European Neuropsychopharmacology (2010) 20, 112–122 www.elsevier.com/locate/euroneuro Potential anxiogenic effects of cannabinoid CB1 receptor antagonists/inverse agonists in rats: Comparisons between AM4113, AM251, and the benzodiazepine inverse agonist FG-7142 K.S. Sink a, K.N. Segovia a, J. Sink a, P.A. Randall a, L.E. Collins a, M. Correa a,c, E.J. Markus a, V.K. Vemuri b, A. Makriyannis b, J.D. Salamone a,⁎ a Dept. of Psychology, University of Connecticut, Storrs, CT 06269-1020, USA b Center for Drug Discovery, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA c Dept. Psicologia, Area de Psicobiologica, University of Jaume I, Castello, Spain Received 11 September 2009; received in revised form 30 October 2009; accepted 10 November 2009 KEYWORDS Abstract Rimonabant; Anxiety; Cannabinoid CB1 inverse agonists suppress food-motivated behaviors, but may also induce Depression; psychiatric effects such as depression and anxiety. To evaluate behaviors potentially related to Cannabinoid; anxiety, the present experiments assessed the CB1 inverse agonist AM251 (2.0–8.0 mg/kg), the Aversive motivation; CB1 antagonist AM4113 (3.0–12.0 mg/kg), and the benzodiazepine inverse agonist FG-7142 Open field; (10.0–20.0 mg/kg), using the open field test and the elevated plus maze. Although all three Plus maze drugs affected open field behavior, these effects were largely due to actions on locomotion. In the elevated plus maze, FG-7142 and AM251 both produced anxiogenic effects. FG-7142 and AM251 also significantly increased c-Fos activity in the amygdala and nucleus accumbens shell. In contrast, AM4113 failed to affect performance in the plus maze, and did not induce c-Fos immunoreactivity. The weak effects of AM4113 are consistent with biochemical data showing that AM4113 induces little or no intrinsic cellular activity. This research may lead to the development of novel appetite suppressants with reduced anxiogenic effects. © 2009 Elsevier B.V. and ECNP. All rights reserved. 1. Introduction Drugs that impair cannabinoid transmission are known to suppress food intake in animals and humans. CB1 inverse ⁎ Corresponding author. Tel.: +1 860 486 4302; fax: +1 860 486 2760. agonists such as SR141716 (rimonabant) and AM251 reduce E-mail address: [email protected] (J.D. Salamone). food intake in a number of different animal models under 0924-977X/$ - see front matter © 2009 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2009.11.002 Potential anxiogenic effects of cannabinoid CB1 receptor antagonists/inverse agonists in rats 113 conditions of food-deprivation and satiation, and after acute 1991; Sheng and Greenberg, 1990). It is a powerful tool for or chronic treatment (Arnone et al., 1997; Colombo et al., mapping neuronal activation in part because of low basal 1998; Williams and Kirkham, 1999; Shearman et al., 2003; level of expression (Hughes et al., 1992), because c-Fos is Wiley et al., 2005; McLaughlin et al., 2003, 2005; Gardner and inducible by a wide array of pharmacological treatments and Mallet, 2006; Salamone et al., 2007a,b). Clinical trials with external stimuli (Morgan and Curran, 1989), and also because CB1 inverse agonists rimonabant and taranabant have shown c-Fos is expressed in a region- and cell-specific manner that these drug to be effective at reducing body weight and waist depends upon the type of stimulus (Wisden et al., 1990). circumference (Curioni and Andre, 2006; Despres et al., 2005; Previous studies reported that injections of 10.0 mg/kg Pi-Sunyer et al., 2006; Van Gaal et al., 2005; Addy et al., 2008). SR141716 (rimonabant) induced c-Fos activation in several However, these same clinical trials also provide evidence of brain structures, including prefrontal cortex, nucleus accum- serious psychiatric side effects including depression and bens shell, central amygdala, paraventricular nucleus of the anxiety. A meta-analysis of rimonabant in obesity (RIO) studies hypothalamus, and central gray area (Alonso et al., 1999; performed by the Food and Drug Administration (FDA) showed Rodriguez de Fonseca et al., 1997). Also, increased c-Fos that 26% of subjects treated with 20 mg rimonabant reported expression within the dorsal striatum and nucleus accumbens adverse psychiatric events versus 14% of those treated with shell, as well as paraventricular, arcuate and lateral placebo (US Food and Drug Administration Advisory Commit- hypothalamic nuclei, has been reported to occur after tee, 2007). The significantly higher incidence of psychiatric injections of AM251 (Rueda-Orozco et al., 2008; Sinnayah events observed with rimonabant was a key factor in the et al., 2008). To date, there have been no studies of c-Fos decision of the FDA to deny approval of the drug for treatment activation using AM4113. The present work compared c-Fos of obesity in the United States. Clinical trials of taranabant, activation produced by the well-characterized CB1 inverse another CB1 inverse agonist, were similarly plagued with agonist AM251 with the novel drug, AM4113, within the adverse psychiatric events. Onset of adverse