Pharmacology, Biochemistry and Behavior 103 (2012) 425–430

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Pharmacology, Biochemistry and Behavior

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Interactions of CB1 and mGlu5 receptor antagonists in food intake, anxiety andmemory models in rats

Balázs Varga 1, Ferenc Kassai ⁎,1, István GyertyánDepartment of Behavioral Pharmacology, Gedeon Richter Plc., 1103 Budapest Gyömrői út 19-21, Hungary

Abbreviations: MTEP, 3-[(2-methyl-1,3-thiazol-4-y6-methyl-2-(phenylazo)-3-pyridinol); Rimonabant,dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazoleUSV, ultrasonic vocalization; s.c., subcutaneously; i.p., intrstandard error of mean; ANOVA, analysis of varianceDihydro-5-methyl-3-(4-morpholinylmethyl)pyrroloyl]-1-napthalenylmethanone; AM251, 1-(2,4-dichlor4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide.⁎ Corresponding author. Tel.: +36 1 889 8504; fax: +

E-mail address: fe.kassai@richter.hu (F. Kassai).1 These authors equally contributed to this work.

0091-3057/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.pbb.2012.09.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 March 2012Received in revised form 19 September 2012Accepted 23 September 2012Available online 28 September 2012

Keywords:MTEPRimonabantFood intakeAnxietyMemory

CB1 receptor antagonists proved to be effective anti-obesity drugs, however, their depressive and anxiogenic ef-fects became also evident. Finding solution to overcome these psychiatric side effects is still in focus of research.Based on the available clinical and preclinical results we hypothesized that the combination of CB1 and mGlu5receptor antagonismsmay result in a pharmacological intervention, where the anxiolytic mGlu5 receptor inhibi-tion may counteract the anxiogenic psychiatric side effects of CB1 antagonism, while CB1 antagonismmay ame-liorate the memory impairing effect of mGlu5 receptor antagonism. Further, the two components willsynergistically interact in blocking food-intake and reducing obesity.For testing the interaction of mGlu5 and CB1 receptor antagonism MTEP [3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pridine; SIB-1757, 6-methyl-2-(phenylazo)-3-pyridinol)] (mGlu5 antagonist) and rimonabant[(5-(4-Chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)hydrochloride] (CB1 antagonist) were used. All experiments were carried out in rats. Effects of the com-pounds on anxiety were tested in two foot shock induced ultrasonic vocalization paradigms, appetite sup-pression was assessed in the food intake test, while memory effects were tested in a context conditionedultrasonic vocalization setup.MTEP abolished the anxiogenic effect of rimonabant, while there was an additive cooperation in suppressingappetite. However, rimonabant did not ameliorate the memory impairing effect of MTEP.By combination of CB1 and mGluR5 antagonism, anxiety related side effects might be attenuated, appetitesuppression maintained, nevertheless, the possible emergence of unwanted memory impairments can over-shadow its therapeutic success.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Obesity is one of the most important health problems not only inthe developed, but also in developing countries (WHO, 2006). Its tra-ditional treatment, low calorie diet and exercise is apparently insuffi-cient, while there is only one long term applicable antiobesitypharmaceutical in the market — the moderately effective Xenical®(orlistat). An alternative option to treat obesity is bariatric surgery,but it should be only considered for obese patients at high risk ofmorbidity (Karmali et al., 2010).

l)ethynyl]pridine; SIB-1757,(5-(4-Chlorophenyl)-1-(2,4--3-carboxamide)hydrochloride;aperitoneally; p.o., orally; SEM,; WIN55212-2, (R)-(+)-[2,3-[1,2,3-de]-1,4-benzoxazin-6-ophenyl)-5-(4-iodophenyl)-

36 889 8400.

rights reserved.

A few years ago, it seemed that a new class of antiobesity drugs – theCB1 antagonists – would be able to alleviate the patients needs(Christensen et al., 2007). However, in 2007 it emerged, that rimonabant,the first drug of this class reaching the market, had serious psychiatricside effects. It induced suicidal ideation, depression and anxiety symp-toms in the patient population (FDA, 2007). Because of these side effects,rimonabant was withdrawn from themarket in 2008.When taranabant,an other CB1 antagonist developed for the treatment of obesity, wasfound to show similar side effects (Addy et al., 2008), clinical studies ofCB1 antagonists were terminated worldwide.

