Central and Peripheral Consequences of the Chronic Blockade of CB1

JOURNAL OF NEUROCHEMISTRY | 2010 | 112 | 1338–1351 doi: 10.1111/j.1471-4159.2009.06549.x

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*Laboratori de Neurofarmacologia, Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra, PRBB, Barcelona, Spain Medimod pharmacology services GmbH, Reutlingen, Germany àInstitut d’Alta Tecnologia PRBB Fundacio´ Privada (IAT), PRBB, Barcelona, Spain

Abstract chronic taranabant treatment in both lean and obese rats. In The endocannabinoid system plays a crucial role in the contrast, chronic treatment with rimonabant did not modify the pathophysiology of obesity. However, the clinical use of can- density of CB1 cannabinoid receptor binding, and decreased nabinoid antagonists has been recently stopped because of its its functional activity to a lower degree than taranabant. Six central side-effects. The aim of this study was to compare the weeks after rimonabant and taranabant withdrawal, CB1 effects of a chronic treatment with the CB1 cannabinoid receptor density and activity recovered to basal levels. These antagonist rimonabant or the CB1 inverse agonist taranabant results reveal differential adaptive changes in CB1 canna- in diet-induced obese female rats to clarify the biological binoid receptors after chronic treatment with rimonabant and consequences of CB1 blockade at central and peripheral taranabant that could be related to the central side-effects levels. As expected, chronic treatment with rimonabant and reported with the use of these cannabinoid antagonists. taranabant reduced body weight and fat content. Interestingly, Keywords: autoradiography, CB1 cannabinoid receptor, a decrease in the number of CB1 receptors and its functional obesity, rimonabant, taranabant, withdrawal. activity was observed in all the brain areas investigated after J. Neurochem. (2010) 112, 1338–1351.

Obesity continues to grow as a worldwide health problem Di Marzo 2009). Animal and clinical studies have revealed and represents a major concern in the health care system in that the overactivity of the endocannabinoid system is a key developed and developing countries (Hagmann 2008). component in the pathophysiological mechanisms leading to Multiple physiological systems are involved in the control obesity and metabolic unbalance (Cota et al. 2003; Ravinet of food intake and metabolism and participate in the et al. 2004; Despres et al. 2005; Van Gaal et al. 2005; pathophysiological mechanisms leading to obesity. In this Vickers and Kennett 2005; Ward and Dykstra 2005; Pagotto sense, several studies have recently identiﬁed the crucial role et al. 2006; Pi-Sunyer et al. 2006; Scheen et al. 2006; played by the endocannabinoid system in the control of Schafer et al. 2008). Therefore, the blockade of CB1 energy balance. The endocannabinoid system constitutes a receptor was considered a potential pharmacological tool ‘silent’ mechanism that is activated in a transitory way to maintain the homeostatic equilibrium (Di Marzo and Matias Received September 11, 2009; revised manuscript received December 9, 2005; Di Marzo 2008). This system includes the cannabi- 2009; accepted December 12, 2009. noid receptors (CB1,CB2 and G-protein-coupled receptor Address correspondence and reprint requests to Rafael Maldonado, 55), the endogenous lipid ligands (endocannabinoids), and Departament de Ciencies Experimentals i de la Salut, Universitat Pom- the enzymatic machinery for their synthesis and inactivation. peu Fabra, PRBB, C/Dr. Aiguader 88, 08003, Barcelona, Spain. E-mail: Obesity seems to be associated with a pathological over- [email protected] 1These authors contributed equally to this work. activation of the endocannabinoid system revealed by an Abbreviations used:CB1, cannabinoid receptor 1; CB2, cannabinoid up-regulation of CB1 receptor and/or an enhancement receptor 2; CD, cafeteria diet; CT, computed tomography; GTPcS, of endocannabinoid levels (Di Marzo and Matias 2005; guanosine 5¢-[c-thio] triphosphate; HU, Hounsﬁeld; SC, standard chow.

2010 The Authors 1338 Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 Central and peripheral effects of rimonabant and taranabant | 1339 to restore the normal endocannabinoid tone under these Winkelmann, Germany). The rats were kept in groups of four pathological conditions. animals in Makrolon type IV cages (60 · 38 · 20 cm) with In view of the promising results reported in the different heightened lids (5 cm) during acclimatization and the ﬁrst 6 weeks of obesity development. Subsequently, they were housed singly in clinical trials, the CB1 receptor antagonist rimonabant initially emerged as an effective treatment of obesity and Makrolon type III cages (43 · 26 · 15 cm) with heightened lids (5 cm) for the remaining period of the experimental sequence. metabolic disorders (Despres et al. 2005; Pi-Sunyer et al. Animals were housed in rooms with controlled temperature 2006; Scheen et al. 2006; Van Gaal et al. 2008). In 2006, the (20 ± 2C) and humidity (40–60%) under a 12 h/12 h light dark European regulatory authorities approved the use of rimo- cycle. Twenty-four animals were assigned to the lean group and nabant in obese patients (BMI ‡ 30 kg/m or > 27 kg/m with were maintained on standard pellet chow (Altromin diet 1324 from complications) and was in the market as Acomplia from Altromin GmbH, Germany, 65% energy from carbohydrates, 24% 2006 to 2008 in more than 40 countries around the world. from protein and 9% from fat with in total 2.850 kcal/g). Another 24

