Antiobesity effects of the novel in vivo neutral cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1 H-1,2,4-triazole – LH 21

Neuropharmacology 51 (2006) 358e366www.elsevier.com/locate/neuropharm

Antiobesity effects of the novel in vivo neutral cannabinoid receptorantagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-

3-hexyl-1H-1,2,4-triazole e LH 21

Francisco Javier Pavon a, Ainhoa Bilbao a,c, Laura Hernandez-Folgado b, Andrea Cippitelli a,c,Nadine Jagerovic b, Gumersindo Abellan b, Ma Isabel Rodrıguez-Franco b, Antonia Serrano a,

Manuel Macias a, Raquel Gomez c, Miguel Navarro c, Pilar Goya b,**,Fernando Rodrıguez de Fonseca a,c,*

a Fundacion IMABIS, Neuropharmacology, Hospital Carlos Haya, Avenida Carlos Haya 82, 7a Planta, Pabellon A, Malaga 29010, Spainb Instituto de Quımica Medica, CSIC, Juan de la Cierva 3, Madrid 28006, Spain

c Instituto Universitario de Drogodependencias, Departamento de Psicobiologıa, Universidad Complutense, Madrid 28223, Spain

Received 10 March 2005; received in revised form 24 March 2006; accepted 27 March 2006

Abstract

The present study evaluates the pharmacological profile of the new neutral cannabinoid CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole -LH-21- on feeding behavior and alcohol self-administration in rats, two behaviors inhibited bycannabinoid CB1 receptor antagonists. Administration of LH-21 (0.03, 0.3 and 3 mg/kg) to food-deprived rats resulted in a dose-dependentinhibition of feeding. Subchronic administration of LH-21 reduced food intake and body weight gain in obese Zucker rats. Acute effects on feedingwere not associated with anxiety-like behaviors, or induction of complex motor behaviors such as grooming or scratching sequences, usuallyobserved after central administration of cannabinoid receptor blockers with inverse agonist properties. LH-21 did not markedly reduce alcoholself-administration (30% reduction observed only at a high dose of 10 mg/kg). This pharmacological pattern partially overlaps that of the referencecannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide, SR141716A,(0.3, 1 and 3 mg/kg) that reduced feeding and alcohol self-administration with similar efficacy. In vitro analysis of bloodebrain barrier permeabil-ity using a parallel artificial membrane permeation assay demonstrated that LH-21 has lower permeation through membranes than SR141716A.That was confirmed in vivo by studies showing lower potency of peripherally injected LH-21 when compared to SR141716A to antagonize motordepression induced by intracerebroventricular administration of the CB1 agonist CP55,940. The neutral antagonist profile and the lower penetra-tion into the brain of LH-21 favour this class of antagonists with respect to reference inverse agonists for the treatment of obesity because theypotentially will display reduced side effects.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Cannabinoid; Food intake; Alcohol; Rat; SR141716A; Self-administration

* Corresponding author. Fundacion IMABIS, Neuropharmacology, Hospital

Carlos Haya, Avenida Carlos Haya 82, 7a Planta, Pabellon A, Malaga 29010,

Spain. Tel.: þ34 6 6942 6548; fax: þ34 9 5103 0447.

** Corresponding author. Tel.: þ34 9 1562 5048.

E-mail addresses: [email protected] (P. Goya), fernando.rodriguez.

[email protected] (F. Rodrıguez de Fonseca).

0028-3908/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2006.03.029

1. Introduction

Since the discovery of the existence of receptors for thepsychoactive compounds of cannabis sativa (Devane et al.,1988; Matsuda et al., 1990) and the isolation of their endoge-nous ligands, anandamide (Devane et al., 1992) and 2-arachidonoylglycerol (Mechoulam et al., 1995; Sugiura et al.,1995), more than 4000 scientific reports have explored indepth the main aspects of the so called ‘‘endocannabinoid

359F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

system’’. This system emerges nowadays as a relevant modula-tor of physiological functions not only in the central nervoussystem but also in the autonomic nervous system, the endocrinenetwork, the immune system, the gastrointestinal tract, the re-productive system and the microcirculation (Piomelli, 2003;Rodriguez de Fonseca et al., 2005). Pharmacological studiesreveal that there are at least two types of cannabinoid receptors,CB1 and CB2, and a wide range of CB1 and CB2 ligands withdiverse chemical structures are now available (Howlett et al.,2002). Several series of compounds have been developed ascannabinoid CB1 receptor antagonists. The most representativeare diarylpyrazoles, substitute benzofuranes, aminoalkylin-doles and triazole derivatives (for review see Rodriguez deFonseca et al., 2005). Diarylpyrazoles include both the firstCB1 receptor antagonist synthesized (SR141716A, Rinaldi-Carmona et al., 1994) and the first CB2 receptor antagonist(SR144528). Both are considered the reference antagonists.Modification of the SR141716A molecule has yielded otherCB1 receptor antagonists with improved properties includingSR147778 and AM281 (Howlett et al., 2002; Rinaldi-Carmonaet al., 2004). However, diarylpyrazoles are not neutral antago-nists since they display significant inverse agonist properties. Inthis context we have recently reported a silent cannabinoid an-tagonist derived from a 1,2,4-triazole which represents a novelentry in cannabinoid chemistry (Fig. 1, Jagerovic et al., 2004).5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole -LH-21- is an in vivo CB1 antagonist with a paradoxiclow affinity in vitro for CB1 receptors, and devoid of inverse ag-onist properties.

