Content uploaded by Colin T Dourish
Author content
All content in this area was uploaded by Colin T Dourish
Content may be subject to copyright.
Psychopharmacology (2003) 167:103–111
DOI 10.1007/s00213-002-1384-8
ORIGINAL INVESTIGATION
S. P. Vickers · L. J. Webster · A. Wyatt · C. T. Dourish ·
G. A. Kennett
Preferential effects of the cannabinoid CB
1
receptor antagonist,
SR 141716, on food intake and body weight gain
of obese (
fa/fa
) compared to lean Zucker rats
Received: 20 October 2002 / Accepted: 6 December 2002 / Published online: 11 March 2003
Springer-Verlag 2003
Abstract Rationale: The selective CB
1
receptor antago-
nist, SR 141716, has been demonstrated to reduce food
consumption in a range of animal species. Objective: To
assess the effect of chronic administration of SR 141716
on body weight and ingestive behaviour of lean and obese
(fa/fa) Zucker rats. Methods: Lean and obese Zucker rats
were orally dosed with SR 141716 (3, 10, 30 mg/kg PO),
sibutramine (5 mg/kg PO) or vehicle for one week. Pair-
fed controls provided insight as to whether the effect of
SR 141716 on body weight was attributable to drug-
induced hypophagia. Subsequently, the effect of chronic
oral administration of SR 141716 (1, 3, 10 mg/kg) was
assessed for 28 days. At the end of this period, all animals
were given vehicle for 14 days. The incidence of wet-dog
shakes, yawning, scratching, and grooming behaviours,
was assessed after acute administration and at weekly
intervals thereafter for 4 weeks. Results: SR 141716 dose-
dependently decreased food intake and body weight gain
in both lean and obese animals. The inhibition of food
intake and body weight gain was greater in obese Zuckers
than in lean Zucker controls. Changes in the body weights
of pair-fed controls closely paralleled those of their drug-
treated counterparts. Chronic 28-day treatment led to a
maintained reduction of body weight gain. Withdrawal of
SR 141716 on day 28 resulted in rebound hyperphagia
and a significant weight gain. On acute administration,
SR 141716 dose-dependently induced motor behaviours
that showed tolerance upon repeated administration.
Conclusion: These data indicate that chronic oral treat-
ment with SR 141716 significantly reduces the food
intake and body weight gain of obese and lean Zucker
rats, an effect that is greater in obese animals and
reversible upon drug withdrawal.
Keywords CB
1
receptors · SR 141716 · Rimonabant ·
Paired feeding · Chronic · Tolerance · Zucker rat
Introduction
Cannabinoid receptors and their endogenous ligands,
including arachidonoyl ethanolamide (anandamide), 2-
arachidonoyl glycerol (2-AG) and 2-arachidonyl glyceryl
ether (noladin ether), are widely distributed throughout
the central nervous system (Devane et al. 1988, 1992;
Matsuda et al. 1990; Mechoulam et al. 1995; Hanus et al.
2001). A role for the central cannabinoid system in the
regulation of ingestive behaviour is now well established
(for review, see Kirkham and Williams 2001). Hence, the
exogenous cannabinoid, D
9
-THC (Williams et al. 1998),
and the endogenous cannabinoids, anandamide (Williams
and Kirkham 1999; Jamshidi and Taylor 2001), and 2-AG
(Kirkham et al. 2002) all increase food intake in rats. This
hyperphagia is likely to be mediated through the activa-
tion of CB
1
receptors since the effects were antagonised
by pre-treatment with the selective CB
1
receptor antag-
onist, SR 141716 (Rinaldi-Carmona 1994; Williams and
Kirkham 1999, 2002; Jamshidi and Taylor 2001).
In contrast to the hyperphagia observed after CB
1
receptor activation, numerous studies have demonstrated
that the blockade of CB
1
receptors with SR 141716
inhibits food intake in laboratory animals (Arnone et al
1997; Colombo et al. 1998; Simiand et al. 1998). In
addition, CB
1
receptor knockout mice eat less than wild-
type controls in response to a fast (Di Marzo et al. 2001).
Interestingly, there is evidence that CB
1
receptor agonists
and antagonists affect food intake through the modulation
of the appetitive and/or consummatory value of food,
perhaps by affecting central reward mechanisms (Arnone
et al. 1997; Kirkham and Williams 2001). Hence, plant
cannabinoids have been reported to increase the con-
sumption of sweet foods in human volunteers (Foltin et al.
1988). Furthermore, the CB
1
receptor antagonist,
SR 141716, selectively decreases the intake of palatable
diets such as sucrose solutions (Arnone et al. 1997;
S. P. Vickers (
)
) · L. J. Webster · A. Wyatt · C. T. Dourish ·
G. A. Kennett
Vernalis Research Limited, Oakdene Court,
613 Reading Road, Winnersh, Wokingham, RG41 5UA, UK
e-mail: S.Vickers@vernalis.com
Tel.: +44-118-9773133
Fax: +44-118-9899300
Simiand et al. 1998). However, the hypophagic effects of
SR 141716 in rodents are not restricted to palatable diets,
since the compound is reported to reduce the intake of
standard laboratory chow (Colombo et al. 1998; Rowland
et al. 2001).