Taranabant, a CB1 receptor inverse agonist, was also animals (obese group) were exposed to a free choice between SC and developed and reached Phase-III clinical trials for the highly caloric palatable pellet diet (CD). This CD was manufactured treatment of obesity (Hagmann 2008). However, the Euro- as previously described (Heyne et al. 2009) by a mix of equal pean Medicines Agency recommended the suspension of the amounts of Bounty, Snickers, Mars and Milka chocolate marketing authorization for rimonabant in 2008 because of prepared as homogenous food pellets (65% energy from carbohydrates, 6.5% from protein and 23% from fat with in total the presence of undesired central side-effects and the clinical 4.846 kcal/g). Both types of food were presented separately on the trials with taranabant were stopped. top of the grid of the cage (food container of the cage) with half of the Animal models are required to investigate the biological space (right/left) containing 150 g of each kind of food (lean group: mechanisms underlying obesity. A new model of compulsive SC + SC or obese group: CD + SC). Water was always available food seeking/taking has been recently validated in rats to ad libitum. After 14 weeks exposed to their corresponding diets, both study obesity (Hansen et al. 2005; Heyne et al. 2009; Shafat lean and obese rats were randomly subdivided into three different et al. 2009). It is based on the exposure to a feeding regime groups (n = 8 each) according to the pharmacological treatment in which rats are offered a free choice of cafeteria diet (CD) assigned (vehicle, rimonabant or taranabant). All protocols were (palatable chocolate-containing food) in addition to their conducted following the standard ethical guidelines of the European standard chow (SC) diet. Under these conditions, animals Communities Directive 86/609/EEC regulating animal research, and highly prefer CD over SC and most of them become obese. according to the internal standard operating procedures of the three laboratories where the experiments were carried out [medimod Furthermore, animals rapidly develop compulsive feeding pharmacology services, Reutlingen, Germany; Departament de Cien- reflected by a lack of flexibility in food taking with rejection cies Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), of SC when CD is transiently not available. This model is an Barcelona, Spain; Institut d’Alta Tecnologia (IAT), Barcelona, Spain]. excellent tool to investigate the neurobiological basis underlying obesity, and to evaluate new therapeutical approaches. Pharmacological treatment The aim of this study was to improve the knowledge of the Rimonabant, taranabant and vehicle were administered sublingually central and peripheral consequences of chronic CB1 block- (application volume 0.5 mL per rat) once per day 1 h before the ade. For this purpose, we have compared the central and beginning of the dark phase. Fresh drug solutions were prepared daily. The selective CB receptor antagonist rimonabant [(Npiperi- peripheral effects of a chronic treatment with the CB1 1 receptor antagonist rimonabant (10 mg/kg, once daily, sub- din-1-yl)-5-(4-chlorophenyl)-1(2,4-dichlorophenyl)-4-methyl-1H- lingual) or with the CB receptor inverse agonist taranabant pyrazole-3-carboxy amide] was administered at a dose of 10 mg/kg/ 1 day. The CB receptor inverse agonist taranabant [MK-0364, N- (3 mg/kg, once daily, sublingual) in this new model of 1 [(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2- compulsive food seeking/taking. Body weight and food methyl-2-{[5-(trifluoromethyl) pyridine-2-yl]oxy}propanamide] was intake were periodically recorded, and both molecular and administered at a dose of 3 mg/kg/day. Both drugs were dissolved in imaging analyses were performed. At the molecular level, a vehicle solution containing 0.5% natrosol (Sigma-Aldrich, Madrid, autoradiography was used to evaluate the functional activity Spain), 0.1% tween-80 (Sigma-Aldrich) and 99.4% distilled water. and density of CB1 receptors in these animals. The possible Rimonabant was purchased from Chemos GmbH (Regenstauf, longitudinal variation in the content and distribution of body Germany) and taranabant was a gift from Solvay Pharmaceuticals fat was also evaluated by using a customized computed (Hannover, Germany). tomography (CT) imaging approach. Study design

Material and methods Experimental part in the German laboratory (Medimod Pharmacology Services) Obesity development phase (14 weeks). After 1 week of acclimati- Animals, housing and food regimen zation, one group was exposed to a free choice between SC and CD The experiments were performed with 48 outbred female Wistar rats (obese group), whereas the other group received SC only (lean group). that were 6 months old at the beginning of the study (Harlan,

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 1340 | E. Martı´n-Garcı´a et al.

Treatment phase I (6 weeks). The same food regimen was nozzle to the background. Fat was determined from the subtracted maintained and the corresponding anti-obesity treatment (vehicle, image as those voxels presenting differences between 15 and 45 in rimonabant or taranabant) was given once daily to each group of HU, and falling within the pre-calculated soft-tissue mask. To animals. Transfer from the medimod pharmacology service to the estimate the total body weight and its distribution, the following Spanish laboratory (UPF and IAT) was carried out at the end of this density values were assigned to the segmented tissues: lungs, 0.43 g/ period. The international shipment was handled by World Courier cm3; soft tissue, 0.73 g/cm3; bone, 1.53 g/cm3; fat, 0.55 g/cm3. following internal standard operating procedures for reducing stress to the animals. Upon arrival, an acclimatization period of four days Brain slicing for autoradiography studies was allowed before starting the next experimental sequence. Animals were killed by decapitation. Brains were removed and quickly frozen by immersion in chilled 2-methyl-butane. Coronal Experimental part in the Spanish laboratories (UPF and IAT) sections 20-lm-thick were cut in a cryostat, thaw-mounted on Treatment phase II (7 weeks). The same food regimen and anti- gelatin/chrome-coated slides, dried briefly at 30C and stored at obesity treatments were maintained as in treatment phase I. )80C until the cannabinoid receptor and guanosine 5¢-[c-thio] Animals were scanned by CT for body fat content determination triphosphate (GTPcS) binding experiments were performed. on the first, third and seventh week upon arrival, which corresponded to days 44, 58 and 85 of treatment. One day after the last CT scan Autoradiography of cannabinoid receptor binding session, 24 of the 48 animals were killed to evaluate the density and the The protocol used was based on the method described before functional activity of CB1 cannabinoid receptors by autoradiography. (Herkenham et al. 1991). Briefly, slide-mounted brain sections were incubated for 2.5 h at 37C in a buffer containing 50 mM Treatment withdrawal phase (6 weeks). For the next 6 weeks, the 24 Tris with 5% bovine serum albumin (fatty acid-free), pH 7.4, and remaining animals were maintained under the same food regimen, 10 nM [3H] CP-55 940 (NEN, Boston, MA, USA) prepared in the but no pharmacological treatment was administered. Their body fat same buffer, in the absence or presence of 10 lM nonlabeled content was evaluated by CT scans performed on weeks 3 and 6 of CP-55,940 (Tocris Bioscience, Ellisville, MO, USA) to determine this phase, which corresponded to days 21 and 39 after treatment total and nonspecific binding, respectively. Following this incuba- withdrawal. After the last CT scan, animals were killed to evaluate tion, slides were washed in 50 mM Tris buffer with 1% bovine the density and functional activity of CB1 cannabinoid receptor by serum albumin (fatty acid-free), pH 7.4, for 4 h (2 · 2h)at0C, autoradiography. dipped in ice-cold distilled water, then dried under a stream of cool dry air. Body weight and food intake control Autoradiograms were generated by apposing the labeled tissues, Body weight was registered twice a week. Data are expressed as the together with autoradiographic standards ([3H] microscales; Amer- percentage of body weight gain taking as reference the weight of sham, UK), to tritium-sensitive film (Amersham Hyperfilm-3H; each animal before the beginning of treatment. Food intake in the Barcelona, Spain) for a period of 10 days and developed for 4 min Spanish laboratory was determined weekly by placing an estab- at 20C. Densitometry determinations were carried out with a GS- lished quantity of 150 g of food (CD and/or SC) at the top of the 800 Calibrated Densitometer (Bio-Rad Laboratories, Hercules, CA, cage every Monday at 12:00 h. One week later, the remaining food USA) and analyzed with Quantity One 1D software (Bio-Rad), was removed, weighed and replaced by a same initial quantity using the standard curve generated from [3H]-standards. Specific (150 g). Food intake was normalized for each animal based on its binding measured in each structure was determined by subtracting body weight using the following equation: [(weekly food intake the non-specific binding image from that of total binding. (g) · (kcal/g))/body weight (g)] · 100. Analysis of WIN-55,212–2-stimulated [35S]-GTPcS binding Computed tomography imaging and data processing The protocol used was based on the method described before (Sim Animals were anesthetized by 2.5% isoflurane inhalation using et al. 1995). Briefly, slide-mounted brain sections were rinsed in oxygen as vehicle and data from two consecutive CT scans at assay buffer (50 mM Tris, 3 mM MgCl2, 0.2 mM EGTA, different voltage settings of the X-ray tube (140 and 80 kV, both at 100 mM NaCl and 0.5% fatty acid-free bovine serum albumin, 100 mA) were acquired using a General Electric Discovery ST pH 7.4) at 25C for 10 min, then pre-treated for 15 min with an device. Images were acquired with a matrix size of 256 · 256 excess concentration (2 mM) of GDP (Sigma Chemical Co., yielding a 0.39 · 0.39 mm pixel size. Slice thickness was 1.25 mm Madrid, Spain) in assay buffer. Afterwards, sections were and the transversal field of view covered about 25 cm, which was incubated at 25C for 2 h in assay buffer containing 0.04 nM enough to image the whole animal, except for the tail. Immediately [35S]-GTPcS (PerkinElmer Espanã SL, Madrid, Spain), 2 mM after the second CT scan, animals were placed back into their cages, GDP, and 10 lM WIN-55,212–2 (Sigma Chemical Co.). Basal allowed to wake up and housed as usual. activity was assessed in the absence of agonist, whereas non- Two composite images were created from the 140 and 80 kV specific binding was measured in the presence of 10 lM unlabeled images: one average image and one subtraction image. The average GTPcS. In pilot experiments, additional brain sections were image was used to segment the background, lungs, soft tissues and incubated in the presence of rimonabant (3 lM) (Rinaldi-Carmona bone by applying appropriate thresholds to the Hounsfield (HU) et al. 1994) in addition to 0.04 nM [35S]-GTPcS, 2 mM GDP and units: background, below )600 HU; lungs, between )600 and )200 10 lM WIN-55,212–2. Slices were rinsed twice in 50 mM Tris HU; soft tissue, between )200 and 200 HU; bone, over 200 HU. A buffer, pH 7.4, at 4C and deionized once in water, then dried mask was created to assign the scanner platform and the anesthesia under a stream of cool dry air. Autoradiograms were generated by

