 μM in Duplicate
oleoyl-L-valinolamide oleoyl-D-valinolamide elaidoyl-L-valinolamide elaidoyl-D-valinolamide stearoyl-L-valinolamide palmitoyl-L-valinolamide
(1)
(2)
(3)
(4)
(5)
(6)
CB1
35
18
14
1
36
5
8
5
7
6
0
7
6
6
21
0
CB2
0
13
FAAH*
TRPV1
PPARα
8
0
−13
−20
2
3
−22
0
−9
0
2
0
a
Percent of competitive binding/inhibition* of a given receptor (enzyme). Sign (−) could be within normal background variation or caused by
physical properties or insolubility.
Table 6. Effect of Compounds 1−6 on POMC, CART, NPY, CAMKK2, 5HT1A, FAAH Gene Expression by Real Time PCR
effect, %
oleoyl-L-valinolamide oleoyl-D-valinolamide elaidoyl-L-valinolamide elaidoyl-D-valinolamide stearoyl-L-valinolamide palmitoyl-L-valinolamide
(1)
36a,*
15*
(2)
(3)
10
−11
(4)
(5)
(6)
POMC
CART
NPY
31*
44*
8
5
43*
2
25
2
−13
−52
15
**
−11*
69
−9
8
−39b
−14
−16
***
**
CAMKK2
5HT1A
FAAH
−18
−20
−33
−2
17
62
**
−9
15
−18
−46
−1
15
***
***
**
−28*
−26
*
**
***
p < 0.01, p < 0.05,
p < 0.001,
Table 7. Weight Gain and % Weight Gain/Loss (Related to Control) of Sabra Mice at Selected Days after Treatment of
Compound 1 and Rimonabant
a
a
oleoyl-L-valinolamide
rimonabant (SR141617A)
day
control, average
weight gain, g
% weight gain/loss
weight gain, g
% weight gain/loss
1
5
100
100
100
105.2 1.0
101.5 0.9
106.2 0.8
107.6 0.6
111.8 1.2
110.6 1.0
115.3 1.5
118.3 1.8
118.6 2.6
122.5 1.8
131.8 3.6
107.1 0.8
102.2 0.6
103.9 1.0
105.2 1.8
106.3 1.5
104.8 1.6
107.3 1.4**
109.4 1.2***
111.5 2.1***
112.4 1.8
116.8 2.4**
+1.8
+0.7
105.6 0.6
104.8 0.9
107.2 0.8
109.4 0.9
113.5 1.2
111.5 0.8
112.8 0.6
114.6 0.7
115.8 0.8
118.2 0.5
130.6 1.3
+0.3
+3.2
+0.9
+1.6
+1.5
+0.8
−2.2
−3.1
−2.4
−3.5
−0.9
8
12
15
29
33
36
43
50
54
71
−2.2
−2.2
−4.9
−5.2
−6.9
−7.5
−6.0
−8.2
−11.4
a
1 mg/kg. Mice were treated on days 8, 12, 43. **p < 0.01. ***p < 0.001.
conclusion, no major changes in the cellular morphology or
tissue architecture were observed, indicating that the
representative compound exhibited no toxic effect on either
the brain or the liver.
Table 8. Effect of Oleoyl-L-valinolamide and Rimonabant
Administered ip to Sabra Mice On Cognitive Function,
Neurological Score, and ALT Levels
a
rimonabant
a
Comparison between Oleoyl-L-valinolamide (1) and
Rimonabant. Sabra mice were treated as described in trial F.
From Table 7 it can be seen that (1) oleoyl-L-valinolamide
significantly reduced body weight while the same dose of
rimonabant only slightly affected weight loss, (2) oleoyl-L-
valinolamide and rimonabant did not change the neurological
score (Table 8), (3) oleoyl-L-valinolamide did not change ALT
levels while rimonabant elevated them significantly (Table 8),
and (4) oleoyl-L-valinolamide improved cognitive function
while rimonabant did not have any effect. Obesity is associated
with a chronic inflammatory response that in turn causes
abnormalities in glucose metabolism. The collection of
impaired glucose metabolism, central obesity, elevated blood
pressure, and dyslipidemia is identified as metabolic syndrome
(MetS). It is estimated that approximately 25% of the world’s
control
oleoyl-L-valinolamide
(SR141716A)
cognitive function
neurological score
−4 (a)
0.1
−13 (a*b)
0.1
−5 (b*)
0.2
alanine
transaminase
(ALT)
47 (a)
55 (b)
165 (a**b**)
a
1 mg/kg. Mice were treated on days 8, 12, 43. Mean values with a
common letter (a or b) differ: *p < 0.05, **p < 0.01.
population has MetS which increases the risk of developing
cardiovascular disease and type 2 diabetes mellitus.1,2 The
underlying risk factors include insulin resistance and abdominal
obesity. Thus, it was important to detect whether compound 1
administered ip reduced, in addition to weight, glucose levels
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Table 9. Weight Gain and % Weight Gain/Loss (Related to Control) and % Glucose in Blood of C57BL/6 Mice at Selected
Days after Treatment with Compound 1 and Rimonabant
a
a
control
oleoyl-L-valinolamide
% weight gain/loss
rimonabant (SR141617A)
% weight gain/loss
day
weight change
weight change
% glucose in blood
weight change
% glucose in blood
1
3
100
100
100
101.21
99.61
100.16
98.34
99.70
100.71
100.53
100.15
99.04
98.86
96.37
95.56*
95.35
96.54
95.08
95.76***
96.54
96.97
−2.6
−3.2
−4.6
−3.0
−3.2
−5.6
−4.7
−3.6
2.1
−2.7
−5.7
100.55
96.36
−0.6
−3.2
−3.5
−4.6
−3.1
−4.5
−4.5
−1.9
−4.5
+0.86
5
−11.0
8
96.67***
93.81
9
−11.8*
−17.3*
−16.2*
−20.7*
11
12
15
17
19
96.60
96.13
−16.3
96.02***
98.22
−15.5*
19.8*
−21.6*
94.57
a
Mice were injected 1 mg/kg on days 1, 4, 5, 7, 14, 15, 19. The results were recorded from day 18 (day 1). *p < 0.05. ***p < 0.001.
and dyslipidemia as well as liver enzymes associated with the
metabolic syndrome (Tables 9 and 10).
Our data (Table 3 and Figure 3) clearly indicate that
compounds 1, 2, 5, 6 significantly decreased body weight of
Sabra mice by 6.0−6.7% whereas compounds 3 and 4 did not
have any effect. The weight loss is associated with decreased
food consumption following administration of compounds 1, 2,
5, 6 (Table 4), while compounds 3 and 4 enhanced it. From a
structural point of view the immediate conclusion is that the
fatty acid residues of acylvalinolamides are probably involved in
the orexigenic−anorexigenic balance. Saturated fatty acids 16:0,
18:0, and Z-18:1-Δ9 had anorexigenic effects associated with
weight loss, while-E-18:1-Δ9 functioned in the opposite
direction. The chirality of the optical active valinol moiety
(Table 3, Figure 3) did not exhibit any distinctive selectivity
between the two enatiomers (S, R). It has been documented by
us previously40 that diet restriction lowers the level of 2-AG in
the hypothalamus. Figure 7 displays a decrease of 45.5% in 2-
AG following treatment with compound 1. To evaluate the
effect of the tested compounds on orexigenic and anorexigenic
pathways in the hypothalamus, real time PCR experiments were
performed. The results demonstrated that among the four
weight reducing compounds oleoyl-L-valinolamide (1), oleoyl-
D-valinolamide (2), stearoyl-L-valinolamide (5), and palmitoyl-
L-valinolamide (6), only the actions of 1 (p < 0.01), 2, and 5 (p
< 0.05) are probably mediated through the POMC cascade
(Table 6) and partially by the CART pathway (compounds 1
and 2 (Table 6), p < 0.05). Down-regulation of NPY mRNA
(Table 6) was initiated by compounds 1, 5, and 6, probably
impairing the ghrelin signaling pathway of starvation. The
suppression of this pathway is also suggested by the reduced
CAMKK2 mRNA levels caused by compounds 1 and 5 (Table
6).34
The duration of action of the above agents is dependent on
the activity of the enzyme fatty acid amide hydrolase (FAAH)
that degrades the amide bonds of fatty acids. It is apparent that
FAAH mRNA was reduced by oleoyl-L-valinolamide (1) (p <
0.001), oleoyl-D-valinolamide (2) (p < 0.05), and stearoyl-L-
valinolamide (5) (p < 0.01) (Table 6). The reduced FAAH
activity is related to the branched ethanolamine residue.
In conclusion, among the four potential weight reducing
compounds only oleoylvalinolamide affects the four anorexi-
genic/orexigenic mediators toward an anorectic route, i.e.,
POMC↑↑ (above 30%), CART↑ (less than 20%), NPY↓,
CAMKK2↓, and decreases FAAH↓↓ expression. In the case of
stearoylvalinolamide only CART mRNA expression was found
to be the same as that of the control (CART0). Therefore, the
weight loss caused by compound 5 is probably due to the
Table 10. Effect of Oleoyl-L-valinolamide and Rimonabant
Administered ip to C57BL/6 Mice on Triglycerides, AST,
and Cholesterol Levels
a
rimonabant
a
control oleoyl-L-valinolamide
(SR141716A)
triglycerides
(mg/dL)
0.92
1.017
1.286*
AST (UI/L)
146.7
86.58
126*
83.01
125.3*
94.16
cholesterol
(mg/dL)
a
Protocol as described in Table 9.
Comparison between Oleoyl-L-valinolamide (1) and
Rimonabant Administered to C75BL/6 Fed a High Fat
Diet. C75BL/6 mice were treated as in trial G. Table 9 shows
that (1) oleoyl-L-valinolamide and rimonabant reduced
significantly body weight and glucose levels 5.6% and 21.6%,
respectively, by compound 1 and 4.6% and 20.7%, respectively,
by rimonabant), (2) oleoyl-L-valinolamide slightly elevated
triglycerides levels (+10.5%) while rimonabant enhanced them
significantly (+39.8%), (3) both compound 1 and rimonabant
reduced significantly AST levels by the same extent, and (4)
both affected slightly cholesterol levels.
DISCUSSION
■
According to Figure 2, food intake and body weight are
regulated by multifactorial systems, with both central and
peripheral mechanisms of actions. The anorexigenic signal
leptin originates from adipocytes following eating and activates
hypothalamic POMC/CART neurons and α-MSH, which
binds to MC3/4 receptors and causes satiety, while the
orexigenic signal ghrelin derived from the stomach, following
starvation, elevates hypothalamic NPY/AGRP neurons, causing
increased food intake. Thus, it is clear that POMC/CART
neurons play an important role in regulation of starvation/
satiety perceptions, and changing the balance between the
anorexigenic/orexigenic signal by promoting POMC/CART
pathway will be highly advantageous in preventing food intake.
Indeed between the various tracks displayed in Table 1 for food
uptake inhibition and body weight loss, POMC/CART neurons
are considered candidates.
