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Psychopharmacology (2006) 184: 26–35
DOI 10.1007/s00213-005-0234-x
ORIGINAL INVESTIGATION
Anne B. Need .Richard J. Davis .
Jesline T. Alexander-Chacko .Brian Eastwood .
Eyassu Chernet .Lee A. Phebus .Dana K. Sindelar .
George G. Nomikos
The relationship of in vivo central CB1 receptor occupancy
to changes in cortical monoamine release and feeding elicited
by CB1 receptor antagonists in rats
Received: 27 April 2005 / Accepted: 3 October 2005 / Published online: 18 November 2005
#Springer-Verlag 2005
Abstract Rationale: Cannabinoid type 1 (CB
1
) receptor
antagonists are reportedly effective in reducing food intake
both preclinically and clinically. This may be due in part to
their effects on monoamine release in the brain. The level of
central CB
1
receptor occupancy underlying these neurobi-
ological effects is unclear. Objectives: We explored the rela-
tionship between in vivo CB
1
receptor occupancy in the
frontal cortex and changes in both monoamine release in the
medial prefrontal cortex (mPFC) and feeding behavior in
rats in response to two orally administered CB
1
receptor
antagonists presently in clinical trials, SR141716A (rimo-
nabant) and SLV319. Methods: CB
1
receptor occupancy
was measured using [
3
H] SR141716A, and these occupan-
cies were related to potencies to mediate increases in do-
pamine (DA) and norepinephrine (NE) release measured
with microdialysis and decreases in consumption of a highly
palatable diet (HP). Results: High receptor occupancy
levels (>65%) were required to detect increases in mono-
amine release that were achieved with 3 and 10 mg/kg of
SR141716A and 10 mg/kg of SLV319 for DA and 10 mg/kg
of SR141716A for NE. Decreases in HP consumption were
seen at occupancies higher than 65% for SR141716A that
were achieved with 3 and 10 mg/kg. In contrast, decreases in
HP consumption were seen at relatively low CB
1
receptor
occupancies (11%) for SLV319. Conclusions: The occu-
pancy method described here is an effective tool for interrelating
central CB
1
receptor occupancy with neurobiological actions of
CB
1
receptor antagonists and for furthering our understanding
of the role of CB
1
receptors in central nervous system
physiology and pathology.
Keywords SR141716A .SLV319 .[
3
H]SR141716A .
Dopamine .Norepinephrine .Microdialysis .Receptor
occupancy .Feeding .Cannabinoid .In vivo binding
Introduction
Primarily in the form of marijuana, cannabinoids have been
used therapeutically and recreationally for centuries al-
though the precise mechanisms underlying a variety of
their effects have been poorly understood. While much
remains to be discovered, significant progress has been
made in recent decades. The identification of Δ
9
-tetrahy-
drocannabinol (THC) as the major psychoactive compo-
nent of marijuana (Gaoni and Mechoulam 1964) was the
first step. Hindered by the lipophilicity of the known
ligands, it was more than 20 years later that investigators
identified specific binding sites. With the synthesis of [
3
H]
CP-55,940, binding sites for cannabinoids were elucidated
in the brain (Devane et al. 1988). Two receptors were then
cloned, cannabinoid type 1(CB
1
; Matsuda et al. 1990),
primarily located in the central nervous system (CNS), and
type 2 (CB
2
; Munro et al. 1993), found mainly in the
peripheral immune system and reportedly in perivascular
microglial cells in the brain (see, e.g., Nunez et al. 2004).
Several endogenous compounds have been found to
activate the CB
1
receptor, including arachidonylethanol-
amide (anandamide) (Devane et al. 1992) and 2-arachidon-
oyl glycerol (2-AG) (Mechoulam et al. 1995). Along with
the synthesis of selective antagonists to the CB
1
receptor,
such as SR141716A (N- (piperidin-1-yl)-5-(4-chlorophe-
nyl)-1-(2,4- dichlorophenyl)-4- methyl-1H-pyrazole-3-car-
boxamide-hydrochloride; rimonabant) (Rinaldi-Carmona
et al. 1994), these fairly recent discoveries have led to an
explosion of research aimed at elucidating the many func-
tions of the endocannabinoid systems in CNS physiology
and pathology.