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 Central and peripheral effects of rimonabant and taranabant | 1341 apposing the labeled tissues to film (Biomax MS, Amersham) for Results a period of 3 days and developed for 4 min at 20C. Developed films were analyzed and quantified in a computerized image Body weight analysis system (MCID, St Catharines, Ontario, Canada). Densi- Body weight gain changes across days during treatment and tometry determinations were carried out with a GS-800 Calibrated treatment withdrawal are shown in Fig. 1. The statistical Densitometer (Bio-Rad) and analyzed with Quantity One 1D software (Bio-Rad). analysis of body weight gain was performed considering three different periods, early treatment period (from day 1 to Statistical analysis 40), late treatment period (from day 42 to 89) and Data from body weight gain, food intake and CT imaging were withdrawal of treatment (from day 8 to 46 after withdrawal). analyzed using three-way ANOVA with diet (lean and obese) and Three-way ANOVA during the early period of treatment treatment (vehicle, rimonabant and taranabant) as between-subjects revealed a significant main effect of ‘diet’ [F(1,42) = 10.60; factors and day as within-subject factor. Data analysis of CB1 p < 0.01] confirming the higher body weight gain in obese 35 receptor binding and of WIN-55,212–2-stimulated [ S]-GTPcS than lean animals. A significant main effect of ‘treatment’ binding was performed for each brain structure by two-way ANOVA was also obtained [F(2,42) = 36.46; p < 0.001], which was with diet and treatment as between-subjects factors. All these dependent on the ‘diet’ [F = 5.23; p < 0.01]. Post hoc analyses were carried out separately for the different periods (2,42) analyses revealed that pharmacological treatments had corresponding to the presence or absence of treatment. Percentage of body weight gain was analyzed the last day of treatment and the last differential effects in reducing body weight in obese and day of the experiment using one-way ANOVA with treatment as lean animals. Thus, rimonabant produced higher reduction of between-subjects factor. The percentage of body weight gain was body weight gain in obese than lean rats (p < 0.01). In calculated taking as reference the value obtained before the contrast, taranabant-induced reduction of body weight gain beginning of the pharmacological treatment. Differences were was similar in both groups of animals. In both lean and considered significant at p < 0.05. Results are expressed as obese animals, body weight gain similarly decreased after mean ± SEM, unless otherwise stated. The statistical analysis was rimonabant (p < 0.001) and taranabant (p < 0.001) treatment performed using the Statistical Package for Social Science program compared with vehicle. A significant main effect of ‘day’ SPSS 15.0 (SPSS Inc, Chicago, IL, USA). was revealed during the early treatment period

(a) (b)

Fig. 1 Mean values of body weight gain evolution in lean (a) and withdrawal phase (from 8 to 46). From beginning of treatment to day obese rats (c) during the treatment and withdrawal periods of the 89 of treatment, the number of rats per group was 8. During the experiment. Mean ± SEM values of last day of treatment or treat- withdrawal phase (from 8 to 46), the number of animals per group ment withdrawal in lean (b) and obese animals (d). The reference was 4. wp < 0.05; wwwp < 0.001 signiﬁcant differences when value used to calculate the percentage of body weight variation was comparing vehicle to taranabant group. p < 0.01 signiﬁcant the value obtained before the beginning of treatment. X-axis (a and differences when comparing vehicle to rimonabant group (Newman- c) indicates day of the treatment period (from 1 to 89) and the Keuls).

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 1342 | E. Martı´n-Garcı´a et al.

