Characterization of a novel and selective cannabinoid CB1 receptor inverse

agonist, Imidazole 24b, in rodents

Lauren P. Shearman a, D. Sloan Stribling a, Ramon E. Camacho a, Kimberly M. Rosko a,Junying Wang a, Sharon Tong a, Yue Feng b, Donald J. Marsh b, Hong Yu b, Xiaoming Guan b,

Stephanie K. Spann a, Douglas J. MacNeil b, Tung M. Fong b, Joseph M. Metzger a,Mark T. Goulet c, William K. Hagmann c, Christopher W. Plummer c, Paul E. Finke c,

Sander G. Mills c, Shrenik K. Shah c, Quang Truong c, L.H.T. Van der Ploeg b,D. Euan MacIntyre a, Alison M. Strack a,⁎

a Department of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, United Statesb Department of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ 07065, United Statesc Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, United States

Received 3 January 2007; received in revised form 11 September 2007; accepted 16 October 2007

Available online 25 October 2007

Abstract

We document in vitro and in vivo effects of a novel, selective cannabinoid CB1 receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-

cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human

and rat CB1 receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB2 receptor with an affinity of 297 nM; in vitro, it

is a receptor inverse agonist at both cannabinoid CB1 and CB2 receptors as it causes a further increase of forskolin-induced cAMP increase. Oral

administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB1 receptor antagonist efficacy in vivo.

Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB1 receptor occupancy (RO) at 2h post-

dosing in rats, indicating that ∼50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b

administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague–Dawley rats attenuated overnight food intake with a minimal effective

dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB1 receptor-deficient mice. DIO rats were dosed orally with vehicle,

Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading

to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated

weight gain. These findings suggest that selective cannabinoid CB1 receptor inverse agonists such as Imidazole 24b have potential for the

treatment of obesity.

© 2007 Elsevier B.V. All rights reserved.

Keywords: Cannabinoid; Food intake; Obesity; Body weight; Diet-induced obesity; Adiposity

1. Introduction

Cannabinoid receptors and associated endocannabinoids are

found in numerous physiological systems and most recently

have received attention for their potential in the pharmacolog-

ical treatment of obesity. The endocannabinoid system is

comprised of several endogenous ligands (anandamide, 2-

arachidonoyl glycerol (2-AG), 2-arachidonyl glyceryl ether and

virodhamine (Devane et al., 1992; Mechoulam et al., 1995)).

Two cannabinoid receptor subtypes, cannabinoid CB1 receptor

(Matsuda et al., 1990) and cannabinoid CB2 receptor (Munro

et al., 1993) have been cloned and characterized. Both receptors

are primarily coupled to Gi/o proteins and act to inhibit adenylyl

cyclase (Howlett, 1995) and other signaling pathways (Boua-

boula et al., 1997, 1995). Cannabinoid CB1 receptors are

Available online at www.sciencedirect.com

European Journal of Pharmacology 579 (2008) 215–224www.elsevier.com/locate/ejphar

⁎ Corresponding author. RY80Y-145, Department of Pharmacology, Merck

Research Laboratories, PO Box 2000, Rahway, NJ 07065, United States. Tel.:

+1 732 594 8367; fax: +1 732 594 3841.

E-mail address: alison_strack@merck.com (A.M. Strack).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.ejphar.2007.10.033

present in the central nervous system (CNS), with high levels of

expression in brain areas implicated in motivation, mood, and

appetite regulation (Devane et al., 1992; Matsuda et al., 1990;

Munro et al., 1993 Westlake et al., 1994). In contrast,

cannabinoid CB2 receptors are predominantly localized in

peripheral tissues and immune cells (Galiegue et al., 1995;

Munro et al., 1993) and in microglial cells (Walter et al., 2003).

There are a growing number of observations, particularly in

brain and isolated heart and blood vessel preparations that

suggest that other cannabinoid receptor subtypes may exist

although the genes that encode them have not been identified

(Breivogel et al., 2001; Jarai et al., 1999).

An increasing body of evidence underscores the role of

cannabinoid CB1 receptor in appetite and in reward aspects of

food consumption. Endocannabinoids and Δ9-tetrahydrocan-

nabinol (Δ9-THC) stimulate food intake in animals and man

(Abel, 1975; Cota et al., 2003a; Foltin et al., 1988).

Hypothalamic endocannabinoid levels are elevated in leptin-

deficient or leptin receptor-deficient mice (Di Marzo et al.,

2001). Anandamide and 2-AG administered into specific brain

nuclei stimulate food intake in rodents (Jamshidi and Taylor,

2001; Williams and Kirkham, 1999). Furthermore, extracellular

concentrations of endocannabinoids fluctuate during fasting and

upon feeding in brain regions that are regulated by the anorectic

peptide leptin (Di Marzo et al., 2001; Kirkham et al., 2002).

Besides these effects on food intake, cannabinoid receptor

agonists also induce hypothermia, ataxia, catalepsy, and

analgesia in rodents (Martin et al., 1991).

Conversely, cannabinoid CB1 receptor antagonists/inverse

agonists (e.g. SR141716A) are anorexigenic and cause weight

loss in rodents (Colombo et al., 1998; Hildebrandt et al., 2003;

Ravinet Trillou et al., 2003) and in humans (Pi-Sunyer et al.,

2006). Blockade of cannabinoid CB1 receptor by SR141716A

decreases food consumption in rats (Arnone et al., 1997;

Rinaldi-Carmona et al., 1994) and marmosets (Simiand et al.,

1998) and attenuates alcohol drinking in rodents (Arnone et al.,

1997; Wang et al., 2003). SR141716A inhibits the orexigenic

effects of endocannabinoids and Δ9-THC in rats (Kirkham

et al., 2002; Williams and Kirkham, 1999, 2002). SR141716A

inhibits feeding in non-deprived rats (Colombo et al., 1998),

genetically obese animals (Bensaid et al., 2003; Di Marzo et al.,

2001; Vickers et al., 2003) and animals raised on high fat diets

(Hildebrandt et al., 2003; Ravinet Trillou et al., 2003).

Deletion of the murine gene coding for cannabinoid CB1

receptor (Cnr1) leads to leanness, resistance to diet-induced

obesity and enhanced leptin sensitivity (Cota et al., 2003b;

Ravinet Trillou et al., 2004). When maintained on standard

chow, cannabinoid CB1 receptor-deficient (Cnr1−/−) mice

exhibit reduced spontaneous food intake and body weight

gain. Cnr1−/− mice also have reduced food intake following a

fast (Di Marzo et al., 2001). The anorexigenic effects of

SR141716A are absent in Cnr1−/− mice (Di Marzo et al., 2001;

Ravinet Trillou et al., 2004). Importantly, hypothalamic

cannabinoid CB1 receptor mRNA is co-expressed in neurons

containing neuropeptides which modulate food intake, partic-

ularly corticotropin-releasing hormone, cocaine–amphetamine

regulated transcript, melanin concentrating hormone, and

prepro-orexin (Cota et al., 2003b), further implicating canna-

binoid CB1 receptor in the pathways known to control appetite.