To overcome the psychiatric side effects different concepts havebeen raised. One of them was developing neutral antagonists, which –

unlike the inverse agonists rimonabant and taranabant – do not affectconstitutive CB1 signaling. This may optimize the benefits of CB1 recep-tor antagonists while minimize the risk of side effects (Janero andMakriyannis, 2009). A different approachwas developing solely periph-erally acting drugs (Pavon et al., 2006). Less attention has been paid oncombining CB1 antagonism with other, “protective” off-target effects.

For this latter approach, one possibility is combining CB1 antago-nism with mGlu5 receptor antagonist effect. Antagonists of mGlu5 re-ceptors displayed anxiolytic-like properties in various animal models

426 B. Varga et al. / Pharmacology, Biochemistry and Behavior 103 (2012) 425–430

(Busse et al., 2004; Klodzinska et al., 2004; Nordquist et al., 2007) andthe non-specific mGlu5 receptor antagonist fenobam decreased anxi-ety in humans (Porter et al., 2005). In vivo data obtained in rodentssuggest that mGlu5 receptors also play a role in feeding regulationand obesity. The mGlu5 receptor knockout mice were resistant todiet induced obesity, and MTEP, a negative allosteric modulator ofthe receptor was able to decrease fasting induced feeding and obesityin rats (Bradbury et al., 2005). However, mGlu5 receptor antagonistsimpair memory in several animal models (Homayoun et al., 2004;Gravius et al., 2005; Steckler et al., 2005; Christoffersen et al., 2008).On the other hand, CB1 antagonists have memory enhancing activity(Zanettini et al., 2011). Immunohistochemical data showed, that thegross distribution of type I metabotropic glutamate receptors(mGlu1 and 5) (Shigemoto and Mizuno, 2000) and CB1 receptors(Howlett et al., 2004) were overlapping in the central nervous sys-tem. Furthermore, it was reported, that when great amount of gluta-mate is released into the synaptic cleft, the postsynaptically locatedmGlu5 receptors can trigger a backward endocannabinoid signal tothe presynaptically located CB1 receptors, which are consequentlyable to attenuate glutamate release (Wilson and Nicoll, 2002). SimilarCB1–mGlu5 receptor interactions were described in the amygdala andin the hippocampus (Chevaleyre et al., 2006), brain areas shown toplay crucial role in stress induced anxiety and memory, respectively.In the hypothalamus, a structure which regulates food intake, directCB1–mGlu5 interaction has to our knowledge not been described sofar. However, the activity of melanin-concentrating hormone andhypocretin synthesizing cells of the hypothalamus, a cell groupwhich plays an important role in food intake, strongly depends onboth mGlu5 receptor (Huang and van den Pol, 2007) and CB1 receptor(Huang et al., 2007) activation.

Thus, the combination of CB1 and mGlu5 receptor antagonismsmay result in a pharmacological intervention, where the anxiolyticmGlu5 receptor inhibition may counteract the anxiogenic side effectsof CB1 antagonism, while the two components would synergisticallyinteract in blocking food-intake. Furthermore, the CB1 antagonismmay prevent the memory impairing effect of mGlu5 antagonism. Ifthese assumptions turned true, that would open the possibility ofthe development of a dual-acting CB1–mGlur5 antagonist compoundwith at least as potent anti-obesity action as that of the CB1 antago-nists, but with diminished anxiogenic or cognitive side effects.

To test experimentally this hypothesis, the interaction of rimonabantand MTEP – as a prototypic CB1 and mGlur5 antagonist, respectively –

was investigated in distinct animal models. Effects of these drugs on anx-iety were tested in ultrasonic vocalization (USV) paradigms in adult rats.As most of the anti-obesity drugs decrease appetite, and the reduction offood intake can predict weight loss efficacy (Thornton-Jones et al., 2006),the effect of CB1 andmGlu5 receptor antagonismwas studied in fasting in-duced food intake assay. Their effect on memory was tested in the acqui-sition of context conditioned USV paradigm.