[F(13,546) = 10.08; p < 0.001] with a signiﬁcant interaction (a) between ‘day’ and ‘diet’ [F(13,546) = 3.25; p < 0.001], as well as an interaction between ‘day’ and ‘treatment’

[F(26,546) = 3.93; p < 0.001], and a significant interaction between ‘day’, ‘diet’ and ‘treatment’ [F(26,546) = 1.51; p < 0.05]. Three-way ANOVA during the late period of treatment also revealed a significant main effect of ‘diet’ [F(1,42) = 15.04; p < 0.001] and ‘treatment’ [F(2,42) = 23.22; p < 0.001], which was dependent on the ‘diet’ [F(2,42) = 2.96; p < 0.05]. Accordingly to the previous period, post hoc analyses revealed that rimonabant produced higher reduction (b) of body weight gain in obese than lean rats (p < 0.01). In contrast, taranabant reduction of body weight gain was similar in both groups of animals. In obese rats, body weight gain similarly decreased after rimonabant (p < 0.001) and taranabant (p < 0.01) treatment compared with vehicle group. In lean animals, both treatments also decreased body weight gain compared with vehicle (p < 0.001), although taranabant was more efficacious than rimonabant (p < 0.05). A significant interaction between ‘day’ and ‘diet’

[F(13,546) = 2.51; p < 0.01] was also revealed. No significant Fig. 2 Mean ± SEM values of energy intake in lean (a) and obese main effect of ‘day’ [F(13,546) = 1.11; n.s.] or interaction rats (b) during first 40 days of treatment. wp < 0.05; wwp < 0.01; between ‘day’ and ‘treatment’ [F(26,546) = 1.49; n.s.] or wwwp < 0.001 significant differences when comparing vehicle to tar- between ‘day’, ‘diet’ and ‘treatment’ [F = 0.63; n.s.] (26,546) anabant group. p < 0.001; p < 0.01 significant differences were revealed during this late treatment period. when comparing vehicle to rimonabant group. #p < 0.05 significant In the treatment withdrawal period, three-way ANOVA did differences when comparing rimonabant to taranabant group not reveal significant main effects of ‘diet’ [F(1,18) = 0.93; (Newman-Keuls) (n = 8 per group). n.s.], ‘treatment’ [F(2,18) = 0.71; n.s.] or interaction between ‘diet’ and ‘treatment’ [F(2,18) = 0.08; n.s.], ‘day’ and ‘treat- Food intake ment’ [F(22,198) = 1.52; n.s.] or between ‘day’, ‘diet’ and Caloric food intake for each group of rats is shown in Fig. 2 ‘treatment’ [F(22,198) = 1.17; n.s.]. Significant main effects of (treatment from day 1 to 40) and table 1 (treatment from day ‘day’ [F(11,198) = 11.67; p < 0.001], and significant inter- 42 to 89 and treatment withdrawal from day 8 to 46). The action between ‘day’ and ‘diet’ [F(11,198) = 8.52; p < 0.001] statistical analysis of caloric food intake was also performed were found in this period. considering three different periods, early treatment period The values obtained on the last day of treatment and the (from day 1 to 40), late treatment period (from day 42 to 89) last day of the experiment were analyzed separately for the and withdrawal of treatment (from day 8 to 46 after two diet groups (lean and obese). In lean animals, one-way withdrawal). Three-way ANOVA during the early period of ANOVA showed a significant main effect of ‘treatment’ treatment showed a significant main effect of ‘diet’

[F(2,21) = 19.17; p < 0.001]. Post hoc Newman-Keuls anal- [F(1,42) = 33.34; p < 0.001], confirming the enhanced caloric ysis showed decreased body weight gain in rimonabant intake in obese compared with lean animals. Significant main (p < 0.001) and taranabant (p < 0.001) treated groups com- effects of ‘treatment’ [F(2,42) = 14.03; p < 0.001] and inter- pared with vehicle. In the obese group, one-way ANOVA also action between these two factors [F(2,42) = 3.37; p < 0.05] showed a main effect of ‘treatment’ [F(2,21) = 6.21; p < 0.01] were also found. Significant main effects of ‘day’ and Newman-Keuls analysis showed decreased body weight [F(7,294) = 47.38; p < 0.001], interaction between ‘day’ and gain in rimonabant (p < 0.001) and taranabant (p < 0.05) ‘diet’ [F(7,294) = 5.84; p < 0.001], ‘day’ and ‘treatment’ groups compared with vehicle. The last day of treatment [F(14,294) = 12.99; p < 0.001] and between ‘day’, ‘diet’ and withdrawal one-way ANOVA showed no significant main ‘treatment’ [F(14,294) = 3.71; p < 0.001] were also revealed. effects of ‘treatment’ in lean [F(2,9) = 0.21; n.s.] or in obese Considering the significant effect of treatment during this animals [F(2,9) = 0.25; n.s.]. On this last day of treatment, period, a more detailed post hoc analysis was performed to rimonabant was more effective in reducing body weight gain determine the time point when CB1 antagonists decreased in obese (13% of reduction) than lean (2% of reduction) rats, food intake. Thus, post hoc Newman-Keuls showed in whereas taranabant was similarly efficacious in both groups lean animals significant lower caloric consumption after of rats (9% vs. 6% of reduction respectively). taranabant administration than after vehicle on days 1

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 Central and peripheral effects of rimonabant and taranabant | 1343

Table 1 Caloric food intake Mean ± SEM

Treatment Treatment withdrawal

Lean Obese Lean Obese

Vehicle 104.46 ± 4.90 107.62 ± 9.86 101.70 ± 7.46 110.22 ± 8.84 Rimonabant 105.46 ± 4.75 115.45 ± 8.18 110.83 ± 8.84 119.25 ± 9.01 Taranabant 107.09 ± 5.17 121.32 ± 6.74 113.85 ± 7.60 136.07 ± 10.54

Food intake was normalized for each animal based on body weight using the following equation: [(weekly food intake (g) · (kcal/g))/body weight (g)] · 100.

(p < 0.001), 2 (p < 0.001), 7 (p < 0.01) and 28 (p < 0.05) of p < 0.01] was obtained indicating that caloric intake evolved treatment. Lean animals under rimonabant treatment also differently in obese and lean animals. No significant presented lower caloric consumption than lean vehicle group interactions between ‘day’ and ‘treatment’ [F(10,90) = 1.27; on days 1 (p < 0.001) and 2 (p < 0.001) of treatment. In obese n.s.] or between ‘day’, ‘diet’ and ‘treatment’ [F(10,90) = 0.35; animals, post hoc Newman-Keuls showed significant lower n.s.] were revealed. caloric consumption after taranabant administration than after vehicle on days 1 (p < 0.001) and 2 (p < 0.01) of treatment. Computed tomography image derived body fat content Obese animals under rimonabant treatment also presented Three-way ANOVA analysis of the body fat content revealed a lower caloric consumption than vehicle group on days 1 significant main effect of ‘diet’ [F(1,41) = 33.26; p < 0.001] (p < 0.001), 2 (p < 0.001), 7 (p < 0.01) and 14 (p < 0.05) of confirming the differences between lean and obese animals treatment. In addition, rimonabant treated animals presented (see Fig. 3 for coronal CT images). A significant main effect lower caloric intake than obese taranabant treated rats on of ‘treatment’ was also found [F(2,41) = 5.21; p < 0.05], days 1 (p < 0.05), 2 (p < 0.05) and 7 (p < 0.05) of treatment. which was dependent on the ‘diet’ [F(2,41) = 3.62; p < 0.05] Three-way ANOVA for caloric food intake during the late (Fig. 4). Newman-Keuls post hoc analyses revealed signif- period of treatment showed a significant main effect of ‘diet’ icantly lower body fat content in obese animals chronically