Clearly, potential pharmacological manipulations of this

system could lead to the development of novel compounds for

the treatment of diseases in which cannabinoid CB1 receptors

are implicated. Here we describe and characterize the in vitro

characteristics, CNS receptor occupancy and in vivo effects on

indices of behaviour and of energy balance in rodents of a

novel, potent and orally active cannabinoid CB1 receptor

inverse agonist, Imidazole 24b (Plummer et al., 2005).

2. Materials and methods

2.1. Animals

All testing protocols described below were reviewed and

approved by the Merck Research Laboratories Institutional

Animals Care and Use Committee in Rahway, NJ. Rats and

mice were maintained in a 12 h/12 h light–dark cycle with free

access to food and water in group housing conditions in a

temperature controlled environment (22 °C).

Sprague–Dawley CD and DIO (Dietary-Induced Obese

Sprague–Dawley CD) rats were obtained from Charles River

Labs (Wilmington, DE). DIO rats received a moderately high

fat, high sucrose diet of 4.41 total kcal/g (D12266B, Research

Diets, Brunswick, NJ; 32% dietary fat, 16% protein, 51%

carbohydrate) starting at 4 weeks of age while chow-fed rats

were maintained on a standard rodent chow of 3.82 total kcal/g

(#7001, Teklad, Madison, WI, 4% dietary fat, 24% protein, 72%

carbohydrate). DIO rats were used at 12–16 weeks of age.

Cannabinoid CB1 receptor-deficient (Cnr1−/−) and wild-type

(Cnr1+/+) male mice (Taconic, USA) weighing 25 + 2 g were

housed at constant temperature (22 °C) and humidity (30–

70%), with food (Teklad Diet #7012, 5% dietary fat; 3.75 kcal/

g) and water available ad libitum. Cnr1−/− mice were licensed

from A. Zimmer (Zimmer et al., 1999). These mice were

backcrossed onto the C57BL/6J genetic background for ten

generations by A. Zimmer prior to N10 homozygous Cnr1−/−

mice being rederived at Taconic Farms onto the C57BL/6N

genetic background. The resulting heterozygous mice were

intracrossed to yield mice of all three possible genotypes

(Cnr1+/+; Cnr1+/−, heterozygous; Cnr1−/−). The resulting

wild-type and CB1 receptor-deficient mice were then used to

set up time-matched homozygous and wild-type intracrosses.

These intracrosses were used to generate age-matched Cnr1+/+

and Cnr1−/− mice. Mice were maintained at all times on an

irradiated rodent chow (Harlan Teklad #7012) except during the

time of study.

2.2. Automated food intake system

Rats were caged individually in an automated food intake

system in Nalgene cages with metabolism feeders attached to

them. The food cups were external to the feeder and were placed

on individual balances. Each balance was connected to a central

computer which collected readings every 5 min (weight of food

in grams to 0.1 g); for visual clarity, data from every 30′ was

216 L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

graphed. These data were analyzed to measure grams of food

consumed. Rats were transferred into the caging with

specialized feeders for at least 3 days before experimentation

to allow for acclimation.

2.3. Compounds

Tween 80 was purchased from Fisher Scientific (Pittsburgh,

PA). CP55940 (5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-

hydroxypropyl)cyclohexyl]phenol) was purchased from Tocris

Cookson (Ellisville, MO). S-(+)-fenfluramine (dexfenflura-

mine) was purchased from Research Biochemicals Inc. (Natick,

MA). The cannabinoid CB1 receptor inverse agonist, Imidazole

24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-

1-methyl-imidazole-2-carboxamide; Fig. 1) was synthesized

and prepared in the Medicinal Chemistry Department at Merck

Research Laboratories, Rahway, NJ (Plummer et al., 2005).

[125I]-AM-2233 (R-2-[125I]iodophenyl-(1-(1-methylpiperidin-

2-ylmethyl)-1H-indol-3-yl)methanone) was synthesized and

labeled in-house.

2.4. Experiments

2.4.1. Radioligand binding

The long form of the human cannabinoid CB1 receptor, the

human cannabinoid CB2 receptor, and the rat cannabinoid CB1

receptor were cloned based on GenBank sequences, and were

stably expressed in clonal CHO cell lines. The binding affinity

of Imidazole 24b was measured by incubating various

concentrations of Imidazole 24b with 0.5 nM [3H] CP55940,

1.5 μg of recombinant cannabinoid CB1 receptor-CHO

membranes (or 0.1 μg of CBR2 CHO membranes) in 50 mM

Tris–HCl pH 7.4, 5 mM MgCl2, 2.5 mM EDTA, 0.5 mg/mL

fatty acid free Bovine Serum Albumin (BSA), 1× proteinase

inhibitor mix (P8340, Sigma), and 1% DMSO. After 1h

incubation at 37 °C, the reaction was stopped by filtration and

bound radioligand was separated from free radioligand by

washing the filter plate. Total specifically bound radiolabel was

10% or less of the total added radiolabel. Inhibitory IC50 values

were calculated through non-linear curve fitting.

2.4.2. Adenylyl cyclase assays

The intrinsic activity of activating or inhibiting Gi by the

cannabinoid CB1 receptor-ligand complex was measured by

incubating recombinant cannabinoid CB1 receptor-CHO cells

with various concentrations of test ligand in the presence of

10 μM forskolin, 200 μM phosphodiesterase inhibitor, 3-

isobutyl-1-methylxanthine (IBMX) in the assay buffer (Earle's

balanced salt solution supplemented with 5 mMMgCl2, 10 mM

HEPES pH7.3, 1 mg/mL BSA) at room temperature for 30 min.

Cells were lysed by boiling and intracellular cAMP level was

determined using an cAMP SPA kit (Amersham). The maximal

CP55940-mediated inhibition of forskolin-stimulated cAMP

increase is defined as 100% receptor agonist efficacy, and the

intrinsic activity of all other compounds is relative to the efficacy

of CP55940. Negative efficacy denotes inverse agonism.

Functional antagonism of receptor agonist response by

Imidazole 24b was determined by the Gi-cAMP assay as above,

except that both the CB receptor agonist 2-AG and Imidazole

24b were present in the incubation. A series of dose response

curves of 2-AG was performed in the absence or presence of

various concentrations of Imidazole 24b. The EC50 values were

determined and a Schild's plot was constructed, from which the

Kb value was determined.