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combination of three factors: POMC↑↑, NPY↓↓, CAMKK2↓. In
the case of oleoyl-D-valinolamide (2) and likewise compound 1,
POMC, and CART are up-regulated; however, the food intake
signals are NPY0↑ (less than 10%) and CAMKK20 (as in
control).
replaced only 6−36%, −2% to 18%, −20% to 6%, and −2% to
2% of corresponding substrates [3H]SR141716A, [3H]WIN-
55,212−2, [3H]resiniferatoxin, and WY14643 bound to CB1,
CB2, TRPV1, and PPARα respectively.
Compounds 1−6 are also poor competitive inhibitors of the
FAAH enzyme. 10 μM tested compounds produced −13% to
14% inhibitory effects on [3H]ethanolamine production from
anandamide (cold) and [3H]anandamide (total 1 μM)
Regarding the peroxisome proliferator-activated receptor α
(PPARα), it has been shown that binding to PPARα is
associated with serious side effects such as liver injury and
skeletal myopathy.46Therefore, lack of such binding by our
compounds is an important safety benefit.
Compound 6 exhibits a distinct signaling pattern of POMC0,
CART↓, NPY↓↓, CAMKK2↑. It is apparent that in this case the
anorexigenic/orexigenic pathways are mainly inhibited
(POMC, CART, and NPY) while CAMKK2 is slightly
activated.
The weight loss exerted by compound 6 seems to be only at
the level of reduction of NPY. Evidence indicates that blocking
NPY signaling reduces food intake and body weight without
cachexia or rebound weight gain.41−43
Structurally related (“endocannabinoid-like”) compounds to
1−6 involved in feeding regulation are the endogenous fatty
acids N-acylethanolamides (NAEs) such as arachidonoyletha-
nol amide (AEA),27,30 also known as anandamide,
linoleoylethanolamide(LEA),47 oleoylethanolamide
(OEA),44,48 stearoylethanolamide (SEA),49 and palmitoyletha-
nol amide (PEA).50 Anandamide binds to the central CB1 and
peripheral CB2 cannabinoid receptors through which it is
thought to exhibit its analgesic and anti-inflammatory effects. It
has been reported to bind with higher affinity to CB1 (Ki of
23.4,51 39.2,52 and 61 nM (with PMSF),53 89 nM (with
PMSF54,47)) than to CB2 (Ki of 1.93 53 and 371 nM 47 in CHO
cells). AEA modulates distinct and diverse physiological
processes, including appetite.37,55 The endogenous levels of
AEA are regulated by food intake so that feeding decreases AEA
levels in the small intestine, whereas fasting increases them.56
AEA is also reported to be an endogenous ligand for the
vanilloid receptor (TRPV1) involved in the transduction of
acute and inflammatory pain signals and to PPARα.
Oleoylethanolamide a non-CB1 receptor-binding analogue of
anandamide, induced satiety and reduced body weight gain in
animals. OEA is a feeding control signal, which activates
PPARα with high binding affinity of EC50 = 120 nM and Kd =
37 nM.48 OEA administration produces anorexic effects by
activating satiety signals forwarded from the vagal afferent
neurons to the paraventricular hypothalamic nucleus and
stimulates peripheral lipolysis by activating PPARα in
adipocytes. In isolated rat adipocytes and hepatocytes, OEA
treatment increased fatty acid oxidation. OEA is synthesized in
astrocytes48 and neurons,57,58 as well as in cells of the small
intestine and adipose tissue where the level of this lipid
mediator is reduced by fasting and increased upon refeeding in
a reciprocal pattern to that exhibited by AEA.59,56,44
PEA is the most widely represented endocannabinoid in
plasma and in many tissues. It is secreted by a large range of
cells such as neurons, immune cells, macrophages, leukocytes,
lymphocytes, and mastocytes. PEA is known to be involved in
immune responsiveness,60 pain relief,61−63 and anti-inflamma-
tion.64−67 Palmitoylethanolamide does not activate CB1/CB2
receptors but binds to the orphan G-protein-coupled receptor
GPR5568 (probably a new cannabinoid receptor) with high
affinity of ED50 = 3.2 nM. Similar to OEA, PEA binds to
PPARα with lower affinity (EC50 ≈ 3 μM),69 and consequently
because of its lower efficacy at this nuclear receptor, it seems
likely that PEA is significantly less potent than OEA in food
intake suppression. However, PEA activity at PPARα is
efficacious enough to induce anti-inflammatory effects.69
Stearoyl ethanolamide (SEA) has been shown to accompany
anandamide in many tissues including rat central neurons,
brain, and testis and mouse neuroblastoma, murine basophils,
To summarize, POMC appears to be the important
modulator of weight loss regarding compounds 1, 2, and 5,
while for compound 6 the diminished levels of NPY probably
play a similar role. Supporting evidence for the involvement of
POMC and/or leptin comes from the experiments on ob/ob
mice which are leptin deficient mice. Since the hypothalamic
POMC/CART expressing neurons are activated by the
adipostatic hormone leptin, no POMC is formed in these
mice. The fact that ob/ob mice treated by oleoyl-L-valinolamide
did not reduce weight while Sabra and C57BL/6 mice did show
weight loss implicates both leptin and POMC in the
anorexigenic response process (Figure 2, Table 1).
Elaidoyl-L-valinolamide (3) and elaidoyl-D-valinolamide (4)
did not reduce weight and even increased food consumption.
The changes in their tested signaling mediators pattern in the
hypothalamus are POMC0↑, CART↓, NPY↑↑, CAMKK2↑↑ and
POMC0↑, CART↑+ (+25%), NPY0↓, CAMKK20↑, respectively.
Therefore, it is conceivable that the increase in food intake
caused by compound 3 is due to the increase of NPY and
CAMKK2 while for compound 4 it is probably a result of a
slight change in the orexigenic−anorexigenic balance. It is
noteworthy that elaidoylethanolamide affects food intake
similarly to oleoylethanolamide which is known to be an
anorexic mediator.44
In addition, only oleoyl-L-valinolamide reduced significantly
5-HT1A levels in the hypothalamus which leads to enhanced
5HT levels in the synaptic cleft causing satiety35 and enhanced
5HT levels (Table 1).
Another parameter contributing to weight loss is energy
expenditure. In this regard the effects of the conjugated fatty
acids are not alike. It is apparent (Figure 5) that activity
increased significantly following administration of compounds 1
and 2 and decreased following compounds 5 and 6. Thus, the
decrease in body weight by compounds 1 and 2 can also be
related to the decrease in food intake and enhanced energy
expenditure, while compounds 5 and 6 reduced weight only by
a decrease in food intake over and above their decreased energy
expenditure. Compounds 3 and 4 did not change body weight;
however, they increased food intake and did not change energy
expenditure probably because of enhanced thermogenesis.45
Regarding cognitive function, it was significantly improved
following 1 mg/kg compound 1 and to some extent by
compound 5 but not in the case of the other compounds
(Figure 6).
Receptor binding experiments done by Ricerca (Table 5)
showed that oleoyl-L-valinolamide (1), oleoyl-D-valinolamide
(2), stearoyl-L-valinolamide (5), and palmitoyl-L-valinolamide
(6) were not mediated by CB1, CB2, TRPV1, or PPARα
receptors. 10 μM fatty acids valinolamide (compounds 1−6)
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and macrophages. Palmitoyl and stearoyl ethanolamides have
been found to be the two most abundant N-acylethanolamides
in most tissues.
The endogenous role of SEA has yet to be fully elucidated. It
does not bind to cannabinoid receptors; however, it can affect
cell signaling and elicit biological effects potentially through
other target receptors.47
the entire mode of action of the conjugates and may affect
various targets along the anorexic−orexigenic pathway. A
comparison between binding affinity of endogenous NEAs and
some NVEs (N-acylvalinol amide) is presented in Table 11.
a
Table 11. Comparison between Binding Potency of
Endogenous NEAs and Some NVEs (N-Acylvalinolamides)
SEA exerts anorexic effects in mice after both ip and oral
administration.70 The anorexic effect of oral administration is
not associated with changes of serum leptin levels or
hematochemical parameters but accompanied by down-
regulation of liver stearoyl-coenzyme A desaturase 1 (SCD-1)
mRNA expression with no changes in liver PPAR (α, β, γ)
expression. SCD-1 is proposed to be a molecular target for
treating obesity, since its deficiency in mice increased energy
expenditure and reduced body adiposity.
Linoleoyl ethanolamide (LEA) has been isolated from mouse
macrophages, neuroblastoma cells, and RBL-2H3 leukocytes. It
binds only weakly to CB1 and CB2 receptors, inhibiting the
binding of [3H]CP-55,940 with Ki values of 10 and 25 μM,
respectively47 In addition, LEA may be involved in the
regulation of food intake by selective prolongation of feeding
latency and postmeal intervals. It appears to be formed locally
in the intestine, where it activates PPARα,30 although it is not
as effective as OEA.
It is therefore apparent that these endogenous NAEs
(besides anandamide) do not have any cannabinoid effects
but affect appetite regulation with numerous other biological
effects such as anti-inflammatory, analgesic, and neuro-
modulation effects. The chain length, degree of saturation,
and conformational flexibility of the conjugated fatty acids affect
their signaling properties and modulate the selectivity and
binding efficacy of the different receptors.
Regarding food regulation, it is well established that AEA
serves as a CB1/CB2 activator to enhance food intake.
However, PEA, OEA, SEA, and LEA bearing the same
ethanolamine headgroup have negligible affinity for CB1R/
CB2R and function in the opposite direction by stimulating
anorexic pathways. Among signaling hormones OEA is known
to exert its hypophagic effect by activating the intestinal PPARα
receptor. PEA and LEA also seem to operate in the same
manner as PPARαR agonists but with much lower potency.
Another molecular target participating in the anorexic effects
of OEA is the transient receptor potential vanilloid type 1
(TRPV1) channel71 which OEA activates with moderate
potency and good efficacy (EC50 of 400 nM 72,73and 2
μM74). It is also generally accepted that the TRPV1 receptor
is activated by AEA (EC50 of 1.15 μM 72 and 158 nM 75,76) and
LEA (EC50 = 630 nM).72 In contrast to the unsaturated NAEs,
the saturated 18:0 N-stearoylethanolamide did not activate
TRPV1 in any of the bioassays used and did not displace the
selective TRPV1 ligand [3H] resiniferatoxin from mouse
cortical slices.77 In addition, the saturated 16:0 NAE N-
palmitoylethanolamine also failed to activate TRPV1 in rat
blood vessels and HEK293 cells.78
on the Anorxigenic/Orexigenic Receptor
AEA
OEA/OVA
SEA/SVA
PEA/PVA
LEA
CB1
++
++
+
−/−
−/−
−/−
−/−
−/−
−/−
−
−/−
−/−
−/−
+
CB2
TRPV1
PPARα
GPR119
GPR55
SCD-1
+
/−
+
+
+
++/−
−
−
++
+
a
++ > + > + > > −.