[F(1,42) = 6.71; p < 0.05], which conﬁrmed the expected treated with both rimonabant (p < 0.05) and taranabant differences in food intake between lean and obese animals. (p < 0.01) when compared with the obese vehicle group on

No significant main effects of ‘treatment’ [F(2,42) = 1.77; the CT scans performed on days 44, 58 and 85 of treatment n.s.] or interaction between these two factors [F(2,42) = 0.84; (Fig. 4b). These results showed that both pharmacological n.s.] were found. This three-way ANOVA also showed treatments were effective in reducing body fat content of significant main effects of ‘day’ [F(5,210) = 2.90; p < 0.05], obese animals when compared with the vehicle treatment. No indicating that all animals increased caloric intake across significant main effect of ‘day of CT scan’ was revealed days but this effect was independent of ‘diet’ or ‘treatment’ during the treatment period [F(2,82) = 0.17; n.s.], but a as shown by the absence of a significant interaction between significant interaction between ‘day of scan’ and ‘diet’ was

‘day’ and ‘diet’ [F(5,210) = 1.16; n.s.], ‘day’ and ‘treatment’ observed [F(2,82) = 4.42; p < 0.05]. These results confirmed [F(10,210) = 1.78; n.s.] or between ‘day’, ‘diet’ and ‘treat- that the body fat content evolved in a different way in lean ment’ [F(10,210) = 0.70; n.s.]. (Fig. 4a) and obese (Fig. 4b) animals during the time that Three-way ANOVA was also performed for the period of animals were treated. No interactions between ‘day’ and treatment withdrawal. The significant main effect of ‘diet’ ‘treatment’ [F(4,82) = 1.27; n.s.] or between ‘day’, ‘diet’ and was maintained [F(1,18) = 13.89; p < 0.01] revealing that the ‘treatment’ [F(4,82) = 0.55; n.s.] were revealed. obese animals maintained a higher caloric consumption than Three-way ANOVA of the CT-estimated body fat content the lean group. A significant main effect of ‘treatment’ was during the withdrawal period showed a significant main also obtained during withdrawal [F(2,18) = 9.81; p < 0.01]. effect of ‘diet’ [F(1,18) = 23.24; p < 0.001], thus confirming Post hoc Newman-Keuls analyses showed significant higher persistent differences in fat content between lean and obese caloric consumption after taranabant withdrawal than after animals. No significant effects of ‘treatment’ [F(2,18) = 0.09; rimonabant (p < 0.05) or vehicle withdrawal (p < 0.001). n.s.] or interaction between ‘treatment’ and ‘diet’

Animals under rimonabant withdrawal also presented higher [F(2,18) = 0.001; n.s.] were found in the withdrawal period. caloric consumption than those of the vehicle group Similarly to what was observed during the ‘treatment’ period, (p < 0.05). No signiﬁcant interaction between ‘diet’ and signiﬁcant effects were obtained for ‘day of scan’

‘treatment’ was obtained [F(2,18) = 1.71; n.s.]. In contrast, a [F(2,36) = 23.33; p < 0.001] and its interaction with ‘diet’ signiﬁcant main effect of ‘day’ [F(5,90) = 12.78; p < 0.001] [F(2,36) = 4.65; p < 0.05] and ‘treatment’ [F(2,36) = 3.51; and interaction between ‘day’ and ‘diet’[F(5,90) = 4.40; p < 0.05], but no signiﬁcant interaction between the three

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Fig. 3 Mosaic image showing coronal CT images of one representative individual for each group under study over the five scans. Amber signal corresponds to the estimated fat distribution. factors was observed [F(2,36) = 1.27; n.s.]. These results tative autoradiograms for cannabinoid receptor binding are indicated that body fat content evolved differently during shown in Fig. 6. In the nucleus accumbens, two-way ANOVA treatment withdrawal in lean and obese animals depending of CB1 receptor binding after treatment revealed a signi- on the previous pharmacological treatment. ficant main effect of ‘treatment’ [F(2,17) = 55.24; p < 0.001], but no significant effect of ‘diet’ [F(1,17) = 1.70; n.s.] or Autoradiography of cannabinoid receptor binding interaction between these two factors [F(2,17) = 2.90; n.s.] The quantitative values of CB1 receptor binding obtained was observed. Subsequent post hoc Newman-Keuls showed after treatment and withdrawal were analyzed for each lower levels of CB1 receptors in animals treated with different brain structure by two-way ANOVA with ‘diet’ and taranabant than in those treated with rimonabant (p < 0.001)

‘treatment’ as between-subjects factors (Fig. 5). Represen- or vehicle (p < 0.001). Two-way ANOVA of CB1 receptor

(a) (b)

Fig. 4 Evolution of the mean values of body fat percentage in (a) lean and (b) obese rats during treatment and withdrawal phase. wp < 0.05; wwp < 0.01 signiﬁcant differences when comparing vehicle to taranabant group. p < 0.05 signiﬁcant differences when comparing vehicle to rimonabant group (Newman-Keuls).

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Fig. 5 Cannabinoid receptor binding in the nucleus accumbens, striatum, hippocampus, hypothalamus, prefrontal cortex and cerebellum after chronic treatment with taranabant and rimonabant (a) and after treatment withdrawal (b). Data are expressed as mean ± SEM. wp < 0.05; wwp < 0.01; wwwp < 0.001 significant differences when compared with vehicle group. p < 0.001 significant differences when comparing taranabant to rimonabant group. #p < 0.05 significant differences when comparing vehicle lean to vehicle obese group (Newman-Keuls) (n = 4 per group). binding after withdrawal of treatment did not show treated with taranabant than in those treated with rimonabant significant main effects of ‘diet’ [F(1,18) = 0.02; n.s.], (p < 0.001) or vehicle (p < 0.001). In this brain structure, ‘treatment’ [F(2,18) = 1.51; n.s.] or interaction between ‘diet’ significant differences between vehicle and rimonabant were and ‘treatment’ [F(2,18) = 1.42; n.s.]. This result indicates also observed (p < 0.05). Two-way ANOVA of CB1 receptor that the changes induced on CB1 receptors binding recover binding after treatment withdrawal did not show significant following withdrawal. main effects of ‘diet’ [F(1,18) = 0.23; n.s.], ‘treatment’ In the striatum, two-way ANOVA of CB1 receptor binding [F(2,18) = 1.16; n.s.], or interaction between ‘diet’ and after treatment showed a significant main effect of ‘treat- ‘treatment’ [F(2,18) = 1.48; n.s.], indicating that changes ment’ [F(2,17) = 43.29; p < 0.001], but no significant effect induced on CB1 receptors binding recover following with- of ‘diet’ [F(1,17) = 3.42; n.s.] or interaction between ‘diet’ drawal. and ‘treatment’ [F(2,17) = 0.58; n.s.]. Post hoc Newman- In the hippocampus, two-way ANOVA of CB1 receptor Keuls analysis showed lower density of CB1 receptors in rats binding after treatment revealed a significant main effect of

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(a) conﬁrmed that the differences in CB1 receptor binding between lean and obese animals were only observed in vehicle animals (p < 0.05).

