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A neutral CB1 receptor antagonist reduces weight gain in rat.
Adam P. Chambers1, V. Kiran Vemuri2, Yan Peng2, JodiAnne T. Wood2, Teresa
Olszewska2, Quentin J. Pittman1, Alexandros Makriyannis2, Keith A. Sharkey1.
1Hotchkiss Brain Institute and Institute of Infection, Immunity and Inflammation,
Department of Physiology & Biophysics, University of Calgary, AB, Canada;
2Center for Drug Discovery, Northeastern University, Boston, MA, USA.
These studies were supported by grants from Canadian Institutes of Health
Research (to QJP and KAS) and from National Institutes of Health, U.S.A
(DA09158, DA7215, DA3801 to AM). APC is an Alberta Heritage Foundation for
Medical Research (AHFMR) Graduate Student. QJP and KAS are AHFMR Medical
Scientists. KAS is the Crohn’s and Colitis Foundation of Canada Chair in
Inflammatory Bowel Disease Research and QJP holds a University Professorship.
*Correspondence should be addressed to these authors at:
Keith A. Sharkey, PhD, Department of Physiology & Biophysics, University of
Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada; Tel.: 1-403-220-
4601; Fax: 1-403-283-3028; Email: ksharkey@ucalgary.ca
Alexandros Makriyannis, PhD, Center for Drug Discovery, 116 Mugar Hall,
Northeastern University, Boston, MA, 02115 USA; Tel: 1-617-373-4200; Fax: 1-
617-373-8886; Email: a.makriyannis@neu.edu
Running Head: AM4113 Reduces Weight Gain In Rat
Page 1 of 40Articles in PresS. Am J Physiol Regul Integr Comp Physiol (October 24, 2007). doi:10.1152/ajpregu.00663.2007
Copyright © 2007 by the American Physiological Society.
AM4113 Reduces Weight Gain In Rat 2
ABSTRACT
Cannabinoid (CB) 1 receptor inverse agonists inhibit food intake in animals and
humans, but also potentiate emesis. It is not clear whether these effects result
from inverse agonist properties or from the blockade of endogenous cannabinoid
signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on
food intake, weight gain and emesis. Neutral antagonist and binding properties
were confirmed in HEK293 cells transfected with human CB1 or CB2 receptors.
AM4113 had no effect on forskolin stimulated cAMP production at concentrations
up to 630 nM. The Ki value of AM4113 (0.80±0.44nM) in competitive binding
assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1
over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in
brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg kg-1)
significantly reduced food intake and weight gain in rat. Compared with AM251,
higher doses of AM4113 were needed to produce similar effects on food intake
and body weight. Unlike AM251 (5 mg kg-1), a highly anorectic dose of AM4113
(10 mg kg-1) did not significantly potentiate vomiting induced by the emetic
morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor
antagonist shares the appetite suppressant and weight loss effects of inverse
agonists. If these compounds display similar properties in humans they could be
developed into a new class of anti-obesity agents.
Keywords: cannabinoid receptor, food intake, inverse agonist, obesity, emesis,
hypothermia.
Page 2 of 40
AM4113 Reduces Weight Gain In Rat 3
INTRODUCTION
Cannabinoid (CB)1 G-protein coupled receptors, along with endogenous
cannabinoids (endocannabinoids), including anandamide and 2-
arachidonylglycerol (2-AG) and the biosynthetic and degradative enzymes for
these ligands are found in pathways involved in feeding and metabolism (33).
These pathways include vagal afferents (5), area postrema and brainstem nuclei
(40; 56), hypothalamus (11; 52), and reward pathways (29; 50). In the periphery,
CB1 receptors and their ligands are also found in the gut (20), liver (43), and
adipose tissue (2; 11; 28). Vagal afferents, area postrema and brainstem are
also involved in emesis (26; 55; 56).
Therapeutically, CB1 receptor agonists are used as anti-emetics and to
promote appetite during serious illness such as cancer (14) and acquired
immune deficiency syndrome (AIDS) (58). In the case of cancer, nausea and
emesis induced by chemo- and radiation therapy is significantly attenuated by the
active ingredient in marijuana, delta-9-tetrahydrocannbinol, and other CB1
receptor agonists (14; 58). CB1 receptor inverse agonists such as SR141716A
and the structurally, and pharmacologically similar compound AM251 are also
therapeutically relevant. These agents suppress appetite, promote weight loss,
and improve cardiometabolic risk factors in humans (17; 46; 54) and rodents (7;
19; 22; 23; 25; 28; 47; 53; 57). However, significant psychological and
physiological concerns in patients treated with CB1 receptor inverse agonists are
also present including increased risks of nausea and vomiting (17; 46; 54).