To date, there are few data detailing the effect of
chronic administration of SR 141716 on body weight. In
one study, a reduction in body weight was observed after
14 days intraperitoneal treatment in lean animals (Colom-
bo et al. 1998). Since SR 141716 is currently undergoing
clinical trials for the treatment of obesity, it is surprising
that few publications detail the chronic effects of the
compound. Furthermore, currently there are no reports
detailing the effects of SR 141716 on the ingestive
behaviour, body weight, and motor behaviours of obese
rodents. Accordingly, the present study assesses the effect
of chronic, oral, administration of SR 141716 on the body
weight, food and water intake of genetically obese (fa/fa)
and lean Zucker rats. Although Colombo and colleagues
(1998) showed that a reduced body weight was main-
tained after administration for 14 days, tolerance to the
effects of SR 141716 on food intake was observed within
5 days. As part of the present series of experiments, a 1-
week study was run to investigate the effect of sub-
chronic drug treatment on food intake. In addition, pair-
fed controls were included to assess whether the effects of
SR 141716 on body weight were likely to be attributable
to maintained hypophagia. The serotonin and noradren-
aline re-uptake inhibitor, sibutramine, was chosen as a
positive control in the present studies since it is effica-
cious in reducing body weight both in animal models of
obesity and in obese patients (Ryan et al. 1995).
In addition to reducing food intake, administration of
SR 141716 leads to the induction of a characteristic set of
behaviours. Hence, the incidence of wet-dog shakes,
scratching sequences, abdominal stretching and grooming
behaviour is markedly increased subsequent to acute
administration of SR 141716 in rodents (Navarro et al.
1997; Darmani and Pandya 2000). The incidence of these
behaviours has been attributed to SR 141716 precipitating
a withdrawal-like state (Aceto et al. 1996), suggesting that
there may be a high level of endogenous tone in the
cannabinoid system. The present study evaluated the
effect of acute and chronic SR 141716 administration on
the incidence of such behaviours.
Materials and methods
Animals
All work reported in this manuscript was performed in accordance
with Home Office regulations as detailed in the Animals (Scientific
Procedures) Act 1986. Male obese (fa/fa) Zucker rats (Harlan Ltd,
Bicester, UK) weighing 443–552 g (28-day study) and 449–602 g
(paired feeding study) and lean (FA/FA or FA/fa) Zucker rats
(Harlan Ltd) weighing 306–415 g were singly housed in an air-
conditioned room maintained under a 12-h light/dark cycle (lights
on: 0800 hours). Ambient temperature was 21€1C. A red light was
the sole source of illumination during the dark period. Except
where stated, animals had continuous access to standard rodent diet
(Bantin & Kingman UK Ltd, Hull) and tap water.
Procedure
Animals were singly housed and randomly allocated to drug
treatment groups in all experiments. In the initial study, obese
animals (n=5) received SR 141716 (3, 10, 30 mg/kg PO),
sibutramine (5 mg/kg PO), or vehicle once daily for a 7-day
period. Pair-fed controls were dosed with vehicle and given a daily
food allotment equal to that consumed by a drug-treated counter-
part over the previous 24-h period. A sham group of animals that
received no treatments but whose weight and food and water intake
were determined daily was included. In parallel, age-matched lean
Zucker rats (n=5–7) also received SR 141716 (3, 10, 30 mg/kg PO),
sibutramine (5 mg/kg PO), or vehicle once daily for a 7-day period.
A sham group of animals that received no treatments but whose
weight and food and water intake were determined daily was
included. No pair-fed controls were included for the lean Zucker rat
experiment. All weighing and dosing took place in the morning.
In the subsequent, 28-day, study animals (n=6) received
SR 141716 (1, 3, 10 mg/kg PO) or vehicle once daily. Animals
were dosed at approximately 08:30 a.m. each day. Body weight,
and food and water consumption were recorded daily for 28 days.
All weighing took place in the morning. Upon completion of the
28-day dosing, the body weights, daily food intake and water intake
of the animals were monitored for the subsequent 2-week period.
Assessment of SR 141716-induced motor behaviours
SR 141716-induced motor behaviours were only assessed in the 28-
day repeated administration experiment. Animals were assessed for
the incidence of wet-dog shakes, scratching behaviour, and
grooming behaviour upon acute administration, and at weekly
intervals thereafter for a 4-week period. Assessment of the motor
behaviours was not carried out during the 2-week period of
antagonist withdrawal. One hour after dosing, animals were placed
individually into clear Perspex cages (40 cm20 cm; Techniplast
UK Ltd) containing a light dusting of sawdust. The number of wet-
dog shakes, scratching episodes, yawns, number of grooming bouts
and grooming bout duration were recorded for the subsequent 20-
min period by observers blind to drug treatment.