To test for potential off-target activities, Imidazole 24b was

tested in 169 assays of receptor binding and enzyme activity by

MDS Pharma Services (King of Prussia, PA).

2.4.3. Receptor occupancy of Imidazole 24b in rats

Sprague–Dawley rats (male, 350 to 400g; n=3/group) were

dosed with Imidazole 24b (3, 10 and 30 mg/kg, p.o.) or vehicle

(10% Tween 80 in water). Two hours later, rats were euthanized

by CO2 asphyxiation. Plasma samples were taken by cardiac

puncture, and brains were quickly dissected and frozen in

isopentane at −40 °C and stored at −80 °C until use. The degree

of brain receptor occupancy (reciprocal of receptor binding) was

determined by quantitative receptor autoradiography in vitro,

using [125I]-AM-2233 as the radioligand. Thin (20 μm) coronal

brain sections encompassing the substantial nigra (20 μm) were

cut on a cryostat at −17 °C, and thaw-mounted onto microslides

and dried completely at room temperature. Receptor binding

was initiated by incubating the brain sections in the binding

buffer (Tris–HCl: 50 mM, pH. 7.3, MgCl2: 2 mM, CaCl2:

1 mM, KCl: 5 mM, BSA: 0.5%) containing 0.5 nM [125I]-AM-

2233 for 30 min at room temperature. Non-specific binding was

defined by similar incubation conditions in the presence of

10 μM Imidazole 24b. Brain sections were washed 3×4 min in

ice-cold washing buffer (Tris–HCl: 50 mM, pH. 7.3, MgCl2:

2 mM, CaCl2: 1 mM, KCl: 5 mM, Triton X-100: 0.05% (v/v)),

and then rinsed briefly in ice-cold distilled water before drying

at room temperature. Brain sections were exposed to X-ray film

overnight, and films were developed following standard

procedures.

Autoradiographic images were captured and analyzed with

an MCID/M2 image analysis system. Relative optical density

was determined for vehicle- or compound-treated groups (n=3

for vehicle or each dose of compound treatment). Specific

binding was obtained by subtracting the non-specific binding

Fig. 1. Structure of cannabinoid CB1 receptor inverse agonist Imidazole 24b

(5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidaz-

ole-2-carboxamide).

217L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

from the corresponding total binding. The maximal binding was

defined by specific binding in the vehicle treated rat brains. Oral

dosing of Imidazole 24b resulted in a dose-dependent reduction

of [125I]-AM-2233 binding to cannabinoid CB1 receptor, and %

receptor occupancy is derived by the reciprocal of % maximal

binding.

2.4.4. Reversal of cannabinoid receptor agonist-evoked

hypothermia

Sprague–Dawley rats (male, average body weight 162g)

were dosed with vehicle (10% Tween 80 in H2O) or Imidazole

24b (3, 10, or 30 mg/kg, p.o.). After 120 min, vehicle treated

animals were dosed with vehicle (0.1% Tween 80/H2O, i.p.) or

the cannabinoid receptor agonist CP-55940 (1 mg/kg, i.p.). All

Imidazole 24b treated animals were challenged with CP-55940.

Rectal temperatures were measured with a thermocouple probe

(BAT 10 Microprobe thermometer and RET-2 temperature

probe; Physitemp Instruments Inc., Clifton, NJ) prior to the p.o.

dose (t=− 120 min), prior to the i.p. dose (t=0 min) and at 45,

60 and 75 min post-CP55940 challenge.

2.4.5. Imidazole 24b effects on mouse food intake

Two experiments to test the suppression of food intake in mice

were performed. First, ad libitum fed male C57BL/6N wild-type

mice (n=9–10 per group, 11 week old, mean body weight 33 g)

were dosed with vehicle or Imidazole 24b (5 or 10 mg/kg, p.o.). In

the second experiment, to test specificity of Imidazole 24b for the

CB1 receptor, ad libitum fed,11-week oldmale wild-type (Cnr1+/+

or cannabinoid CB1 receptor-deficient (Cnr1−/−)mice were dosed

with vehicle or Imidazole 24b (10 or 16 mg/kg, p.o.).

In both experiments, Imidazole 24b was dissolved or

dispersed as a fine homogeneous suspension in 0.225%

methylcellulose/10% Tween 80 in water for subsequent oral

dosing. All mice were weighed and vehicle or Imidazole 24b

was administered by oral gavage to male mice ∼30 min prior to

the onset of the dark phase of the light cycle. A pre-weighed

aliquot of a highly palatable moderate high fat diet (Research

Diets D12266Bi; 25% kcal from sucrose, 32% kcal from fat,

4.41 kcal/g) was provided in the food hopper of the wire cage

top∼5 min prior to the onset of the dark phase of the light–dark

cycle and weighed 2 and 18 h after the onset of the dark cycle.

Additionally, all mice were weighed 18 h after the onset of the

dark phase of the light cycle. All dosing studies were of

crossover design.

2.4.6. Imidazole 24b effects on acute food intake in DIO rat

At 1h prior to dark cycle onset, male DIO rats (n=6–8 per

group, 536 g average body weight) were orally gavaged with

vehicle (10% Tween 80 in H2O) or the cannabinoid CB1

receptor inverse agonist Imidazole 24b (1, 3 or 10 mg/kg). Rats

were fed milled diet during acclimation to the caging and during

the experiment. Overnight food intake and body weight changes

were measured.

2.4.7. Imidazole 24b pharmacokinetics in DIO rat

A pharmacokinetic study with Imidazole 24b was performed

with male DIO rats (n=4). Rats were dosed at 3 mg/kg, p.o. in

10% tween/H2O. Blood samples were collected at 0.25, 0.5, 1,

2, 4, 6 and 8 h post-dosing. Plasma compound concentrations

were measured by Liquid Chromatography/Mass Spectrometry.

2.4.8. Imidazole 24b 14-day study in DIO rat

Baseline body composition was determined by DEXAscan

(Fan Beam X-Ray Bone Densitometer, QDR4500; Hologics,

Inc., Waltham, MA) 7 days prior to day 1 of dosing. Male DIO

rats (∼550 g avg. body weight; 22% body fat) were dosed orally

with vehicle (10% Tween80 in water), Imidazole 24b (1, 3 or

10 mg/kg), or S-(+)-fenfluramine (dexfenfluramine; 3 mg/kg).

Rats were dosed 1h before the onset of the dark cycle. Food

intake and body weight were recorded daily. Upon termination

on day 14, body composition was again determined by

DEXAscan analysis and brains and blood were collected.

White adipose tissues (WAT; retroperitoneal, mesenteric,

epididymal) were removed and weighed.