In the prefrontal cortex, two-way ANOVA of CB1 receptor binding after treatment showed a signiﬁcant main effect of

‘treatment’ [F(2,18) = 9.98; p < 0.01], but no signiﬁcant effect of ‘diet’ [F(1,18) = 0.003; n.s.] or interaction between ‘diet’ and ‘treatment’ [F(2,18) = 0.74; n.s.]. Post hoc Newman- Keuls showed lower levels of CB receptor in animals treated (b) 1 with taranabant than in those treated with rimonabant

(p < 0.001) or vehicle (p < 0.01). Two-way ANOVA of CB1 receptor binding after treatment withdrawal did not show

significant main effects of ‘diet’ [F(1,18) = 0.22; n.s.], ‘treatment’ [F(2,18) = 0.27; n.s.] or interaction between ‘diet’ and ‘treatment’ [F(2,18) = 1.20; n.s.], revealing the recovery of the changes induced on CB1 receptors binding after treatment withdrawal. Fig. 6 Representative autoradiograms for cannabinoid receptor Finally, in the cerebellum two-way ANOVA of CB1 receptor binding of coronal brain sections from obese animals treated with binding after treatment showed a significant main effect of vehicle, rimonabant or taranabant during treatment period and treatment withdrawal phase. The sections shown are from (a) the striatum ‘treatment’ [F(2,18) = 14.38; p < 0.001], but not significant and nucleus accumbens, and (b) hippocampus and hypothalamus. effect of ‘diet’ [F(1,18) = 0.19; n.s.] or interaction between ‘diet’ and ‘treatment’ [F(2,18) = 2.01; n.s.]. Post hoc New- man-Keuls showed lower levels of CB1 receptors in animals ‘treatment’ [F(2,17) = 10.70; p < 0.01], whereas no significant treated with taranabant than in those treated with rimonabant effect of ‘diet’ [F(1,17) = 0.49; n.s.] or interaction between (p < 0.001) or vehicle (p < 0.01). Two-way ANOVA of CB1 these two factors [F(2,17) = 0.25; n.s.] was obtained. Again, receptor binding after withdrawal of the treatment did not subsequent post hoc Newman-Keuls revealed lower levels of show significant main effects of ‘diet’ [F(1,18) = 0.23; n.s.], CB1 receptors in rats treated with taranabant than in those ‘treatment’ [F(2,18) = 0.03; n.s.] or interaction between ‘diet’ treated with rimonabant (p < 0.001) or vehicle (p < 0.01). and ‘treatment’ [F(2,18) = 0.08; n.s.]. This result indicates a Two-way ANOVA of CB1 receptor binding after treatment recovery of the changes induced on CB1 receptors binding withdrawal did not show significant main effects of ‘diet’ after treatment withdrawal. [F(1,18) = 0.44; n.s.], ‘treatment’ [F(2,18) = 3.32; n.s.] or inter- 35 action between ‘diet’ and ‘treatment’ [F(2,18) = 1.97; n.s.]. Analysis of WIN-55,212–2-stimulated [ S]-GTPcS Similarly, a recovery of the changes induced on CB1 receptors binding binding by the pharmacological treatment was observed. The quantitative values of WIN-55,212–2-stimulated [35S]- In the hypothalamus, two-way ANOVA of CB1 receptor GTPcS binding obtained after treatment and withdrawal binding revealed a significant main effect of ‘treatment’ were analyzed for each different brain structure by two-way [F(2,17) = 76.02; p < 0.001], but no significant effect of ‘diet’ ANOVA with ‘diet’ and ‘treatment’ as between-subjects factors [F(1,17) = 0.97; n.s.] or interaction between ‘diet’ and ‘treat- (Fig. 7). Representative autoradiograms for cannabinoid ment’ [F(2,17) = 1.74; n.s.]. Subsequent post hoc Newman- receptor functional activity are shown in Fig. 8. After Keuls analysis revealed again lower levels of CB1 receptors treatment, two-way ANOVA of WIN-55,212–2-stimulated in animals treated with taranabant than in those treated with [35S]-GTPcS binding in the nucleus accumbens showed a rimonabant (p < 0.001) or vehicle (p < 0.001). During the significant main effect of ‘treatment’ [F(2,18) = 25.09; treatment withdrawal period, two-way ANOVA of CB1 recep- p < 0.001] but no significant effect of ‘diet’ [F(1,18) = 0.02; tor binding showed significant main effects of ‘diet’ n.s.] or interaction between ‘diet’ and ‘treatment’

[F(1,18) = 6.64; p < 0.05] and obese animals showed higher [F(2,18) = 0.39; n.s.]. Subsequent post hoc Newman-Keuls 35 density of CB1 receptors than lean rats. No significant main showed lower WIN-55,212–2-stimulated binding of [ S]- effects of ‘treatment’ [F(2,18) = 1.49; n.s.] were obtained, GTPcS in animals treated with taranabant than in those which indicates a recovery of the changes induced on CB1 treated with rimonabant (p < 0.01) or vehicle (p < 0.001). A receptors binding after treatment withdrawal. Furthermore, lower binding of [35S]-GTPcS was also observed in animals a significant interaction between ‘diet’ and ‘treatment’ treated with rimonabant than in animals treated with vehicle [F(2,18) = 4.60; p < 0.05] was obtained indicating that the (p < 0.01). Two-way ANOVA after withdrawal of the treatment differences between lean and obese animals depended on the did not show significant main effects of ‘diet’ [F(1,16) = 0.01; previous treatment. Subsequent post hoc Newman-Keuls test n.s.], ‘treatment’ [F(2,16) = 1.32; n.s.] or interaction between

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 Central and peripheral effects of rimonabant and taranabant | 1347

Fig. 7 WIN-55,212–2-stimulated [35S]-GTPcS binding in the nucleus accumbens, striatum, hippocampus, hypothalamus, prefrontal cortex and cerebellum after chronic treatment with taranabant and rimonabant (a) and after treatment withdrawal (b). Data are expressed as mean ± SEM, wp < 0.05; wwp < 0.01; wwwp < 0.001 signiﬁcant differences compared with vehicle group. p < 0.05; p < 0.01 p < 0.001 sig- niﬁcant differences when comparing taranabant to rimonabant group (Newman- keuls).