Page 3 of 40
AM4113 Reduces Weight Gain In Rat 4
Previously, CB1 receptor inverse agonists were shown to promote emesis in
ferret (56) and shrew (13), and promote nausea (37) in rat.
At CB1 receptors, agonist activity decreases adenylyl cyclase and the
conversion of adenosine triphosphate (ATP) to cyclic adenosine 3’,5’-
monophosphate (cAMP) (18; 45). Like an antagonist, inverse agonists block
receptor binding and activation by a competitive agonist, but in addition, they also
inhibit intrinsic or spontaneous receptor signaling (42). The result is an effect
opposite to that produced by a given agonist and is termed inverse agonism.
Although no valid method for assessing inverse agonist activity in vivo exists
(42), the inhibition of intrinsic CB1 receptor transduction can be shown in vitro by
the increased expression of adenylyl cyclase and cAMP in cultured cells
transfected with CB1 receptors (35; 39; 51). Whether the physiological and
behavioral effects of CB1 receptor inverse agonists result from the ability of these
compounds to inhibit intrinsic receptor activity, or, from the pharmacological
blockade of endogenous cannabinoid signaling, or, both, is not known. However,
a recent study by Sink et al, 2007 (51) describing a novel CB1 receptor
antagonist, AM4113, with no inverse agonist properties shows that such
determinations are now possible. These authors demonstrated that AM4113
reduced food intake in rat with no effect on conditioned gaping, a specific marker
of nausea in that species (51), suggesting that pharmacological blockade of CB1
receptors alone is sufficient to reduce food intake independent of nausea.
However, in that study food intake was only examined during a 30 min
observation period (51). The effect of a neutral CB1 receptor antagonist on daily
Page 4 of 40
AM4113 Reduces Weight Gain In Rat 5
food intake and body weight is not known. Given that in some cases (2; 10; 23;
28; 47; 49), but not others (7; 53; 57), weight loss induced by CB1 receptor
inverse agonists is unrelated to their effect on feeding, studies that examine the
effect of neutral CB1 receptor antagonism on body weight are crucial. In addition,
rats lack the neuronal pathways required for vomiting, and the effect of a neutral
CB1 receptor on emesis, which is distinct from nausea, is also unknown. In the
present study, we confirm the neutral antagonist and binding properties of
AM4113. We then test the ability of AM4113 to antagonize hypothermia induced
by the non-selective cannabinoid CP55,940 in rat. We chose this assay because
the mild hypothermia caused by cannabinoid agonists is mediated by CB1
receptors in brain (4; 21), enabling us to determine if AM4113 crossed the blood
brain barrier. We also examine the effect of AM4113 on food intake and body
weight in rats treated daily for 5 d and compare the acute effect of the antagonist
on food intake with the inverse agonist AM251 directly. Finally, we examine the
effect of AM4113 on emesis in ferrets treated with the emetic morphine-6-
glucoronide (M6G) using vehicle and AM251 treated ferrets for comparison.
MATERIALS & METHODS
Forskolin-Stimulated cAMP Assay
Intracellular cAMP levels were measured with a competitive protein-
binding assay using intact HEK293 cells expressing hCB1 or hCB2 receptors, and
a cAMP immunoassay kit from Sigma (St. Louis, MO, USA). In short, forskolin
stimulated cells were incubated with various concentrations of AM4113 or
Page 5 of 40
AM4113 Reduces Weight Gain In Rat 6
AM251, cAMP antibody and cAMP conjugate for 2 hours at ambient temperature.
The reaction was stopped by emptying the wells followed by the addition of p-
NPP substrate and incubation for 1 hour. Absorbance intensity, detected at 405
nm, was inversely proportional to the concentration of cAMP produced by the
cells. The results are expressed as the percent stimulation of forskolin-
stimulated cAMP accumulation.
[3H]CP55,940 Competitive Binding Assay
Compounds were tested for their CB1 and CB2 receptor affinity using
membrane preparations from rat brain (CB1 receptors) or HEK293 cells
expressing human CB2 (hCB2) receptor, respectively, and [3H]CP55,940 as
previously described (30; 32; 35; 41). Stock solutions of the compounds [10 mM
in dimethyl sulfoxide (DMSO)] were diluted in TME buffer (50 mM Tris-HCL, 3
mM MgCl2, 100 mM NaCl, 0.2 mM EDTA, pH 7.4) with 0.1% BSA and
transferred to 96 well plates containing [3H]CP55,940 (specific activity 128
Ci/mmol; NIDA) at a final concentration of 0.76 nM. Non-specific binding was
assessed in the presence of 100 nM CP55,940. The binding reaction was
initiated with the addition of the respective membrane suspension (~50 µg
membrane protein) followed by incubation at 30°C with gentle agitation in a
shaking water bath for 60 minutes. Binding was terminated by rapid filtration of
the membrane suspension over Unifilter GF/B-96 Well Filter Plates (Packard
Instruments) using a Packard Filtermate-196 Cell Harvester. The filter plates
were washed with ice-cold wash buffer (50 mM Tris-base, 5 mM MgCl2 with 0.5%
Page 6 of 40
AM4113 Reduces Weight Gain In Rat 7
BSA) and bound radioactivity was determined using a Packard TopCount
Scintillation Counter. All data were in duplicate with IC50 and Ki values
determined from at least two independent experiments.