The interest to develop cannabinoid antagonists derives fromthe multiple functions in which the endogenous cannabinoidsystem is engaged, some of which are relevant for major humandiseases (Piomelli et al., 2000). The endogenous cannabinoidsystem controls motivation for appetitive stimuli, includingfood and drugs (Arnone et al., 1997; Colombo et al., 1998;Gomez et al., 2002; Navarro et al., 2001; Rodriguez de Fonsecaet al., 1999). The homeostatic control of motivated behaviorsand the regulation of emotions warrant special attention becauseof the impact on the health systems of entities such as feedingdisorders (including obesity), addiction (especially alcoholismand tobacco smoking) or mood alterations such as anxiety anddepression. Following this rationale, clinical trials of the effectsof the cannabinoid receptor antagonist SR141716A on obesity

NN

N

Me

Cl

ClCl

LH-21

Fig. 1. Chemical structure of the new in vivo cannabinoid CB1 receptor

antagonist 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-

triazole (LH-21).

and tobacco smoking are currently under way (Cleland et al.,2004; Fernandez and Allison, 2004). Because of the inverseagonism properties of the cannabinoid antagonist SR141716A(MacLennan et al., 1998; Mato et al., 2002) the evaluation ofa neutral cannabinoid CB1 receptor antagonist such as LH-21will help to clarify whether the inhibitory effects on feeding oralcohol self-administration are derived from receptor blockadeand not from the intrinsic activity of these compounds.

In the present study we tested LH-21 on models of motorbehavior, anxiety, feeding and alcohol self-administration. Be-cause of the structure of the triazole motif of LH-21 that mightreduce its crossing through biological membranes, andbecause of the neutral antagonistic properties of this com-pound, we hypothesized that the pharmacological profile ofLH-21 will be different from that of reference cannabinoidreceptor antagonists with inverse agonist properties such asSR141716A. A neutral cannabinoid receptor antagonist withpoor penetration in the brain will potentially have lessuwanted effects, and thus display a better safety profilewhen used as a therapy for obesity. This is relevant because theinhibition of food intake is mediated both centrally (Jamshidiand Taylor, 2001; Kirkham et al., 2002; Hanus et al., 2003);and peripherally (Gomez et al., 2002) while the effects on re-ward/addiction, mood and motor control are mediated throughcannabinoid receptors located in central circuits (Rodriguez deFonseca et al., 2005). Our results confirm that the neutral an-tagonist LH-21 has clear antiobesity effects and has poor pen-etration in the brain, reducing side effects of reference inverseagonists such as motor alterations and anxiety.

2. Materials and methods

2.1. Animals

All experiments were performed in male Wistar rats, weighing 175e225 g

at the start of the experiments, or in obese Zucker rats aged 12 to 16 weeks

(Panlab, Balona). Animals were housed in groups of two in a temperature

and humidity controlled vivarium on a reverse 12-h light/dark cycle (on

6:00 PM; off 6 AM). All training and experimental sessions were conducted

during the dark phase of the cycle. Standard National Institutes of Health lab-

oratory rat chow and water were available ad libitum in the home cage, except

as noted in ‘‘Behavioral Training and Testing Procedures’’. All the procedures

were conducted in adherence with the European Community Council Directive

86/609/EEC regulating animal research.

2.2. Surgery and intracerebroventricularadministration of drugs

For intracerebroventricular injections, stainless steel guide cannulas aimed

at the lateral ventricle were implanted in the rats. The animals were anesthe-

tized with equithesin and placed in a David Kopf Instruments (Tujunga, CA)

stereotaxic instrument with the incisor bar set at 5 mm above the interaural

line. A guide cannula (7 mm, 23 gauge) was secured to the skull by using

two stainless steel screws and dental cement and was closed with 30 gauge ob-

turators (Gomez et al., 2002). The implantation coordinates were 0.6 mm pos-

terior to bregma, �2.0 mm lateral, and 3.2 mm below the surface of the skull.

These coordinates placed the cannula 1 mm above the ventricle. After a 7 d

postsurgical recovery period, cannula patency was confirmed by gravity flow

of isotonic saline through an 8-mm-long, 30 gauge injector inserted within

the guide to 1 mm beyond its tip. This procedure allowed the animals to be-

come familiar with the injection technique.