In addition we have tested the weight loss of Sabra mice
treated with oleoyl-L-valinolamide compared to the endogenous
oleoylethanolamide (OEA) and two related compounds,
oleoyl-L-alaninolamide and oleoyl-N-methylethanolamide, de-
scribed in the literature in the context of obesity (Table 12).38
From Table 12 it is evident that oleoylvalinolamide significantly
reduced weight up to 7.3% on day 39 while the other tested
compounds reduced weight on day 39 by up to 3.4−3.7%. Only
oleoylethanolamide reduced significantly weight on day 39 by
3.4% (p < 0.05). It is also apparent that application of 1 mg/kg
oleoylvalinolamide reduced weight on day 24 to a higher extent
than other compounds on day 39. Furthermore, in the case of
oleoyl-L-valinol the percentage of weight reduction increased
gradually from day 12 to day 39, while in the case of
oleoylethanolamide and oleoyl-N-methylethanolamide it seems
that there was a lag in time before their weight reduction
potential emerged. A plausible explanation may be the
possibility that part of the anorexigenic effect of the latter
compounds is attributed to the accumulation of oleic acid
formed by FAAH. The contribution of oleic acid can also be
ascribed to the activity of oleoylethanolamide. However, our
data indicate that in such a case the oleic acid contribution will
not exceed the range of 25−30%. In addition, compound 1
(OVA) reduced significantly glucose levels in C57BL/6 fed
high fat diet while OEA did not reduce glucose levels in obese
Zucker rats.80 Thus, between these four compounds
oleoylvalinolamide is the most active substance.
In comparison to rimonabant, experiments performed on
Sabra mice (Table 7) showed that compound 1 administered at
1 mg/kg on days 8, 12, 43 reduced body weight even on day 71
by 11.4% while rimonabant at the same dose did not have any
effect. Furthermore, compound 1 improved cognitive function
(Table 8) while rimonabant had no impact. Regarding ALT
levels, it can be seen (Table 8) that compound 1 did not affect
the ALT levels while rimonabant elevated them significantly. In
C57BL/6 mice fed by high fat diet, Table 9 revealed that the
effect of oleoyl-L-valinolamide and rimonabant at 1−5 mg/kg
was similar. After 19 days, both reduced weight significantly and
blood glucose levels by 21.6% and 19.8%, respectively. Both
reduced slightly body weight by 3.1% and 4.5%, respectively,
and both did not change the levels of triglycerides, AST, or
cholesterol (Table 10).
Recently, it has been suggested that the effects of OEA on
food intake and body weight gain may also be mediated in part
by an orphan G-protein-coupled receptor (GPR119).79 In
comparison to other NAEs, OEA is the most potent (EC50
=
3.2 μM),79 whereas other NAEs are significantly less potent in
the order OEA ≫ PEA > SEA ≫ AEA. Our data, however,
indicate that replacing the ethanolamine residue of NAE for a
nonendogenous substituted ethanolamine (NASE) may change
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Table 12. Effect of Oleoyl-L-valinolamide, Oleoyl-L-alaninolamide, Oleoyl-N-methylethanolamide, and Oleoylethanolamide on
a
Percentage of Body Weight (Compared to Control) during a Period of 39 Days
% weight gain (+)/loss (−) at selected days
compd
D-12
D-19
D-22
D-24
D-26
D-29
D-33
D-36
D-39
oleoyl-L-valinolamide (1)
oleoyl-L-alaninolamide
oleoylethanolamide
−1.79
−1.80
−0.23
−0.61
−1.85
−2.24
−1.17
−0.48
−3.06*
−2.24
−1.17
−0.48
−4.24*
−1.57
−0.18
0.03
−4.05*
−0.50
1.43
−5.98*
−3.66
−0.20
−1.44
−4.00*
−1.63
−0.15
−0.30
−5.49**
−2.56
−2.57
−7.31**
−3.58
−3.42*
−3.67
oleoyl-N-methylethanolamide
−0.50
−3.28
a
Values are the mean SEM (%); N = 10 mice per group. *p < 0.05. **p < 0.01. Mice were treated ip with either vehicle (control) or 1 mg/kg of
the above compounds on days 9, 10, 11, 12, 24, 29.
to 3 mM in a total volume of 200 μL. The wavelength of the maximal
amplitude (Amax) is denoted as λmax
CONCLUSIONS
■
.
From our data it is apparent that compounds 1, 2, 5, and 6
could be used for the treatment of obesity. These compounds
are not toxic, and no mortality occurred during any of the
experiments. Only oleoyl-L-valinolamide affects the four
anorexigenic/orexigenic mediators toward an anorectic route
POMC↑↑, CART↑, NPY↓, CAMKK2↓ and decreases FAAH↓.
Oleoyl-L-valinolamide, the lead compound, displayed a dose-
dependent response on weight, cognitive function, and POMC
gene expression and was active by both ip and oral
administration. Oleoyl-L-valinolamide reduced significantly the
level of glucose in C57BL/6 mice fed a high fat diet. Since there
is a link between obesity and diabetes (type II), this
phenomenon is a benefit in treating obesity and its associated
complications. Oleoyl-L-valinolamide does not change ALT
levels, histopathology, neurological score, or survival.
Oleoyl-L-valinolamide resembles the effect of moderate
caloric restriction (to 60%) which has been shown to be
beneficial with respect to cognitive function and longevity.19,81
It may be given orally (either by tip or gavage) and produces
weight reduction by affecting both energy intake and
expenditure in Sabra and C57BL/6 mice.
Oleoyl-L-valinolamide, Oleoyl-D-valinolamide [(R or S)-N-[(1-
Hydroxymethyl)-2-methylpropyl]-(Z)-9-octadecenamides] (1
or 2). In a round-bottom flask, 144 mg (1.4 mmol) of L(D)-valinol
and 282 mg (1 mmol) of oleic acid were dissolved in 25 mL of DMF.
The solution was stirred, and an amount of 135 mg (1 mmol) of
HOBt followed by 230 mg (1.2 mmol) of EDC·HCl was added. After
the mixture was stirred for 2 h at room temperature, an additional 80
mg of EDC was added. The mixture was left to stir overnight. The
progress of the reaction was evaluated by TLC (100% ethyl acetate).
Following the completion of the reaction, the solution was transferred
to a separating funnel and an amount of 60 mL of DCM/hexane (1:1)
was added. The solution was washed 3 times with water (40 mL),
followed by 3 × 0.5 N NaOH, 3 × 0.5 N HCl (40 mL each), and
brine. The organic phase was dried on MgSO4 and evaporated under
reduced pressure to attain a yellowish viscous oil. The resulting
product was dissolved in 25 mL of DCM, and the desired oleoyl-L(D)-
valinolamide was purified on silica column (dichloromethane/ethyl
acetate, 1:1, v/v) to yield 274 mg (75%) of off-white ointment for
compound 1 and 264 mg (72%) for compound 2.
Elaidoyl-L-valinolamide (3), Elaidoyl-D-valinolamide (4),
Stearoyl-L-valinolamide (5), and Palmitoyl-L-valinolamide (6).
Compounds 3−6 were prepared according to the above procedure,
applying 282 mg (1 mmol) of elaidic acid, 284 mg (1 mmol) of stearic
acid, and 256 mg (1 mmol) of palmitic acid as starting materials. After
purification on silica column chromatography (dichloromethane/ethyl
acetate, 1:1, v/v), the yellow-brown gummy crude of compounds 3
and 4 resulted in a white and a light yellow solid, respectively, while
compounds 5 and 6 were acquired as a white solid.
EXPERIMENTAL SECTION
■
Materials. Fatty acids oleic, palmitic, stearic, and eliadic acid were
purchased from Sigma-Aldrich Co. L-Valinol and D-valinol were
purchased from GL Biochem, China. Solvents, reagents, and
deuterated solvents CDCl3 and DMSO-d6 were purchased from
ACROS Chemicals Ltd. and were used without further purification.
Silica gel and thin layer chromatography (TLC) sheets were purchased
from Merck Ltd.
Analytical HPLC for assessing purity was performed on a Varian
HPLC system equipped with a UV detector at 220 nm using a RP-C18
column ((Phenomenex, Luna, 250 mm × 4.6 nm, 5 μm, 100 Å). The
purities of all final compounds were at least >95%.
Oleoyl-L-valinolamide (1). (S)-N-[(1-Hydroxymethyl)-2-methyl-
propyl]-(Z)-9-octadecenamide. Yield = 75% (0.274 g). TLC [100%
Pyridine was dried by keeping it over KOH pellets for at least 24 h
1
ethyl acetate]: Rf = 0.53. CD, λmax = 217.8 nm, Amax =9.73 mdeg. H
prior to usage.
1H NMR and 13C NMR. All NMR spectra were recorded using a
Varian Unity Inova 500 MHz spectrometer (1H, 499.7 MHz; 13C,
125.6 MHz) equipped with either a 5 mm switchable probe or a 5 mm
triple-resonance inverse-detection probe. Data were processed using
NMR (499.7 MHz, CDCl3) δ: 5.68 (bd,1H), 5.39−5.28 (m, 2H),
3.77−3.60 (m, 3H), 2.85 (s, 1H), 2.21 (t, J = 7.6 Hz, 2H), 2.03−1.97
(m, 4H), 1.94−1.80 (m,1H), 1.63 (quin, 2H, J = 7.12 Hz), 1.34−1.26
(m, 20H), 0.96 (d, J = 6.84 Hz, 3H), 0.93 (d, J = 6.84 Hz, 3H), 0.87 (t,
J = 6.84 Hz, 3H). 13C NMR (125.6 MHz, CDCl3) δ: 174.43 (CO),
130.14 ((CC), 129.85 ((CC), 64.32 (CH2OH), 57.25
(NHCHCH2), 37.12 (CH2CH2CO), 32.04 (CH3CH2 CH2),
29.91, 29.87, 29.67, 29.47, 29.41, 29.33, 29.28, 29.21, (CH2)4−7 and
(CH2)12−15, 29.13 (CH3)2CH), 27.31, 27.36, (CH2CHCHCH2),
26.01 (CH2CH2CO), 22.82 (CH3CH2), 18.97, 19.64 ((CH3)2CH),
14.25 (CH3CH2). HRMS calcd for C23H46NO2 (M + H)+ 368.3529,
found 368.3519.
Oleoyl-D-valinolamide (2). (R)-N-[(1-Hydroxymethyl)-2-methyl-
propyl]-(Z)-9-octadecenamide. Yield = 72% (0.264 g). TLC [100%
ethyl acetate]: Rf = 0.53. CD, λmax =217.1 nm, Amax = −9.47 mdeg. 1H
NMR (499.7 MHz, CDCl3) δ: 5.70 (bd,1H), 5.39−5.28 (m, 2H),
3.77−3.61 (m, 3H)), 2.22 (t, J = 7.58 Hz, 2H), 2.03−1.97 (m, 4H),
1.93−1.82 (m,1H), 1.63 (quin, 2H, J = 7.10 Hz), 1.34−1.26 (m, 20H),
0.96 (d, J = 7.10 Hz, 3H), 0.93 (d, J = 7.10 Hz, 3H), 0.87 (t, J = 6.7
Hz, 3H). 13C NMR (125.6 MHz, CDCl3) δ: 174.49 (CO), 130.14
1
the VNMR software. For H NMR chemical shifts are reported in
parts per million (ppm, δ) downfield from tetramethylsilane (TMS).