Behavioral Experiments
All experimental protocols were approved by the University of Calgary Animal
Care Committee, and were carried out in accordance with the guidelines of the
Canadian Council on Animal Care. Different concentrations of drugs were
administered i.p. in a 1 ml kg-1 volume, except M6G, which was given s.c. in a 0.1
ml kg-1 volume.
Antagonism by AM4113 at CB1 receptors in brain
We examined hypothermia induced by the CB1 agonist CP55,940 (0.3 mg
kg-1) (15) in male Sprague Dawley rats (Charles River, Montreal, Qc, Canada)
pre-treated 45 min earlier with either vehicle, AM251 (5 mg kg-1, n=5) (7), or
AM4113 (5 mg kg-1, n=5). Briefly, silicone coated temperature data loggers
(SubCue Inc, Calgary, Alta, Canada) were surgically implanted into the
abdominal cavity of rats (450-500g) under isoflurane anesthesia (4% induction; 2-
2.5% maintenance). Rats recovered for 3 days before being acclimatized to
testing and handling procedures for an additional seven days. During the
experiment core body temperature readings were sampled every 5 min for 300
min. Apart from the injection procedure the rats were not handled during the
experiment.
Page 7 of 40
AM4113 Reduces Weight Gain In Rat 8
Food Intake Studies
Male Sprague-Dawley rats weighing between 330-380 g at the start of the
study were used to examine the effect of AM4113 on food intake and body
weight. Animals were fed strawberry flavored Ensure Plus® liquid diet (53.3%
carbohydrate, 29% fat, and 16.7% protein; 1.41 kcal g-1) (Abbott Laboratories,
Abbott Park, IL, USA) to promote food intake (1) and control for spillage. Rats
were habituated to testing and handling procedures, daily, for seven days prior to
testing. Food and water were presented in drip-free inverted glass bottles
attached to the outside of the cage. Food was available for 18 h each day starting
at 16:00 h (12 h light-dark cycle; lights off 16:00 h). Bottles were removed and
washed at 09:00 h, a time during the light-dark cycle when rats are generally
inactive.
Experiment 1
In experiment 1 we examined the effect of AM4113 (1 mg kg-1; 5 mg kg-1)
in rats treated daily for 5 days with the neutral antagonist. Doses were chosen
based on our thermoregulation data and previous work by our group with the
inverse agonist AM251 (7; 8; 36; 37). The day before the experiment rats were
assigned to one of three treatment groups: vehicle [mean bodyweight ± standard
error of the mean (SEM); (363±10 g, n=5)], AM4113 1 mg kg-1 (365±5 g, n=5),
AM4113 5 mg kg-1 (361±9 g, n=5). Food intake and body weight were monitored
daily for 5 d.
Experiment 2
Page 8 of 40
AM4113 Reduces Weight Gain In Rat 9
In experiment 2 the effect of AM4113 (10; 20 mg kg-1) on food intake and
body weight were compared to rats treated with vehicle or the inverse agonist
AM251 (5 mg kg-1). On the day before the experiment rats were randomly
assigned to one of four treatment groups: vehicle (492±8 g, n=6), AM251 5 mg
kg-1 (496±11 g, n=6), AM4113 10 mg kg-1 (491±7 g, n=6), or AM4113 20 mg kg-1
(489±9 g, n=6). Rats were injected between 15:00 and 16:00 h on the day of the
experiment. Treatments were counterbalanced to avoid order effects. After
treatment food was made available as described in experiment 1. Food intake
was measured 1.5, 3, 5, and 18 h after treatment. After the experiment food
intake and body weight were monitored for another five days to assess the
sustained effect of each compound after a single treatment.