360 F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

For intracerebroventricular administration, the obturator was removed

from the guide cannula and an 8 mm injector (30 gauge stainless steel tubing)

that was connected to 70 cm of calibrated polyethylene-10 tubing was lowered

into the ventricle. The tubing was then raised until flow began, and 5 ml of a so-

lution containing 10 micrograms of CP 55,940 was infused over a 30e60 s pe-

riod. The injector was left in the guide cannula for an additional 30 s and then

removed. The stylet was immediately replaced. Animals were tested 5 min af-

ter injections. The intracerebroventricular cannula placements were evaluated

after each experiment by dye injection. Only rats with proper intracerebroven-

tricular placements were included in the data analysis.

2.3. Drugs

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole -LH-21-

(Fig. 1) was synthesized in the laboratory as previously described (Jagerovic et al.,

2004). N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-

3-carboxamide, SR141716A, was a gift from Sanofi-Aventis, Montpellier, France.

(�)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cy-

clohexanol (CP 55,940) was obtained through Tocris Cookson (Avonmouth, UK).

The drugs were suspended with 2e3 drops of Tween 80 in saline as vehicle and

administered intraperitoneally (i.p.) at doses of 0.03, 0.3 and 3 mg/kg (LH-21) or

0.3, 1 and 3 mg/kg (SR141716A).

2.4. Food intake studies

The acute effects of drugs on feeding behavior were analyzed in animals

deprived of food for 24 h and habituated to handling (Gomez et al., 2002).

To habituate the animals, 72 h before the testing with drugs, animals were

food-deprived for 24 h. Then, the bedding material was removed from the

cage and a small can containing weighed food pellets was placed inside the

cage for 4 h and the amount of food eaten registered. After the initial test,

the animales were under a free-feeding period of 48 h. Then, the animals

were food-deprived for 24 h again, with access to water ad libitum. Fifteen

minutes before the start of the test drugs were administered i.p., the animals

were returned to their home cage, where a can with a measured amount of

food (usually 30e40 gm) and a bottle containing 250 ml of fresh water were

placed again. Food pellets and food spillage were weighed at 30, 60 and

120 min after starting the test, and the amount of food eaten was recorded.

At the end of the test, the amount of water consumed was also measured.

For analyzing the subchronic (8 days of treatment) effects of LH-21 in an

obesity model, free-feeding Zucker rats were daily injected with LH-21 (0.3

and 3 mg/kg) at the start of the dark period of the light cycle. The amount

of food eaten and the body weight was registered daily.

2.5. Open-field test

Motor behaviors in the open field were studied in an opaque open field

(100 � 100 � 40 cm) as described previously (Beltramo et al., 2000). The

field was illuminated using a ceiling halogen lamp regulated to yield

350 lux at the center of the field. Rats were habituated to the field for

10 min the day before testing. On the experimental day, the animals were

treated and 30 min later placed in the center of the field, and locomotor activ-

ity (number of lines crossed or distance travelled in cm) and rearing and

grooming behavior (number of rearings and time spent grooming) were scored

for 5 min at 5, 30, 60, and 120 min after drug injection. Scratching sequences,

a behavior elicited by cannabinoid receptor antagonism (Navarro et al., 1997)

were also monitored. Behavior was scored by trained observers who were un-

aware of the experimental conditions.

2.6. Elevated plus-maze

As previously described (Navarro et al., 1997), the elevated plus-maze was

made of opaque plastic, with two opposite open arms (45 � 10 cm) and two

opposite closed arms of the same size and 50-cm-high walls. The arms were

connected by a central square (10 � 10 cm). The entire apparatus was elevated

75 cm above a white floor and exposed to dim illumination (70 lux). Rats were

randomly placed in the central square of the maze, facing an open arm. The

number of entries onto and time spent on each arm were scored, through

the use of a video monitor placed in an adjacent room, for the first 5 min

(an arm entry was defined as all four feet in the arm). At the end of the

test, each rat was returned to its home cage. Final data are expressed as per-

centage of time spent on the exposed or the closed arms of the maze.