‘diet’ and ‘treatment’ [F(2,16) = 1.18; n.s.] indicating a recovery of the changes induced on CB1 receptors functional (a) activity by the pharmacological treatment. 35 In the striatum, two-way ANOVA of [ S]-GTPcS binding after treatment showed a signiﬁcant main effect of ‘treat-

ment’ [F(2,18) = 43.29; p < 0.001], but no signiﬁcant effect of ‘diet’ [F(1,18) = 3.42; n.s.] or interaction between these two factors [F(2,18) = 0.58; n.s.]. In the same way, post hoc Newman-Keuls showed lower WIN-55,212–2-stimulated [35S]-GTPcS binding in animals treated with taranabant than in those treated with rimonabant (p < 0.05) or vehicle (b) (p < 0.001). Lower WIN-55,212–2-stimulated [35S]-GTPcS binding was also observed after treatment in animals receiving rimonabant than in those treated with vehicle (p < 0.05). Two-way ANOVA after withdrawal of the treatment

did not show significant effects of ‘diet’ [F(1,16) = 0.03; n.s.], ‘treatment’ [F(2,16) = 0.17; n.s.], or interaction between ‘diet’ and ‘treatment’ [F(2,16) = 0.19; n.s.], indicating a recovery of the changes induced on CB1 receptors functional activity by the pharmacological treatment. 35 Fig. 8 Representative autoradiograms for cannabinoid receptor In the hippocampus, two-way ANOVA of [ S]-GTPcS functional activity in coronal brain sections from obese animals binding after treatment showed a significant main effect of treated with vehicle, rimonabant or taranabant during treatment ‘treatment’ [F(2,18) = 8.84; p < 0.01] without significant period and treatment withdrawal. The sections shown are from (a) effect of ‘diet’ [F(1,18) = 0.30; n.s.] or interaction between the striatum and nucleus accumbens, and (b) hippocampus and ‘diet’ and ‘treatment’ [F(2,18) = 0.25; n.s.]. Again, post hoc hypothalamus.

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Newman-Keuls revealed lower WIN-55,212–2-stimulated which could be explained by the preferential inverse agonist [35S]-GTPcS binding in animals treated with taranabant than proﬁle of taranabant. Indeed, taranabant acts as a highly in those treated with rimonabant (p < 0.05) or vehicle potent and selective CB1 receptor inverse agonist that exerts (p < 0.01). Similarly to previous results two-way ANOVA the opposite pharmacological effects to CB1 receptor agon- after withdrawal of the treatment did not show signiﬁcant ists (Fong et al. 2007). CB1 receptors are coupled to Gi effects of ‘diet’ [F(1,16) = 0.08; n.s.], ‘treatment’ [F(2,16) = proteins and their activation produces a decrease of intracel- 0.31; n.s.] or interaction between these two factors lular cAMP concentrations (Devane et al. 1988; Howlett

[F(2,16) = 0.32; n.s.], indicating a recovery of the changes et al. 1990). Therefore, CB1 receptor inverse agonism results induced on CB1 receptors functional activity by the in an increase of cAMP levels, whereas neutral antagonists pharmacological treatment. do not induce any signal transduction effects of their own 35 In the hypothalamus, two-way ANOVA of [ S]-GTPcS (Lange and Kruse 2008). In this context, rimonabant was binding after treatment showed a significant main effect of initially defined as a selective CB1 receptor antagonist ‘treatment’ [F(2,17) = 18.81; p < 0.001], but no significant (Rinaldi-Carmona et al. 1994, 1995; Pagotto et al. 2006). effect of ‘diet’ [F(1,17) = 0.51; n.s.] or interaction between However, several studies have reported that rimonabant those factors [F(2,17) = 0.09; n.s.]. Post hoc Newman-Keuls might function as an inverse agonist mainly when adminis- showed lower WIN-55,212–2-stimulated [35S]-GTPcS bind- tered at high doses (Xie et al. 2007). In this study, the ing in animals treated with taranabant than in those treated decreased functional activity after rimonabant treatment was with rimonabant (p < 0.001) or vehicle (p < 0.001). Two- lower than after taranabant, and the density of central CB1 way ANOVA after withdrawal of the treatment showed receptors was only decreased after taranabant administration. significant main effects of ‘diet’ [F(1,16) = 6.03; p < 0.05] One possible explanation for this result could be that being higher WIN-55,212–2-stimulated [35S]-GTPcS bind- rimonabant preferentially acts as a neutral antagonist at the ing in lean animals. Furthermore, significant main effects of dose tested, while taranabant mainly acts as an inverse

‘treatment’ [F(2,16) = 6.27; p < 0.05] without signiﬁcant agonist in these experimental conditions. The chronic interaction between ‘diet’ and ‘treatment’ [F(2,16) = 3.02; blockade of the activity of the endocannabinoid system n.s.] were observed. Post hoc Newman-Keuls revealed lower produced by rimonabant would decrease CB1 receptor WIN-55,212–2-stimulated [35S]-GTPcS binding in animals functional activity whereas the chronic inverse agonist treated with vehicle than in those treated with rimonabant effects of taranabant would also decrease CB1 receptor (p < 0.01) or taranabant (p < 0.01) indicating a recovery of density. On the other hand, it has been demonstrated that the changes induced on CB1 receptors functional activity by rimonabant occupies 50% of central CB1 receptors 8 h after the pharmacological treatment. oral administration, whereas taranabant (Rinaldi-Carmona

et al. 1995) ocupies 25–30% of central CB1 receptors 24 h after oral administration (Fong et al. 2007) when using Discussion similar doses employed in this study. Therefore, the

In this study, a decrease in the density and functional activity predominant changes on CB1 receptor density observed after of CB1 cannabinoid receptor binding was observed in the taranabant administration were not related to a higher nucleus accumbens, striatum, hippocampus and hypotha- occupancy of central CB1 receptors by this antagonist since lamus after chronic treatment with the inverse agonist rimonabant also occupies a high percentage of central CB1 taranabant (3 mg/kg) in both lean and obese rats. In contrast, receptors after oral administration. The undesired side-effects chronic treatment with rimonabant (10 mg/kg) only de- of rimonabant and taranabant could be related with the creased the functional activity of CB1 receptors in the adaptive changes on CB1 receptors revealed in this study in nucleus accumbens and striatum without decreasing the different brain structures involved in reward and motivation, density of cannabinoid receptor binding. This down-regula- such as the nucleus accumbens, hippocampus and prefrontal tion and/or decreased functional activity disappeared after cortex. Thus, CB1 receptors are present in brain areas treatment withdrawal, indicating a recovery of the changes in involved in the regulation of energy balance such as the all the brain areas studied. These biochemical results parallel hypothalamus, but also play an important role in the the effects of both drugs on the reduction of body weight emotional control by acting on the prefrontal cortex, gain and total amount of fat content observed during chronic hippocampus and limbic system (Breivogel and Sim-Selley treatment, both of which recovered after treatment with- 2009). The changes induced by these CB1 antagonists on drawal. CB1 cannabinoid receptors in the hypothalamus could be Chronic blockade of CB1 receptors after antagonist or related to adaptive changes on food intake after chronic inverse agonist treatment decreased binding and/or functional taranabant treatment and after withdrawal of rimonabant and activity of central CB1 cannabinoid receptors. Different taranabant administration. The changes produced by these effects on CB1 receptor density and functional activity were antagonists on CB1 receptors in the prefrontal cortex, revealed after chronic rimonabant and taranabant treatment, hippocampus and nucleus accumbens could explain the