Emesis Studies
We examined how AM4113 (10 mg kg; n=5) affected vomiting in ferrets
(Marshall Farms, North Rose, NY, USA) using vehicle (n=6) and AM251 (5 mg
kg-1) treated ferrets for comparison. Each treatment was given 15 min prior to
receiving the emetic M6G (0.05 mg kg-1) (56) . Ferrets were lightly anaesthetized
with halothane for each injection. Data were videotaped and analyzed by an
observer blinded to the conditions of each treatment group. During 60 min of
observation the number of vomiting (emetic) episodes were counted, and activity
or sleeping time was noted. In a second experiment the effect of two additional
doses of AM4113 (5; 20 mg kg-1; n=5/5) on M6G induced emesis were
examined. An additional group of vehicle (n=6) treated ferrets were used for
Page 9 of 40
AM4113 Reduces Weight Gain In Rat 10
comparison. Experiments were carried out using a counterbalance design to
avoid order effects.
Statistics
EC50 curves were generated from the forskolin-stimulated cAMP assay
data by non-linear regression with the use of GraphPad Prism software
(GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego
California USA). Data are expressed as mean % change in forskolin stimulated
cAMP accumulation ± SEM. Results from the competitive binding assay were
analyzed using nonlinear regression to determine the actual IC50 and the Ki
values of the ligand (GraphPad Prism) (9). Data are expressed as mean Ki in nM
± standard deviation. Food intake and thermoregulation data were analyzed
using a 2-way mixed design ANOVA with time as the repeated measure; Food
intake data are expressed as mean food intake in kilocalories (kcal) ± SEM. Body
temperature data are expressed in °C ± SEM. Significant differences were
followed up with 1-way independent measures ANOVA at each time point.
Significant differences between treatments were further analyzed using Newman-
Keuls multiple comparisons test. The emesis data comparisons between vehicle,
AM4113 (10 mg kg-1) and AM251 (5 mg kg-1) were made using a one-way
analysis of variance. Significant differences were followed up using Newman-
Keuls multiple comparisons test. Data examining the effect of additional doses of
AM4113 (0-20 mg kg-1) on emesis were analyzed using a one-way analysis of
Page 10 of 40
AM4113 Reduces Weight Gain In Rat 11
variance, and then by linear regression analysis. Emesis data are expressed as
mean number of vomiting episodes ± SEM.
Compounds
AM4113; N-piperidin-1-yl-2,4-dichlorophenyl-1H-pyrazole-3-carboxamide
analog (partial designation). AM4113 is a pyrazole congener of AM251 with a
very similar molecular weight. AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide. AM251 and AM4113 were
synthesized at Northeastern University. CP55,940 (-)-cis-3-[2-Hydroxy-4-(1,1-
dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; Tocris Cookson
Inc., Ellisville, MO, USA or NIDA, USA), and morphine-6-glucoronide (M6G,
Lipomed Inc., Arlesheim, BL, Switzerland). All compounds were dissolved in
DMSO using gentle heating and sonication before being diluted with Tween 80
and saline (4% DMSO; 1% Tween 80; 95% saline), and given in a volume of 1 ml
kg-1. Differences between baseline and vehicle (4% DMSO; 1% Tween 80; 95%
saline) conditions were non-significant at all times.
RESULTS
Forskolin-Stimulated cAMP Assay
Consistent with results reported by others (51) AM4113 did not change the
forskolin-stimulated cAMP accumulation in CB1 transfected HEK cells at
concentrations up to 630 nM and is therefore considered to be a CB1 neutral
antagonist (data not shown). In comparison, the inverse agonist AM251
Page 11 of 40
AM4113 Reduces Weight Gain In Rat 12
increased forskolin stimulated cAMP production in a concentration dependent
manner (EC50 56.4 nM; 95% confidence interval 5.5-573.8 nM, R2=0.49). The
results are from one assay performed in duplicate.
[3H]CP55,940 Competitive Binding Assay
In competitive receptor binding assays against [3H]CP55,940 AM4113
showed selectivity for the CB1 receptor compared to CB2. AM4113 bound to
cannabinoid receptors with a CB1 Ki of 0.80 nM and a CB2 Ki of 97 nM, indicating
that AM4113 exhibits at least a 100-fold selectivity for CB1 versus CB2 receptors
consistent with data by others (51). In comparison, AM251 bound to cannabinoid
receptors with a CB1 Ki of 3.43 nM and a CB2 Ki of 124 nM (data not shown).
AM4113 in Brain: Thermoregulation Assay
To determine whether AM4113 antagonized CB1 receptors in brain we
examined hypothermia induced by CP55,940 in rats pretreated with either
vehicle, AM251 (5 mg kg-1), or AM4113 (5 mg kg-1). Figure 1a shows changes in
body temperature over time. A 2-Way ANOVA performed on the body
temperature data showed a significant treatment by time interaction, F=4.4,
P<0.001. Differences between treatments were analyzed using a 1-way ANOVA
at selected time points (Figure 1b). Prior to treatment with CP55,940, differences
in body temperature between groups were non significant, P>0.05, indicating
that AM4113 and AM251 alone had no effect on core body temperature. As
expected, body temperature rapidly fell in vehicle treated rats after the
Page 12 of 40
AM4113 Reduces Weight Gain In Rat 13
administration of CP55,940 (15). In comparison, the hypothermic effect of
CP55,940 was completely blocked in rats treated with the neutral antagonist
AM4113, P<0.05, and significantly attenuated in rats pre-treated with inverse
agonist AM251, P<0.05.