Evidence is now well established for a role of the CB
1
receptor in feeding. One of the best-known effects of
marijuana use is to increase appetite, and this has been
A. B. Need .R. J. Davis .J. T. Alexander-Chacko .
B. Eastwood .E. Chernet .L. A. Phebus .
D. K. Sindelar .G. G. Nomikos (*)
Lilly Research Laboratories,
Lilly Corporate Center,
Indianapolis, IN 46285-0510, USA
e-mail: nomikos_george@lilly.com
Tel.: +1-317-4332541
Fax: +1-317-2765546
confirmed in controlled settings (see, e.g., Abel 1971).
Cannabinoid agonists have been prescribed for the treat-
ment of nausea during chemotherapy and to improve ap-
petite of patients suffering from AIDS (Plasse et al. 1991).
Genetically obese mice have elevated anandamide and 2-
AG levels in the hypothalamus, and intravenous adminis-
tration of leptin, a key regulator of food intake, suppresses
hypothalamic levels of endocannabinoids in these mice, as
well as in normal rats (Di Marzo et al. 2001). SR141716A
administration to rodents decreases sucrose consumption
(Arnone et al. 1997) and reduces the intake of a highly
palatable diet in marmosets (Simiand et al. 1998). It was also
shown in nondeprived rats to decrease the intake of all food,
regardless of macronutrient composition (Verty et al. 2004).
Moreover, CB
1
receptor knockout mice display decreased
neuropeptide Y (NPY)-induced overeating (Poncelet et al.
2003) and are resistant to diet-induced obesity (Ravinet
Trillou et al. 2004).
SR141716A also decreases self-administration of various
drugs, such as ethanol (Arnone et al. 1997), nicotine (Cohen
et al. 2002), and heroin (Navarro et al. 2001) and reduces
responding for rewarding brain stimulation (Deroche-
Gamonet et al. 2001). Cohen et al. (2002) also found that
SR141716A reduces the dopamine-releasing properties of
both nicotine and alcohol in the nucleus accumbens. CB
1
receptor knockout mice have been shown to exhibit reduced
ethanol and sucrose consumption (Poncelet et al. 2003).
These and other studies indicate a role for the cannabinoid
system in reward function in the brain. There is also ev-
idence for a role of the cannabinoid system in learning and
memory (Lichtman 2000; reviewed in Robinson and Riedel
2004), sleep/arousal (Mechoulam et al. 1997; Santucci et al.
1996), analgesia (reviewed in Walker and Huang 2002),
movement disorders (reviewed by De Fonseca et al. 1998),
neuroprotection (reviewed in Grundy 2002), and anxiety
(Haller et al. 2004). Some of the aforementioned physio-
logical, psychotropic, and potentially therapeutic actions of
SR141716A have been attributed to its selective effects on
monoamine release in the medial prefrontal cortex (mPFC;
Tzavara et al. 2003), although the level of CB
1
receptor
occupancy in the brain that is required to detect these effects
in animal models and in humans is presently not known.
Although others have measured in vivo CB
1
receptor
occupancy with a derivative of SR141716A, [
131
I]AM281,
relating this occupancy to changes in locomotor activity
(Cosenza et al. 2000), we sought to evaluate central CB
1
receptor occupancy using [
3
H]SR141716A in relation to
physiological and neurochemical changes elicited by leading
CB
1
receptor antagonists that are directly relevant to their
therapeutic potential in the clinic. Specifically, dose–occu-
pancy curves were generated for two orally administered
CB
1
receptor antagonists, SR141716A and SLV319 ((4S)-
(-)-3- (4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)sulfo-
nyl] -4-phenyl- 4,5-dihydro -1H-pyrazole-1- carboxamidine)
(Lange et al. 2004). Preclinical studies have shown that these
two compounds exhibit similar in vitro binding affinity for
CB
1
receptors and in vivo CB
1
receptor antagonistic activity
(Lange et al. 2004). Importantly, SR141716A is in Phase III
clinical trials for obesity (see Van Gaal et al. 2005)and
smoking cessation, and SLV319 is in Phase I clinical testing
for obesity and metabolic disorder (Smith and Fathi 2005).
With this in mind, we compared the CB
1
receptor occupancy
in rats required to generate release of the monoamines
dopamine (DA) and norepinephrine (NE) in the mPFC and
changes in feeding behavior (consumption of a highly
palatable diet).