2. Materials and methods

2.1. Animals

The experiments were carried out on male Hannover Wistar rats.For the ultrasonic vocalization tests animals weighing 175–200 g onarrival were purchased from Charles River, while for the food intaketest animals weighing 180–220 g on arrival were purchased fromToxiccop Hungary. The animals were housed in groups of four inplastic cages with a wire grid top in a temperature — andlight-controlled laboratory animal care unit (22±2 °C, 12-h light/dark cycle, light on at 6:00 AM) with ad libitum access to commercialpellet rat feed (ssniff R/M+H produced by Spezialdiäten GmbH,Germany; autoclaved at 105 °C) and tap water. All procedures carriedout on animals had been approved by the local ethical committee andconformed to the rules and principles of the National Institutes of

Health guidelines with regard to the use of animals in research andthe 86/609/EEC Directive (the operative regulation at the time ofthe study).

2.2. Drugs

MTEP and rimonabantwere synthesized at GedeonRichter (Budapest,Hungary). Rimonabant was suspended in 5%tween80/distilled water inall of the experiments. MTEP was dissolved in saline except in the foodintake test where it was dissolved in 10%tween80/saline following theprotocol of Bradbury et al. (2005). Depending on the experimental condi-tions and based on prior literature data, drugs were administered subcu-taneously (s.c.), intraperitoneally (i.p.) or orally (p.o.) at a volume of 1, 2or 5 ml/kg, respectively.

2.3. The anxiolysis and anxiogenesis USV test

In stressful conditions adult rats emit 22–24 kHz ultrasonic vocaliza-tions. The duration of ultrasonic vocalizations can be reduced by anxiolyt-ic drugs (Jelen et al., 2003; Sanchez, 2003) or increased by anxiogenicdrugs (Jelen et al., 2003; Roche et al., 2007). Accordingly, two foot shockinduced USV protocols were applied, one for testing anxiogenesis andone for testing anxiolysis. In the anxiogenesis protocol, rats were placedinto a 30×30×20 cm sound attenuated shocking chamber. After 30 s ha-bituation, a single shock of 1 s duration and 0.6 mA intensity was deliv-ered. This mild shocking elicits a low level USV that can be increased byanxiogenic drugs. In the anxiolysis experimental setup, following the30 s habituation period, rats received six shocks of 1 s duration and0.6 mA intensity. The inter-shock intervals were 10 s. This high intensityshocking elicits a high level USV that can be reduced by anxiolytic drugs.Total time spent on vocalizing wasmeasured right after the last shock for10 min in case of bothprotocolswith anUltravox™ system(Noldus Infor-mation Technology, The Netherlands).

In the anxiogenesis protocol it was tested, whether MTEP couldreduce the anxiogenic effect of rimonabant. In the first experimentrimonabant was tested alone in a dose range from 0.3 to 20 mg/kg.In the second experiment (on a different cohort of animals),rimonabant was coadministered at doses of 1.25 and 5 mg/kg with3 mg/kg MTEP.

In the anxiolysis protocol it was tested, whether rimonabant couldreduce the anxiolytic effect of MTEP. In the first experiment MTEPwas administered alone at a dose range from 3 to 24 mg/kg, whilein the second experiment MTEP was coadministered at 3 mg/kgdose with 1.25 of rimonabant. In both setups, both drugs were admin-istered i.p. 30 min prior to habituation.

2.4. The food intake assay

The test protocol was adopted from the literature (Bradbury et al.,2005) with some modification to fit to our current purposes. Fourdays before the test session, rats were habituated to the diet and tothe test protocol with vehicle treatments. On the test day, rats werefasted for 10 h before the end of the light phase. Ten minutes afterlights off, palatable diet (58V8 [45% of energy content from fat])was given into the cages. Food consumption was measured 1 h afterre-feeding, and boxes have been checked for spillage. Treatmentgroups were counterbalanced for body weights.