Spin multiplicities are described as s (singlet), bd (broad doublet), t
(triplet), q (quartet), and m (multiplet). For 13C NMR the chemical
shifts are reported in parts per million (ppm, δ) with the chemical shift
of CDCl3 or DMSO-d6 as the internal reference.
High Resolution Mass Spectrometry (HRMS). High resolution
mass spectrometry was carried out with Orbi-trap (ThermoFinnigen)
using a nanospray attachment. The peptide Met-Arg-Phe-Ala was used
as internal standard. Data were processed using the BIOWORKS 3.3
package.
Circular Dichroism (CD). CD spectra of the acyloylvalinolamides
were recorded on a JASCO J-810 spectrophotometer (JASCO, Japan)
using the supplied Spectra-Manager software. The temperature was
kept at ∼25 °C. Stock solution was made by dissolving samples in n-
butanol . Prior to each measurement a sample from stock was diluted
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(CC), 129.86 (CC), 64.32 (CH2OH), 57.25 (NHCHCH2),
37.12 (CH2CH2CO), 32.04 (CH3CH2 CH2), 29.91, 29.85, 29.65,
29.46, 29.40, 29.39, 29.29, 29.21, (CH2)4−7 and (CH2)12−15, 29.14
((CH3)2CH), 27.31, 27.36, (CH2CHCHCH2), 26.01
(CH2CH2CO), 22.82 (CH3CH2), 19.64, 18.97 ((CH3)2CH),
14.25 (CH3CH2). HRMS calcd for C23H46NO2 (M + H)+ 368.3529,
found 368.3546.
Organism Unit) of the Hebrew University Hadassah Medical School,
Jerusalem. Mice were sacrificed after each treatment by decapitation.
Experimental Protocols. Trial A: The Effect of Compounds
1−6 on Food Consumption, Body Weight, Motor Activity,
Cognitive Function, and Gene Expression in the Hypothal-
amus and of Compound 1 on 2-AG Levels in the
Hypothalamus and Brain and Liver Histopathology in Sabra
Mice. Female Sabra mice, 12 weeks old (30−40 g weight), were
randomized (5 mice per cage) into 7 different treatment groups:
control and 6 treated groups, 10 mice per group. Mice were fed ad
libitum with 2018SC (Harlan Teklad). The treated mice were injected
intraperitoneally (ip) with 1 mg/kg oleoyl-L-valinolamide (1) or
oleoyl-D-valinolamide (2) or stearoyl-L-valinolamide (5) or palmitoyl-
L-valinolamide (6) or elaidoyl-L-valinolamide (3) or elaidoyl-D-
valinolamide (4). Prior to injection the compounds were dissolved
in ethanol/Chremophor/saline, 1:1:18. Control mice were injected
with the corresponding vehicle alone. Mice were injected on days 8, 9,
10, 11, 14, 18, 24, and 38. Body weight was measured every 2−3 days
and food intake every day throughout the experimental period till day
39. Cognitive function was assessed by the eight arm maze,
neurological score by the NSS, and motor activity by the open field
test during the second week of the experiment. Mice were sacrificed on
day 40. Gene expression in the hypothalamus was evaluated by RT-
PCR. 2-AG levels in the hypothalamus and brain and liver
histopathology were evaluated after the administration of oleoyl-L-
valinolamide.
Elaidoyl-L-valinolamide (3). (S)-N-[(1-Hydroxymethyl)-2-meth-
ylpropyl]-(E)-9-octadecenamide. Yield = 65.2% (0.239 g), mp = 51
°C. TLC [100% ethyl acetate]: Rf = 0.42. CD, λmax = 217.3 nm, Amax
=
1
4.41 mdeg. H NMR (499.7 MHz, CDCl3) δ: 5.68 (bd,1H), 5.44−
5.30 (m, 2H), 3.77−3.61 (m, 3H)), 2.21 (t, J = 7.60 Hz, 2H), 1.98−
1.92 (m, 4H), 1.94−1.80 (m,1H), 1.63 (quin, 2H, J = 7.12 Hz), 1.34−
1.26 (m, 20H), 0.96 (d, J = 6.92 Hz, 3H), 0.94 (d, J = 6.92 Hz, 3H),
0.87 (t, J = 6.73 Hz, 3H). 13C NMR (125.6 MHz, CDCl3) δ: 174.46
(CO), 130.61 (CC), 130.32 (CC), 64.34 (CH2OH), 57.26
(NHCHCH2), 37.12 (CH2CH2CO), 32.04 (CH3CH2CH2), 29.80,
29.73, 29.63, 29.45, 29.40, 29.35, 29.33, 29.18, (CH2)4−7 and
(CH2)12−15, 29.15 ((CH3)2CH), 32.75, 32.70 (CH2CHCHCH2),
26.02 (CH2CH2CO), 22.83 (CH3CH2), 19.64, 18.98 (CH3)2CH,
14.26 (CH3CH2). HRMS calcd for C23H46NO2 (M + H)+ 368.3529,
found 368.3546.
Elaidoyl-D-valinolamide (4). (R)-N-[(1-Hydroxymethyl)-2-meth-
ylpropyl]-(E)-9-octadecenamide. Yield = 73.4% (0.269g), mp = 45 °C.
TLC [100% ethyl acetate]: Rf = 0.42. CD, λmax = 218.3 nm, Amax
=
1
−6.01 mdeg. H NMR (499.7 MHz, CDCl3) δ: 5.62 (bd,1H), 5.44−
5.31 (m, 2H), 3.77−3.61 (m, 3H), 2.22 (t, J = 7.58 Hz, 2H), 1.98−
1.92 (m, 4H), 1.94−1.80 (m,1H), 1.63 (quin, 2H, J = 6.93 Hz), 1.34−
1.25 (m, 20H), 0.96 (d, J = 6.92 Hz, 3H), 0.94 (d, J = 6.92 Hz, 3H),
0.87 (t, J = 6.57 Hz, 3H). 13C NMR (125.6 MHz, CDCl3) δ: 174.47
(CO), 130.61 (CC), 130.32 (CC), 64.41 (CH2OH), 57.29
(NHCHCH2), 37.13 (CH2CH2CO), 32.05 (CH3CH2CH2), 29.79,
29.73, 29.61, 29.42, 29.41, 29.35, 29.31, 29.18, (CH2)4−7 and
(CH2)12−15, 29.13 ((CH3)2CH), 32.76, 32.70, (CH2CHCHCH2),
26.02 (CH2CH2CO), 22.83 (CH3CH2), 19.64, 18.98 ((CH3)2CH),
14.27 (CH3CH2). HRMS calcd for C23H46NO2 (M + H)+ 368.3529,
found 368.3539.
Stearoyl-L-valinolamide (5). (S)-N-[(1-Hydroxymethyl)-2-
methylpropyl]octadecanamide. Yield = 90% (0.332g), mp = 72 °C.
TLC [100% ethyl acetate]: Rf = 0.85. CD, λmax = 217.8 nm, Amax = 5.52
mdeg. 1H NMR (499.7 MHz, CDCl3) δ: 5.66 (bd,1H), 3.75−3.63 (m,
3H), 2.21 (t, J = 7.58 Hz, 2H), 1.94−1.84 (m,1H), 1.64 (quin, 2H, J =
7.12 Hz), 1.29−1.25 (m, 28H), 0.96 (d, J = 6.70 Hz, 3H), 0.94 (d, J =
6.80 Hz, 3H), 0.88 (t, J = 6.80 Hz, 3H). 13C NMR (125.6 MHz,
CDCl3) δ: 174.39 (CO), 64.34 (CH2OH), 57.29 (NHCHCH2),
37.15 (CH2CH2CO), 32.07 (CH3CH2CH2), 29.84−29.46,
(CH2)4−15, 29.22 ((CH3)2CH), 26.04 (CH2CH2CO), 22.82
(CH3CH2), 19.64, 18.98 (CH3)2CH, 14.22 (CH3CH2). HRMS calcd
for C23H48NO2 (M + H)+ 370.3685, found 370.3731.
Palmitoyl-L-valinolamide (6). (S)-N-[(1-Hydroxymethyl)-2-
methylpropyl]hxadecanamide. Yield 87% (0.296 g), mp = 63 °C.
TLC [100% ethyl acetate]: Rf = 0.62. CD, λmax = 216.8 nm, Amax = 7.63
mdeg. 1H NMR (499.7 MHz, CDCl3) δ: 5.67 (bd,1H), 3.75−3.63 (m,
3H)), 2.21 (t, J = 7.58 Hz, 2H), 1.91−1.84 (m,1H), 1.63 (quin, 2H, J
= 7.10 Hz), 1.29−1.25 (m, 28H), 0.96 (d, J = 6.70 Hz, 3H), 0.94 (d, J
= 6.80 Hz, 3H), 0.88 (t, J = 6.80 Hz, 3H). 13C NMR (125.6 MHz,
CDCl3) δ: 174.39 (CO), 64.34 (CH2OH), 57.29 (NHCHCH2),
37.15 (CH2CH2CO), 32.07 (CH3CH2CH2), 29.84−29.43,
(CH2)4−13, 29.17 ((CH3)2CH), 26.03 (CH2CH2CO), 22.82
(CH3CH2), 19.64, 18.98 ((CH3)2CH), 14.27 (CH3CH2). HRMS
calcd for C21H44NO2 (M + H)+ 342.3372, found 342.3398.
Animal Studies. In all experiments the principles of laboratory
animal care were followed in accordance with NIH guidelines and the
protocols were authorized by the local animal care facility board ethics
committee research number MD 87.04-2. The cages contained wood-
chip bedding and were placed in a temperature controlled room at 22
°C on a 12 h light/dark cycle (lights on at 07:00). The mice had free
access to water 24 h a day. The food provided was 2018SC (Harlan
Teklad) or high fat diet TD95217 (Harlan Teklad) Food and the
animals were maintained in the animal facility (Specific Pathogen Free
Trial B: Oral Application of Oleoyl-L-valinolamide (1) by
Gavage. Female Sabra mice of 12 weeks old (30−40 g weight) were
randomized (5 mice per cage) into 2 different treatment groups:
control and treated group, 10 mice per group. Mice were fed ad
libitum by 2018SC (Harlan Teklad). The treated mice were
administered by gavage with 10 mg/kg oleoyl-L-valinolamide on
days 6, 18, 32 during 51 days (10 mice per group). Prior to
administration the compound was dissolved in ethanol/Chremophor/
saline, 1:1:18. Control mice were administered with the corresponding
vehicle alone. Body weight was measured every 2−3 days throughout
the experimental period. Mice were sacrificed on day 52.
Trial C: Dose Response Studies of Oleoyl-L-valinolamide (1).
Female Sabra mice 12 weeks old (30−40 g weight) were randomized
(5 mice per cage) into 3 different treatment groups: control and 2
treated groups. Mice were fed ad libitum with 2018SC (Harlan
Teklad) and injected intraperitoneally with 1.0 or10.0 mg/kg oleoyl-L-
valinolamide (10 mice per group) on days 6−10 (10 mice per group).