2.7. Operant training for liquid reinforcers

Training and testing were conducted in standard operant chambers located

in sound-attenuating, ventilated environmental cubicles. Each chamber was

equipped with a drinking reservoir (volume capacity: 0.10 ml) positioned

4 cm above the grid floor in the center of the front panel of the chamber,

and two retractable levers were located 3 cm to the right and left of the drink-

ing receptacle. Auditory and visual stimuli were presented via a speaker and

a light located on the front panel. A microcomputer controlled the delivery

of fluids, presentation of auditory and visual stimuli, and recording of the

behavioral data. Rats were trained to self-administer 10% [volume/volume

(vol./vol.)] ethanol in daily 30-min sessions on a fixed-ratio 1 schedule of re-

inforcement, where each response resulted in delivery of 0.1 ml (Rodriguez de

Fonseca et al., 1999). Briefly for the first 3 days of training, water availability

in the home cage was restricted to 2 h/day in order to facilitate acquisition of

operant responding for a liquid reinforcer. During this time, lever pressing re-

inforced by 0.2% (w/v) saccharin solution was established. At this point water

was made freely available, and saccharin self-administration training contin-

ued until animals reached stable baseline responding. Rats were then trained

to self-administer ethanol by using a modification of the sucrose-fading proce-

dure (Samson, 1986) that used saccharin instead of sucrose (Weiss et al.,

1993). During the first 6 days of this ethanol initiation phase a 5% (w/v) eth-

anol solution containing 0.2% saccharin (w/v) was available to the rats. Start-

ing on day 7, the concentration of ethanol was gradually increased from 5.0%

to 8.0% and finally to 10.0% (w/v), whereas the concentration of saccharin

was correspondingly decreased to 0%. At the beginning of the saccharin-fad-

ing procedure a second, but inactive lever was introduced. During all training

and testing phases responses at this lever, which had no programmed conse-

quences, were recorded as a measure of non-specific behavioral activation.

2.8. Ethanol self-administration: effect ofLH-21 and SR141716A

Following completion of the saccharin fading procedure rats were trained

in 30 min session/day to lever-press for 10% ethanol (0.1 ml/response) until

a stable baseline level of responding was reached. The CB1 receptor antago-

nists LH-21 (0.03, 0.3, 3 and 10 mg/kg) or SR141716A (0.0, 0.3, 1.0 and

3.0 mg/kg) were administered i.p. 30 min prior to the self-administration ses-

sion. The experiment was conducted every fourth day using a Latin square

counterbalanced design. Responding at the inactive lever was recorded

throughout the experiment to monitor non-specific behavioral effects.

2.9. Prediction of the brain penetration of LH-21

Prediction of the brain penetration was performed using a parallel artificial

membrane permeation assay (PAMPA), in a similar manner as described pre-

viously (Di et al., 2003). Commercial drugs, phosphate buffered saline solu-

tion at pH 7.4 (PBS), and dodecane were purchased from Sigma, Aldrich,

Acros, and Fluka. Ethanol was reagent grade from Merck. The Millex filter

units (PVDF membrane, diameter 25 mm, pore size 0.45 mm) were acquired

from Millipore. The porcine brain lipid (PBL) was obtained from Avanti Polar

Lipids. The donor microplate was a 96-well filter plate (PVDF membrane,

pore size 0.45 mm) and the acceptor microplate was an indented 96-well plate,

both from Millipore.

The acceptor 96-well microplate was filled with 170 mL of PBS: ethanol

(70:30) and the filter surface of the donor microplate was impregnated with

4 mL of PBL in dodecane (20 mg ml�1). Compound LH-21 and commercial

drugs of known CNS permeability were dissolved in PBS: ethanol (70:30)

at 1 mg ml�1, filtered through a Millex filter, and then added to the donor wells

(170 mL). The donor filter plate was carefully put on the acceptor plate to form

361F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

a sandwich, which was left undisturbed for 120 min at 25 �C. After incubation,

the donor plate was carefully removed and the concentration of compounds

in the acceptor wells was determined by UV spectroscopy.

Every sample was analyzed in four wells and the average of the runs is re-

ported, including quality control standards of known permeability to validate

the analysis set.

2.10. Statistics

Statistical significance of behavioral studies was assessed by analysis of

variance (ANOVA). All the studies were performed in between subjects. Fol-

lowing a significant F value, post hoc analysis (Student-NewmaneKeuls) was

performed to assess specific comparisons between dose groups.

3. Results

3.1. Effects of LH-21 on food intake

The administration of LH-21 (0.03, 0.3 and 3 mg/kg) resultedin dose-dependent reduction in feeding behavior of food-deprived animals (Fig. 2). Doses of 0.3 and 3 mg/kg suppressedfeeding with a high degree of efficacy, an effect that lasted up to4 h. This effect was significant (P < 0.05, NewmaneKeuls) atall the time points tested for the 3 mg/kg dose.

3.2. Effects of subchronic administration of LH-21 onfood intake on obese Zucker rats

The daily administration of LH-21 for 8 days reduced foodintake (Fig. 3A) and body weight gain (Fig. 3B) in obeseZuker rats. This effect was only observed with the highestdose tested (3 mg/kg). As described previously in Zuckerrats treated with the cannabinoid receptor antagonist

30 60 120 2400

2

4

6

8

VEHICLE

0.03

0.3

3

TIME AFTER INJECTION (min)

FOO

D IN

TAKE

(g)

** * *

*

*

*

LH-21 (mg/kg)

Fig. 2. Acute administration of the in vivo cannabinoid CB1 receptor antago-

nist LH-21 (0.03, 0.3 or 3 mg/kg) to 24-h food-deprived rats resulted in dose

dependent reduction in feeding behavior. Data are means � SEM of 8 determi-

nations per group. (*)P < 0.05, versus vehicle-treated animals.