2.4.9. Data analysis

Data are presented as mean + S.E.M. For the in vivo

pharmacological studies, statistical analysis was performed

using Student's t-test or 1-way ANOVA followed by Dunnett's

t-test for post hoc analysis. Two-way ANOVA was used to

analyze the effects of Imidazole 24b in Cnr1+/+ and Cnr1−/−

mice and for the hypothermia study; a Bonferroni post hoc

analysis was used in conjunction with the 2-way ANOVA.

Significance level was set at Pb0.05.

3. Results

3.1. Radioligand binding

The binding affinity of Imidazole 24b for the recombinant

human or rat cannabinoid CB1 receptor or human cannabinoid

CB2 receptor was determined by measuring its ability to inhibit

the binding of [3H] CP55940. Imidazole 24b binds to human

and rat cannabinoid CB1 receptor with 4 and 10 nM affinity,

respectively. Imidazole 24b is selective for cannabinoid CB1

receptor, and it binds to the human cannabinoid CB2 receptor

with a binding affinity of 297 nM (Table 1).

As shown in Fig. 2 and Table 1, Imidazole 24b is an inverse

agonist at both cannabinoid CB1 receptor and cannabinoid CB2

receptor. It causes a further increase of forskolin-induced cAMP

increase. In contrast, a typical receptor agonist such as CP55940

Table 1

Binding IC50 value and receptor inverse agonist EC50 value of Imidazole 24b

IC50, nM EC50, nM (%act)

Human cannabinoid CB1 receptor 4 (n=16) 17 (−110%) (n=4)

Human cannabinoid CB2 receptor 297 (n=6) 165 (−74%) (n=2)

Rat cannabinoid CB1 receptor 10 (n=4) 4 (−110%) (n=2)

Binding assays were run in CHO membranes containing stably expressed

recombinant cannabinoid receptor. 0.5 nM [3H] CP55940 was used for

competition. To test functional inverse agonism, Imidazole 24b was tested in

adenylyl cyclase assays in cannabinoid receptor-containing CHO cells.

CP44940-mediated inhibition of forskolin-stimulated cAMP was used to define

100% receptor agonist efficacy. Data represent mean values with the number of

independent measurements indicated in parentheses.

218 L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

inhibits the forskolin-induced cAMP increase. These data

demonstrate that Imidazole 24b possesses intrinsic receptor

inverse agonist activity in the absence of receptor agonist.

In the presence of receptor agonist, the competitive receptor

antagonist activity of Imidazole 24b was demonstrated by

determining the dose response curve of the endogenous receptor

agonist 2-AG in the presence of various concentrations of

Imidazole 24b. Imidazole 24b shifted the receptor agonist dose

response to the right, and the Kb value from Schild's plot (4 nM)

was consistent with the binding affinity of Imidazole 24b (Fig. 3).

These data demonstrate that Imidazole 24b is a competitive

receptor antagonist when receptor agonist is present.

Imidazole 24b was tested in 169 assays of receptor binding

and enzyme activity by MDS Pharma Services (King of Prussia,

PA). In addition to its binding activity at cannabinoid CB1

receptor and cannabinoid CB2 receptor, Imidazole 24b affected

only 4 targets with IC50b10 μM (rat A3 receptor=0.5 μM;

rabbit VMAT2=0.3 μM; human HERG=4 μM; human

pp60SRC=8.7 μM). Thus, Imidazole 24b is a highly selective

cannabinoid CB1 receptor inverse agonist.

3.2. Receptor occupancy study in rats

Oral administration of Imidazole 24b resulted in a dose-

dependent increase in brain cannabinoid CB1 receptor occu-

pancy (23±1, 49±1, 66±18% receptor occupancy for 3, 10 and

30 mg/kg Imidazole 24b, respectively) at 2h post-dosing.

3.3. Imidazole 24b temperature study

The in vivo pharmacological antagonism of Imidazole 24b for

brain cannabinoid receptors was studied in a hypothermia model

(Fig. 4). The cannabinoid receptor agonist CP-55940 evoked

significant reductions in temperature relative to vehicle at 45, 60

and 75 min with a maximal decrease of 4.2 °C at 75 min post-

treatment (Dose: F(1.47)=263, Pb0.001; time: F(3, 47)=81.4,

Pb0.001; interaction: F(3, 47)=74.8, Pb0.001)). Imidazole

24b inhibited CP-55940-induced hypothermia in a dose-related

manner (Dose: F(3,95)=7.5, Pb0.001; time: F(3, 95)=273,

Pb0.001; interaction: F(9, 95)=5.6, Pb0.001)). Imidazole 24b

was not tested alone in this study. Historically, other CB1 receptor

inverse agonists that we have tested have not altered temperature

in the rat.

3.4. C57BL/6 and Cnr1−/− mouse food intake studies

In C57BL/6 mice, Imidazole 24b (10 mg/kg, p.o.) signi-

ficantly reduced spontaneous food intake at 2 and 18 h (2 h:

Fig. 3. Competitive receptor antagonist effect of Imidazole 24b. (A) Effect of

Imidazole 24b on the dose response curves of the receptor agonist 2-AG.

Specified concentrations of Imidazole 24b were included in each dose response

curve of 2-AG. (B) Schild's plot derived from (A).

Fig. 4. Imidazole 24b treatment inhibits cannabinoid CB1 receptor agonist-

induced hypothermia in rats. Imidazole 24b (0, 3, 10, or 30 mg/kg, p.o. in 10%

tween/H2O) was administered 2 h prior to the cannabinoid CB1 receptor agonist,

CP-55940 (1 mg/kg, IP in 1% tween/H2O). ⁎=Pb0.05 vs. the vehicle/CP-

55940 group. #=Pb0.05 for CP-55940 vs. the all vehicle/vehicle group at their

respective time points.

Fig. 2. Inverse receptor agonist activity of Imidazole 24b at the human

cannabinoid CB1 receptor expressed in CHO cells. To test functional inverse

agonism, Imidazole 24b was tested in adenylyl cyclase assays in cannabinoid

receptor-containing CHO cells. CP44940-mediated inhibition of forskolin-

stimulated cAMP was used to define 100% agonist efficacy.

219L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

F(29)=7.2, P=0.003; 18 h: F(29)=16.1, Pb0.001; Table 2).

Dose-dependent reductions in overnight body weight gain were

observed following treatment with 5 and 10 mg/kg of Imidazole

24b (F (29)=21.3, Pb0.001; Table 2). Imidazole 24b treatment

dose dependently reduced 2-hour (phenotype: F (1,56)=1.7,

P=0.2; dose: F(2,56)=7.8, P=0.001; interaction: F(2,56)=

8.0, Pb0.001) and overnight food intake (phenotype: F (1,56)=

1.1, P=0.3; dose: F(2,56)=3.8, P=0.03; interaction: F(2,56)=

7.2, Pb0.01) in Cnr1 +/+ mice but was without effect in Cnr1−/−

mice (Fig. 5A and B). Imidazole 24b treatment only elicited

dose-dependent reductions in body weight gain in Cnr1 +/+ mice

(Fig. 5C; phenotype: F (1,56)=0.3, P=0.6; dose: F(2,56)=7.4,

P=0.001; interaction: F(2,56)=4.7, P=0.014)).