The rest of the protocol was as in trial A above. Mice were sacrificed
on day 16.
Trial D: Effect of Oleoyl-L-valinolamide (1) on Food
Consumption, Body Weight, Motor Activity, and Cognitive
Function in C57BL/6 Mice Fed a High Fat Diet (40% Calories of
Fat). Female C57BL/6 mice 12 weeks old (20−25 g weight) were
randomized (5 mice per cage) into 2 different treatment groups:
control and treated group (10 mice per group). Mice were fed ad
libitum by high fat diet TD95217 (Harlan Teklad) and injected
intraperitoneally with 1.0 mg/kg oleoyl-L-valinolamide on days 3, 6, 8,
9, 10. The rest of the protocol was as in trial A above. Mice were
sacrificed on day 10.
Trial E: Effect of Oleoyl-L-valinolamide (1) on ob/ob Mice.
Female ob/ob mice 9 weeks old (30−35 g weight) were randomized
(5 mice per cage) into 2 different treatment groups: control and
treated group. Mice were fed ad libitum with 2018SC (Harlan Teklad)
and administered by gavage with 1 mg/kg oleoyl-L-valinolamide on
days 1, 4, 7 and 11, 10 mg/kg on days 14, 18, and 1 mg/kg on days 28,
29, 32, 35 (ip). Body weight was measured every 2−3 days and food
consumption every day throughout the experimental period (60 days).
Trial F: Comparison between the Effects of Oleoyl-L-
valinolamide (1) and Rimonabant (SR141716A) on Body
Weight, Cognitive Function, Neurological Score, and ALT
Levels in Sabra Mice. Female Sabra mice 12 weeks old (30−40 g
weight) were randomized (5 mice per cage) into 3 different treatment
groups: control and 2 treated groups. Mice (10 mice per group) were
fed ad libitum with 2018SC (Harlan Teklad) and injected intra-
peritoneally with 1.0 mg/kg oleoyl-L-valinolamide on days 8, 12, and
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Scheme 2
43. The rest of the protocol was as in trial A above. Mice were
sacrificed on day 74, and ALT levels were measured.
were made into all eight arms or until 24 entries were completed. Each
mouse had to enter all the eight different arms of the maze in the least
number of entries and with the ability to try up to 24 times. Data were
evaluated using the following formula:19,33 AUC (area under curve) =
(day 2 + day 3 + day 4 + day 5) − 4(day 1). On the first day the mice
explore the maze and make many entries in order to enter all eight
arms to get the reward (without compounds or vehicle admin-
istration). Hence, the lower the score, the better is the cognitive
function. The scores are averaged for each group.
Locomotor Activity Test. Activity was assessed in the open field
(20 cm × 30 cm field divided into 12 squares of equal size) as
previously described.33,84 Two mice were observed simultaneously for
3 min to lower possible effects of stress. Locomotor activity was
recorded by counting the number of crossings by the mice at 1 min
intervals. Results are presented as the mean number of crossings per
minute.
Neurological Score Test. Neurological function was assessed by a
10-point scale based on reflexes and task performance: (1) exiting
from a circle 1 m in diameter in less than 1 min, (2) seeking, (3)
walking a straight line, (4) startle reflex, (5) grasping reflex, (6)
righting reflex, (7) placing reflex, (8) corneal reflex, (9) maintaining
balance on a beam 3, 2, and 1 cm in width, (10) climbing onto a
square and a round pole. For each task failed or abnormal reflex
reaction, a score of 1 was assigned. Thus, a higher score indicates
poorer neurological function.33,84,85
Trial G: Comparison between the Effects of Oleoyl-L-
valinolamide (1) Rimonabant (SR141716A) and Oleic Acid on
Body Weight, Glucose Levels, Cholesterol, Triglycerides and
Liver Enzymes in C57BL/6 mice. Female C57BL/6 mice 12 weeks
old were weighted (20−25 g) and randomized (5 mice per cage) into
4 different treatment groups: control and 3 treated groups (oleoyl-L-
valinolamide, rimonabant, and oleic acid), 10 mice/group. Each group
was fed ad libitum with a high fat diet TD95217 (Harlan Teklad) for 9
days, and then they were transferred to normal diet 2018SC (Harlan
Teklad). Mice were injected on days 1, 4, 5, 7, 14, 15, and 19 starting
with 1 mg/kg and from day 20 and onward with 5 mg/kg. The results
were recorded from day 18 (day 1) starting with 1 mg/kg and from
day 20 and onward with 5 mg/kg. The dose of 5 mg (ip) was on days
20, 21, 25, and 35 (days 3, 4, 8, and 18 in Table 9). Prior to
administration, the compound was dissolved in ethanol/Chremophor/
saline, 1:1:18. Control mice were administered with the corresponding
vehicle alone.
Body weight was measured every 2−3 days and food consumption
every day throughout the experimental period (36 days). Glucose
levels were evaluated during the experiment. Cognitive function by the
eight arm maze, neurological score by the NSS, and motor activity by
the open field were measured during the second week of the
experiment. Mice were sacrificed on day 36, and total cholesterol,
triglycerides, and liver enzymes were evaluated.
Scheme 2 describes the doses and time course regimen of the tested
compounds.
Biochemical Analyses. Determination of 2-Arachidonoyl
Glycerol (2-AG). Determination of 2-AG was performed at the end
of the study as described by Hanus
̌
et al.40 In brief, immediately after
Chemicals Used. RNA isolation was performed by Trireagent
(Sigma). Reverse transcription was performed with dNTP mix
(Promega), RQ-1 DNase (Promega), M-MLV reverse transcriptase
(Promega), rRNAsin (Promega), random primers (Syntezza), Power
SYBR Green PCR Master Mix (Applied Biosystems, U.K.),
rimonabant (Cayman Chemical Co., U.S.), 2-arachdonoylglycerol (2-
AG) (Cayman Chemical Co., U.S.), and monoarachidin (Nu-Chek
Prep, Inc., U.S.).
Administration of the Compounds. The compounds were
dissolved in ethanol/Chremophor/saline (1:1:18 v/v/v) (vehicle). All
injections were administered as indicated either intraperitonially in a
volume of 0.1 mL per mouse at 30 min before maze testing or activity
trials. The injection (0.1 mL ip) timing was based on previous
experiments conducted by our group.19 Gavage administration was at
10 mg/kg with the same vehicle.
decapitation, the hypothalamus was weighed and placed in glass tubes
containing 7 mL of chloroform−methanol (2:1) and manually
homogenized for 1−2 min. Vacuum filtration was carried out. The
solvents were evaporated to dryness. Precoated TLC plates were used
to separate 2-AG and monoarachidin, as internal standard from other
lipids. Synthetic 2-AG and monoarachidin were applied alongside the
unknown lipid, dissolved in chloroform, to help in their identification
in the lipid sample.
Determination of Liver Enzymes, Triglycerides, and Total
Cholesterol. Blood for liver enzymes, serum for alanine transaminase
(ALT), aspartate transaminase (AST), cholestrol, and triglycerides
were obtained at the time of sacrifice, centrifuged, and analyzed on the
day of sampling using a Kone progress selective chemistry analyzer
(Kone Instruments, Espoo, Finland). All serum samples were
processed in the same laboratory using the same methods and the
same reference values as previously described.86
Evaluation of Glucose Levels. Glucose levels were detected
using Contour “glucometer” and strips (Bayer Co.). A drop of tail
blood was obtained on the days indicated.
GC−MS Analysis. For quantitative analysis the samples were
analyzed by GC−MS in a Hewlett-Packard G1800A GCD system with
a HP-5971 gas chromatograph with electron ionization detector as
described previously.40
Quantitative RT-PCR Analysis. On the final day of the
experiment mice were injected ip with the compounds 10 min before
sacrifice. Brains were dissected. The hypothalamus was separated,
transferred to liquid nitrogen, and kept at −80 °C. Total hypothalamic
RNA was extracted using Tri reagent according to the manufacturer’s
instructions. RNA concentration was evaluated using a ND-1000
spectrophotometer of NanoDrop Technologies, Inc., U.S., and DNA
left in the sample was degradated by DNase and reverse transcribed.
Food Consumption. Food was provided ad libitum and weighted
every day. The amount of food consumed in grams was evaluated by
the following formula:
week
⎡
⎤
n+1
× 100 (treatment group)
⎢
⎣
⎥
⎦
week1
week
⎡
⎤
n+1
× 100 (control group)
⎢
⎣
⎥
⎦
week1
Eight-Arm Spatial Maze: Cognition Studies. Cognitive
function studies were performed 7 days after initiation of ad libitum
feeding. The mice were placed in an eight-arm maze, which is a scaled-
down version of that developed for rats.82,83 We used water
deprivation and a reward of 50 μL of water presented at the end of
each arm. Water deprivation was achieved by limiting water
consumption for 3 h before the experiment. Mice were randomized
to each treatment group and tested in a maze between 11:00 and
16:00 each day for 5 days. Observations were recorded until entries
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Table 13. Primers Employed for the Various Genes Tested in RT-PCR
gene
forward primer
reverse primer
GAPDH
POMC
5HT1A
CAMKK2
SIRT-1
NPY
5′-CTCTGCTCCTCCCTGTTCCA-3′
5′-GGCCACTGAACATCTTTGTCC-3′
5′-GACAGGCGGCAACGATACT-3′
5′-AAAGGGCTCCTATGGTGTTGTC-3′
5′-TATAGATACCTTGGAGCAGGTTGC-3′
5′-CTCCGCTCTGCGACACTACA-3′
5′-TAATTGCTGTGTTGCCTCTCC-3′
5′-GCTTCCGGCTGCAGAATC −3′
5′-GCTCAAGAGTAAACGCATTCCG-3′
5′-CTGGCACTGCACAAGAAGATG-3′
5′-GACCCCTGTCGCGGAAA-3′
5′-CCAAGGAGCCGATGAGATAGTT-3′
5′-GTATTGTCATTTTCATTGTAGGCCA-3′
5′-TGTCTCCACGAACAGCTTCAC-3′
5′-AATCAGTGTCTCAGGGCTGGA-3′
5′-TCCGATCCAGAACATCAGGTA-3′
5′-CCCCACTCTGTAACTTCTGTACCA-3′
5′-GCTCTCCAGCGTCACACATG-3′
CB-1
FAAH
CART
Quantitative RT-PCR was carried out with Power SYBR Green
PCR Master Mix (Applied Biosystems, U.K.) in a 7900HT instrument
(Applied Biosystems). Reaction volume was 15 μL, and GAPDH was
used as endogenous control. Threshold cycle (Ct) was determined by
SDS software for each of the samples tested, and the average Ct was
calculated for each triplicate. ΔCt of each target gene was calculated by
subtracting the average Ct for GAPDH of a given sample from the
average Ct for the target gene of the same sample. Average ΔCt of a
certain target gene in the sham group was subtracted from ΔCt of the
same gene in samples from the treated groups to yield ΔΔCt of this
gene in the sample (group treated with oleoylethanolamide minus
group treated with saline). The quantity of a specific target gene in a
certain sample relative to the control group was calculated as 2−ΔΔΔCt
using ΔΔCt determined for that sample.33 All primers were
synthesized by Syntezza (Jerusalem, Israel). Specific primers for each
gene are detailed in Table 13.