An episode of scratching behaviour was defined according to
previously published studies using SR 141716 (Darmani and
Pandya 2000; Janoyan et al. 2002). Specifically, scratching
behaviour was defined as one or more repetitive scratches directed
to the head, neck, or lateral body area by either hind limb. Where
the interval between successive scratches exceeded 2 s, a separate
scratching episode was counted. If alternate hind limbs were used
to scratch, then each scratch was regarded as a separate episode.
Grooming duration was determined using a hand-held stopwatch.
Drugs
1H-Pyrazole-3-carboxamide 5-(4-chlorophenyl)-1-(2,4-dichloro-
phenyl)-4-methyl-N-1-piperidinyl-HCl (SR 141716) and sibu-
tramine HCl were synthesised in the Department of Chemistry,
Vernalis Research Ltd. SR 141716 was given orally in a suspension
of 1% methyl cellulose. Sibutramine was dissolved in 1% methyl
cellulose. Both compounds were administered orally by gavage in a
volume of 2 ml/kg. All drug doses are expressed as free base.
Statistical analysis
All data are presented as mean€SEM. In the initial study, body
weight and food intake data were analysed by three-way analysis of
variance (ANOVA) where phenotype and dose were between-
subjects factors and day a within-subjects factor. Significant
104
interactions were succeeded by the performance of ANOVA at each
level of time. Dunnett’s test was used to test for significant
differences between treatment groups and controls. Newman-Keuls
tests were used to test for significant differences between pheno-
types at each level of dose. Data from the 28-day study were
analysed by two-way ANOVA with dose as a between-subjects and
day a within-subjects factor. Where appropriate, one-way ANOVA
were run to investigate the effect of drug treatment on a particular
day. A significant difference from control was determined using
Dunnett’s test. All data analysis was performed using Statistica 6.0
(Statsoft Inc.).
Results
Paired feeding study
SR 141716 dose-dependently reduced body weight gain
over the 7-day study in both lean and obese Zucker rats
[see Table 1; dosetime interaction in obese Zuckers
F(35,168)=10.5, P<0.001; lean Zuckers F(35,182)=14.7,
P<0.001]. The effects of SR 141716 on body weight gain
were greater in obese compared to lean Zucker rats
[Table 1; significant dosephenotype interaction
F(5,350)=9.72, P<0.001]. The minimum effective dose
for reducing body weight and food intake in lean Zucker
rats was 10 mg/kg, compared to 3 mg/kg in obese
animals. The change in body weight gain observed at the
highest dose of SR 141716 was significantly greater in
obese animals than it was in lean counterparts (Table 1;
P<0.01). In contrast, although significant effects on the
body weight gain of obese and lean Zucker rats were also
observed with sibutramine, at the dose tested the
compound did not preferentially affect the body weight
of obese Zucker rats.
Daily food intake data are illustrated in Fig. 1. Acute
administration of SR 141716 (3, 10, 30 mg/kg) dose-
dependently reduced food intake in both genotypes. In the
three-way ANOVA, there were highly significant
dosephenotype [F(5,350)=7.91, P<0.001, and dosephe-
notypeday interactions, F(5,350)=2.31, P<0.001]. Lean
animals developed rapid tolerance to the hypophagia
induced both by SR 141716 and sibutramine. After 5 days
of treatment, only sibutramine-treated animals exhibited a
statistically significant hypophagia (Fig. 1a). In contrast,
the same doses of SR 141716 and sibutramine signifi-
cantly reduced the food consumption of the obese Zucker
rats for the duration of the 7-day study (Fig. 1b).
Pair-fed obese controls exhibited a similar drop in
body weight to drug-treated obese counterparts (Table 1).
The final body weights of pair-fed animals were also
similar to drug-treated counterparts [main effect of
feeding condition on final body weight gain
F(1,32)=0.51, NS; dosefeeding condition interaction
F(3,32)=0.47, NS].
Drug treatment had a preferential effect on the mean
daily water intake [Table 2; significant dosephenotype
interaction F(5,350)=4.62, P<0.01] of obese (fa/fa) com-
pared to lean Zucker rats. The reduction in water
consumption observed in drug-treated obese Zucker rats
tended to be greater than that observed in pair-fed animals
at all doses [Table 2; main effect of feeding condition on
mean daily water intake F(1,32)=5.94, P<0.05].
Chronic (28-day) administration of SR 141716
Administration of SR 141716 for 28 days significantly
reduced the body weight gain of obese (fa/fa) Zucker rats
[Fig. 2; dosetime interaction: F(123,820)=2.94,
P<0.001]. Although 3 mg/kg SR 141716 significantly
reduced body weight gain during the initial week of
administration, only the 10 mg/kg dose led to a statisti-
cally significant and maintained reduction in body weight
(P<0.05) over the 28-day period. Upon drug withdrawal,
body weight increased such that at the end of the study all
experimental groups had a mean body weight similar to
controls. Animals treated with 3 mg/kg SR 141716
showed a body weight gain in excess of vehicle-treated
controls (Fig. 2); however, this increase was not statis-
tically significant.