3.5. Imidazole 24b acute DIO rat food intake study

Imidazole 24b, at 10 mg/kg, significantly reduced cumulative

overnight food intake by 28% (Fig. 6A). Reductions in food

intake were seen as early as 10 h post-dosing with 10 mg/kg

Imidazole 24b. At 18 h, food intake was significantly reduced

F(3,28)=4.8, Pb0.01). Neither Imidazole 24b at 1 nor 3 mg/kg

altered overnight food intake significantly, although a trend

towards food intake reduction, ∼10%, was observed at 3 mg/

kg. Vehicle treated rats gained an average of 5g of body weight

overnight. Dose-related reductions in overnight weight gain

were observed F(3,28) = 4.8, P=0.014); Imidazole 24b

Table 2

Food intake and body weight effects of Imidazole 24b in mice

Treatment Food intake (g) Overnight change in

body weight (g)

2 h Overnight

Vehicle 0.96±0.08 4.41±0.09 1.5±0.11

5 mg/kg 0.79±0.09 4.22±0.15 0.97±0.15

10 mg/kg 0.37±0.14 a 2.58±0.52 a−0.57±0.47 a

Imidazole 24b decreases food intake and body weight in C57Bl/6 mice

significantly (Pb0.01) by 1-way ANOVA. See text for detailed statistics.a Represents difference from vehicle of Pb0.05 by Dunnett's post hoc

analysis.

Fig. 5. Cannabinoid CB1 receptor-mediated food intake suppression by

Imidazole 24b decreases food intake in Cnr1+/+ but not Cnr1−/− mice at

(A) 2 h and (B) 24 h. C. Similarly, body weight loss evoked by Imid24 b is

observed in Cnr1+/+ but not Cnr1−/− mice. Cnr1+/+, n=10; Cnr1−/−, n=9.⁎Pb0.05 vs. corresponding vehicle; #Pb0.05 from Cnr1+/+ vehicle group.

Fig. 6. (A) Acute Imidazole 24b treatment 1 h prior to dark onset (Time 0)

reduces cumulative overnight food intake in DIO rats in a dose-related manner.

Data points in the 10 mg/kg group are statistically lower than vehicle treatment

from 10 h post-treatment onward. (B) Acute Imidazole 24b treatment reduces

overnight body weight gain in DIO rats. ⁎Pb0.05 vs. vehicle by Dunnett's post

hoc analysis; n=6 rats/group.

220 L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

treatment at 3 mg/kg led to no net change in body weight, while

10 mg/kg evoked a ∼2 g weight loss (Fig. 6B).

3.6. Pharmacokinetics of Imidazole 24b in DIO rats

Oral administration of Imidazole 24b led to peak plasma

levels of 502±190 nM and an 8 h area under the curve of 1.57±

0.34 μM⁎h.

3.7. Imidazole 24b 14-day study in DIO rats

3.7.1. Body weight

Dose-related decreases in body weight gain were observed

(Fig. 7A). Daily body weight significantly decreased on all days

by Imidazole 24b as analyzed by 1-way ANOVA; only the

10 mg/kg group was significantly different from vehicle by a

Dunnett's post hoc analysis and was different on all days except

day 1. The highest dose of Imidazole 24b, 10 mg/kg, evoked a

significant weight loss of∼9% relative to the final weight of the

vehicle treated rats. Dexfenfluramine evoked decreases in body

weight on all days (final t (12) 2.2, P=0.01).

3.7.2. Food intake

Consistent with the body weight changes, a dose-related

suppression of food intake was observed (Fig. 7B). Daily food

intake was decreased by Imidazole 24b as measured by 1-way

ANOVA on all days except days 9, 11, and 14. At 3 mg/kg,

Imidazole 24b reduced daily food intake on days 2, 5, and 13

when measuring by Dunnett's post hoc analysis; Imidazole 24b

at 10 mg/kg reduced food intake on days 1 through 8 and days

12 and 13. Cumulative 14-day intake for vehicle treated rats was

303±5 g. Imidazole 24b dose dependently decreased cumula-

tive food intake (F (27)=10.7, Pb0.001) to 302±12, 264±12,

and 234±10 g for 1, 3, and 10 mg/kg, respectively resulting in a

suppression of 13% and 23% respectively for the 3 and 10 mg/

kg groups, both of which were less than vehicle (Pb0.05) by

Dunnett's analysis. The control, dexfenfluramine, evoked daily

suppression of food intake on all days and a cumulative

suppression in 14 day food intake of 26% (t (8.8), Pb0.001).

3.7.3. Body composition

All groups averaged 22% fat mass at the start of the study.

Imidazole 24b dose dependently reduced fat mass from day 7

(day of first DEXAscan) to day 14 (F (3,27)=8.5, Pb0.001;

Fig. 7C), resulting in the highest dose group, 10 mg/kg having a

final fat percentage of 16%. Dexfenfluramine-treated rats

finished the study with a final % adiposity of 17%. (t (12)=

2.7, P=0.02). Lean mass was not altered by Imidazole 24b or

dexfenfluramine treatment (data not shown). When examining

individual fat pads, retroperitoneal, epididymal, and mesenteric,

Imib24b decreased mesenteric white adipose tissue weights

only, (∼19%; F(3,27)=3.7, P=0.025; Dunnett's analysis

demonstrated a post hoc reduction in the 10 mg/kg group only;

data not shown). Dexfenfluramine did not significantly reduce fat

pad weights.

4. Discussion

Here we describe in vitro and in vivo characteristics of a

novel, potent, selective and orally bioavailable cannabinoid

CB1 receptor inverse agonist, Imib24b. Imidazole 24b proves to

be a useful tool for probing the neurobiological mechanisms

underlying the role of the endocannabinoid system in various

physiological functions. In vitro, Imidazole 24b potently binds

the recombinant human and rat cannabinoid CB1 receptors and

is N50× selective for cannabinoid CB1 receptor over the human

Fig. 7. 14-day Imidazole 24b treatment evokes body weight loss and food intake

suppression in DIO rats. (A) Daily body weight significantly decreased on all

days by Imidazole 24b; the 10 mg/kg group was significantly different from

vehicle by a Dunnett's post hoc analysis on all days except day 1.

Dexfenfluramine evoked significant decreases in body weight on all days.