Materials and methods
Drugs
SR141716A and SLV319 were synthesized at Eli Lilly and
Company, Indianapolis, IN, USA. These were administered
by oral gavage in a vehicle comprised of 1% sodium car-
boxymethylcellulose (Hercules, Wilmington, DE, USA),
0.5% sodium lauryl sulfate (Sigma Chemical Co. St. Louis,
MO, USA), 0.085% Povidone K30 (International Specialty
Products, Wayne, NJ, USA), 0.05% Antifoam 1510-US
(Dow Corning, Midland, MI, USA) (all w/w). Both drugs
were prepared fresh and administered as an even and fine
suspension. [
3
H]SR141716A, used as a tracer to determine
in vivo CB
1
receptor occupancy, was purchased from
Amersham Biosciences (Piscataway, NJ, USA) at a specific
activity of 103 mCi/mg.
Animals
Male Wistar rats (240–300 g; Harlan Sprague–Dawley,
Indianapolis, IN, USA, for feeding studies and Charles
Rivers Laboratories, Wilmington, MA, USA, for occupancy
and microdialysis studies) had ad libitum access to normal
rat chow and water unless indicated below. Two different
vendors were used as a source for animals, as each method
followed in the present study was conducted according to
conditions previously established (see below), i.e., for
reasons of continuity and comparison. All studies were
performed in accordance with National Institutes of Health
guidelines under protocols approved by the Animal Care
and Use Committee of Eli Lilly and Company.
Receptor occupancy determinations
Vehicle or CB
1
receptor antagonist test compound was
administered orally (p.o.) at various doses ranging from 0.01
to 30 mg/kg to groups of four rats. Either 1 or 3 h later,
10 μCi [
3
H]SR141716A was administered intravenously
(i.v.) in the lateral tail vein. After an additional hour, rats
were killed by cervical dislocation, and a portion of the
frontal cortex was removed and placed on ice. The frontal
cortex was chosen due to the density of CB
1
receptors in this
area, as well as its relevance to the psychotropic effects of
27
CB
1
receptor antagonists and potentially their therapeutic
applicability (see, e.g., Tzavara et al. 2003; Tataranni et al.
1999). In one dose–occupancy experiment, the brain stem
was also collected in order to examine specific binding in
this brain structure. The tissues were solubilized with 2 ml
Soluene 350 (Perkin Elmer, Wellesley, MA, USA), and
samples were agitated slowly on a shaker table. After ap-
proximately 24 h, 5 ml of scintillation fluid (Ready Solv HP,
Beckman Coulter, Fullerton, CA, USA) was added, and the
samples were shaken for an additional 24 h. Samples were
counted using liquid scintillation spectroscopy, and the
results were expressed as disintegrations per minute per
milligram tissue. Tracer levels in the frontal cortex re-
presented total binding, the sum of specific and nonspecific
binding sites. In order to calculate receptor occupancy, the
level of tracer that represented nonspecific binding needed
to be determined. To accomplish this, we used an intrave-
nous dose of 10 mg/kg unlabeled SR141716A that we had
previously demonstrated, through the generation of i.v. dose
occupancy curves, was well on the dose–occupancy curve
plateau, with higher doses not further suppressing tracer
binding. In each experiment, we included an additional
group of four rats dosed with SR141716A at 10 mg/kg, i.v.,
followed by the tracer 15 min later. The level of tracer in the
cortex of vehicle-pretreated animals represents the sum of
nonspecific and specific binding and is assigned the value of
0% occupancy (all receptors available to the tracer). The
lower level of tracer in animals pretreated with the very high
intravenous dose of SR141716A, the positive control group,
represents the nonspecific binding and is assigned the value
of 100% occupancy (no receptors available to the tracer).
Levels of tracer in the cortex following oral CB
1
receptor
antagonist treatment were interpolated linearly between
these two extremes in order to determine the percent CB
1
receptor occupancy. Data were expressed as means±SEM
(N=4/group) calculated using Prism version 3.0 (GraphPad
Software Inc., San Diego, CA, USA). The ED
50
values were
obtained by fitting the data to a sigmoidal curve using
nonlinear regression. To compare the occupancy curves
generated with the cortex and brain stem, the relative
potency was estimated by simultaneously fitting a four-
parameter logistic equation to the cortex and brain stem
occupancy dose-responses assuming common top, bottom,
and slope parameters using SAS/STAT version 8 (SAS
Institute, Cary, NC, USA). To account for the correlation
between occupancies measured on the same animal, the
multivariate nonlinear regression methods outlined in
Gallant (1987) were used to estimate the correlation and
refit the results with the data transformed to eliminate the
correlation.