psychiatric striatum, hypothalamus, and amygdala. These structures events occurred early in the clinical trials, and dropouts were examined because of their roles in food intake, related to psychiatric events were frequent (Addy et al., locomotor activity, and anxiety, all functions that are 2008). In the FDA's meta-analysis of clinical trials using modified by drugs that act on CB1 receptors (Sink et al., rimonabant, the most commonly reported adverse psychiatric 2008a,b, 2009; McLaughlin et al., 2003, 2005; Chambers event was anxiety (US Food and Drug Administration Advisory et al., 2007; Jarbe et al., 2008). The anxiogenic benzodiaz- Committee, 2007). Several animal models of anxiety have epine inverse agonist, FG-7142, was also included in this been utilized in the testing of various CB1 inverse agonists. experiment for comparison of activation patterns within Although the outcomes from these tests are inconsistent (see structures related to anxiety. Akinshola et al., 1999; Haller et al., 2002), many of these Based on the results of previous studies of anxiety-related animal studies have found CB1 inverse agonists to produce behavior and c-Fos expression using CB1 inverse agonists, behavioral signs of anxiety (Arevalo et al., 2001; McGregor and in vitro evidence that AM4113 has little intrinsic activity et al., 1996; Navarro et al., 1997; Rodriguez de Fonseca et al., at CB1 receptors compared to AM251 (Sink et al., 2008a; 1996; Haller et al., 2004; Rodgers et al., 2005). Chambers et al., 2007), it was hypothesized that AM251 A novel CB1 antagonist, AM4113, was shown to induce less would produce some anxiety-like behavior and moderate intrinsic biological activity in comparison with CB1 inverse neural activation, and that the overall patterns of behavioral agonists such as AM251 and rimonabant as demonstrated by and neural activation effects produced by AM4113 would be in vitro cAMP accumulation assays (Chambers et al., 2007; weaker than those elicited by AM251. Sink et al., 2008a). Like CB1 inverse agonists, AM4113 attenuated feeding and food-motivated behaviors (Sink 2. Experimental procedures et al., 2008a,b, 2009). AM4113 has also been assessed in behavioral tests of spontaneous locomotion and induction of 2.1. Animals nausea (Sink et al., 2008a; Chambers et al., 2007; Jarbe et al., 2008). However, the effects of AM4113 on anxiety- A total of 297 animals were used for these experiments. Adult male related behaviors have not yet been characterized. Thus, the Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN; present experiments were undertaken to evaluate the 300–325 g, 3–4 months age) were pair-housed in a colony main- potential anxiogenic behavioral effects of AM4113 and tained at 23 °C, with a 12-h light/dark cycle (lights on 07:00). Food AM251. For evaluation of anxiety-related behavioral effects, and water were available ad libitum in the home cages. Animal two exploration-based tests of anxiety were employed: the protocols were approved by the University of Connecticut Institu- elevated plus maze, and the open field. Anxiety-related tional Animal Care and Use Committee, and the studies were behavioral effects evoked by AM4113 and AM251 were conducted according to NIH guidelines for animal care and use. compared with those produced by a well-characterized anxiogenic drug, the benzodiazepine inverse agonist, FG- 2.2. Drugs and selection of doses 7142 (Evans and Lowry, 2007). A second goal of these studies was to compare the neural activation patterns induced by All studies used intraperitoneal (IP) injections, with a total volume of 1.0 mL/kg. AM251 and AM4113 (synthesized at the Center for Drug AM251 and AM4113, as measured using c-Fos expression, Discovery, Northeastern University) were dissolved in a vehicle of within anxiety- and feeding-related brain areas. c-Fos is an dimethylsulfoxide (DMSO; Fisher, Waltham, MA, USA), Tween-80 immediate early gene product that encodes a transcription (Fisher), and 0.9% saline in a 1:1:8 ratio. This mixture also served as factor involved in the coupling of extracellular signals to vehicle for elevated plus maze and open field experiments. FG-7142 long-term phenotypic cellular changes by regulating the (β-carboline-3-carboxylic acid N-methylamide) obtained from Sigma expression of later-onset gene products (Morgan and Curran, Chemical (St. Louis, MO) was suspended in a solution containing 0.9% 114 K.S. Sink et al. saline and 2% Tween-80. ±Fenfluramine was obtained from Sigma anxiety levels (Hogg, 1996). Number of head dips while on open arms Chemical Co. (St. Louis, Mo., USA). Although the+isomer is thought is an ethological measure also thought to relate to anxiety level to be the more active isomer, several studies have shown that the (Hogg, 1996; Wall and Messier, 2001). A pilot study (n=29) was racemic mixture is active in suppressing food intake (see, for conducted to determine the effect of AM251 on various ethological example, Francis et al., 1997; Voigt et al., 2000; Salamone et al., measures in the plus maze, including head dips, scanning, risk 2002).