SR 141716 dose-dependently reduced food consump-
tion [Fig. 3; dosetime interaction: F(123,820)=4.84,
P<0.001]. Statistically significant effects on food con-
sumption were seen only after administration of 3 and
10 mg/kg SR 141716. Tolerance to the effects of
Table 1 Effect of 1 week administration of SR 141716 (3, 10,
30 mg/kg PO) sibutramine (5 mg/kg) or vehicle, once daily, on
body weight change of genetically obese (fa/fa) Zucker rats and
lean counterparts (FA/FA or FA/fa). Sham animals received no
treatments. Groups of pair-fed obese controls were included to
assess whether the effects of drug treatment on body weight gain
were attributable to effects on food intake. Data are mean (€SEM)
Obese Total weight change over 7 days (g)
Pair-fed obese Lean
Sham 10.0 (1.2) – 6.2 (1.5)
Vehicle 12.6 (3.5) – 6.1 (1.0)
3 mg/kg 8.2 (3.7)
**
7.8 (3.5)** –1.0 (2.8)
10 mg/kg 13.0 (1.8)
**
14.2 (2.2)
**
–10.0 (2.3)
**
30 mg/kg 34.0 (3.7)
**,$
31.8 (2.6)
**
–10.6 (2.2)
**
Sibutramine 13 (6.0)
**
13.0 (7.2)
**
–19.4 (2.5)
**
*
P<0.05,
**
P<0.01 Dunnett’s test from vehicle-treated controls.
$
P<0.01 Newman-Keuls test compared to lean controls
Table 2 Effect of 1 week administration of SR 141716 (3, 10,
30 mg/kg) sibutramine (5 mg/kg) or vehicle, once daily, on mean
daily water intake of genetically obese (fa/fa) Zucker rats and lean
counter parts (FA/FA or FA/fa) over the 7-day period. Sham
animals received no treatments. Data are mean (€SEM)
Obese (fa/fa) Mean daily water intake (ml)
Pair-fed (fa/fa) Lean
Sham 36.2 (0.6) – 25.4 (0.8)
Vehicle 42.6 (1.3) – 23.6 (0.9)
3 mg/kg 24.0 (1.0)
**
25.6 (1.1)
*
23.4 (0.8)
10 mg/kg 23.7 (1.5)
**
27.8 (2.7)
*
23.6 (0.9)
30 mg/kg 18.1 (1.7)
**
26.8 (3.7)
*
22.1 (1.3)
Sibutramine 24.7 (1.4)
*
27.2 (1.9)
*
19.9 (1.3)
*
*
P<0.05,
**
P<0.01 Dunnett’s test from vehicle-treated controls
105
Fig. 1 Effect of once daily oral
administration of SR 141716 (3,
10, 30 mg/kg) on the daily food
intake of genetically obese (fa/
fa)(b) and lean (a) Zucker rats
over a 7-day period.
*
P<0.05,
**
P<0.01 significant difference
from vehicle-treated animals as
assessed by Dunnett’s test after
significant ANOVA
Fig. 2 Obese (fa/fa) Zucker rat body weight gain (mean€SEM; g)
during chronic, once daily, oral administration of SR 141716 (1, 3,
10 mg/kg). The 10 mg/kg dose significantly reduced body weight
gain compared to vehicle-treated controls for the duration of
treatment. Withdrawal from treatment, illustrated by the vertical
line on the figure, resulted in a significant weight gain in
SR 141716-treated rats. *P<0.05, **P<0.01 significant difference
from vehicle-treated animals (Dunnett’s test). Dosing occurred on
day 1. Day 0 refers to the day before dosing and day 0 data
illustrate the weight gain from baseline (0 g)
106
SR 141716 on food intake was observed such that from
day 13 drug treatment did not induce a statistically
significant hypophagia. Upon drug withdrawal on day 28,
the food intake of animals treated with 3 and 10 mg/kg
SR 141716 increased above that of controls (Fig. 3);
however, this increase was not statistically significant.
Chronic treatment with SR 141716 significantly
reduced water intake [Fig. 4; dosetime interaction
F(123,779)=2.24, P<0.001]. Note, due to technical errors,
water intake data for two animals were lost on 2 separate
days. Accordingly, the data for these animals were not
assessed by two-way ANOVA and differences in the
degrees of freedom reflect this. Partial tolerance was
observed to the hypodipsic effect of SR 141716 treatment
upon chronic administration, although this was less
marked than for the food intake data.