Microdialysis studies
Two weeks prior to the microdialysis experiments, rats were
anaesthetized with a mixture of chloral hydrate and
pentobarbital (170 and 36 mg/kg in 30% propylene glycol
and 14% ethanol) and implanted with a guide cannula
(BAS) aimed at the mPFC (AP +3.2, ML +0.6, DV -2.2)
according to Paxinos and Watson (1998). Twenty-four
hours before testing, a 4-mm concentric microdialysis probe
was inserted in the mPFC (BAS, BR-4). Microdialysis ex-
periments were conducted in freely moving animals es-
sentially as described by Tzavara et al. (2003) and briefly
outlined below. The appropriate location of the probes was
verified histologically at the end of the experiment.
On the day of the experiment, a modified Ringer’s so-
lution (150 mM NaCl, 3 mM KCl, 1.7 mM CaCl
2
, 0.9 mM
MgCl
2
) was perfused at a rate of 1.0 μl/min. Samples were
collected in the presence of an antioxidant (3 mM L-cysteine,
10 mM EDTA) every 30 min into a refrigerated fraction
collector and analyzed the same day with high performance
liquid chromatography (HPLC) coupled to electrochemical
detection as previously described (Perry and Fuller, 1997).
Briefly, catecholamines were separated on a 250×2-mm C-
18 carbon polymer column (BDS Hypersil, ThermoHy-
persil, USA) (mobile phase: 75 mM NaH
2
PO
4
,0.5mM
EDTA, 1.6 mM SOS, 8% CH
3
CN, 75% THF) and detected
on dual carbon electrodes with the potentials set at +680
and + 100 mV. After about 3 h of baseline sampling, vehicle
or drugs were administered p.o., and samples were col-
lected for an additional 4-h period. In this manner, con-
centrations of the catecholamines DA and NE and their
metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC)
and homovanillic acid (HVA), were quantitated in the
dialysate from the mPFC, reflecting their extracellular
concentrations as a result of release or efflux.
Data obtained in response to vehicle and two doses of
each of the compounds were expressed as mean multifold
changes over baseline (±SEM, N=5–8 rats/group), which is
the average of the three basal values collected immediately
before treatment, and were analyzed with a two-way
ANOVA (dose/treatment × time) followed by a Newman–
Keuls multiple comparisons analysis using Statistica soft-
ware (StatSoft, Inc., Tulsa, OK, USA). Data were also
presented as overall (average) mean (±SEM) multifold
changes over baseline and analyzed with one-way ANOVA
followed by the Newman–Keuls test.
Feeding studies
Animals were exposed to a highly palatable, high-fat diet
(HP, 40% fat, 41.3% carbohydrate, 4.25 kcal/g; Teklad,
Madison, WI, USA) at the same time each day for 1 h over a
period of 4 days. Normal rodent chow (Purina 5001, PMI
Nutrition International, Brentwood, MO, USA) was avail-
able at all times. One or three hours prior to HP diet pre-
sentation, rats were dosed p.o. with vehicle on days 1 to 3
and either vehicle, 1, 3, or 10 mg/kg of a CB
1
receptor
antagonist on day 4. The studies were conducted in the light
phase to minimize the impact of nocturnal feeding bouts.
Data were expressed as means (±SEM; N=7 rats/group).
Comparisons between vehicle- and drug-treated groups
28
were performed using one-way ANOVAs followed by
Tukey’s test using Prism version 3.0 (GraphPad Software
Inc.), with a pvalue of<0.05 considered significant.
Results
Receptor occupancy determinations
Dose–occupancy curves were generated for the two CB
1
receptor antagonists with either 1- or 3-h pretreatment.
SR141716A administration generated similar curves at both
time points, with absolute ED
50
values of 1.0 mg/kg at 1 h
and 1.1 mg/kg at 3 h (Fig. 1). The dose–occupancy curves
generated for SLV319 were also similar, with absolute ED
50
values of 5.7 and 7.8 mg/kg at 1 and 3 h, respectively
(Fig. 2). The SLV319 occupancy curve generated in the rat
brain stem confirmed the presence of specific binding sites
in this brain structure. A statistical comparison of the curves
obtained simultaneously from cortical and brain stem tissue
demonstrated no significant difference (relative potency of
0.64, 95% CI=0.38–1.05, p=0.078) (Fig. 3).