In a preliminary test, 10 mg/kg MTEP was given s.c. 8, 4 and 1 hbefore the beginning of the dark phase (following Bradbury et al.,2005), to estimate the time-course of appetite suppressive effect ofthe compound (data not shown). In subsequent experiments, MTEPwas administered with 4 h pretreatment time. Rimonabant was ad-ministered p.o. 60 min before the end of fasting. Effect of rimonabantand MTEP on food intake was tested alone and in combination.

Fig. 1. Effect of rimonabant administered alone (A) and coadministered with MTEP(B) in the anxiogenesis USV test paradigm. Mean values are plotted, error bars indi-cate SEM. N — sample sizes per group; **pb0.01 — significant difference comparedto the vehicle treated group (Dunnett post hoc test); ###pb0.001 — effect of MTEPtreatment (two-way ANOVA).

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2.5. The context conditioned USV test

The effect of rimonabant andMTEP onmemorywas tested in the ac-quisition of context conditioned USV paradigm. In this test setup ani-mals were shocked on two consecutive conditioning days in the sameshocking box described at the anxiolysis and anxiogenesis protocols.The daily shocking session was identical to the one applied in case ofthe anxiolysis setup (30 s exploration time, 6 shocks, 0.6 mA shock in-tensity, 1 s shock duration, 10 s inter-shock intervals, measurement ofUSV for 10 min). On the third day animals were replaced into theshocking box but no shock was delivered. USV was measured for10 min right after the animals were replaced into the shocking cham-ber. Animals that spent less than 100 s on vocalizing on the secondconditioning day were omitted from the experiment. Drugs wereadministered before the shocking sessions on both shocking days. Effectof rimonabant and MTEP on the acquisition of context conditioned USVwas tested in two separate experiments on different animals. In thethird experiment the compounds were coadministered. Rimonabantwas administered orally with 60 min pretreatment time, while MTEPi.p. with 30 min pretreatment time.

2.6. Statistical analysis

At the USV protocols mean duration of USV and SEM was calculat-ed for each group. In those experiments, where drugs were adminis-tered alone one-way ANOVA with Dunnett's post hoc test was usedand where drugs were coadministered two-way ANOVA were usedfor statistical analysis. In case of the context conditioned USV proto-col, at the coadministration experiment, percental impairment com-pared to the vehicle treated control was also calculated. Data of thefood intake test were analyzed with one-way ANOVA followed byDuncan post hoc test for pair-wise comparison. Dose–response effectswere plotted based on the percental food intake inhibition comparedto control.

3. Results

3.1. Effect of MTEP and rimonabant on anxiety related behavior

In the anxiogenesis test, rimonabant displayed a bell-shape dose–response curve, and significantly increased USV [F(7,135)=2.75,pb0.05] (Fig. 1A). Its anxiogenic effect was completely abolishedby MTEP [rimonabant: F(2,89)=1.88 p=0.16, MTEP: F(1,89)=33.5 pb0.001, interaction: F(2,89)=1.63 p=0.2] (Fig. 1B).

In the anxiolysis test, MTEP dose dependently reduced USV[F(4,67)=8.26, pb0.001] (Fig. 2A). This marked anxiolytic activitywas not influenced by rimonabant [rimonabant: F(1,68)=0.01 p=0.656, MTEP: F(1,68)=44.1 pb0.001, interaction: F(1,68=1.67 p=0.20] (Fig. 2B).