Receptor Binding. Receptor binding activity of compounds 1−6
to CB1, CB2, TRPV1, and PPARα receptors, which are involved in
body weight regulation,30 was tested by Ricerca Biosciences LLC. In
addition the percent of competitive inhibition of anandamide bound to
FAAH by compounds 1−6 was also examined. All the experiments
were carried out in duplicate in the presence of 10 μM tested
compound.
Peroxisome Proliferator-Activated Receptor PPARA
(PPARα)89 (Human). Human recombinant peroxisome prolifera-
tors-activated receptor α (PPARA, PPARα, hPPAR, NR1C1)
receptors expressed in E. coli are used. Test compound and/or vehicle
is preincubated with 25 nM receptors and binding buffer in modified
Tris-HCl buffer, pH 7.4, for 16 h at 4 °C. Determination of the
amount of complex formed is read spectrofluorimetrically at 337 nm/
665 nm. Test-compound-induced increase of fluorescence by 50% or
more (≥50%) relative to the 100 μM WY14643 response indicates
possible PPARα receptor agonist activity. Test-compound-induced
inhibition of 30 μM WY14643-induced fluorescence response by 50%
or more (≥50%) indicates receptor antagonist activity. Compounds
were screened at 10 μM. The calculation was based on the following
definition: % response = [(c − a)/(b − a)] × 100 where a is the
fluorescence intensity of vehicle control, b is the fluorescence intensity
of the positive control (100 μM WY14643), and c is the fluorescence
intensity of the test compound. Membrane protein may change from
lot to lot, so the concentration was adjusted if necessary.
Fatty Acid Amide Hydrolase (FAAH).90 Rat brain FAAH is used.
Test compound and/or vehicle was preincubated with 25 μg/mL
enzyme in Tris-HCl buffer, pH 7.6, for 15 min at 37 °C. The reaction
was initiated by addition of 1 μM anandamide and 3.3 nM
[3H]anandamide for another 20 min incubation period. An aliquot
was extracted by methanol/chloroform and counted to determine the
amount of [3H]ethanolamine formed. Compounds were screened at
10 μM. The FAAH enzyme assay was designed for inhibitors to inhibit
enzyme activity. The detailed calculation of test compounds is
described as below.
Cannabinoid CB1 (Human) Receptors.87 This assay measures
binding of [3H]SR141716A to human cannabinoid CB1 receptors. Rat
hematpoetic Chem-1 cells stably transfected with a plasmid encoding
the human recombinant cannabinoid CB1 receptors are used to
prepare membranes in HEPES buffer, pH 7.4 (50 mM HEPES, 5 mM
MgCl2, 1 mM CaCl2, 0.2% BSA), using standard techniques. A 10 μg
aliquot of membrane is incubated with 2 nM [3H]SR141716A for 90
min at 37 °C. Nonspecific binding is estimated in the presence of 10
μM R(+)-WIN-55,212-2. Membranes are filtered and washed 4 times,
and the filters are counted to determine [3H]SR141716A specifically
bound. Compounds are screened at 10 μM. Membrane protein may
change from lot to lot, and the concentration used was adjusted if
necessary.
Results of test compounds were expressed as a percent inhibition
referenced to the control group. A positive control of trifluoromethyl
ketone (IC50 = 0.029 μM) was carried out in parallel.
The percentage of inhibition of enzyme activity by the test
compounds was calculated according to the following formula:
inhibition (%) = {1 − (c − a)/(b − a)} × 100, where a is the
average cpm of minimum (blank), b is the average cpm of maximum,
and c is the cpm in the presence of test or reference substance. Note
that the minimum (blank) measurement was defined as in the absence
of enzyme in the reaction. Maximum measurement was defined as in
the presence of enzyme and the absence of test substance in the
reaction. Usually, the setting of the significance criterion is ≥50%
inhibition. Since enzyme activity may change among batches, the
concentration was adjusted as necessary.
Brain and Liver Histology. Possible toxicity of oleoyl-L-
valinolamide was assessed by histopathology of brain and liver
samples.84,86 Briefly, at necropsy, mouse brain and liver tissues were
fixed in 10% neutral-buffered formalin. The brain was cut along the
midline and separated into two pieces containing brain and cerebellum
hemispheres. Both pieces were embedded en bloc in paraffin, and 6
mm sagittal sections were adhered on slides. Serial sections were taken
in 15 groups of slides (10 slides each, three pairs of sections per slide)
at 100 mM intervals. From each group of slides, two were randomly
selected and processed for hematoxylin and eosin staining for
morphological examination (a total of 30 slides). Fixed midsections
of the left, median, caudate, and right liver lobe were embedded in
paraffin, cut at 5 μm, and stained with hematoxylin and eosin. All
Cannabinoid CB2 (Human) Receptors.87 Human recombinant
cannabinoid CB2 receptors expressed in CHO-K1 cells are used in
HEPES buffer, pH 7.0 (20 mM HEPES, 5 mg/mL BSA). A 30 μg
aliquot is incubated with 2.4 nM [3H]WIN-55,212-2 for 90 min at 37
°C. Nonspecific binding is estimated in the presence of 10 μM R(+)-
WIN-55,212-2. Membranes are filtered and washed. The filters are
then counted to determine [3H]WIN-55,212-2 specifically bound.
Compounds were screened at 10 μM. Membrane protein may change
from lot to lot, and the concentration used was adjusted where
necessary.
Vanilloid Receptors.88 This assay measures binding of [3H]-
resiniferatoxin to rat vanilloid receptors. Spinal cords of Wistar derived
rats weighing 175 25 g are prepared in modified HEPES buffer, pH
7.4. A 5 mg aliquot of membrane is incubated with 0.2 nM
[3H]resiniferatoxin for 60 min at 37 °C. Nonspecific binding is
estimated in the presence of 0.1 μM resiniferatoxin. Membranes are
filtered and washed. The filters are then counted to determine
[3H]resiniferatoxin specifically bound. Compounds were screened at
10 μM. Membrane protein may change from lot to lot, and the
concentration used was adjusted if necessary.
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sections were examined by light microscopy (Zeiss Axioplan 2,
Oberkochen, Baden-Wuerttemberg, Germany).
Statistical Analysis. Results are expressed as mean values plus/
minus standard errors. Statistical analysis was performed using two
tailed Student’s t test. Differences were considered significant if p <
0.05.
(10) Schwartz, M. W.; Woods, S. C.; Porte, D.; Seeley, R. J.; Baskin,
D. G. Central nervous system control of food intake. Nature 2000,
404, 661−671.
(11) Gardiner, J. V.; Jayasena, C. N.; Bloom, S. R. Gut hormones: a
weight off your mind. J. Neuroendocrinol. 2008, 20, 834−841.
(12) Farooqi, I. S. Genetic, molecular and physiological insights into
human obesity. Eur. J. Clin. Invest. 2011, 41, 451−455.
(13) Rucker, D.; Padwal, R.; Li, S. K.; Curioni, C.; Lau, D. C.W. Long
term pharmacotherapy for obesity and overweight: updated meta-
analysis. BMJ 2007, 335, 1194−1199.
AUTHOR INFORMATION
Corresponding Author
■
*For Y.A.: phone, 972-2-6757547; fax, 972-2-6431105; e-mail,
yosefa@md.huji.ac.il. For Y.N.: telefax, 970-2-2790413; e-mail,
ynajajreh@pharm.alquds.edu.
(14) Heal, J.; Gosden, J.; Smith, S. L. What is the prognosis for new
centrally-acting anti-obesity drugs? Neuropharmacology 2012, 63, 132−
146.
(15) Kang, J. G.; Park, C. H. Anti-obesity drugs: a review about their
effects and safety. Diabetes Metab. J. 2012, 36, 13−25.
(16) Pandit, R.; de Jong, J. W.; Vanderschuren, L. J.; Adan, R. A.
Neurobiology of overeating and obesity: the role of melanocortins and
beyond. Eur. J. Pharmacol. 2011, 660, 28−42.
Author Contributions
⊥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
(17) Morton, G. J.; Schwartz, M. W. Leptin and the central nervous
system control of glucose metabolism. Physiol. Rev. 2011, 91, 389−
411.
ABBREVIATIONS USED
■
2-AG, 2-arachidonoylglycerol; AEA, anandamide; AgRP,
agouti-related protein; ALT, alanine transaminase; ANA,
anandamide; ARC, arcuate hypothalamic nuclei; AST,
Aspartate transaminase; BDNF, brain-derived neurotrophic
factor; CAMKK2, calcium/calmodulin-dependent protein
kinase kinase 2; CART, cocaine and amphetamine regulated
transcript; CB1/2, cannabinoid receptor type 1/2; DCC, N,N,-
dicyclohexylcarbodiimide; DIC, N,N,-diisopropylcarbodiimide;
D C M , d i c h l o r o m e t h a n e ; E D C , 1 - e t h y l - 3 ( 3 -
dimethylaminopropyl)carbodiimide; FAAH, fatty acid amide
hydrolaze; GapDH, glyceraldehyde 3-phosphate dehydrogen-
ase; GC−MS, gas chromatography−mass spectrometry; GPR,
G-protein-coupled receptor; GPR55, orphan G-protein-coupled
receptor; GPR119, orphan G-protein-coupled receptor; HOBt,
N-hydroxybenzotriazole; HRMS, high resolution mass spec-
trometry; LEA, linoleoylethanolamide; α-MSH, α-melanocyte-
stimulating hormone; MetS, metabolic syndrome; NHS, N-
hydroxysuccinimide; NMR, nuclear magnetic resonance; NPY,
neuropeptide Y; OEA, oleoylethanolamide; OVA, oleoylvali-
nolamide; PEA, palmitoylethanolamide; POMC, pro-opiome-
lanocortin; PVA, palmitoylvalinolamide; rt, room temperature;
RT-PCR, real time polymerase chain reaction; SVA,
stearoylvalinolamide; TEA, triethylamine; THC, tetrahydro-
cannabinol; TRPV1, transient receptor potential vanilloid 1
receptor; TMS, trimethylsilane
(18) Ahima, R. S.; Osei, S. Y. Adipokines in obesity. Front. Horm. Res.
2008, 36, 182−197.
(19) Dagon, Y.; Avraham, Y.; Magen, I.; Gertler, A.; Ben-Hur, T.;
Berry, E. M. Nutritional status, cognition, and survival: a new role for
leptin and AMP kinase. J. Biol. Chem. 2005, 280, 42142−42148.
(20) Dagon, Y.; Avraham, Y.; Berry, E. M. AMPK activation regulates
apoptosis, adipogenesis, and lipolysis by eIF2alpha in adipocytes.
Biochem. Biophys. Res. Commun. 2006, 340, 43−47.