SR141716A (rimonabant), the effects on weight gain weremore evident and prolongued than the reduction on feeding,suggesting the induction of metabolic adaptions (Vickerset al., 2003).

3.3. Effect of LH-21 on ethanol self-administration

We tested LH-21 on ethanol self-administration usinga broad range of doses, since we have recently observed thatsuppression of ethanol self-administration by SR141716A isa central effect (Hansson et al., in press). Using a Latin-squarecounterbalanced design we observed only a small decrease inethanol self-administration at the highest dose tested (10 mg/kg, Fig. 4). This lack of activity on ethanol self-administrationof LH-21 contrasts with the high potency displayed in sup-pressing feeding behavior (Figs. 2 and 3).

3.4. Open field: effects of LH-21 and SR141716A onlocomotor activity and motor behaviors

LH-21 (3 and 10 mg/kg) did not affect locomotor activity(Fig. 5A) nor did it induce complex motor sequences (such

1 2 3 4 5 6 7 850

60

70

80EFECTS OF LH-21 ON DAILY FOOD INTAKE

IN OBESE ZUCKER RATS

DAY OF TREATMENT

1 2 3 4 5 6 7 8DAY OF TREATMENT

g/kg

of b

ody

wei

ght

** *

** *

*

*

*

0

20

40

60

80VEHICLE0.3 mg/kg3 mg/kg

VEHICLE0.3 mg/kg3 mg/kg

EFFECTS OF LH-21 ON RELATIVE WEIGHT GAININ OBESE ZUCKER RATS

REL

ATIV

E W

EIG

HT

GAI

N(g

/kg

body

wei

ght)

**

* **

A

B

Fig. 3. Subchronic (8 days) administration of the in vivo cannabinoid CB1

receptor antagonist LH-21 (0.3 or 3 mg/kg) reduced relative food intake (g/kg

of body weight) and relative weight gain (g/g of body weight at day 1). Data

are means � SEM of 8 determinations per group. (*)P < 0.05, versus vehicle-

treated animals.

362 F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

as grooming behavior or scratching sequences, Table 1) as it istypically observed after the injection of the reference cannabi-noid receptor antagonist/inverse agonist SR141716A (3 mg/kg, Navarro et al., 1997). Although the highest dose of LH-21 (10 mg/kg) apparently reduced locomotor activity during

0 0.03 0.3 3 100

10

20

30

40

50

LH-21 (mg/kg)

RES

PON

SES

*

Fig. 4. Acute intraperitoneal injection of LH-21 (0.03 to 10 mg/kg) 30 min be-

fore the testing procedure reduced alcohol self-administration in male Wistar

rats only at the highest dose tested (10 mg/kg). Data are means � SEM of 8e

10 determinations per group. (*)P < 0.05, versus vehicle-treated animals.

0

20

40

60

80

100

TIM

E (%

)

CLOSED ARMS EXPOSED ARMS

5 30 600

50

100

150

200 VEHICLE

LH-21 3 mg/kg

LH-21 10 mg/kg

TIME (min)

CR

OSS

ING

S (O

PEN

FIE

LD)

A

B

Fig. 5. A. Acute administration of LH-21 did not affect locomotor activity on

the open field or B. performance of the animals in the elevated plus-maze,

a standard anxiety test in rodents. Animals were injected i.p. 30 min before

the test. Data are means � SEM of 8 determinations per group. (*)P < 0.05,

versus vehicle-treated animals.

the first time interval of the open field test (5 min after startingthe trial), the difference was not statistically significant.

3.5. Effects of LH-21 on the elevated plus-maze

The administration of the reference cannabinoid receptorantagonist SR141716A or its related compound AM251(Rodgers et al., 2005) is associated with decreased explorationon the elevated plus-maze (Navarro et al., 1997). This anxiety-like behavior was not observed when LH-21 (3 and 10 mg/kg)was administered to drug-naive animals (Fig. 5B).

3.6. Effects of SR141716A on feeding behavior andethanol self-administration

The i.p. administration of SR141716A (0.3, 1 and 3 mg/kg)to food-deprived animals resulted in a dose-dependent reduc-tion in food intake, which was constantly observed with thedoses of 1 and 3 mg/kg (Fig. 6A). With respect to ethanolself-administration, SR141716A was less potent than LH-21,although a sustained inhibition of this behavior was observedwhen the dose of 3 mg/kg was injected into the animals30 min prior to testing (Fig. 6B).

