0022-3565/05/3152-711–721$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 89839/3054932 JPET 315:711–721, 2005 Printed in U.S.A. Fenobam: A Clinically Validated Nonbenzodiazepine Anxiolytic Is a Potent, Selective, and Noncompetitive mGlu5 Receptor Antagonist with Inverse Agonist Activity Richard H. P. Porter, Georg Jaeschke, Will Spooren, Theresa M. Ballard, Bernd Bu¨ttelmann, Sabine Kolczewski, Jens-Uwe Peters, Eric Prinssen, Ju¨rgen Wichmann, Eric Vieira, Andreas Mu¨hlemann, Silvia Gatti, Vincent Mutel,1 and Pari Malherbe Pharma Division, Discovery Research CNS (R.H.P.P., W.S., T.M.B., A.M., E.P., A.M., S.G., V.M., P.M.) and Chemistry (G.J., B.B., S.K., J.-U.P., J.W., E.V.), F. Hoffmann-La Roche, Basel, Switzerland Downloaded from Received May 19, 2005; accepted July 19, 2005 ABSTRACT Ј Ϯ 3 Fenobam [N-(3-chlorophenyl)-N -(4,5-dihydro-1-methyl-4-oxo- 31 4 nM, respectively. MPEP inhibited [ H]fenobam binding jpet.aspetjournals.org Ϯ 1H-imidazole-2-yl)urea] is an atypical anxiolytic agent with un- to human mGlu5 receptors with a Ki value of 6.7 0.7 nM, known molecular target that has previously been demonstrated indicating a common binding site shared by both allosteric both in rodents and human to exert anxiolytic activity. Here, we antagonists. Fenobam exhibits anxiolytic activity in the stress- report that fenobam is a selective and potent metabotropic induced hyperthermia model, Vogel conflict test, Geller-Seifter glutamate (mGlu)5 receptor antagonist acting at an allosteric conflict test, and conditioned emotional response with a mini- modulatory site shared with 2-methyl-6-phenylethynyl-pyridine mum effective dose of 10 to 30 mg/kg p.o. Furthermore, feno- (MPEP), the protypical selective mGlu5 receptor antagonist. bam is devoid of GABAergic activity, confirming previous re- Fenobam inhibited quisqualate-evoked intracellular calcium re- ports that fenobam acts by a mechanism distinct from at ASPET Journals on January 24, 2020 ϭ Ϯ sponse mediated by human mGlu5 receptor with IC50 58 benzodiazepines. The non-GABAergic activity of fenobam, 2 nM. It acted in a noncompetitive manner, similar to MPEP and coupled with its robust anxiolytic activity and reported efficacy demonstrated inverse agonist properties, blocking 66% of the in human in a double blind placebo-controlled trial, supports mGlu5 receptor basal activity (in an over expressed cell line) the potential of developing mGlu5 receptor antagonists with an ϭ Ϯ 3 with an IC50 84 13 nM. [ H]Fenobam bound to rat and improved therapeutic window over benzodiazepines as novel Ϯ human recombinant receptors with Kd values of 54 6 and anxiolytic agents. Anxiety disorders are the most common mental illnesses in agents, especially for short term use, but they suffer from dose- the Western world. Although effective treatment exists in the limiting side effects, including sedation, memory impairment, form of the benzodiazepines and SSRI/norepinephrine reuptake ataxia, abuse potential, an interaction with ethanol, and phys- inhibitors, there is still considerable room for improvement. The ical dependence (Woods et al., 1992). The SSRIs/norepinephrine benzodiazepines are generally regarded as the most efficacious reuptake inhibitor as well as other serotonergic agents (such as buspirone) are commonly prescribed for the longer term treat- 1 Current address: Addex Pharmaceuticals S.A., Plan Les Ouates, ment of anxiety but in addition to a delayed onset of action of 3 Switzerland. to 6 weeks and sometimes an increased level of agitation for the Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. first few weeks, they also suffer from side effects in terms of doi:10.1124/jpet.105.089839. sexual dysfunction, nausea, anorexia, and headache (Vaswani ABBREVIATIONS: SSRI, serotonin reuptake inhibitor; mGlu, metabotropic glutamate; GPCR, G protein-coupled receptor; TM, transmembrane; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; HTS, high-throughput screening; FLIPR, fluorometric imaging plate reader; fenobam, N-(3-chloro- phenyl)-NЈ-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea; MTEP, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pridine; SIB-1757, 6-methyl-2-(phe- nylazo)-3-pyridinol); SIB-1893, (E)-2-methyl-6-(2-phenylethenyl)pyridine; NPS 2390, N-adamantan-1-yl-2-quinoxaline-carboxamide-2-quinoxa- line-carboxamide-N-adamantan-1-yl; S-4-CPG, S-4-carboxyphenylglycine; HEK, human embryonic kidney; PBS, phosphate-buffered saline; RT, 2ϩ ␥ room temperature; IP, inositol phosphates; hGlu, human metabotropic glutamate; [Ca ]i, intracellular calcium; DMSO, dimethyl sulfoxide; GTP S, guanosine 5Ј-O-(3-thio)triphosphate; S-4C3HPG, S-4-carboxy-3-hydrophenylglycine; S-DHPG, S-dihydrophenylglycine; S-3HPG, 3-hydroxyphe- nylglycine; 2Me4CPG, 2-methyl-4-carboxyphenylglycine; -4-CPG, S-4-carboxyphenylglycine; (ϩ)-MCPG, (ϩ)-␣-methyl-4-carboxyphenylglycine; CHPG, RS-2-chloro-5-hydroxyphenylglycine; L-AP4, 2-amino-4-phsophonobutyric acid; CHO, Chinese hamster ovary; RIA, radioimmunoassay; CER, conditioned emotional response; SR, suppression