Food Intake & Body Weight Studies
In experiment 1 we examined how daily treatment with AM4113 (1, 5, mg
kg-1) affected food intake over 5 days (Figure 2a). A 2-way ANOVA performed on
the food intake data showed that there was a significant treatment by time
interaction, F= 3.3, P<0.001. There was also a significant effect of dose, F=3.6,
P< 0.001. 1-way ANOVA performed between treatments showed that food intake
was significantly reduced in rats treated with 5 mg kg-1 of AM4113 on days 3, 4,
and 5 compared with vehicle and 1 mg kg-1 treated rats, P<.05, Newman-Keuls
Multiple Comparison Test.
Reductions in food intake by AM4113 (5 mg kg-1) were accompanied by
significant reductions in weight gain (Figure 2b). A 2-way ANOVA revealed a
significant effect of dose F=15.2, P<0.001, and, time F=15.4, P<0.001. Rats
treated with 5 mg kg-1 of AM4113 gained significantly less weight than rats
treated with 1 mg kg-1 of the neutral antagonist on day 4 (-16.9 kcal), P<0.05, and
significantly less weight than vehicle treated rats on day 4 (-22.0 kcal), day 5 (-
30.7 kcal), and day 6 (-33.4 kcal). Although rats treated daily with the 1 mg kg-1
dose of AM4113 tended to eat less, and gain less weight, than vehicle treated
rats such differences were not significant, P>0.05. The results demonstrate that
Page 13 of 40
AM4113 Reduces Weight Gain In Rat 14
repeated administrations of a 5 mg kg-1 dose of the neutral antagonist AM4113
significantly reduced food intake and weight gain in rat.
In experiment 2, we examined the acute effect of AM4113 on food intake
by evaluating two additional doses of the neutral antagonist (10; 20 mg kg-1)
compared with vehicle treated rats, and rats treated with the inverse agonist
AM251 (5 mg kg-1). Figure 3a shows the early effect of AM4113 and AM251 on
food intake. Results from a 2-way ANOVA performed on the data revealed a
significant treatment by time interaction, F=18.1, P<0.001. At each time point
food intake was reduced in rats treated with AM4113 (10; 20 mg kg-1) compared
to vehicle treated rats; 1.5 h (-20.2 kcal, -21.2 kcal), 3 h (-28.2 kcal, -30.0 kcal), 5
h (-32.1 kcal, -42.4 kcal), and 18 h (-59.2 kcal, -85.4 kcal), P<0.05. As expected
(8), AM251 produced a similar effect on feeding, P<0.05. Differences in food
intake between 10 and 20 mg kg-1 treatment groups were significant 18 h after
treatment, P<0.001. Differences between rats treated with AM251 and AM4113
were significant at 3, 5, and 18 h time points, P<0.05. The results demonstrate
that the neutral receptor antagonist AM4113 produced early, and dose-
dependent, reductions in food intake in rat.
Furthermore, continued monitoring revealed that the anorectic effect of the
neutral antagonist AM4113 (10; 20 mg kg-1) was sustained for several days after
treatment. Figure 3b shows the mean daily food intake in each group before and
after the experiment. A 2-way ANOVA performed on the daily food intake data
revealed a significant treatment by time interaction, F=12.7, P<0.001. Prior to
treatment, differences in food intake between groups were non-significant,
Page 14 of 40
AM4113 Reduces Weight Gain In Rat 15
P>0.05. After treatment, food intake was reduced in rats given AM251 or
AM4113 relative to vehicle treated rats, P<0.05. The sustained effect of an acute
administration of AM251 has been reported previously by our group (7; 8). Here
we report that reductions in food intake by the neutral antagonist AM4113 were
similar in duration to the anorectic effect of AM251.
Figure 3c shows that reductions in food intake by AM251 and the neutral
antagonist AM4113 (10; 20 mg kg-1) were also associated with significant
reductions in weight gain. Results from a 2-way ANOVA performed on the weight
change data showed that there was a significant treatment by time interaction,
F=10.9, P<0.001. Weight gain was significantly reduced in rats treated with
AM4113 or AM251 for several days after the experiment; P<0.05, Newman-Keuls
Multiple Comparisons Test. A 2-way ANOVA performed on the weight change
data also showed a significant effect of dose, F=27.9, P<0.001. Differences in
weight gain between 10 and 20 mg kg-1 AM4113 treatment groups were
significant on day 5 (-12.3 g), P<0.05. The results demonstrate that a single
administration of the neutral antagonist AM4113 (10;20 mg kg-1) produced a
dose-dependent reduction in weight gain and food intake.