Microdialysis studies
Basal dialysate concentrations of either DA or NE in the
various pretreatment groups were pooled, as they were not
statistically different. The overall mean (±SEM) values of
DA and NE were 0.54±0.05 and 1.00±0.10 pmol/ml, re-
spectively. Both CB
1
receptor antagonists increased extra-
cellular DA levels in the mPFC. SR141716A appeared to be
more potent, causing a significant increase following the 3-
and 10-mg/kg doses, while SLV319 only caused a signif-
icant increase at 10 mg/kg (Fig. 4). The time course of the
effects of the two compounds at the 10 mg/kg dose appeared
to be similar, although a more pronounced biphasic effect on
DA release was seen with SLV319, with peak effects at 30
and 180 min (Fig. 4); this may be due to the pharmacokinetic
characteristics of this compound that influence plasma and
brain concentrations, accordingly. Specifically, significant
dose (F
2,14
=12.96, p<0.001), time (F
9,126
=8.30, p<0.001),
and interaction (F
18,126
=2.57, p<0.01) effects were found in
response to SR141716A, and multiple comparisons re-
vealed significant (p<0.05) differences in the effects of each
0.01 0.1
1 3 10 30
-40
-20
0
20
40
60
80
100
120
0.3
SR141716A (mg/kg, p.o.)
% occupancy
Fig. 1 In vivo CB1 receptor
dose–occupancy curves in the
frontal cortex of Wistar rats by
orally administered
SR141716A, as measured with
[3H]SR141716A tracer.
SR141716A was dosed 1 h
(squares)or3h(triangles) prior
to intravenous tracer adminis-
tration. Animals were killed 1 h
after tracer
0.01 0.1 110
-20
0
20
40
60
80
100
120
3300.30.03
SLV319 (mg/kg, p.o.)
% occupancy
Fig. 2 In vivo CB1 receptor
dose–occupancy curves in the
frontal cortex of Wistar rats by
orally administered SLV319, as
measured with [3H]SR141716A
tracer. SLV319 was dosed 1 h
(squares)or3h(triangles) prior
to intravenous tracer adminis-
tration. Animals were killed 1 h
after tracer
29
dose of SR141716A from the effects of vehicle at several
time points (Fig. 4a). In addition, when the data were
expressed as overall effects, a significant (F
2,16
=15.09,
p<0.001) treatment effect was found, as well as significant
(p<0.05) differences of the effect of each dose compared to
the effects of vehicle (Fig. 4c). Similarly, significant dose
(F
2,9
=8.71, p<0.01), time (F
9,81
=3.83, p<0.001), and inter-
action (F
18,81
=3.45, p<0.001) effects were found after
administration of SLV319, and multiple comparisons
revealed significant (p<0.05) differences of each dose of
SVL319 compared to vehicle at specific time points
(Fig. 4b). In addition, when the data were expressed as
0.01 0.1 1 10
-40
-20
0
20
40
60
80
100
3300.30.03
SLV319 (mg/kg, p.o., 1 hr)
% occupancy
Fig. 3 In vivo CB1 receptor
dose–occupancy curves in the
frontal cortex (squares) and
brain stem (triangles) of Wistar
rats by orally administered
SLV319, as measured with [3H]
SR141716A tracer. One hour
after SLV319 administration,
intravenous tracer was injected.
Animals were killed 1 h after
tracer
-90 -60 -30 0 30 60 90 120 150 180 210 240
0.5
1.0
1.5
2.0
vehicle
SR141716A (10 mg/kg)
*
SR141716A (3 mg/kg)
p.o.
*
*
*
*
*
*
*
*
*
Time (min)
DA efflux (fold over baseline)
vehicle
SLV319 (10 mg/kg)
p.o.
*
*
***
*
SLV319 (3 mg/kg)
*
0.50
0.75
1.00
1.25
1.50
1.75
*
*
vehicle SR141716A
(
10 m
g
/k
g)
SR141716A
(
3 m
g
/k
g)
67-74%
88-107%
Overall DA efflux (fold over baseline)
*
11-30%
69%
a
-90 -60 -30 0 30 60 90 120 150 180 210 240
0.5
1.0
1.5
2.0
Time (min)
DA efflux (fold over baseline)
b
c
0.50
0.75
1.00
1.25
1.50
1.75
vehicle
(
10 m
g
/k
g)
SLV319 SLV319
(
3 m
g
/k
g)
Overall DA efflux (fold over baseline)
d
Fig. 4 The CB
1
receptor antagonists SR141716A and SLV319 dose-
dependently increased extracellular DA concentrations (efflux) in the
medial prefrontal cortex of Wistar rats. aand bTime course of DA
changes in the dialysate expressed as multifold change from baseline.