(21) Borges, B. C.; Rorato, R.; Avraham, Y.; da Silva, L. E.C. M.;
Castro, M.; Vorobiav, L.; Berry, E. M.; Antunes-Rodrigues, J.; Elias, L.
L. K. Leptin resistance and desensitization of hypophagia during
prolonged inflammatory challenge. Am. J. Physiol.: Endocrinol. Metab.
2011, 300, E858−E869.
(22) Quarta, C.; Mazza, R.; Obici, S.; Pasquali, R.; Pagotto, U. Energy
balance regulation by endocannabinoids at central and peripheral
levels. Trends Mol. Med. 2011, 17, 518−526.
(23) Di Marzo, V.; Piscitelli, F.; Mechoulam, R. Cannabinoids and
endocannabinoids in metabolic disorders with focus on diabetes.
Handb. Exp. Pharmacol. 2011, 203, 75−104.
(24) Mechoulam, R.; Berry, E. M.; Avraham, Y.; Di Marzo, V.; Fride,
E. Endocannabinoids, feeding and sucklingfrom our perspective. Int.
J. Obes. 2006, 30, S24−S28.
(25) Berry, E. M.; Mechoulam, R. Tetrahydrocannabinol and
endocannabinoids in feeding and appetite. Pharmacol. Ther. 2002,
95, 185−190.
(26) Avraham, Y.; Ben-Shushan, D.; Breuer, A.; Zolotarev, O.; Okon,
A.; Fink, N.; Katz, V.; Berry, E. M. Very low doses of delta 8-THC
increase food consumption and alter neurotransmitter levels following
weight loss. Pharmacol., Biochem. Behav. 2004, 77675−77684.
(27) Hao, S.; Avraham, Y.; Mechoulam, R.; Berry, E. M. Low dose
anandamide affects food intake, cognitive function, neurotransmitter
and corticosterone levels in diet-restricted mice. Eur. J. Pharmacol.
2000, 392, 147−156.
(28) Reggio, P. H. Endocannabinoid binding to the cannabinoid
receptors: what is known and what remains unknown. Curr. Med.
Chem. 2010, 17, 1468−1486.
(29) Hornby, P. J.; Prouty, S. M. Involvement of cannabinoid
receptors in gut motility and visceral perception. Br. J. Pharmacol.
2004, 141 (8), 1335−1345.
REFERENCES
■
(1) Nguyen, D. M.; El-Serag, H. B. The epidemiology of obesity.
Gastroenterol. Clin. North Am. 2010, 39, 1−7.
(2) Fry, J.; Finley, W. The prevalence and costs of obesity in the EU.
Proc. Nutr. Soc. 2005, 64, 359−362.
(3) Dubnov-Raz, G.; Berry, E. M. The dietary treatment of obesity.
Med. Clin. North Am. 2011, 95, 939−952.
(4) Kaluski, D. N.; Berry, E. M. Prevalence of obesity in Israel. Obes.
Rev. 2005, 6, 115−116.
(5) Hussain, S. S.; Bloom, S. R. The pharmacological treatment and
management of obesity. Postgrad. Med. 2011, 123, 34−44.
(6) Field, B. C. T.; Chaudhri, O. B.; Bloom, S. R. Obesity treatment:
novel peripheral targets. Br. J. Clin. Pharmacol. 2009, 68, 830−843.
(7) Suzuki, K.; Simpson, K. A.; Minnion, J. S.; Shillito, J. C.; Bloom,
S. R. The role of gut hormones and the hypothalamus in appetite
regulation. Endocr. J. 2010, 57, 359−372.
(8) Park, A. J.; Bloom, S. Neuroendocrine control of food intake.
Curr. Opin. Gastroenterol. 2005, 21, 228−233.
(9) Murphy, K. G.; Bloom, S. R. Gut hormones and the regulation of
energy homeostasis. Nature 2006, 444, 854−859.
(30) Hansen, H. S.; Diep, T. A. N-Acylethanolamines, anandamide
and food intake. Biochem. Pharmacol. 2009, 78, 553−560.
(31) Zhu, Z.; Luo, Z.; Ma, S.; Liu, D. TRP channels and their
implications in metabolic diseases. Pfluegers Arch. 2011, 461, 211−223.
(32) Starowicz, K.; Cristino, L.; Di Marzo, V. TRPV1 receptors in the
central nervous system: potential for previously unforeseen therapeutic
applications. Curr. Pharm. Des. 2008, 14, 42−54.
(33) Avraham, Y.; Saidian, M.; Burston, J. J.; Mevorach, R.; Vorobiev,
L.; Magen, I.; Kunkes, E.; Borges, B.; Lichtman, A. H.; Berry, E. M.
1827
dx.doi.org/10.1021/jm300484d | J. Med. Chem. 2013, 56, 1811−1829

Journal of Medicinal Chemistry
Article
Fish oil promotes survival and protects against cognitive decline in
severely undernourished mice by normalizing satiety signals. J. Nutr.
Biochem. 2011, 22, 766−776.
(34) Anderson, K. A.; Ribar, T. J.; Lin, F.; Noeldner, P. K.; Green, M.
F.; Muehlbauer, M. J.; Witters, L. A.; Kemp, B. E.; Means, A. R.
Hypothalamic CaMKK2 contributes to the regulation of energy
balance. Cell Metab. 2008, 7, 377−388.
(35) Li, D. L.; Simmons, R. M.; Iyengar, S. 5HT1A receptor
antagonists enhance the functional activity of fluoxetine in a mouse
model of feeding. Brain Res. 1998, 781, 119−126.
(36) Gardier, A. M. Mutant mouse models and antidepressant drug
research: focus on serotonin and brain-derived neurotrophic factor.
Behav. Pharmacol. 2009, 20, 18−32.
high-affinity binding site involved in the transport of endocannabi-
noids. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17852−17857.
(52) Sheskin, T.; Hanus, L.; Slager, J.; Vogel, Z.; Mechoulam, R.
Structural requirements for binding of anandamide-type compounds to
the brain cannabinoid receptor. J. Med. Chem. 1997, 40, 659−667.
(53) Lin, S.; Khanolkar, A. D.; Fan, P.; Goutopoulos, A.; Qin, C.;
Papahadjis, D.; Makriyannis, A. Novel analogues of arachidonyletha-
nolamide (anandamide): affinities for the CB1 and CB2 cannabinoid
receptors and metabolic stability. J. Med. Chem. 1998, 41, 5353−5361.
(54) Adams, I. B.; Ryan, W.; Singer, M.; Thomas, B. F.; Compton, D.
R.; Razdan, R. K.; Martin, B. R. Evaluation of cannabinoid receptor
binding and in vivo activities for anandamide analogs. J. Pharmacol.
Exp. Ther. 1995, 273, 1172−1181.
(37) Borrelli, F.; Izzo, A. A Role of acylethanolamides in the
gastrointestinal tract with special reference to food intake and energy
balance. Best Pract. Res., Clin. Endocrinol. Metab. 2009, 23, 33−49.
(38) Astarita, G.; Di Giacomo, B.; Gaetani, S.; Oveisi, F.; Compton,
T. R.; Rivara, S.; Tarzia, G.; Mor, M.; Piomelli, D. Pharmacological
characterization of hydrolysis-resistant analogs of oleoylethanolamide
with potent anorexiant properties. J. Pharmacol. Exp. Ther. 2006, 318,
563−570.
(39) Chang, W. S.; Won, K. H.; Lee, J.;Y; Kim, E. T.; Kweon, H. J.
The relationship between obesity and the high probability of dementia
based on the body mass index and waist circumference. Korean J. Fam.
Med. 2012, 33, 17−24.
(55) Lambert, D. M.; Muccioli, G. G. Endocannabinoids and related
N-acylethanolamines in the control of appetite and energy
metabolism: emergence of new molecular players. Curr. Opin. Clin.
Nutr. Metab. Care 2007, 10, 735−744.
(56) Gomez, R.; Navarro, M.; Ferrer, B.; Trigo, J. M.; Bilbao, A.; Del
Arco, I.; Cippitelli, A.; Nava, F.; Piomelli, D.; De Fonseca, F. R. A
peripheral mechanism for CB1 cannabinoid receptor-dependent
modulation of feeding. J. Neurosci. 2002, 22, 9612−9617.
(57) Di Marzo, V.; Fontana, A.; Cadas, H.; Schinelli, S.; Cimino, G.;
Schwartz, J. C.; Piomelli, D. Formation and inactivation of endogenous
cannabinoid anandamide in central neurons. Nature 1994, 372, 686−
691.
(40) Hanus, L.; Avraham, Y.; Ben-Shushan, D.; Zolotarev, O.; Berry,
E. M.; Mechoulam, R. Short-term fasting and prolonged semi-
starvation have opposite effects on 2-AG levels in mouse brain. Brain
Res. 2003, 983, 144−151.
(41) Broberger, C.; Hokfelt, T. Hypothalamic and vagal neuropeptide
circuitries regulating food intake. Physiol. Behav. 2001, 74, 669−682.
(42) Gropp, E.; Shanabrough, M.; Borok, E.; Xu, A. W.; Janoschek,
R.; Buch, T.; Plum, L.; Balthasar, N.; Hampel, B.; Waisman, A.; Barsh,
G. S.; Horvath, T. L.; Bruning, J. C. Agouti-related peptide-expressing
neurons are mandatory for feeding. Nat. Neurosci. 2005, 8, 1289−
1291.
(58) Cadas, H.; Tomaso, E.; Piomelli, D. Occurrence and
biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl
phosphatidylethanolamine, in rat brain. J. Neurosci. 1997, 17, 1226−
1242.
(59) Fu, J.; Astarita, G.; Gaetani, S.; Kim, J.; Cravatt, B. F.; Mackie,
K.; Piomelli, D. Food intake regulates oleoylethanolamide formation
and degradation in the proximal small intestine. J. Biol. Chem. 2007,
282, 1518−1528.
(60) Berdyshev, E. V. Cannabinoid receptors and the regulation of
immune response. Chem. Phys. Lipids 2000, 108, 169−190.
(61) Calignano, A.; La Rana, G.; Giuffrida, A.; Piomelli, D. Control of
pain initiation by endogenous cannabinoids. Nature 1998, 394, 277−
281.
(62) Petrosino, S.; Iuvone, T.; Di Marzo, V. N-Palmitoyl-ethanol-
amine: biochemistry and new therapeutic opportunities. Biochimie
2010, 92, 724−727.
(63) Calignano, A.; La Rana, G.; Piomelli, D. Antinociceptive activity
of the endogenous fatty acid amide, palmitylethanolamide. Eur. J.
Pharmacol. 2001, 419, 191−198.
(64) Hoareau, L.; Buyse, M.; Festy, F.; Ravanan, P.; Gonthier, M..P.;
Matias, I.; Petrosino, S.; Tallet, F.; d’Hellencourt, C. L.; Cesari, M.; Di
Marzo, V.; Roche, R. Anti-inflammatory effect of palmitoylethanola-
mide on human adipocytes. Obesity 2009, 17, 431−438.