3.7. Prediction of the brain penetration of LH-21

Because the different profile of LH-21 and SR141716A, thereference cannabinoid receptor antagonist, we suspected thatboth drugs may have a differential permeability through thebloodebrain barrier. To this end we characterized this perme-ability in an in vitro model, and then, we studied it on an invivo test. The in vitro permeability of LH-21 and ten commer-cial drugs through a lipid extract from porcine brain was deter-mined using a PAMPA test, and the results are gathered inTable 1. Assay validation was made comparing the experimen-tal permeability with the reported values of the commercialdrugs (Di et al., 2003), which gave a good linear correlation(Fig. 7). From the straight line equation and taking intoaccount the pattern determined by Di et al. (2003) forbloodebrain barrier (BBB) permeation prediction to classifycompounds, we established the ranges of this assay as follows:

Compounds of high BBB permeation: Pe (10�6 cm s�1)> 5.30.

Table 1

Effects of acute administration of the cannabinoid receptor antagonists

SR141716A and LH-21 on behaviors recorded in the open field

VEHICLE LH-21

3 mg/kg

LH-21

10 mg/kg

SR1417161A

3 mg/kg

BehaviorTime spent

grooming (s)

29.1 � 18.6 37.9 � 17.1 23.3 � 11.9 124.3 � 26.5*

Rearings 62.5 � 6.2 65.5 � 5.8 54.4 � 8.6 41.3 � 15.1

Scratching

sequences

0.1 � 0.1 1.6 � 1.3 0.2 � 0.1 10.0 � 3.7*

Data are means � SEM of 8e10 determinations per group. (*)P < 0.05, ver-

sus vehicle-treated animals.

363F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

Compounds of low BBB permeation: Pe (10�6 cm s�1)< 3.10.

As can seen in Table 2, the assay predicted the control com-pounds correctly and showed that compound LH-21 had lowbloodebrain barrier permeation. Data obtained for the refer-ence cannabinoid receptor antagonist SR141716A revealedan experimental permeability above 3.77, higher than that ob-served for LH-21, but intermediate between drugs with high orlow bloodebrain barrier permeability. These results suggestthat LH-21 has a lower penetration into the brain, when com-pared with the reference cannabinoid receptorantagonistSR141716A.

3.8. Antagonism of central actions of the cannabinoidCB1 receptor agonist CP55,940 after peripheraladministration of LH-21 or SR141716A

In order to validate the in vitro permeability assay, weconducted an in vivo experiment on which we tested whetherLH-21 or SR141716A were able of antagonize the motor

30 60 1200

2

4

6

8

10

12

14

VEHICLE

0.3

1

3

TIME AFTER INJECTION (min)

FOO

D IN

TAKE

(g)

RES

PON

SES

* * **

*

* * SR141716A (mg/kg)

VEH 0.3 1 30

10

20

30

40

50

60

SR141716A (mg/kg)

*

A

B

Fig. 6. A. Acute administration of SR141716A reduced feeding in 24 h food-

deprived male Wistar rats. B. Acute administration of SR141716A reduced

ethanol self-administration in Wistar rats at the dose of 3 mg/kg. Animals

were injected i.p. 30 min before the test. Data are means � SEM of 8e10 de-

terminations per group. (*)P < 0.05, versus vehicle-treated animals.

depression induced by central administration (i.c.v.) of a can-nabinoid receptor agonist (CP 55940). Either vehicle, LH-21(0.3, 3 and 3 mg/kg) or SR141716A (3 mg/kg) were injectedi.p. 30 min before the administration of mg of CP 55,940 onthe lateral ventricles of the rat. CP 55,940 dramatically re-duced locomotion (Fig. 8A) and rearing activity (Fig. 8B).Pretreatment with SR141716A reversed this effect. HoweverLH-21 only exhibited a weak antagonistic activity on locomo-tion at the highest dose tested (10 mg/kg) and was devoided of

Expe

rimen

tal P

e (1

0-6 c

m s

-1)

Described Pe (10-6 cm s-1)

y = 1.109x + 0.8751R2 = 0.9729

0

5

10

15

20

0 5 10 15 20

Fig. 7. Lineal correlation between experimental and reported permeability of

ten commercial drugs using the parallel artificial membrane permeation assay.

Details of compounds and permeability values are described in Table 2.