cand dThe average changes from baseline over the entire experiment
(overall effects). SR141716A (aand c) or SLV319 (band d) were
administered orally (p.o., indicated by arrows). Percentages in boxes
represent mean CB
1
receptor occupancy at each dose as determined in
in vivo occupancy experiments. *p<0.05 compared to vehicle
30
overall effects, a significant (F
2,13
=7.69, p<0.01) treatment
effect was found that was due to a significant (p<0.05)
difference of the effect of the 10-mg/kg dose compared to
the effect of vehicle (Fig. 4d). The 10-mg/kg dose of
SR141716A also increased extracellular NE levels in this
brain area, while neither dose of SLV319 caused a sig-
nificant NE increase (Fig. 5). Specifically, significant dose
(F
2,15
=5.76, p<0.05) and time (F
9,135
=3.41, p<0.001)
effects were only found in response to SR141716A; for
this reason, one-way ANOVAs were performed at each time
point, and multiple comparisons thereafter revealed signif-
icant (p<0.05) differences in the effects of the 10-mg/kg
dose of SR141716A from the effects of vehicle at several
time points (Fig. 5a). In addition, when the data were
expressed as overall effects, a significant (F
2,16
=6.20,
p<0.05) treatment effect was found that was due to sig-
nificant (p<0.05) differences of the effect of the 10-mg/kg
dose compared to the effects of vehicle (Fig. 5c). When the
NE data in response to SLV319 were analyzed, only a
significant time effect reached statistical significance
(F
9,90
=2.44, p<0.05), whereas neither dose affected NE
levels (Fig. 5b,d). As previously shown with intraperitoneal
administration (Tzavara et al., 2003), SR141716A adminis-
tered orally significantly increased extracellular concentra-
tions of DOPAC and HVA after both doses, while SLV319
administration only caused a significant increase in these
metabolites after the 10-mg/kg dose (data not shown).
Feeding studies
As can be seen in Fig. 6, prior to antagonist treatment (days 1
to 3), the rats consumed an increasing amount of the HP diet
during their 1 h of exposure. Then on the treatment day
(day 4), there was a significant (p<0.05) treatment effect at
both time points with either SR141716A (F
3,27
=9.08,
pretreatment of 1 h; F
3,27
=8.66, 3 h) or SLV319
(F
3,27
=12.41, 1 h; F
3,27
=6.32, 3 h) resulting in a decrease
in HP diet intake compared to vehicle-treated animals.
Significant (p<0.05) decreases were seen with both CB
1
receptor antagonists at the 3- and 10-mg/kg doses at 1 h
pretreatment, as well as at 3 h pretreatment. As this dif-
ference was not compensated for by an increase in normal
chow consumption, there was a significant (p<0.05) dose
effect resulting in a decrease in total kilocalories per
kilogram (caloric intake) consumed for the 3- and 10-mg/kg
vehicle
SR141716A (10 mg/kg)
SR141716A (3 mg/kg)
p.o.
*
*
*
**
vehicle
SLV319 (10 mg/kg)
p.o.
SLV319 (3 mg/kg)
*
67-74%
88-107%
11-30%
69%
-90 -60 -30 0 30 60 90 120 150 180 210 240
0.5
1.0
1.5
2.5
2.0
Time (min)
NE efflux (fold over baseline)
0.5
1.0
1.5
2.0
vehicle SR141716A
(
10 m
g
/k
g)
SR141716A
(
3 m
g
/k
g)
Overall NE efflux (fold over baseline)
a
-90 -60 -30 0 30 60 90 120 150 180 210 240
0.5
1.0
1.5
2.5
2.0
Time (min)
NE efflux (fold over baseline)
b
c
0.5
1.0
1.5
2.0
vehicle
(
10 m
g
/k
g)
SLV319 SLV319
(
3 m
g
/k
g)
Overall NE efflux (fold over baseline)
d
Fig. 5 SR141716A (10 mg/kg, p.o.) increased extracellular NE
efflux in the medial prefrontal cortex of Wistar rats. aand bTime
course of NE changes in dialysate expressed as multifold change
from baseline. cand dThe average changes from baseline over the
entire experiment (overall effects). Rats were orally administered
(indicated by arrows) SR141716A (aand c) or SLV319 (band d).
Percentages in boxes represent mean CB
1
receptor occupancy at
each dose as determined in in vivo occupancy experiments. *p<0.05
compared to vehicle
31
groups with both compounds at both time points
[SR141716A: 1 h 227±4, 194±7, 137±8 (F
3,27
=42.52) and
3 h 231±7, 203±8, 165±12 (F
3,27
=11.36); SLV319: 1 h 215±
4, 200±3, 171±3 (F
3,27
=29.51) and 3 h 219±8, 194±5, 187±
10 (F
3,27
=6.33) kcal/kg for the vehicle, 3- and 10-mg/kg
groups, respectively].