Emesis Studies
We examined how AM4113 (10 mg kg; n=5) affected emesis in ferrets
compared with vehicle (n=6) treated ferrets, or, ferrets treated with the inverse
agonist AM251 (5 mg kg-1). Each ferret was given M6G 15 min after treatment to
induce emesis (Figure 4a). Doses were chosen based on work from our feeding
Page 15 of 40
AM4113 Reduces Weight Gain In Rat 16
studies showing that a 10 mg kg-1 dose of AM4113 produced roughly the same
effect on food intake as a 5 mg kg-1 dose of AM251 (Figure 3). A 1-way ANOVA
performed on the data showed that there was a significant effect of treatment,
F=4.1, P<0.05, caused by increased vomiting in AM251 treated ferrets compared
to vehicle treatment, P<0.05, Newman-Keuls Multiple comparison test. No
significant difference in vomiting episodes between AM4113 and vehicle treated
ferrets was observed. However, there was also no significant difference between
ferrets treated with AM4113 and AM251, P>0.05. In order to test the effect of
AM4113 on emesis more rigorously, two additional doses of AM4113 (0;5;20 mg
kg-1) were examined and compared to a new group of vehicle treated ferrets
(Figure 4b). A 1-way ANOVA performed on the data revealed no significant effect
of treatment, P>0.05. However, when the data was analyzed using linear
regression analysis a significant relationship between the dose of AM4113 and
number of vomiting episodes was shown, r2=0.96, P=0.04. The results suggest
that although AM4113 is less pro-emetic than the inverse agonist, AM251,
pharmacological blockade of endogenous cannabinoid signaling is capable of
affecting emesis in ferret.
DISCUSSION
We compared the effects of a novel CB1 receptor neutral antagonist,
AM4113, on food intake and body weight with those of the well characterized
inverse agonist AM251. Neutral antagonist properties were established in
HEK293 cells transfected with human CB1 receptors. We confirmed that AM4113
Page 16 of 40
AM4113 Reduces Weight Gain In Rat 17
acted as a neutral antagonist in vitro in HEK cells transfected with human CB1
receptors. Consistent with results by others (51), AM4113 had no effect on cAMP
at concentrations up to 630 nM and displayed low Ki values in competitive
binding assays with the CB1 agonist [3H]CP55,940. In vivo, AM4113 antagonized
the hypothermic effect of the CB1 receptor agonist CP55,940 and significantly
reduced food intake and weight gain in rat. Compared with AM251, higher doses
of the neutral antagonist AM4113 were needed to produce similar effects on food
intake and weight gain. Unlike AM251, AM4113 did not significantly increase
vomiting induced by the emetic M6G, however, there was a positive correlation
between dose and vomiting in ferrets treated with the neutral antagonist.
The fact that AM4113 blocked the hypothermic effect of a CB1 agonist
demonstrates that the neutral antagonist crosses the blood-brain barrier,
because the hypothermic effect of cannabinoid agonists is specific to activity at
CB1 receptors in the anterior hypothalamus (4; 21). Consistent with these
observations, preliminary studies performed on mice in our lab show that 0.4-
0.7% (Initial Dose/g brain) of an intravenously injected 2 mg/kg dose is found in
brain 15 minutes post-injection (data not shown). Together with a high brain
(µg/g) to plasma (µg/mL) ratio (0.49 ± 0.029), these data show that AM4113
crosses the blood-brain-barrier to affect CB1 receptors in the CNS. Moreover,
both AM4113 and AM251, alone, had no effect on core body temperature in the
45 min prior to treatment with the CB1 agonist CP55,940. These data
demonstrates that endogenous cannabinoid tone is not involved in
thermoregulation under the conditions we described, even with the inhibition of
Page 17 of 40
AM4113 Reduces Weight Gain In Rat 18
intrinsic CB1 receptor activity by AM251. Similar effects on temperature with CB1
receptor inverse agonists were shown previously (15; 38).
In contrast to our thermoregulation assay, larger doses of the neutral
antagonist AM4113 were needed to elicit the same effect on food intake as
AM251. AM4113 only affected daily food intake and body weight in rat at a 5 mg
kg-1 dose after several treatments. Whereas the same dose of the inverse
agonist, AM251, produced an immediate and sustained anorectic effect that
lasted for several days. If reductions in food intake by these agents were
produced solely from pharmacological blockade at CB1 receptors alone, then
matching doses of each compound should produce similar effects on feeding.