(B) Daily food intake was suppressed on all days by Imidazole 24b except days 9,

11, and 14. Differences of individual doses of Imidazole 24b by a Dunnett's post

hoc analysis for 3 mg/kg are observed on days 2, 5, and 13, and for 10 mg/kg on

days 1–8 and 12 and 13. Dexfenfluramine evoked decreases in food intake on all

days. For clarity's sake, the statistical analyses are not denoted on the graphs but

are detailed further in the text. (C) 14-day Imidazole 24b and dexfenfluramine

treatment reduce fat mass of DIO rats (1-way ANOVA, followed by a Dunnett's

post hoc analysis or by t-test (dexfenfluramine). n=7 rats/group.

221L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

cannabinoid CB2 receptor. It is a receptor inverse agonist at both

cannabinoid CB1 receptor and cannabinoid CB2 receptor and in

the presence of a cannabinoid receptor agonist, Imidazole 24b is a

competitive receptor antagonist. In vivo, Imidazole 24b occupies

CNS cannabinoid CB1 receptor in a manner correlative with

Imidazole 24b functional inhibition of cannabinoid receptor

agonist modulated hypothermia and suppresses food intake and

modulates energy balance in rodents.

Endocannabinoids and synthetic cannabinoid receptor ago-

nists produce a panoply of cannabinoid-induced physiological

effects, one of which is hypothermia (Martin et al., 1991). We

demonstrated that oral (and intravenous, data not shown)

administration of Imidazole 24b attenuated cannabinoid

receptor agonist (CP-55940)-induced hypothermia, indicating

that Imidazole 24b is an effective cannabinoid CB1 receptor

antagonist in vivo. As cannabinoid receptor agonist-induced

hypothermia is believed to be centrally mediated (Fitton and

Pertwee, 1982; Rawls et al., 2002), these results also indicate

sufficient cannabinoid CB1 receptor occupancy and brain

penetration by Imidazole 24b. Indeed, using a novel ex vivo

autoradiographic method, oral administration of Imidazole 24b

resulted in a dose-dependent increase in brain cannabinoid CB1

receptor occupancy (ranging from 23 to 66% receptor

occupancy for 3, 10 and 30 mg/kg Imidazole 24b) at 2 h

post-dosing in rats, with ∼50% receptor occupancy needed for

significant attenuation of cannabinoid receptor agonist-induced

hypothermia in the rat. Others using [131I]AM281 as an in vivo

tracer/radioligand have suggested that the locomotor effects of

cannabinoids in mouse occur at ∼35–40% receptor occupancy

(Cosenza et al., 2000). In both these instances, administration of

receptor inverse agonists led to pharmacological effects at low

to moderate occupancy of the receptor. In contrast, Need (Need

et al., 2006) found ligand-specific relationships between

receptor occupancy were required for catecholaminergic

changes induced by two cannabinoid CB1 receptor inverse

agonists. Dopamine and norepinephrine efflux in the prefrontal

cortex was observed only above 65% receptor occupancy with

either compound, suggesting a very high occupancy require-

ment. However, when suppression of palatable food intake was

examined, food intake suppression by SLV319 was observed at

11–30% receptor occupancy, whereas when SR141716A was

studied, food intake suppression was only observed at receptor

occupancy of 67% or greater. Hence, generalization about

occupancy required may depend on factors such as ligand of

use, brain regions and the specific endpoint being measured.

The endocannabinoid system regulates appetitive behavior.

Cannabinoid CB1 receptors are present in brain regions

implicated in feeding and reward pathways. Our present work

demonstrates that Imidazole 24b reduces appetite in a dose-

dependent manner in both mice and DIO rats. Imidazole 24b

(10 mg/kg, p.o.) reduced food intake significantly in lean mice

at both 2 and 18 h after administration. Dose-dependent

reductions in overnight body weight were observed following

treatment with 5 and 10 mg/kg of Imidazole 24b in chow-fed

mice. Importantly, the lack of efficacy of acute Imidazole 24b

(up to 16 mg/kg, p.o.) administration in Cnr1−/− mice demon-

strates that the anorexigenic effects of Imidazole 24b are

receptor-mediated. Imidazole 24b was ineffective at 1 and 3 mg/

kg on food intake or body weight in DIO rats, but at 10 mg/kg

did inhibit food intake by ∼28%. Significant reductions in food

intake were observed as early as 5 h after administration of

10 mg/kg which led to a significant overnight body weight loss.

Furthermore, we demonstrated that when chronically

administered, Imidazole 24b evokes long-lasting dose-related

reductions in food intake, body weight gain and adiposity in

DIO rats. Chronic administration of Imidazole 24b at 3 mg/kg

reduced food intake, resulting in sustained effects on body

weight gain in DIO rats. Chronic Imidazole 24b treatment at

10 mg/kg evoked a significant decrease in body weight and

adiposity (e.g., decreased fat mass and white adipose tissue

weights). The observation that cannabinoid CB1 receptor

inverse agonists have sustained effects on body weight despite

partial tachyphylaxis of food intake inhibition have led to the

suggestion that the anti-obesity effects of cannabinoid CB1

receptor inverse agonists involve mechanisms in addition to

anorexigenesis, such as increased energy expenditure. In

support of this hypothesis, SR141716 has been shown to

increase oxygen consumption in ob/ob mice (Liu et al., 2005).

Thus, treatment of DIO rats for 14 days with the cannabinoid

CB1 receptor inverse agonist Imidazole 24b reduced body

weight gain and adipose tissue weight (particularly mesenteric

fat) without effects on lean mass and thus, likely results from

both food intake and energy expenditure changes. These

findings are consistent with the effects of chronic pharmaco-

logical blockade of cannabinoid CB1 receptors by SR141716 or

its analog, AM251 in rats (Colombo et al., 1998; Vickers et al.,

2003) and in mice (Hildebrandt et al., 2003; Ravinet Trillou

et al., 2003).

Cannabinoid CB1 receptors reportedly modulate energy

balance by direct peripheral effects in addition to the known

CNS regulation. Cannabinoid CB1 receptor mRNA is observed

in adipocytes isolated from epididymal white adipose tissue in

Cnr1+/+, but not Cnr1−/− mice. The cannabinoid CB1 receptor

agonist, WIN-55,212 at high doses stimulates lipoprotein lipase

activity in mouse adipocytes, activity which is blocked by

SR141716 (Cota et al., 2003b). Treatment of obese Zucker rats

with SR141716 has been shown to increase adiponectin

(Acrp30) expression in adipocytes (Bensaid et al., 2003). In

turn, adiponectin secreted from adipocytes, has been shown to

induce free fatty acid oxidation and decrease body weight in

mice (Fruebis et al., 2001; Masaki et al., 2003). We did not

measure serum adiponectin levels in the chronic Imidazole 24b

study and do not know if levels had been modulated by

Imidazole 24b treatment. Thus, we cannot eliminate the

possibility that a component of the effects on energy balance

may derive in part from contributions by peripheral cannabinoid

CB1 receptor mechanisms.