Discussion
The CB
1
receptor antagonists SR141716A and SLV319
dose-dependently occupied CB
1
receptors in the frontal
cortex of rats after oral administration. There was little
difference in ED
50
values between the 1- and 3-h post-
injection time intervals for either compound, showing that
occupancy was relatively stable within that time frame. One
hour after SLV319 treatment, the only point where brain
stem binding was examined, there was no statistically
significant difference in the receptor occupancy curves gen-
erated simultaneously with the brain stem and the cortex,
despite differences in CB
1
receptor densities between these
two areas (see, e.g., Tsou et al. 1998). The microdialysis data
demonstrated that orally administered SR141716A (at 3 and
10 mg/kg) and SLV319 (at 10 mg/kg) increased extracel-
lular DA concentrations in the mPFC. SR141716A also
increased NE efflux in the mPFC at 10 mg/kg, while none of
the tested doses of SLV319 affected NE concentrations.
Both compounds, administered at 3 and 10 mg/kg, caused a
significant decrease in HP diet consumption at both the 1- and
3-h postinjection intervals. Although it appeared that
SR14716A was more potent than SLV319 in occupying
cortical CB
1
receptors and in increasing cortical release of
monoamines, but not in reducing HP food intake, any claim
for actual differences in in vivo potency of these compounds
would require additional work, taking into consideration
solubility, exposure, and other pharmacokinetic variables.
The occupancy method described here offers certain
methodological advances compared to the study by
Cosenza et al. (2000). Specifically, in the latter study, the
nonspecific binding was estimated using an area thought to
contain very low or no CB
1
receptor density, the brain
stem. We found that the use of the brain stem as a surrogate
0 1 2 3 4
0.0
2.5
5.0
7.5
10.0
12.5
vehicle
SR141716A (1 mg/kg)
SR141716A (3 mg/kg)
SR141716A (10 mg/kg)
*
*
51%
67%
88%
days
grams consumed
vehicle
SR141716A (1 mg/kg)
SR141716A (3 mg/kg)
SR141716A (10 mg/kg)
*
*
48%
74%
107%
vehicle
SLV319 (1 mg/kg)
SLV319 (3 mg/kg)
SLV319 (10 mg/kg)
*
*
12%
30%
69%
vehicle
SLV319 (1 mg/kg)
SLV319 (3 mg/kg)
SLV319 (10 mg/kg)
*
*
3%
11%
69%
a
01234
0.0
2.5
5.0
7.5
10.0
12.5
days
grams consumed
b
01234
0.0
2.5
5.0
7.5
10.0
12.5
da
y
s
grams consumed
c
01234
0.0
2.5
5.0
7.5
10.0
12.5
da
y
s
grams consumed
d
Fig. 6 SR141716A and SLV319 dose-dependently reduced intake
by Wistar rats of highly palatable chow (HP) during 1 h of daily
exposure. Animals were orally administered vehicle prior to HP
presentation on days 1–3. On day 4, they were dosed with the CB
1
receptor antagonist. aand cData after 1 h pretreatment with
SR141716A (a) or SLV319 (c). band dData after 3 h pretreatment
with SR141716A (b) or SLV319 (d). Percentages in boxes represent
mean CB
1
receptor occupancy at each dose as determined in in vivo
occupancy experiments. *p<0.05 vs vehicle
32
region for nonspecific binding is not appropriate in rats.
Instead, a high dose of a receptor-blocking drug was used
for this purpose (Goeders and Kuhar 1985). This method
of estimating nonspecific binding has the advantage in
that both total and nonspecific binding are determined in
the same brain area, obviating the need for assuming uni-
form nonspecific binding throughout the brain. Employing
this positive control method to determine nonspecific bind-
ing, we generated dose occupancy curves for the CB1
antagonists, SR141716A and SLV319.
As can be seen in Fig. 4,CB
1
receptor occupancy of
approximately 65% was sufficient to increase DA efflux in
the mPFC. This was attained by the 3- and 10-mg/kg doses
of SR141716A and the 10-mg/kg dose of SLV319. When
looking at the NE efflux data (Fig. 5), 65% occupancy was
not sufficient to cause an increase with either antagonist.