This was not the case in our study. Instead, our data suggest that the inverse
agonist properties of AM251 may contribute to the ability of this compound to
affect feeding pathways either directly or, possibly indirectly through feelings of
nausea and malaise (37; 51). However, we previously showed that rats do not
avoid flavored food pellets associated with the effects of AM251 at this dose (7)
even when alternative flavors paired with vehicle treatment are available
(conditioned taste aversion). However, the fact that AM251 increases
conditioned gaping (nausea) in rat (37; 51), combined with higher instances of
nausea in humans treated with the CB1 receptor inverse agonist SR141716A (17;
46; 54), shows that we cannot completely rule this out as a possibility. Both
AM251 (37) and the inverse agonist SR141716A (16) are capable of producing
conditioned taste aversion. Regardless, the fact that a neutral antagonist is
capable of producing reductions in food intake and body weight, independent of
Page 18 of 40
AM4113 Reduces Weight Gain In Rat 19
nausea and emesis, demonstrates that energy balance is controlled, in part, by
the release and action of endogenous cannabinoids. Because of the inverse
agonist properties of AM251, it has been previously impossible to draw this
conclusion. We note that the effect of another potential CB1 antagonist, LH-21,
on food intake and body weight has recently been reported (44), but that the
neutral antagonist properties of this compound have yet to be established (27).
The sustained effect of AM251 on food intake reported previously (7; 8)
and in the present study may result from the long half-life (~22 h) of this
compound in rat (36). One explanation for the delayed onset of the anorectic
effect of AM4113 in our 5 d feeding study is that sufficient quantities of the
compound needed to accumulate in order to become effective. If reductions in
food intake by AM4113 resulted from the build up of the neutral antagonist over
successive treatments, then rats treated acutely with higher doses of the
compound should show an immediate and sustained reduction in feeding.
Consistent with this hypothesis, results from experiment 2 show that higher
doses of AM4113 produced an immediate reduction in food intake, and, later
weight gain that persisted for several days after treatment. In our study, the time
course with which AM4113 reduced food intake and body weight was similar to
that seen with the inverse agonist AM251. Currently, there is no data available
regarding the half-life of AM4113.
CB1 receptor inverse agonists may cause nausea and are proemetic in
humans (17; 46; 54) and animals (13; 37; 56). We induced emesis in ferrets pre-
treated with the neutral antagonist AM4113 and found that there was a positive
Page 19 of 40
AM4113 Reduces Weight Gain In Rat 20
correlation between the dose and the number of vomiting episodes produced by
the emetic. These and other data (26; 55; 56) show that emesis likely stimulates
the release of endogenous cannabinoids which in turn serve to inhibit emesis in a
negative feedback manner. We also show that the inverse agonist properties of
AM251 likely contribute to its effect on emesis. AM4113 antagonized the
hypothermic effect of CP55,940 as effectively as AM251 in rat, but was
substantially less proemetic than the inverse agonist, in so far as AM4113 did not
significantly increase the mean number of vomiting episodes relative to vehicle
treatment. These data suggest that the inverse agonist properties of AM251 do in
fact contribute to its effect on emesis, and is consistent with results by Sink et al,
2007 showing a similar effect on nausea in rat. In this way, a neutral antagonist
such AM4113 may be better tolerated than inverse agonists as antiobesity
agents.
The CB1 receptor inverse agonist SR141716A (Acomplia/Rimonabant™)
has already been shown to significantly improve central obesity, cholesterol
profiles, circulating triglyceride levels and insulin resistance in overweight and
obese humans (17; 46; 54). In humans, circulating levels of the endogenous
cannabinoid 2-AG are positively correlated with intra-abdominal obesity, (3; 12),
and CB1 receptor message is downregulated in visceral adipose tissue of obese
subjects (3). These and other studies (6; 22-25; 28; 29; 31; 34; 43; 46; 48; 53; 54;
57) support the view that changes in endogenous cannabinoid signaling may in
fact be a fundamental aspect of obesity and the metabolic syndrome, and
suggest that treatment with a neutral CB1 receptor antagonist will likely improve
Page 20 of 40
AM4113 Reduces Weight Gain In Rat 21
the metabolic consequences of obesity in addition to reducing body weight.
Future studies designed to assess the effect of neutral CB1 receptor antagonists
on other parameters of the metabolic syndrome such as cholesterol levels,
insulin resistance, and circulating triglycerides are needed in order to confirm this
hypothesis.