Together the in vitro and in vivo characteristics of Imidazole

24b demonstrate its usefulness for elucidating the roles of

cannabinoid CB1 receptor. Our demonstration of cannabinoid

CB1 receptor occupancy in the CNS and its correlation with a

physiological measure (i.e., attenuation of hypothermia)

establish that Imidazole 24b is a valuable tool to further

illuminate the CNS roles of the cannabinoid CB1 receptor and of

222 L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

endocannabinoids. Indeed, another cannabinoid CB1 receptor

inverse agonist has been recently demonstrated to reduce body

weight ∼5% in humans after a year of treatment (Pi-Sunyer et

al., 2006). Thus, these results further confirm that cannabinoid

CB1 receptor inverse agonists may represent an additional

approach to the pharmacotherapy of obesity and the better

understanding of CNS regulation of energy homeostasis.

Acknowledgments

The authors would like to thank Andreas Zimmer for

supplying the Cnr1−/− mice.

References

Abel, E., 1975. Cannabis: effects on hunger and thirst. Behav. Biol. 15,

255–281.

Arnone, M., Maruani, J., Chaperon, F., Thiebot, M., 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. Psychophar-

macology (Berl.) 132, 104–106.

Bensaid, M., Gary-Bobo, M., Esclangon, A., Maffrand, J., Le Fur, G., Oury-

Donat, F., Soubrie, P., 2003. The cannabinoid CB1 receptor antagonist

SR141716 increases Acrp30 mRNA expression in adipose tissue of obese

fa/fa rats and in cultured adipocyte cells. Mol. Pharmacol. 63, 908–914.

Bouaboula, M., Poinot-Chazel, C., Bourrie, B., Canat, X., Calandra, B., Rinaldi-

Carmona, M., Le Fur, G., Casellas, P., 1995. Activation of mitogen-activated

protein kinases by stimulation of the central cannabinoid receptor CB1.

Biochem. J. 312, 637–641.

Bouaboula, M., Perrachon, S., Milligan, L., Canat, X., Rinaldi-Carmona, M.,

Portier, M., Barth, F., Calandra, B., Pecceu, F., Lupker, J., Maffrand, J.-P., Le

Fur, G., Casellas, P., 1997. A selective inverse agonist for central cannabinoid

receptor inhibits mitogen-activated protein kinase activation stimulated by

insulin or insulin-like growth factor. J. Biol. Chem. 272, 22330–22339.

Breivogel, C., Griffin, G., Di Marzo, V., Martin, B., 2001. Evidence for a new G

protein-coupled cannabinoid receptor in mouse brain. Mol. Pharmacol. 60,

155–163.

Colombo, G., Agabio, R., Diaz, G., Lobina, C., Reali, R., Gessa, G., 1998.

Appetite suppression and weight loss after the cannabinoid antagonist SR

141716. Life Sci. 63, PL113–PL117.

Cosenza, M., Gifford, A., Gatley, S., Pyatt, B., Liu, Q., Makriyannis, A.,

Volkow, N., 2000. Locomotor activity and occupancy of brain cannabinoid

CB1 receptors by the antagonist/inverse agonist AM281. Synapse 38,

477–482.

Cota, D., Marsicano, G., Lutz, B., Vicennati, V., Stalla, G., Pasquali, R., Pagotto,

U., 2003a. Endogenous cannabinoid system as a modulator of food intake.

Int. J. Obes. Relat. Metab. Disord. 27, 289–301.

Cota, D., Marsicano, G., Tschop, M., Grubler, Y., Flachskamm, C., Schubert,

M., Auer, D., Yassouridis, A., Thone-Reineke, C., Ortmann, S., Tomassoni,

F., Cervino, C., Nisoli, E., Linthorst, A.C.E., Pasquali, R., Lutz, B., Stalla,

G.K., Pagotto, U., 2003b. The endogenous cannabinoid system affects

energy balance via central orexigenic drive and peripheral lipogenesis.

J. Clin. Invest. 112, 423–431.

Devane, W., Hanus, L., Breuer, A., Pertwee, R., Stevenson, L., Griffin, G.,

Gibson, D., Mandelbaum, A., Etinger, A., Mechoulam, R., 1992. Isolation

and structure of a brain constituent that binds to the cannabinoid receptor.

Science 258, 1882–1884.

Di Marzo, V., Goparaju, S., Wang, L., Liu, J., Batkai, S., Jarai, Z., Fezza, F.,

Miura, G., Palmiter, R., Sugiura, T., Kunos, G., 2001. Leptin-regulated

endocannabinoids are involved in maintaining food intake. Nature 410,

822–825.

Fitton, A., Pertwee, R., 1982. Changes in body temperature and oxygen

consumption rate of conscious mice produced by intrahypothalamic and

intracerebroventricular injections of delta 9-tetrahydrocannabinol. Br. J.

Pharmacol. 75, 409–414.

Foltin, R., Fischman, M., Byrne, M., 1988. Effects of smoked marijuana on food

intake and body weight of humans living in a residential laboratory. Appetite

11, 1–14.

Fruebis, J., Tsao, T., Javorschi, S., Ebbets-Reed, D., Erickson, M., Yen, F.,

Bihain, B., Lodish, H., 2001. Proteolytic cleavage product of 30-kDa

adipocyte complement-related protein increases fatty acid oxidation in

muscle and causes weight loss in mice. Proc. Natl. Acad. Sci. U. S. A. 98,

2005–2010.

Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carriere, D., Carayon, P.,

Bouaboula, M., Shire, D., Le Fur, G., Casellas, P., 1995. Expression of

central and peripheral cannabinoid receptors in human immune tissues and

leukocyte subpopulations. Eur. J. Biochem. 232, 54–61.

Hildebrandt, A., Kelly-Sullivan, D., Black, S., 2003. Antiobesity effects of

chronic cannabinoid CB1 receptor antagonist treatment in diet-induced

obese mice. Eur. J. Pharmacol. 462, 125–132.

Howlett, A.C., 1995. Pharmacology of cannabinoid receptors. Annu. Rev.

Pharmacol. Toxicol. 35, 607–634.

Jamshidi, N., Taylor, D., 2001. Anandamide administration into the ventrome-

dial hypothalamus stimulates appetite in rats. Br. J. Pharmacol. 134,

1151–1154.