3.2. Effect of MTEP and rimonabant on food-intake

In the interaction test, one-way ANOVA revealed significant differ-ences among groups [F(17,150)=8.59, pb0.001]. Both rimonabantand MTEP decreased daytime fasting-induced food intake (Fig. 3).Low dose of MTEP (3 mg/kg) was without significant effect, while thehigh dose (10 mg/kg) decreased food intake significantly (Fig. 3A).The rimonabant-induced food intake suppression reached its maximalefficacy of ~65% at 3 mg/kg. Food intake of groups treated with 1, 3and 10 mg/kg rimonabant and co-treated with 3 mg/kg MTEP wassignificantly lower, than food intake of the group treated only with3 mg/kg MTEP. In case of group co-treated with 10 mg/kg rimonabantand 10 mg/kg MTEP food intake was significantly lower than in the10 mg/kg MTEP treated group, while food intake of groups co-treatedwith 0.1 and 1 mg/kg rimonabant and 10 mg/kgMTEPwas significantlylower than the groups treated only with rimonabant at the same doses

(Fig. 3A). The dose response relationships could be approximated withsigmoid curves (Fig. 3B) The coadministration of 3 and 10 mg/kgMTEPinduced a slight leftward shift in the dose–response curve ofrimonabant. The effect seemed to be additive to rimonabant in the0.1–1 mg/kg dose range (ascending part of rimonabant alone sigmoidcurve), but did not influence the maximal efficacy plateau (Fig. 3B).

3.3. Effect of MTEP and rimonabant on the acquisition of fear relatedmemory

In the single drug tests, MTEP significantly impaired acquisition ofcontext conditioning [F(4,44)=3.7, p=0.01] (Fig. 4A), while rimonabantshowed an impairing tendency [F(6,97)=0.83, pb0.54] (Fig. 4B). In theco-administration test both drugs significantly impaired acquisition[rimonabant: F(1,88)=7.98 pb0.01, MTEP F(1,88)=14.25 pb0.001].Percental impairment in case of the co-treated group was higher, thanin the single treated groups, however the interaction was not significant[F(1,88)=1.95 p=0.16] (Fig. 5).

4. Discussion

In this study, rimonabant and MTEP showed different types ofinteractions in the food intake, USV and fear conditioning assays.

In concordance with literature data (Klodzinska et al., 2004), MTEPshowed anxiolytic profile in the anxiolysis USV protocol. To our knowl-edge, data on the effects of rimonabant administration in adult ratsexposed to the USV test have not been previously published. Similarlyto the results reported in the rat pup ultrasonic vocalization test

Fig. 2. Effect ofMTEP administered alone (A) and coadministeredwith rimonabant (B) in theanxiolysis USV test paradigm. Mean values are plotted, error bars indicate SEM. N— samplesizes per group; *pb0.05 ***pb0.001— significant difference compared to the vehicle treatedgroup (Dunnett post hoc test); ###pb0.001— effect of MTEP treatment (two-way ANOVA).

Fig. 3. Effect of rimonabant and MTEP administered alone and in combination in thefood intake test. A Mean values. Error bars indicate SEM. B Percental inhibitions com-pared to the control. N=8 per group, except 10 mg/kg MTEP N=16 and controlN=24. *pb0.05, **pb0.01, ***pb0.001 — significant difference from control group(Duncan post hoc test); +pb0.05, +++pb0.001 — significant difference from grouptreated only with the same MTEP dose (Duncan post hoc test); #pb0.05 — significantdifference from group treated only with the same rimonabant dose (Duncan post hoctest). The control group received the vehicles of both MTEP and rimonabant — as theMTEP and rimonabant only groups also received the vehicle of the other.

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(McGregor et al., 1996), rimonabant displayed anxiogenic-like effect inthe anxiogenity USV paradigm in the adult rat. In the anxiolysis testrimonabant did not influence the anxiolytic effect of MTEP, while inthe anxiogenesis test MTEP completely reduced the anxiogenic effectof rimonabant. Thus the combination was free from the anxiogenicside effect.

We confirmed literature data (Bradbury et al., 2005; Christensenet al., 2007) about the food intake suppressive effects of MTEP andrimonabant. Rimonabant yielded a sigmoid dose response curvereaching a plateau at 3 mg/kg. As it was previously reported(Bradbury et al., 2005), MTEP dose dependently decreased food in-take at 4 h pretreatment time. The combination of rimonabant andMTEP synergistically decreased food intake. The synergistic interac-tion seems to be additive at lower doses of rimonabant, however,MTEP was not able to increase the maximal efficacy of rimonabant.This may have resulted from an independent mechanism restrictingfurther decrease in food intake, or alternatively, mGlu5 and CB1 antag-onists might influence food intake through a common pathway,which becomes saturated by the 3 mg/kg dose of rimonabant. Never-theless, the observed additive effect at low doses seems to be enoughto allow decreasing the dose of rimonabant in a combination whilemaintaining efficacy. This way the risk of side effects could be re-duced and the therapeutic range may be broadened.