In conclusion, we show that AM4113 is a neutral, high affinity, CB1
receptor antagonist. Our thermoregulation assay demonstrates that this
compound acts centrally to antagonize the effects of a CB1 receptor agonist. The
fact that AM4113 dose-dependently reduces food intake and body weight in rat
supports the view that the endogenous cannabinoid system plays a physiological
role in energy balance. Furthermore, our results suggest that AM4113 may be
substantially less pro-emetic than the CB1 receptor inverse agonist AM251.
Perspectives and Significance
In mammals, several overlapping systems ensure survival by providing the
drive to maintain adequate food intake. These systems converge in the brain,
though many of them lie outside of the CNS, in the gut, liver and adipose tissue.
Endocannabinoids act through central and peripheral CB1 receptors to coordinate
food intake, metabolism and energy expenditure. The lean phenotype of the CB1
receptor deficient mouse (11) supports the idea that this receptor system plays a
central role in these processes. These findings have led to the development of
CB1 receptor compounds for the treatment of obesity. An unresolved issue was
whether activation of the receptor by an inverse agonist contributes to the action
Page 21 of 40
AM4113 Reduces Weight Gain In Rat 22
of these compounds. Here we show that a compound with no intrinsic activity
also reduces food intake and body weight in rats. The relative contributions of
central versus peripheral CB1 receptors to these responses remains to be
determined, as does the role of specific neuronal populations possessing central
CB1 receptors. Further development of novel cannabinoid receptor molecules
will allow these questions to be addressed in order to advance our understating
of the physiology of endocannabinoids and their role in food intake and
metabolism.
DISCLOSURE STATEMENT: AM4113 is currently being patent protected by
AM.
ACKNOWLEDGEMENTS
We wish to thank Lorraine Oland for her excellent technical assistance. These
studies were supported by grants from Canadian Institutes of Health Research
(to QJP and KAS) and from National Institutes of Health, U.S.A (DA09158,
DA7215, DA3801 to AM). APC is an Alberta Heritage Foundation for Medical
Research (AHFMR) Graduate Student. QJP and KAS are AHFMR Medical
Scientists. KAS is the Crohn’s and Colitis Foundation of Canada Chair in
Inflammatory Bowel Disease Research and QJP holds a University
Professorship.
Page 22 of 40
AM4113 Reduces Weight Gain In Rat 23
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AM4113 Reduces Weight Gain In Rat 35
Figure legends
Figure 1. a. Body temperature (mean temp°C ± SEM) in rats pretreated with
either vehicle (n=5), AM251 (5 mg kg-1, n=5), or AM4113 (5 mg kg-1; n=5),
followed by treatment with the CB1 agonist CP55,940 (0.3 mg kg-1) at time 0. b.
The neutral antagonist AM4113 (5 mg kg-1, n=5) and inverse agonist AM251 (5
mg kg-1, n=5) both block hypothermia induced by CP55,940, P<0.05, Newman-
Keuls Multiple Comparison Test.
Figure 2 a. The effect of daily treatment with the neutral antagonist AM4113 (1; 5
mg kg-1, n=5/5) on food intake (mean ± SEM; kcal) in rat. b. Weight gain (mean ±
SEM; g) in rats treated daily with either vehicle or AM4113 (1; 5 mg kg-1), P<0.05,
Newman-Keuls Multiple Comparison Test.* significantly different than vehicle, #
significantly different than 1 mg kg-1.
Figure 3. The effect of AM4113 (10; 20 mg kg-1, n=6/6) on food intake and
weight gain compared with vehicle treated rats (n=6), or, rats treated with the
inverse agonist AM251 (n=6). a. The acute effect of each compound at 1.5, 3, 5,
and 18 h, P<0.05, Newman-Keuls Multiple Comparison Test. b. Daily food intake
(mean ± SEM; kcal) and (c) cumulative weight change (mean ± SEM; g) are
shown before and after the experiment. P<0.05. * significantly different than
vehicle, # significantly different than AM4113 10 mg kg-1.
Page 35 of 40
AM4113 Reduces Weight Gain In Rat 36
Figure 4. a. Emesis (mean number of vomiting episodes ± SEM) in ferrets
treated with either vehicle (n=6), AM4113 (10 mg kg-1, n=5), or AM251 (5 mg kg-
1) followed by the emetic M6G (0.5 mg kg-1). The inverse agonist AM251
significantly increased vomiting compared with vehicle treated ferrets, *P<0.05
Newman-Keuls multiple comparisons test. Differences between vehicle treated
ferrets and ferrets treated with the neutral CB1 receptor antagonist, AM4113,
were non-significant. b. The effect of AM4113 (0;5;10;20 mg kg-1) on M6G
induced emesis. Linear regression analysis revealed a positive correlation
between dose and vomiting episodes, P<0.05, however, differences in the
number of vomiting episodes between vehicle and AM4113 treated ferrets was
again not significant.
Page 36 of 40
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