Antiobesity effects of chronic cannabinoid CB1 receptor antagonist treatment in diet-induced obese mice

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Abstract

We determined the effect of a cannabinoid CB1 receptor antagonist (AM-251; N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) on food intake, body weight and adipose tissue mass in Western diet-induced obese (DIO) mice using a chronic, interrupted, oral dosing paradigm. The dosing paradigm was 2 weeks on treatment (treatment 1), 2 weeks off-treatment, followed by 2 weeks on treatment (treatment 2). During treatment 1 and treatment 2, food intake and body weight were reduced after a single dose. At 30 mg/kg/day, anorectic efficacy was maintained through 12 days (treatment 1) and 7 days (treatment 2). Body weight of AM-251-treated mice remained less than vehicle-treated mice throughout treatment 1 and treatment 2. Administration of AM-251 reduced inguinal subcutaneous, retroperitoneal and mesenteric adipose tissue mass. Antiobesity effects of AM-251 were lost during the off-treatment period, and hyperphagia was observed in treated animals. With re-initiation of AM-251 treatment, mice again responded to the effects of the compound. These results support the hypothesis that chronic treatment of obese individuals with cannabinoid CB1 receptor antagonists is a viable pharmacologic approach to sustained weight loss.

Introduction

Obesity is increasingly recognized as a global health care problem epidemic in proportion (James et al., 2001). In the United States, where the epidemic is particularly evident, the number of approved drug treatments available to treat the disease has been reduced, rather than increased, over the last 5 years with the withdrawal of dexfenfluramine and fenfluramine. These two drugs were commonly prescribed and were associated with valvular heart disease (Connolly et al., 1997) which lead to their withdrawal. However, although the incidence of valvular heart disease was significantly higher in patients taking these drugs, it may not be as high as initially considered (Derry and Pritchard-Copley, 2002) and indeed in certain cases may spontaneously resolve over time (Vagelos et al., 2002). Nevertheless, current prescribed anti-obesity drug therapy is limited in the United States to orlistat (a gastrointestinal lipase inhibitor) or sibutramine (an anorectic). Given the heterogeneity of the etiology of human obesity and limitations of currently available drugs (precluded concomitant use of sibutramine in hypertensive patients, for example), there is a therapeutic need for very safe and effective compounds to treat obesity (Van der Ploeg, 2000). In this respect, the endocannabinoid system has received significant attention for its potential for pharmacologic manipulation to treat obesity.

The endocannabinoid system comprises endogenous ligands (anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glyceryl ether (noladin ether), virodhamine) and two cannabinoid receptor subtypes (CB1 and CB2) Hanus et al., 2001, Howlett et al., 2002, Porter et al., 2002. The cannabinoid CB1 receptor is of interest with respect to appetite regulation. The exogenous agonist Δ9-tetrahydrocannabinol (the principal psychoactive component of marijuana) is hyperphagic in rodents (Williams et al., 1998) and man (Foltin et al., 1988). Experimental studies in rodents have also demonstrated hyperphagic effects of the endogenous cannabinoids anandamide and 2-arachidonoyl glycerol (Williams and Kirkham, 1999). Further evidence that endogenous cannabinoids are involved in central nervous system control of appetite is derived from the observations that (i) direct administration of anandamide into the ventromedial hypothalamus (an area rich in cannabinoid CB1 receptors) stimulates food intake (Jamshidi and Taylor, 2001), and (ii) concentrations of 2-arachidonoyl glycerol in the limbic forebrain and hypothalamus are positively correlated with stimulation of food intake in rats (Kirkham et al., 2002). Furthermore, following temporary food restriction, cannabinoid CB1 receptor knockout mice eat less than their wild-type littermates, and cannabinoid CB1 receptor antagonist treatment reduces food intake in wild-type but not knockout mice Di-Marzo et al., 2001, Van der Ploeg, 2000. Thus, there is rationale to seek a cannabinoid CB1 receptor antagonist as an antiobesity drug.

Discovery of the first selective cannabinoid CB1 receptor antagonist was reported several years ago (Rinaldi-Carmona et al., 1994). This compound, SR141716A (N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), has subsequently been shown to have anorectic efficacy in a number of very short-term (acute) food intake studies in rats (Arnone et al., 1997), mice (Di-Marzo et al., 2001) and monkeys (Simiand et al., 1998). These short-term studies investigated anorectic activity after a single dose of the compound. Two longer-term SR141716A efficacy studies in rats (Colombo et al., 1998) and mice (Ravinet et al., 2003) have shown the compound to be transiently anorectic but produce a sustained reduction in body weight compared to a control group.

To further investigate anorectic and weight loss pharmacology of cannabinoid CB1 receptor antagonists, we used a clinically relevant administration route (oral) and a relevant Western diet-induced obese (DIO) mouse model. Furthermore, we investigated the chronic efficacy of a cannabinoid CB1 receptor antagonist, AM-251, in mice using a chronic interrupted dosing paradigm. AM-251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) is a structural analog to SR141716A differing by the halogen substitution of I for Cl. The interrupted dosing paradigm permitted efficacy assessment after a 2-week treatment period and then again after a 2-week off-treatment period. During the 2-week inter-treatment period, the appetitive behavioral response of mice after abrupt withdrawal of treatment was studied. Efficacy was determined by daily monitoring of food intake, body weight and, after each treatment period, adipose tissue mass. This study represents the first longer duration preclinical assessment of cannabinoid CB1 receptor antagonist in an interrupted dosing paradigm.

Section snippets

Animal care, handling and compound administration

All in vivo animal work conducted in this study conformed to the Guide for the Care and Use of Laboratory Animals published by the Institute of Laboratory Animal Resources (National Research Council, 1996). Male C57BL/6 mice were obtained from Charles River (Wilmington, MA) and housed under standard conditions (12-h light/dark cycle, 22 °C). Animals were acclimated to single housing upon arrival, and fed a pelleted high fat diet (45% kcal from fat, D12451 Research Diets, New Brunswick, NJ).

Results

The effect of AM-251 on food intake during the first treatment period, the off-treatment period and the second treatment period is shown in Fig. 2(A–C). During the first treatment period (Fig. 2A), there was an immediate and significant dose-dependent decrement in food intake in AM-251-treated male DIO mice. The anorectic effect of AM-251 waned over the 2-week treatment period; however, food intake remained significantly reduced through 8 days at the low dose (3 mg/kg/day) and through 12 days

Discussion

The anorectic efficacy of the cannabinoid CB1 receptor antagonist SR141716A is established Arnone et al., 1997, Colombo et al., 1998, Di-Marzo et al., 2001, Ravinet et al., 2003, Simiand et al., 1998; however, anorectic effects of AM-251 have not been previously reported. Our current study sought to determine if AM-251 (different from SR141716A by virtue of a single halogen switch, I for Cl) (Lan et al., 1999) demonstrated antiobesity efficacy in a chronic, interrupted dosing paradigm. AM-251

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