Only the 10-mg/kg dose of SR141716A, with occupancy
of 85%, caused an increase in cortical NE release, while no
doses of SLV319 used in the microdialysis experiments
met this level. The reasons why a higher level of CB
1
receptor occupancy is required to elicit changes in NE than
for DA are not clear. It should be noted that the SR
141716A (administered i.p.) mediated neurotransmitter
changes appear to be brain-region-specific (Tzavara et al.
2003), i.e., marked increases were only found in the mPFC
and not in a subcortical dopaminergic area, the nucleus
accumbens, and even if the CB
1
receptor occupancy is
similar in different parts of the brain, depending on the
circuitry involved, the overall functional consequences
may well differ regionally.
The increases in extracellular DA and NE measured in
the mPFC after administration of SR141716A may be
relevant to its apparent effectiveness in regulating food-
and drug-induced motivation and reward and, in turn, to its
therapeutic actions in obesity and smoking cessation. In
this regard, it is noteworthy that neuronal activity within
the prefrontal cortex appears to play an important role in
the regulation of eating behavior and satiety control
(Tataranni et al. 1999). Similar to SR141716A, bupropion,
an atypical antidepressant, increased extracellular DA and
NE in the prefrontal cortex (Li et al. 2002) and was the first
FDA-approved non-nicotine-based pharmacotherapy for
smoking cessation. Its effectiveness has been linked to
decreases in craving (Durcan et al. 2002).
The feeding data showed little difference between the
1- and 3-h time intervals for either CB
1
receptor anta-
gonist, as would be expected from the occupancy data.
There was no difference in potency and efficacy between
the compounds with respect to the HP feeding effect, with
both antagonists reaching significant decreases at 3 mg/kg,
in spite of an apparent difference in cortical CB
1
receptor
occupancy. There are many possible explanations for this.
For example, occupancy in other CB
1
-receptor-containing
areas such as the nucleus accumbens, hippocampus, endo-
penduncular nucleus, and the hypothalamus that are in-
volved in motivational, hedonic, and metabolic aspects of
eating may be different than in the frontal cortex and may
be more directly involved in the feeding effect. However,
CB
1
receptor occupancy curves generated for at least
SLV319 using frontal cortex, an area with relatively high
CB
1
receptor density, and brain stem, with much lower
density, dissected from the same rats were very similar
(see Fig. 3).
The apparent discrepancy could also be due to the
possibility that these antagonists have effects in the
periphery as well. Several lines of evidence for a peripheral
role of CB
1
receptors in feeding exist, including increased
anandamide in small intestine but not brain of rats with
food deprivation and loss of hyperphagic and hypophagic
responses of anandamide and SR141716A, respectively,
after deafferentation by capsaicin (Gomez et al. 2002). It
was recently shown that SR141716A reduced levels of
plasma ghrelin (Cani et al. 2004). As ghrelin stimulates
appetite, CB
1
receptor antagonists may work in part by
decreasing ghrelin production in the periphery. We mea-
sured central CB
1
receptor occupancy, which is dependent
on brain penetration. SLV319 may have higher peripheral
occupancy, while SR141716A may have higher brain
penetration and therefore higher central occupancy.
Additionally, this incongruity between central CB
1
recep-
tor occupancy and potency in the feeding studies may be
due to other actions of SLV319 not shared with
SR141716A. The regulation of feeding is extremely
complex, with multiple signals and pathways involved.
SLV319 may hypothetically interact with another regula-
tory pathway, in addition to its actions on the cannabinoid
system, leading to a larger decrease in consumption than
that anticipated by its CB
1
receptor occupancy alone.
Nevertheless, it should be emphasized that, if anything,
there is evidence in support of the notion that SR141716A
has off-target, non-CB
1
-receptor-related activities (see,
e.g., Breivogel et al. 2001; Monory et al. 2002), although
the significance of these findings in food intake regulation
is not readily apparent.
While occupancy measurements have previously been
reported using [
131
I]AM281 (Cosenza et al. 2000), we have
described here a more advantageous in vivo central CB
1
receptor occupancy method, this one using commercially
available [
3
H]SR141716A. Employing this method, we
demonstrated that SR141716A and SLV319 occupy cor-
tical CB
1
receptors consistently between 1 and 3 h after
dosing. They are both able to increase extracellular DA
levels in the frontal cortex at CB
1
receptor occupancies
exceeding 65%. SR141716A also increases extracellular
NE levels in this brain region when occupancy exceeds
85%. Both compounds are effective at decreasing intake of
a highly palatable diet, although the effectiveness of
SLV319 at such low CB
1
receptor occupancies may sug-
gest it exerts at least some of its effects through mech-
anisms other than central CB
1
receptor occupancy.
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