Assessment of SR 141716-induced motor behaviours
Acute treatment with SR 141716 caused a dose-related
increase in the incidence of wet-dog shakes [Fig. 5a; main
effect of dose F(3,20)=5.10, P<0.01] and scratching
behaviour [Fig. 5b; main effect of dose F(3,20)=6.80,
P<0.01]. Upon acute administration, and at the 1-week
time point, the incidence of wet-dog shakes showed a
bell-shaped dose response curve. The incidence of both
behaviours decreased with subsequent administration of
SR 141716 leading to significant dosetime interactions
in the two way ANOVA [wet-dog shakes: F(12,80)=2.83,
P<0.001; scratching: F(12,80)=4.05, P<0.001].
Upon initial administration, SR 141716 increased the
number of grooming bouts [main effect of dose
F(3,20)=5.48, P<0.01] with effects at the top dose
reaching statistical significance (Fig. 5c). Statistically
significant increases in grooming behaviour were ob-
served only after acute administration. Drug treatment did
Fig. 3 Daily food consumption
of obese (fa/fa) Zucker rats
(mean€SEM; g) during chronic,
once daily, oral administration
of SR 141716 (1, 3, 10 mg/kg).
SR 141716 significantly re-
duced food intake compared to
vehicle-treated controls at all
doses up to day 10 of the study.
Withdrawal from treatment, il-
lustrated by the vertical line on
the figure, resulted in increased
food intake compared to vehi-
cle-treated animals. *P<0.05,
**P<0.01 significant difference
from vehicle-treated animals
Fig. 4 Daily water intake of
obese (fa/fa) Zucker rats
(mean€SEM; ml) during
chronic, once daily, oral ad-
ministration of SR 141716 (1, 3,
10 mg/kg). Treatment with
SR 141716 significantly re-
duced water intake over the first
2 weeks of drug treatment.
*
P<0.05,
**
P<0.01 significant
difference from vehicle-treated
animals
107
not significantly increase grooming duration (Fig. 5d).
Although yawning behaviour appeared to be increased in
animals treated with SR 141716, the incidence of this
behaviour was very low, variable, and not statistically
significant (data not shown).
Discussion
The present studies demonstrate that sub-chronic, once
daily, treatment with the selective CB
1
receptor antago-
nist, SR 141716, preferentially reduces the food intake
and body weight gain of obese (fa/fa) Zucker rats
compared to lean Zucker controls. Furthermore, over a
1-week period, the effects of SR 141716 treatment on
body weight gain were attributable to the effects of the
drug on daily food intake. During the 7-day study,
significant reductions in water intake were observed as
well though these were likely to be prandial in nature
since pair-fed animals also exhibited marked hypodipsia
compared to controls. In agreement with previous studies
(Colombo et al. 1998), tolerance was observed to the
hypophagia induced by SR 141716 in lean Zucker rats
such that from day 4 onwards drug treatment did not
significantly reduce food intake. However, in the same 7-
day study, obese animals did not exhibit tolerance to
SR 141716 induced hypophagia at any of the doses tested.
This suggests that obese animals may be resistant to the
tolerance to the reduction in food intake induced by
repeated SR 141716 administration compared to their lean
counterparts.
In a subsequent 28-day chronic oral study, once daily
treatment with SR 141716 significantly reduced the body
weight gain of genetically obese (fa/fa) Zucker rats. When
animals were withdrawn from SR 141716 treatment,
increases in daily food and water intake were observed
and body weights increased rapidly to control levels. Such
data, including the findings upon drug withdrawal, are in
agreement with published studies in lean rats (Colombo et
al. 1998) and overweight or obese men (Heshmati et al.
2001). The data are also in agreement with a preliminary
study in mice with diet-induced obesity (Black et al.
2002). However, in this study the only data reported were
obtained using a single high dose of the CB
1
receptor
antagonist, AM251. Interestingly, in the present study,
there is a trend for the body weight of animals withdrawn
from the 3 mg/kg dose of SR 141716 to exceed vehicle
levels, though the effect is not statistically significant. An
increased withdrawal period would allow an assessment
of such withdrawal effects at the 10 mg/kg dose.
Furthermore, a longer withdrawal period would determine
whether withdrawal from SR 141716 leads to long-term
increases in body weight, perhaps as a result of CB
1
receptor upregulation during chronic drug exposure.
In contrast to findings during the 7-day study, toler-
ance was observed to the hypophagia induced by
SR 141716 in obese Zucker rats during 28-day adminis-
tration such that from day 13 onwards drug treatment did
not significantly reduce food intake (Colombo et al. 1998;
Black et al. 2002) although the food consumption of
animals treated with SR 141716 was always below that of
vehicle-treated counterparts. Accordingly, despite the
apparent tolerance to the effects of SR 141716 on food
intake, it is possible that the maintained effects of drug
treatment on body weight are attributable to effects on
food consumption. Paired-feeding or thermogenesis stud-
ies examining the effects of SR141716 over a 28-day
period should address this issue. Interestingly, the present
profile is similar to that seen with chronic administration
of the preferential 5-HT
2C
receptor agonist, mCPP
(Vickers et al. 2000).