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 Central and peripheral effects of rimonabant and taranabant | 1349 presence of psychiatric side-effects described in the literature taranabant treatment, an increase in food intake was observed after chronic rimonabant and taranabant administration that which was associated with a recovery of body weight to include anxiety and depression (Akbas et al. 2009). These values reached by obese rats receiving chronic vehicle. This changes induced by CB1 receptor antagonist/inverse agonist increase in food intake after treatment withdrawal was seem to be transitory since a complete recovery of CB1 parallel to the increased functional activity of CB1 cannab- receptor density and functional activity appeared after inoid receptors in the hypothalamus, the main brain structure treatment withdrawal. involved in the control of food intake. The enhancement of

The successful reduction in body weight observed in the CB1 receptor activity in the hypothalamus after treatment present work during chronic CB1 antagonist/inverse agonist withdrawal could explain the presence of this rebound effect treatment is in agreement with the results previously on food intake that contributes to the rapid recovery of body published in animal studies and clinical trials (Despres et al. weight. These ﬁndings are consistent with previous reports 2005; Di Marzo and Matias 2005; Pi-Sunyer et al. 2006; highlighting the relevance of endocannabinoid levels in Kirkham 2008). In animal studies, mice chronically treated limbic forebrain and hypothalamus in the regulation of with rimonabant (Ward and Dykstra 2005) or lacking the appetite (Di Marzo et al. 2001; Kirkham et al. 2002).

CB1 cannabinoid receptor (Cota et al. 2003; Ravinet et al. CB1 receptors have been identified in multiple central and 2004) are leaner, have lower food-motivation, and present a peripheral tissues involved in the regulation of metabolism, transitory lower caloric intake than their corresponding including adipocites, liver, muscle and pancreas (Cota 2007). controls. In humans, rimonabant administration produces The psychiatric side-effects produced by CB1 antagonists are weight reduction and improves several cardiometabolic risk because of brain CB1 receptor occupancy, whereas their factors (Bellocchio et al. 2006). In all these studies, the effects on weight-loss and metabolic unbalance are also effects of rimonabant on weight gain were reported to be because of the occupancy of CB1 receptors located in more pronounced and prolonged than the reduction in food peripheral tissues (Despres 2007; Addy et al. 2008; Belloc- intake (Vickers et al. 2003). In contrast to the differential chio et al. 2008; Di Marzo 2008, 2009). The important role effects of rimonabant and taranabant on central CB1 played by peripheral CB1 receptors on the metabolic effects receptors, both CB1 ligands reduced body weight gain and of CB1 antagonists opens new interesting strategies. Indeed, fat content in obese rats suggesting a similar efficacy on the the design of CB1 antagonists with predominant effects on central and peripheral mechanisms leading to this metabolic CB1 peripheral receptors remains an interesting experimental unbalance. However, rimonabant was more effective in obese approach to develop new compounds devoid of the psychi- (13% of reduction) than lean (2% of reduction) rats, whereas atric side-effects that were associated to the first generation taranabant was similarly efficacious in both groups of of CB1 antagonists. animals (9% vs. 6% of reduction respectively). The activity In conclusion, this study reveals differential effects after of the endogenous cannabinoid system is enhanced in the chronic rimonabant and taranabant treatment in the adaptive hypothalamus (Di Marzo et al. 2001) and in several periph- changes occurring in CB1 cannabinoid receptors in the eral tissues such as the adipose tissue, liver, skeletal muscle central nervous system. Chronic treatment with taranabant and pancreas during obesity, which plays a major role in the decreased the density and functional activity of CB1 physiopathology of the metabolic unbalance (Matias et al. cannabinoid receptor in the brain, whereas chronic rimona-

2006; Pagotto et al. 2006). Therefore, the present results bant only decreased functional activity of central CB1 suggest that taranabant might act as an inverse agonist in vivo receptors without modifying the density. These results are and it does not need an underlying overactivity of the important for a better understanding of the central side- endocannabinoid system to reduce body weight in lean effects that have been associated to the anti-obesity drugs animals, whereas rimonabant could preferentially act as a acting on the endocannabinoid system. neutral antagonist under these experimental conditions. Food intake was decreased only at the beginning of chronic Acknowledgements rimonabant and taranabant treatment under the present experimental conditions, in agreement with the transitory This work was supported by the USA National Institutes of Health – effects previously reported on food intake during chronic National Institute of Drug Abuse (NIH-NIDA) (no. 1R01-DA01 6768-0111), the DG Research of the European Commission CB1 receptor blockade (Colombo et al. 1998; Carai et al. (PHECOMP, no. LHSM-CT-2007-037669 and GENADDICT, no. 2005). In addition, adult CB1 knockout mice feed ad libitum presented similar levels of food intake to wild-type litter- LSHM-CT-2004-05166), the Spanish ‘Instituto de Salud Carlos III’ (no. RD06/001/001), the Spanish ‘Ministerio de Educacio´n y mates (Di Marzo et al. 2001). The transitory reduction of Ciencia’ (no. SAF2007-64062), the Catalan Government food intake observed in this study can be due to the (SGR2009-00131), the ICREA Foundation (ICREA Academia- decreased CB1 receptor function found in hypothalamus and/ 2008) and the Polish Ministry of Science and Higher Education or nucleus accumbens during chronic rimonabant and subsidiary grant 478/6. PR UE/2007/7. Elena Martı´n-Garcia was taranabant treatment. After withdrawal of rimonabant and

2010 The Authors Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 112, 1338–1351 1350 | E. Martıń-Garcıá et al. supported by a post-doctoral fellowship from the Spanish ‘Instituto Di Marzo V., Goparaju S. K., Wang L. et al. (2001) Leptin-regulated de Salud Carlos III’. We thank Mª Dolors de la Fuente, and Begonã endocannabinoids are involved in maintaining food intake. Nature Penãlba for invaluable technical assistance and Dr Patricia Robledo 410, 822–825. for critical reading and revision of the manuscript. Fong T. M., Guan X. M., Marsh D. J. et al. (2007) Antiobesity efficacy of a novel cannabinoid-1 receptor inverse agonist, N-[(1S,2S)-3- (4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2- Conflict of interest statement [[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364), in rodents. J. Pharmacol. Exp. Ther. 321, 1013–1022. Rafael Maldonado has received research grants from Sanofi-Aventis, Hagmann W. K. 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