In the context conditioning paradigm, rimonabant 10 mg/kg im-paired memory acquisition and enhanced the memory impairmentinduced by MTEP, however this latter effect was not statistically sig-nificant. Although it is generally accepted view that CB1 agonists im-pair while antagonists improve memory (Zanettini et al., 2011),literature data on the effect of CB1 ligands in fear- associated memorytests are equivocal. To our knowledge, CB1 ligands were not tested inthe context conditioned USV paradigm before. A similar method is the

classical contextual fear conditioning. In this test freezing (lack of anymovements) is registered as a measure of fear response. The CB1agonist WIN55212-2 was reported to impair (Pamplona andTakahashi, 2006), while the inverse agonist AM251 was shown to en-hance acquisition of contextual fear (Sink et al., 2010). However, inthe cue-conditioned versions of the test AM251 impaired acquisitionand memory consolidation in most studies (Arenos et al., 2006; Sinket al., 2010; Tan et al., 2010), though opposite findings were alsoreported (Reich et al., 2008), while rimonabant (Marsicano et al.,2002) did not display any effect on acquisition. Furthermore, in stud-ies where CB1 ligands were microinjected into different brain regions,antagonists were found to impair fear-related memory acquisitionand consolidation either after intraamygdalar (Bucherelli et al.,2006; Campolongo et al., 2009), intrahippocampal (De Oliveira etal., 2006, 2008) or intracortical (Laviolette and Grace, 2006; Tan etal., 2010) administration. These data indicate that CB1 antagonismmay result in impairment of fear-related memory which is in accor-dance with our results.

Even though we tried to conduct our experiments with the mostwell known representatives of the CB1 and mGluR5 antagonists, themolecular characteristics and functional peculiarities of these com-pounds may limit the generalization of the result to “CB1 antagonism”

and “mGluR5 antagonism”. Rimonabant is described as an inverseagonist, while MTEP is known as an allosteric mGluR5 inhibitor. Itcannot be excluded that the combination of other types of inhibitory

Fig. 4. Effect of MTEP (A) and rimonabant (B) administered alone in the acquisitionof context conditioned USV paradigm. Mean values are plotted, error bars indicateSEM. N — sample sizes per group;*pb0.05, **pb0.01 — significant difference com-pared to the vehicle treated group (Dunnett post hoc test).

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CB1 and mGlu5 receptor ligands might yield different results. Thus,more extended investigations (on the therapeutic potential) of thisinteraction are warranted.

5. Conclusions

In summary, although the combination of CB1 and mGlu5 receptorantagonists displayed a synergistic effect in reducing food intake, andmGluR5 antagonism abolished the anxiety-like state induced by CB1

antagonism, the memory impairing effect of mGluR5 antagonism

Fig. 5. Effect of rimonabant co-administered withMTEP in the acquisition of context con-ditioned USV paradigm. Mean values are plotted, error bars indicate SEM. N — samplesizes per group; **pb0.01— effect of rimonabant (two-wayANOVA); ###pb0.001— effectof MTEP (two-way ANOVA).

was not successfully ameliorated by concurrent CB1 antagonism.This interaction profile indicates that combining CB1 and mGlu5 an-tagonism is a promising option for development of novel, highly effi-cacious anti-obesity drugs free from the typical anxiogenic side effectof CB1 antagonists. However, the augmentedmemory impairment ob-served after coadministration of rimonabant and MTEP indicates therisk of mnemonic side effects. Clarifying the clinical relevance of thislatter issue needs further research.

Acknowledgments

The authors would like to acknowledge Gabriella Perjés and AnitaBérces for their excellent technical assistance in this study.

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