Tolerance to the effects of SR 141716 on food intake is
more rapid in lean Zucker rats than in obese Zucker rats,
suggesting that the obese rats are more resistant to
SR 141716-induced tolerance. Interestingly, although less
marked than that observed with SR 141716, such an effect
on food intake seems to be evident with sibutramine. On
each day tested, the effects of SR 141716 on food intake
Fig. 5 Effect of SR 141716 (1,
3, 10 mg/kg) on the incidence of
a wet-dog shakes, b scratching
behaviour, c grooming bouts
and d grooming duration. Ani-
mals were assessed after acute
administration and at weekly
intervals thereafter for a 4-week
period. Animals were assessed
for 20 min, 1 h after drug
administration.
**
P<0.01 signif-
icant difference from vehicle-
treated animals
108
were greater in magnitude in obese animals compared to
lean controls. Obese Zucker rats appear to be more
sensitive to the effects of SR 141716 on body weight gain
since each dose of SR 141716 was more efficacious in
obese Zucker rats than in lean controls. These effects are
unlikely to be attributable solely to the fact that the
animals are heavier, providing a higher baseline for
reductions in food intake and body weight to be observed,
since at the dose tested, sibutramine did not preferentially
affect body weight gain of obese animals. Interestingly,
obese Zucker rats have elevated hypothalamic levels of
the endocannabinoid, 2-arachidonoyl glycerol, compared
to lean controls (Di Marzo et al. 2001). Accordingly, it is
possible that such differences in endocannabinoid tone
within the hypothalamus, and its blockade by SR 141716,
could account for the differences observed in the present
study. It is not known whether levels of endocannabinoids
are increased in obese humans. If this were the case,
however, it is possible that cannabinoid CB
1
receptor
antagonists could be a highly efficacious treatment for
obesity. An alternative explanation of the enhanced
efficacy of SR 141716 in obese animals could be due to
an altered pharmacokinetic profile of the drug in the
obese animals. SR 141716 is a highly lipophilic molecule
of low aqueous solubility and may have more stable
plasma levels in obese compared to lean animals. Further
studies measuring plasma and brain levels of SR 141716
at pre-determined times after administration are required
to address this issue. Measurement of drug levels during a
chronic study may also provide insight into whether the
tolerance seen with SR 141716 on food intake, water
intake and motor behaviours is metabolic in nature.
Specifically, with repeated administration, is SR 141716
cleared more readily? An alternative approach in ad-
dressing this question might be to investigate the effect of
SR 141716 in obese and lean Zucker rats when admin-
istered via osmotic mini-pumps where circulating levels
of compound would be less affected by metabolic
tolerance. If the preferential effects of SR 141716 on
the body weight of obese animals are attributable to an
altered pharmacokinetic profile of the compound, this is
of importance since it would be expected that such an
altered profile would also be evident in obese humans.
Repeated treatment with SR 141716 dose-dependently
reduced daily water intake. This finding appears to
contrast with the findings of a previous study that
reported no effect of a hypophagic dose of SR 141716
on water intake (Rowland et al. 2001). However, in this
study, it was reported that a dose of SR 141716 that
reduced food intake by 31%, reduced water intake by
18%, although the effect on drinking was not statistically
significant. It is possible, therefore, that the present study
possessed greater statistical power and provided greater
sensitivity to potential hypodipsic effects of SR 141716.
Interestingly, in the present study, the daily water
consumption of obese controls was greater than generally
observed with this strain in our laboratory. A large
component of SR 141716-induced hypodipsia in the
obese (fa/fa) Zuckers is likely to be attributable to the
reduced daily food intake induced by the compound since
pair-fed animals also showed marked reductions in daily
water intake compared to controls. However, the effects
of SR 141716 on water intake are unlikely to be entirely
prandial in nature, since SR 141716-treated animals
tended to drink less water than pair-fed controls. This
discrepancy may be due to differences between the
temporal nature of feeding behaviour in drug-treated
animals compared to pair-fed animals that were observed
to begin eating their daily food ration almost immediately
upon its presentation.
Previous reports have suggested that SR 141716
selectively decreases the intake of palatable diets such
as sucrose solutions (Arnone et al. 1997; Simiand et al.
1998). The present studies used standard laboratory chow
and confirm that SR 141716 reduces the intake of
relatively bland diets and that the effects of the compound
on food intake are not specific to palatable diets
(Colombo et al. 1998). However, it would be of interest
to determine whether increased efficacy of SR 141716 on
body weight gain, or reduced tolerance to the effects of
the compound on food intake, is observed when animals
are allowed access to palatable diets.
Animals treated chronically with the CB
1
receptor
agonist, D
9
-THC, and then subjected to withdrawal,
exhibit a number of behaviours including scratching,
wet-dog shakes and chewing (Aceto et al. 1996).