Jarai, Z., Wagner, J., Varga, K., Lake, K., Compton, D., Martin, B., Zimmer, A.,

Bonner, T., Buckley, N., Mezey, E., Razdan, R., Zimmer, A., Kunos, G.,

1999. Cannabinoid-induced mesenteric vasodilation through an endothelial

site distinct from CB1 or CB2 receptors. Proc. Natl. Acad. Sci. U. S. A. 96,

14136–14141.

Kirkham, T., Williams, C., Fezza, F., Di Marzo, V., 2002. Endocannabinoid

levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding

and satiation: stimulation of eating by 2-arachidonoyl glycerol. Br. J.

Pharmacol. 136, 550–557.

Liu, Y., Connoley, I., Wilson, C., Stock, M., 2005. Effects of the cannabinoid

CB1 receptor antagonist SR141716 on oxygen consumption and soleus

muscle glucose uptake in Lep(ob)/Lep(ob) mice. Int. J. Obes. (Lond). 29,

183–187.

Martin, B., Compton, D., Thomas, B., Prescott, W., Little, P., Razdan, R.,

Johnson, M., Melvin, L., Mechoulam, R., Ward, S., 1991. Behavioral,

biochemical, and molecular modeling evaluations of cannabinoid analogs.

Pharmacol. Biochem. Behav. 40, 471–478.

Masaki, T., Chiba, S., Yasuda, T., Tsubone, T., Kakuma, T., Shimomura, I.,

Funahashi, T., Matsuzawa, Y., Yoshimatsu, H., 2003. Peripheral, but not

central, administration of adiponectin reduces visceral adiposity and

upregulates the expression of uncoupling protein in agouti yellow (Ay/a)

obese mice. Diabetes 52, 2266–2273.

Matsuda, L., Lolait, S., Brownstein, M., Young, A., Bonner, T., 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, N.,

Schatz, A., Gopher, A., Almog, S., Martin, B., Compton, D., Pertweee, R.,

Griffine, G., Bayewitchf, M., Bargf, J., Vogel, Z., 1995. Identification of an

endogenous 2-monoglyceride, present in canine gut, that binds to

cannabinoid receptors. Br. J. Pharmacol. 50, 83–90.

Munro, S., Thomas, K., Abu-Shaar, M., 1993. Molecular characterization of a

peripheral receptor for cannabinoids. Nature 365, 61–65.

Need, A., Davis, R., Alexander-Chacko, J., Eastwood, B., Chernet, E., Phebus,

L., Sindelar, D., Nomikos, G., 2006. The relationship of in vivo central CB1

receptor occupancy to changes in cortical monoamine release and feeding

elicited by CB1 receptor antagonists in rats. Psychopharmacology (Berl.)

184, 26–35.

Pi-Sunyer, F.X., et al., for the RIO-North America Study Group, 2006. Effect of

rimonabant, a cannabinoid-1 receptor blocker, on weight and cardiometa-

bolic risk factors in overweight or obese patients: RIO-North America: a

randomized controlled trial. JAMA 295, 761–775.

Plummer, C., Finke, P., Mills, S., Wang, J., Tong, X., Doss, G., Fong, T., Lao, J.,

Schaeffer, M., Chen, J., Shen, C., Stribling, D., Shearman, L., Strack, A.,

Van der Ploeg, L., 2005. Synthesis and activity of 4,5-diarylimidazoles as

human CB1 receptor inverse agonists. Bioorg. Med. Chem. Lett. 15,

1441–1446.

Ravinet Trillou, C., Arnone, M., Delgorge, C., Gonalons, N., Keane, P.,

Maffrand, J., Soubrie, P., 2003. Anti-obesity effect of SR141716, a CB1

223L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224

receptor antagonist, in diet-induced obese mice. Am. J. Physiol., Regul.

Integr. Comp. Physiol. 284, R345–R353.

Ravinet Trillou, C., Delgorge, C., Menet, C., Arnone, M., Soubrie, P., 2004.

CB1 cannabinoid receptor knockout in mice leads to leanness, resistance to

diet-induced obesity and enhanced leptin sensitivity. Int. J. Obes. Relat.

Metab. Disord. 28, 640–648.

Rawls, S.M., Cabassa, J., Geller, E.B., Adler, M.W., 2002. CB1 receptors in the

preoptic anterior hypothalamus regulate WIN 55212-2 [(4,5-dihydro-2-

methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo

[3,2,1ij]quinolin-6-one]-induced hypothermia. J. Pharmacol. Exp. Ther.

301, 963–968.

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.,

Breliere, J., Le Fur, G., 1994. SR141716A, a potent and selective antagonist

of the brain cannabinoid receptor. FEBS Lett. 350, 240–244.

Simiand, J., Keane, M., Keane, P., Soubrie, P., 1998. SR 141716, a CB1

cannabinoid receptor antagonist, selectively reduces sweet food intake in

marmoset. Behav. Pharmacol. 9, 179–181.

Vickers, S., Webster, L., Wyatt, A., Dourish, C., Kennett, G., 2003. Preferential

effects of the cannabinoid CB1 receptor antagonist, SR 141716, on food

intake and body weight gain of obese (fa/fa) compared to lean Zucker rats.

Psychopharmacology (Berl.) 167, 103–111.

Walter, L., Franklin, A., Witting, A., Wade, C., Xie, Y., Kunos, G., Mackie, K.,

Stella, N., 2003. Nonpsychotropic cannabinoid receptors regulate microglial

cell migration. J. Neurosci. 23, 1398–1405.

Wang, L., Liu, J., Harvey-White, J., Zimmer, A., Kunos, G., 2003.

Endocannabinoid signaling via cannabinoid receptor 1 is involved in

ethanol preference and its age-dependent decline in mice. Proc. Natl. Acad.

Sci. U. S. A. 100, 1393–1398.

Westlake, T., Howlett, A., Bonner, T., Matsuda, L., Herkenham, M., 1994.

Cannabinoid receptor binding and messenger RNA expression in human

brain: an in vitro receptor autoradiography and in situ hybridization

histochemistry study of normal aged and Alzheimer's brains. Neuroscience

63, 637–652.

Williams, C., Kirkham, T., 1999. Anandamide induces overeating: mediation by

central cannabinoid (CB1) receptors. Psychopharmacology (Berl.) 143,

315–317.

Williams, C., Kirkham, T., 2002. Reversal of delta 9-THC hyperphagia by

SR141716 and naloxone but not dexfenfluramine. Pharmacol. Biochem.

Behav. 71, 333–340.

Zimmer, A., Zimmer, A.M., Hohmann, A.G., Herkenham, M., Bonner, T.I.,

1999. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid

CB1 receptor knockout mice. Proc. Natl. Acad. Sci. U. S. A. 96, 5780–5785.

224 L.P. Shearman et al. / European Journal of Pharmacology 579 (2008) 215–224