(65) Zhu, C.; Solorzano, C.; Sahar, S.; Realini, N.; Fung, E.; Sassone-
Corsi, P.; Piomelli, D. Proinflammatory stimuli control N-acylphos-
phatidylethanolamine-specific phospholipase D expression in macro-
phages. Mol. Pharmacol. 2011, 79, 786−792.
(66) Darmani, N. A.; Izzo, A. A.; Degenhardt, B.; Valenti, M.;
Scaglione, G.; Capasso, R.; Sorrentini, I.; Di Marzo, V. Involvement of
the cannabimimetic compound, N-palmitoyl-ethanolamine, in inflam-
matory and neuropathic conditions: review of the available pre-clinical
data, and first human studies. Neuropharmacology 2005, 48, 1154−
1163.
(43) Luquet, S.; Perez, F. A.; Hnasko, T. S.; Palmiter, R. D. NPY/
AgRP neurons are essential for feeding in adult mice but can be
ablated in neonates. Science 2005, 310, 683−685.
(44) Rodriguez de Fonseca, F.; Navarro, M.; Gomez, R.; Escuredo,
L.; Nava, F.; Fu, J.; Murillo-Rodriguez, E.; Giuffrida, A.; LoVerme, J.;
Gaetani, S.; Kathuria, S.; Gall, C.; Piomelli, D. An anorexic lipid
mediator regulated by feeding. Nature 2001, 414, 209−212.
(45) Dulloo, A. G. The search for compounds that stimulate
thermogenesis in obesity management: from pharmaceuticals to
functional food ingredients. Obes. Rev. 2011, 12, 866−883.
(46) Faiola, B.; Falls, J. G.; Peterson, R. A.; Bordelon, N. R.; Brodie,
T. A.; Cummings, C. A.; Romach, E. H.; Miller, R. T. PPAR alpha,
more than PPAR delta, mediates the hepatic and skeletal muscle
alterations induced by the PPAR agonist GW0742. Toxicol. Sci. 2008,
105, 384−394.
(47) Ezzili, C.; Otrubova, K.; Boger, D. L. Fatty acid amide signaling
molecules. Bioorg. Med. Chem. Lett. 2010, 20, 5959−5968.
(48) Fu, J.; Gaetani, S.; Oveisi, F.; Lo Verme, J.; Serrano, A.; de
Fonseca, F. R.; Rosengarth, A.; Luecke, H.; Di Giacomo, B.; Tarzia, G.;
Piomelli, D. Oleylethanolamide regulates feeding and body weight
through activation of the nuclear receptor PPAR-alpha. Nature 2003,
425, 90−93.
(49) Terrazzino, S.; Berto, F.; Carbonare, M. D.; Fabris, M.; Guiotto,
A.; Bernardini, D.; Leom, A. Stearoylethanolamide exerts anorexic
effects in micevia down-regulation of liver stearoyl-coenzyme A
desaturase-1 mRNA expression. FASEB J. 2004, 18, 1580−1582.
(50) Walter, L.; Franklin, A.; Witting, A.; Moller, T.; Stella, N.
Astrocytes in culture produce anandamide and other acylethanola-
mides. J. Biol. Chem. 2002, 277, 20869−20876.
(51) Moore, S. A.; Nomikos, G. G.; Dickason-Chesterfield, A. K.;
Schober, D. A.; Schaus, J. M.; Ying, B.-P.; Xu, T. C.; Phebus, L.;
Simmons, R. M. A.; Li, D.; Iyengar, S.; Felder, C. C. Identification of a
(67) Lambert, D. M.; Vandevoorde, S.; Jonsson, K. O.; Fowler, C. J.
The palmitoylethanolamide family: a new class of anti-inflammatory
agents? Curr. Med. Chem. 2002, 9, 663−674.
(68) Godlewski, G.; Offertaler, L.; Wagner, J. A.; Kunos, G.
Receptors for acylethanolamides-GPR55 and GPR119. Prostaglandins
Other Lipid Mediators 2009, 89, 105−111.
(69) Lo Verme, J.; Fu, J.; Astarita, G.; La Rana, G.; Russo, R.;
Calignano, A.; Piomelli, D. The nuclear receptor peroxisome
proliferator-activated receptor-alpha mediates the anti-inflammatory
actions of palmitoylethanolamide. Mol. Pharmacol. 2005, 67, 15−19.
1828
dx.doi.org/10.1021/jm300484d | J. Med. Chem. 2013, 56, 1811−1829

Journal of Medicinal Chemistry
Article
(70) Terrazzino, S.; Berto, F.; Dalle Carbonare, M.; Fabris, M.;
Guiotto, A.; Bernardini, D.; Leon, A. Stearoylethanolamide exerts
anorexic effects in mice via down-regulation of liver stearoyl-coenzyme
A desaturase-1 mRNA expression. FASEB. J. 2004, 18, 1580−1582.
(71) Bradshaw, H. B.; Walker, J. M. The expanding field of
cannabimimetic and related lipid mediators. Br. J. Pharmacol. 2005,
144, 459−465.
(72) Movahed, P.; Jonsson, B. A.; Birnir, B.; Wingstrand, J. A.;
Jorgensen, T. D.; Ermund, A.; Sterner, O.; Zygmunt, P. M.; Hogestatt,
E. D. Endogenous unsaturated C18 N-acylethanolamines are vanilloid
receptor (TRPV1 agonists.). J. Biol. Chem. 2005, 280, 38496−38504.
(73) Wang, X. B.; Miyares, R. L.; Ahern, G. P. Oleoylethanolamide
excites vagal sensory neurones, induces visceral pain and reduces short-
term food intake in mice via capsaicin receptor TRPV1. J. Physiol.
2005, 564, 541−547.
(74) Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson,
L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.;
Mechoulam, R. Isolation and structure of a brain constituent that
binds to the cannabinoid receptor. Science 1992, 258, 1946−1949.
(75) Ahern, G. P. Activation of TRPV1 by the satiety factor
oleoylethanolamide. J. Biol. Chem. 2003, 278, 30429−30434.
(76) Price, T. J.; Patwardhan, A.; Akopian, A. N.; Hargreaves, K. M.;
Flores, C. Modulation of trigeminal sensory neuron activity by the dual
cannabinoid-vanilloid agonists anandamide, N-arachidonoyl-dopamine
and arachidonyl-2-chloroethylamide. Br. J. Pharmacol. 2004, 141,
1118−1130.
receptors: insights gained from (E)- and (Z)-naphthylidene indenes. J.
Med. Chem. 1998, 41, 5177−5187.
(88) Szallasi, A.; Goso, C.; Blumberg, P. M.; Manzini, S. Competitive
inhibition by capsazepine of [3H]resiniferatoxin binding to central
(spinal cord and drosal root ganglia) and peripheral (urinary bladder
and airways) vanilloid (capsaicin) receptors in the rat. J. Pharmacol.
Exp. Ther. 1993, 267, 728−733.
(89) Zhou, G.; Cummings, R.; Li, Y.; Mitra, S.; Wilkinson, H. A.;
Elbrecht, A., D.; Hermes, J. D.; Schaeffer, J. M.; Smith, R. G.; Moller,
D. E. Nuclear receptors have distinct affinities for coactivators:
characterization by fluorescence resonance energy transfer. Mol.
Endocrinol. 1998, 12, 1594−1604.
(90) Tiger, G.; Stenstrom, A.; Fowler, C. J. Pharmacological
properties of rat brain fatty acid amidohydrolase in different subcellular
fractions using palmitoylethanolamide as substrate. Biochem. Pharma-
col. 2000, 59, 647−653.
(77) Maccarrone, M.; Cartoni, A.; Parolaro, D.; Margonelli, A.;
Massi, P.; Bari, M.; Battista, N.; Finazzi-Agro, A. Cannabimimetic
activity, binding, and degradation of stearoylethanolamide within the
mouse central nervous system. Mol. Cell. Neurosci. 2002, 21, 126−140.
(78) Zygmunt, P. M.; Petersson, J.; Andersson, D. A.; Chuang, H. H.;
Sorgard, M.; Di Marzo, V.; Julius, D.; Hogestatt, E. D. Vanilloid
receptors on sensory nerves mediate the vasodilator action of
anandamide. Nature 1999, 400, 452−457.
(79) Overton, H. A.; Babbs, A. J.; Doel, S. M.; Fyfe, M. C. T.;
Gardner, L. S.; Griffin, G.; Jackson, H. C.; Procter, M. J.; Rasamison,
C. M.; Tang-Christensen, M.; Widdowson, P. S.; Williams, G. M.;
Reynet, C. Deorphanization of a G protein-coupled receptor for
oleoylethanolamide and its use in the discovery of small-molecule
hypophagic agents. Cell Metab. 2006, 3, 167−175.
(80) Fu, J.; Oveisi, F.; Gaetani, S.; Lin, E.; Piomelli, D.
Oleoylethanolamide, an endogenous PPAR-alpha agonist, lowers
body weight and hyperlipidemia in obese rats. Neuropharmacology
2005, 48, 1147−1153.
(81) Avraham, Y.; Bonne, O.; Berry, E. M. Behavioral and
neurochemical alterations caused by diet restriction-the effect of
tyrosine administration in mice. Brain Res. 1996, 732, 133−144.
(82) Olton, D. S.; Samuelson, R. J. Remembrance of places passed:
spatial memory in rats. J. Exp. Psychol. 1976, 2, 97−116.
(83) Pick, C. G.; Yanai, J. Eight arm maze for mice. Int. J. Neurosci.
1983, 21, 63−66.
(84) Avraham, Y.; Grigoriadis, N. C.; Magen, I.; Poutahidis, T.;
Vorobiav, L.; Zolotarev, O.; Ilan, Y.; Mechoulam, R.; Berry, E. M.
Capsaicin affects brain function in a model of hepatic encephalopathy
associated with fulminant hepatic failure in mice. Br. J. Pharmacol.
2009, 158, 896−906.
(85) Chen, Y.; Constantini, S.; Trembovler, V.; Weinstock, M.;
Shohami, E. An experimental model of closed head injury in mice:
pathophysiology, histopathology, and cognitive deficits. J. Neurotrauma
1996, 13, 557−568.
(86) Avraham, Y.; Zolotarev, O.; Grigoriadis, N. C.; Poutahidis, T.;
Magen, I.; Vorobiav, L.; Zimmer, A.; Ilan, Y.; Mechoulam, R.; Berry, E.
M. Cannabinoids and capsaicin improve liver function following
thioacetamide-induced acute injury in mice. Am. J. Gastroenterol. 2008,
103, 3047−5306.
(87) Reggio, P. H.; Basu-Dutt, S.; Barnett-Norris, J.; Castro, M. T.;
Hurst, D. P.; Seltzman, H. H.; Roche, M. J.; Gilliam, A. F.; Thomas, B.
F.; Stevenson, L. A.; Pertwee, R. G.; Abood, M. E. The bioactive
conformation of aminoalkylindoles at the cannabinoid CB1 and CB2
1829
dx.doi.org/10.1021/jm300484d | J. Med. Chem. 2013, 56, 1811−1829