Accordingly, the increased incidence of wet-dog shakes,
scratching and grooming behaviours observed after the
administration of SR 141716 has been attributed to the
compound precipitating a withdrawal-like state in rodents
(Aceto et al. 1996). In the present study, acute treatment
with SR 141716 led to a dose-related induction of
scratching behaviour, wet-dog shakes and an increase in
the number grooming bouts. Such a finding is in
agreement with other reports (Navarro et al. 1997;
Darmani and Pandya 2000). Interestingly, a bell-shaped
dose response curve was observed for the incidence of
wet-dog shakes. This curve may, in part, be attributable to
response competition since upon acute administration
higher doses of SR 141716 also induce a large number of
scratching episodes and increased grooming behaviour.
However, the bell-shaped nature of the wet dog shake
behaviour dose-response curve was not observed in the
study of Darmani and Pandya (2000), although this study
was performed in mice and species differences may
account for the apparent discrepancy. Tolerance was
observed with repeated administration of SR 141716 such
that no statistically significant effects on motor be-
haviours were observed 1 week later. However, although
the frequency of behaviours was not statistically signif-
icant, the incidence of wet-dog shakes and scratching
behaviour was increased in a dose-dependent manner for
the remaining test sessions. This apparent tolerance
resembles that observed for food intake. Specifically,
marked effects are seen initially which, upon repeated
SR 141716 administration, reduce in magnitude such that
they are no longer statistically significant. Nevertheless,
rapid tolerance to motor effects but not to hypophagia in
109
obese rats argues against metabolic tolerance and in
favour of real sensitivity differences between obese and
lean animals. This finding also demonstrates that the
effects of SR 141716 on food intake are not secondary to
the induction of non-specific behavioural effects.
Interestingly, a recent study suggests that peripheral,
but not central, administration of SR 141716 reduces food
intake in rats (Gomez et al. 2002). Furthermore, 24-h food
deprivation led to significant increases in the concentra-
tion of anandamide in intestinal tissue whereas brain
levels were unaffected. The authors concluded an impor-
tant role for peripheral CB
1
receptors in the regulation of
ingestive behaviour (Gomez et al. 2002). Accordingly,
peripheral CB
1
receptors may have a key role in
mediating the effects detailed in the present study.
In conclusion, the present series of experiments
demonstrate that oral administration of SR 141716
reduces the body weight gain of both genetically obese
(fa/fa) Zucker rats and lean Zucker rats. This effect is
maintained upon chronic treatment and is reversible on
drug withdrawal. The results of our pair-feeding studies
indicate that effects on body weight are attributable to
drug-induced reductions in food intake. Importantly, the
effects of SR 141716 on body weight gain and food intake
were observed to be greater in obese animals than in age-
matched lean controls. SR 141716 has been widely
demonstrated to behave as an inverse agonist in a broad
range of pharmacological assays (for review see Howlett
et al. 2002). In the absence of a silent CB
1
receptor
antagonist, it remains to be seen whether the effects of
SR 141716 on food intake, body weight parameters and/or
the induction of withdrawal-like behaviours are at-
tributable to the blockade of endogenous cannabinoid
tone, or to inverse agonist properties of the compound.
The potential therapeutic utility of CB
1
receptor antago-
nists for the treatment of obesity is reinforced by the
present series of experiments since the data suggest that
such an approach would be preferentially efficacious in
obese subjects.
References
Aceto MD, Scates SM, Lowe JA, Martin BR (1996) D
9
-Tetrahy-
drocannabinol: studies on precipitated and abrupt withdrawal. J
Pharmacol Exp Ther 278:1290–1295
Arnone M, Maruani J, Chaperon F, Thiebot MH, Poncelet M,
Soubrie P, Le Fur G (1997) Selective inhibition of sucrose and
ethanol intake by SR 141716, an antagonist of central
cannabinoid (CB1) receptors. Psychopharmacology 132:104–
106
Black SC, Hildebrandt AL, Kelly-Sullivan DM (2002) Determina-
tion of CB
1
receptor antagonist anorectic efficacy vs adipose
tissue mass reduction. Symposium on the cannabinoids,
Burlington, Vt., International Cannabinoid Research Society
Colombo G, Agabio R, Diaz G, Lobina C, Reali R, Gessa GL
(1998) Appetite suppression and weight loss after the canna-
binoid antagonist SR141716. Life Sci 63:PL113–117
Darmani NA, Pandya DK (2000) Involvement of other neurotrans-
mitters in behaviours induced by the cannabinoid CB
1
receptor
antagonist SR 141716A in naive mice. J Neural Transm
107:931–945
Devane WA, Dysfarz FA, Johnson MR, Melvin LS, Howlett AC
(1988) Determination and characterization of a cannabinoid
receptor in rat brain. Mol Pharmacol 34:605–613
Devane WA, Hanus L, Breuer A, Pertwee RG, tevenson LA,
Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R
(1992) Isolation of a brain constituent that binds to the
cannabinoid receptor. Science 258:1946–1949
Di Marzo V, Goparaju SK, Wang L, Liu J, Batkai S, Jaral Z, Fezza
F, Miura GI, Palmiter RD, Sugiura T, Kunos G (2001) Leptin-
regulated endocannabinoids are involved in maintaining food
intake. Nature 410:822–825
Foltin RW, Fischmann MW, Byme MF (1988) Effects of smoked
marijuana on food intake and body weight of humans living in a
residential laboratory. Appetite 11:1–14
Gomez R, Navarro M, Ferrer B, Trigo JM, Bilbao A, Del Arco I,
Cippitelli A, Nava F, Piomelli D, Rodriguez de Fonseca (2002)
A peripheral mechanism for CB1 cannabinoid receptor-depen-
dent modulation of feeding. J Neurosci 22:9612–9617
Hanus L, Abu-Lafi S, Fride E, Breuer A, Vogel Z, Shalev DE,
Kustanovich I, Mechoulam R (2001) 2-Arachidonyl glyceryl
ether, an endogenous agonist of the cannabinoid CB1 receptor.