ratio; ANOVA, analysis of variance; PG, phenylglycine; MED, minimum effective dose. 711 712 Porter et al. et al., 2003). Numerous recent preclinical and clinical studies tionally, fenobam has been reported to be active in outpa- have demonstrated the involvement of metabotropic glutamate tients with severe anxiety (Pecknold et al., 1980) and in a (mGlu) receptors in the anxiety and stress disorders (Swanson single blind study (Lapierre and Oyewumi, 1982). In all three et al., 2005). studies, fenobam was reported to have a good safety profile The mGlu5 receptor is one of eight G protein-coupled recep- and no oversedation, no muscle relaxant, and no interaction tors (GPCRs) activated by glutamate, in addition to the well with ethanol, further supporting a mechanism of action that characterized ionotropic glutamate receptors (Conn and Pin, is distinct from GABA-ergic potentiation (Pecknold et al., 1997). The eight mGlu receptors are divided into three groups 1980, 1982; Lapierre and Oyewumi, 1982). However, feno- on the basis of their sequence similarities, signal transduction, bam has also been reported to be inactive in one phase II and agonist rank order of potency. Group I (mGlu1 and 5) are outpatient trial where side effects of a psychostimulant na- coupled to the activation of phospholipase C, and group II ture were reported (Friedmann et al., 1980). At the time, (mGlu2 and 3) and group III (mGlu4, 6, 7, and 8) are negatively fenobam was discontinued from further development as an coupled to cAMP production (Conn and Pin, 1997). The mGlu anxiolytic, with indications that the molecule required fur- receptors are members of class C family of GPCRs, having a ther refinement (Friedmann et al., 1980; Wu et al., 1995). large extracellular amino-terminal domain for agonist binding Given the recent interest in mGlu5 receptors for anxiety (venus flytrap model) (Kunishima et al., 2000) and the 7TM and our recent discovery of fenobam’s mode of action via helical segments that form a binding pocket for the novel class mGlu5 receptor antagonism, we have characterized fenobam of compounds acting as allosteric modulators (Kew, 2004). Be- Downloaded from in a broad range of in vitro pharmacology tests and in vivo cause mGlu5 receptors are highly expressed in brain regions rodent models of anxiety. In the present study, we show that known to be involved in emotional processes such as the limbic fenobam is a potent, subtype-selective, and noncompetitive brain structures (prefrontal cortex, amygdala, basal ganglia, antagonist of mGlu5 receptors and that fenobam has inverse and hippocampal regions) (Shigemoto et al., 1993; Romano et agonist activity similar to MPEP (Pagano et al., 2000). Fur- al., 1995), the mGlu5 receptors have attracted considerable thermore, we demonstrate that fenobam has potent antianx- attention for psychiatric disorders (Spooren et al., 2001). In- jpet.aspetjournals.org deed, with the notable exception of the benzodiazepines, the iety properties in rodent models of anxiety tests, including prototypical noncompetitive antagonist MPEP (Gasparini et al., stress-induced hyperthermia, Vogel conflict, Geller-Seifter 1999) and its close analog MTEP (Cosford et al., 2003) (Fig. 1) conflict, and conditioned emotional response. have been reported to be active in the broadest range of pre- clinical anxiety tests, confirming a critical involvement of this receptor in mood control. Materials and Methods During a functional high-throughput screening (HTS) cam- at ASPET Journals on January 24, 2020 Materials paign (fluorometric imaging plate reader; FLIPR) of F. Hoff- mann-La Roche’s small-molecule library for mGlu5 modula- Fenobam and MTEP were synthesized at F. Hoffmann-La Roche tors, we identified fenobam as a potent antagonist of mGlu5 (Basel, Switzerland). SIB-1757, SIB-1893, 7-hydroxyiminocyclopro- pan[b]chromen-1a-carboxylic acid ethyl ester, and NPS 2390 are receptors. Interestingly, fenobam (McN-3377) was originally 3 developed as a nonbenzodiazepine anxiolytic (by McNeil Lab- commercially available. [ H]Fenobam (specific activity, 24.6 Ci/ mmol) was synthesized by Dr. P. Huguenin (Hoffmann-La Roche oratories, 1978–1982), with an unknown molecular target. In chemical and isotope laboratories). MPEP (specific activity, 36 Ci/ preclinical studies, fenobam is reported to be active in rat mmol; TRK 1070), L-quisqualic acid (0188), and S-4-CPG (0323) were models of anxiety (conflict tasks including Geller-Seifter and obtained from Tocris Cookson Inc. (Bristol, UK). MPEP was obtained Vogel) (Patel et al., 1982), and this activity was not blocked from Sigma (Buchs, Switzerland). myo-[2-3H]Inositol (TRK 911) and by a benzodiazepine antagonist (Goldberg et al., 1983). yttrium silicate RNA binding beads (RPNQ0013) were purchased In a double blind placebo-controlled clinical trial, fenobam from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Glu- had an efficacy and onset of action comparable with diaze- tamate pyruvate transaminase (catalog number 737 127) was from pam (HAM-A/HSRC/HAM-D) (Pecknold et al., 1982).