Table 2

Prediction of the bloodebrain barrier (BBB) penetration using a parallel arti-

ficial membrane permeation assay (PAMPA)

Compound Reported

permeability

Pe (10�6 cm s�1)a

Experimental

permeability

Pe (10�6 cm s�1)b

BBB

classification

Verapamil 16.0 17.83 � 0.60 CNS þImipramine 13.0 16.03 � 0.54 CNS þDesipramine 12.0 13.03 � 0.56 CNS þProgesterone 9.3 10.84 � 0.46 CNS þPromazine 8.8 13.23 � 0.47 CNS þHydrocortisone 1.9 2.04 � 0.17 CNS �Enoxacin 0.9 1.42 � 0.24 CNS �Ofloxacin 0.8 1.00 � 0.04 CNS �Norfloxacin 0.1 1.35 � 0.04 CNS �Theophylline 0.1 1.74 � 0.29 CNS �LH-21 2.78 � 0.20 CNS �SR141716A 3.77 � 0.21 CNS þ/�Experimental PAMPA results for tested compounds are compared with those

described by Di et al. (2003). Bloodebrain barrier penetration was classified

as positive (CNSþ), intermediate (CNS þ/�) or negative (CNS�), depending

on the value of experimental permeability: compounds of high BBB perme-

ation display values of Pe (10�6 cm s�1) > 5.30 and compounds of low

BBB permeation values of Pe (10�6 cm s�1) < 3.10.a Taken from Di et al. (2003).b Values are represented as the mean � SEM.

364 F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

antagonistic activity on the rearing suppression induced by CP55,940. These results indicate that LH-21 has a lower penetra-tion through the bloodebrain barrier than the reference agonistSR141716A, compound that did reach a brain concentrationsufficient to reverse the acute actions of CP55,940.

4. Discussion

Three major conclusions derive from the present study.First, LH-21, an in vivo neutral cannabinoid CB1 receptor an-tagonist, has a different behavioral profile than the reference

VEH CP 0.3 3 10 SR 3 +CP

0

10

20

30

40

50

LH-21 + CP

**

**

VEH CP 0.3 3 10 SR 3 +CP

0

1000

2000

3000

4000

5000

6000

LH-21 + CP

*

*

**

A

B

DIS

TAN

CE

TRAV

ELLE

D

TREATMENT

TREATMENT

REA

RIN

GS

Fig. 8. Effects of peripheral (i.p.) administration of cannabinoid CB1 receptor

antagonist LH-21 (LH, 0.3., 3 and 10 mg/kg) or SR141716A (SR, 3 mg/kg) on

the acute motor depression induced by central administration (i.c.v.) of CP

55,940 (10 mg in 5 ml) in male Wistar rats. SR141716A but not LH-21 antag-

onized the effects of CP, suggesting a better brain penetration of SR141716A.

A. Cummulative locomotion score (distance travelled in cm) along a 2-h test-

ing. B. Cummulative number of rearings along a 2-h testing. Animals were in-

jected the antagonist i.p. 30 min before the administration of CP 55,940.

Testing started 5 min after CP injection. Data are means � SEM of 8 determi-

nations per group. (*)P < 0.05, versus vehicle-treated animals.

cannabinoid antagonist/inverse agonist SR141716A, but re-tains its ability to reduce fod intake and weight gain in obeseanimals. Second, LH-21 has a lower penetrability through bi-ological membranes than the reference antagonist SR141716A(Table 2), suggesting that the effects on feeding behavior havemajor peripheral components. We cannot discard central ac-tions of LH-21 since we could observe a partial decrease in al-cohol self administration and a partial antagonism of centralmotor depressant actions of the cannabinoid agonist CP55,940 at high doses of LH-21 (10 mg/kg). Third, the high ef-ficacy of LH-21 as an inhibitor of feeding and the low efficacyof this compound in suppressing ethanol self-administrationare consistent with previous reports that suggest that whilethe inhibitory actions of cannabinoid receptor antagonists onfeeding have both peripheral and central components, the ef-fects on reward/drug self-administration are mediated via cen-tral cannabinoid CB1 receptors (Maldonado and Rodriguez deFonseca, 2002; Hansson et al., in press). In fact, when com-pared to feeding suppression we needed doses 10e30 timesgreater of LH-21 to observe central actions such as inhibitionof alcohol self-administration. The relevance of central effectsof LH-21 is further supported by the absence of two behavioralmarkers of brain cannabinoid receptor blockade, the inductionof anxiety-like responses in the elevated pluz-maze and the in-crease in complex motor sequences, such as grooming orscratching sequences (Navarro et al., 1997; Rodriguez de Fon-seca et al., 1997) (Table 1). Concerning stress reactions ob-served after cannabinoid receptor antagonism, since thiscontroversial effect of cannabinoid receptor blockade is de-pendent on of both the species being used and the test selectedfor emotional reactivity our results need to restricted to the ratspecies. Although in gerbils cannabinoid antagonism displayan axiolytic-like profile (Poncelet et al., 1999; Griebel et al.,2005), in mice there are reports of both, anxiolytic actions(Rodgers et al., 2003) and anxiogenic responses (Halleret al., 2004). For avoiding this discrepancy we have selectedonly one species (rat) on which potentiation of endocannabi-noid transmission in the brain by either blockade of ananda-mide degradation (Kathuria et al., 2002) or anandamidetransport inhibition (Bortolato et al., 2006) is anxyolitic,whereas blockade of cannabinoid receptors is anxiogenic(Navarro et al., 1997). There is no study addressing whetherthe anxogenic responses are dependent on the inverse agonismproperties of certain cannabinoid receptor blocker. However,we do have conclussive studies on the effects of cannabinoidreceptor deletion in mice, with data showing basal and con-text-dependent anxiogenic profile derived of central absenceof cannabinoid receptors (Haller et al., 2002; Rodgers et al.,2005). With the data available we can conclude that in therat the lack of anxiogenic actions of LH-21 can at least par-tially derived of its poor penetration in the brain.