Proc Natl Acad Sci USA 98:3662–3665
Heshmati HM, Caplain H, Bellisle F, Mosse M, Fauveau C, Le Fur
G (2001) SR141716, a selective cannabinoid CB1 receptor
antagonist, reduces hunger, caloric intake, and body weight in
overweight or obese men. Obesity Res 9:O69
Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane
WA, Felder CC, Herkenham M, Mackie K, Martin BR,
Mechoulam R, Pertwee RG (2002) International Union of
Pharmacology. XXVII. Classification of cannabinoid receptors.
Pharmacol Rev 54:161–202
Janoyan JJ, Crim JL, Darmani NA (2002) Reversal of SR 141716A-
induced head-twitch and ear-scratch responses in mice by D
9
-
THC and other cannabinoids. Pharmacol Biochem Behav
71:155–162
Jamshidi N, Taylor DA (2001) Anandamide administration into the
ventromedial hypothalamus stimulates appetite in rats. Br J
Pharmacol 134:1151–1154
Kirkham TC, Williams CM (2001) Endogenous cannabinoids and
appetite. Nutr Res Rev 14:65–86
Kirkham TC, Williams CM, Fezza F, Di Marzo V (2002)
Endocannabinoid levels in rat limbic forebrain and hypothal-
amus in relation to fasting, feeding and satiation: stimulation of
eating by 2-arachidonoyl glycerol. Br J Pharmacol 136:550–
557
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI
(1990) Structure of a cannabinoid receptor and functional
expression of the cloned cDNA. Nature 346:561–564
Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski
NE, Schatz AR, Gopher A, Almog S, Martin BR, Compton DR,
Pertwee RG, Griffin G, Bayewitch M, Barg J, Vogel Z (1995)
Identification of an endogenous 2-monoglyceride, present in
canine gut, that binds to cannabinoid receptors. Biochem
Pharmacol 50:83–90
Navarro M, Hernandez E, Munoz RM, del Arco I, Villanua MA,
Carrera MRA, Rodriguez de Fonseca F (1997) Acute admin-
istration of the CB1 receptor antagonist SR 141716A induces
anxiety-like responses in the rat. NeuroReport 8:491–496
Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B,
Congy C, Martinez S, Maruani J, Neliat G, Caput D, Ferrara P,
Soubrie P (1994) SR141716A, a potent and selective antagonist
of the brain cannabinoid receptor. FEBS Lett 350:240–244
Rowland NE, Mukherjee M, Robertson K (2001) Effects of the
cannabinoid receptor antagonist SR 141716, alone and in
combination with dexfenfluramine or naloxone, on food intake
in rats. Psychopharmacology 159:111–116
Ryan DH, Kaiser P, Bray GA (1995) Sibutramine: a novel new
agent for obesity treatment. Obesity Res 3:553S–559S
Simiand J, Keane M, Keane PE, Soubrie P (1998) SR 141716, a
cannabinoid receptor antagonist, selectively reduces sweet food
intake in marmoset. Behav Pharmacol 9:179–181
110
Vickers SP, Benwell KR, Porter RH, Bickerdike MJ, Kennett GA,
Dourish CT (2000) Comparative effects of continuous infusion
of mCPP, Ro 60-0175 and d-fenfluramine on food intake, water
intake, body weight and locomotor activity in rats. Br J
Pharmacol 130:1305–1314
Williams CM, Kirkham TC (1999) Anandamide induces overeat-
ing: mediation by central cannabinoid receptors. Psychophar-
macology 143:315–317
Williams CM, Kirkham TC (2002) Reversal of D
9
-THC hyperpha-
gia by SR141716 and naloxone but not dexfenfluramine.
Pharmacol Biochem Behav 71:341–348
Williams CM, Rogers PJ, Kirkham TC (1998) Hyperphagia in pre-
fed rats following oral D
9
-THC. Physiol Behav 65:343–346
111