Interestingly, although SR141716A has higher permeabilitythrough lipid membranes than LH-21, its experimental perme-ability also suggests that it may have less penetration in thebrain than originally suspected, an observation which has re-cently been proposed after an initial clinical trial of the effectsof SR141716A in schizophrenics (Meltzer et al., 2004). In this

365F.J. Pavon et al. / Neuropharmacology 51 (2006) 358e366

study, the authors discussed the lack of effects of this cannabi-noid receptor antagonist in relation to the low dosage (20 mg,daily) and poor penetrability through the bloodebrain barrier.Further support for this comes from the observation of the dif-ferent potency of SR141716A and LH-21 on food intake andethanol self-administration. While suppression of feeding in-duced by cannabinoid antagonists can be mediated by CB1 re-ceptors located in peripheral nerve terminals (Gomez et al.,2002) or in brain areas with an open bloodebrain barrier suchas the hypothalamus (Jamshidi and Taylor, 2001; Cota et al.,2003; Hanus et al., 2003), ethanol self-administration is regu-lated by central cannabinoid receptors located in the ventralstriatum and the prefrontal cortex (Maldonado and Rodriguezde Fonseca, 2002; Rodriguez de Fonseca et al., 2005; Hanssonet al., in press). Both LH-21 and SR141716A display more ef-ficacy as feeding inhibitors than as suppressants of ethanol self-administration. When compared, LH-21 and SR141716A havesimilar efficacies with respect to feeding inhibition butSR141716A, which has better penetration through lipid bila-yers, is at least three times more potent when a central testsuch as inhibition of alcohol self-administration or antagonismof central-cannabinoid agonist-induced motor depression(Fig. 8) is used. Moreover, the induction of grooming, a complexbehavior regulated by both central cannabinoid CB1 and dopa-mine D1 receptors located in the ventral striatum (Rodriguezde Fonseca et al., 1998), was observed after the administrationof SR141716A but not after LH-21, again supporting the differ-ent route of access of the two compounds to the central nervoussystem. Following this rationale, and as a last example, LH-21failed to inhibit exploration in the elevated plus-maze. Anxietyand anxiety-like behaviors are adaptive responses regulatedby limbic circuits, especially those involving the amygdalarcomplex. Endocannabinoids and cannabinoid receptors modu-late anxiety-like responses in close association with the anxio-genic amygdalar neuropeptide corticotropin-releasing factor(Caberlotto et al., 2004; Rodriguez de Fonseca et al., 1996,1997). The lack of effects of LH-21 on anxiety tests (elevatedplus-maze as well as time spent in the center of the open field,data not shown) again suggest a lack of central actions. How-ever, despite the suggestive set of experimental data, an addi-tional source of difference between both compounds maycome from the inverse agonist properties of SR141716A (forreview see Pertwee, 2005), a pharmacological profile not foundin LH-21 which is a neutral antagonist (Jagerovic et al., 2004).

In conclusion, because of the neutral antagonist profile ofLH-21 and thanks to its lower penetration in the central ner-vous system when compared with reference antagonists/in-verse agonists available, this new class of drugs offersa potential advantage for the treatment of obesity, becauseof reduced unwanted side effects. The described role forperipheral cannabinoid CB1 receptors in the regulation of ap-petite, glucose homeostasis and lipid metabolism, indicatesthat peripheral targets are of major interest when designinga cannabinoid-receptor acting drug therapy for obesity. Ifneutral antagonism is sufficient to reduce appetite and im-prove metabolism, we believe that this class of drugs canbe considered as an improved therapy for obesity and obesity

associated disorders, further developing the line opened with therecent clinical observations in humans receiving the referencecannabinoid receptor antagonist/inverse agonist SR141716A(Van Gaal et al., 2005).

Acknowledgements

This work has been supported by The European 5th Frame-work Programme, grants QLRT-2001-01048 (TargAlc) andQLRT-2000-01691, MEC SAF 2004/07762, MEC SAF 2003/08003/C02, MEC SAF 2003/02262, Plan Nacional SobreDrogas, FIS 02/001, Redes C03/06, C03/08, G03/028 andG03/05. L. H.-F. is recipient of I3P fellowship from the C.S.I.C.

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