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 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 . 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--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- (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 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). Addi- Roche Diagnostics (Rotkreuz, Switzerland).

Fig. 1. Chemical structures of the noncompetitive mGlu5 antagonists (fenobam, MPEP, MTEP, SIB-1893, and SIB-1757), the competitive group I mGlu antagonist (S-4-CPG), and al-

losteric GABAA receptor agonist (di- azepam). Fenobam: A Noncompetitive Antagonist of mGlu5 Receptors 713

Plasmids, Cell Culture, and Membrane Preparation addition of 5 ␮Ci of myo-[2-3H]inositol at the time of plating (24 h before assay) in an inositol/glutamate-free DMEM with 10% dialyzed cDNAs encoding the rat mGlu1a, 2, 4a, 5a, 7a, and 8a receptors in fetal bovine serum, 1% penicillin/streptomycin, and 1 mM gluta- pBlueScript II was obtained from Prof. S. Nakanishi (Kyoto Univer- mate. On the day of assay, cells were washed three times, incubated sity, Kyoto, Japan). The cDNA encoding human mGlu5a was isolated in the presence of glutamate pyruvate transaminase and sodium as described previously (Malherbe et al., 2002). The cDNA for rat pyruvate for 1 h before the addition of agonists or antagonists in high-affinity glutamate transporter EAAC1 (AC: U39555) was iso- assay buffer (Hanks’ balanced salt solution in 20 mM HEPES plus 8 lated by reverse transcription-polymerase chain reaction from a rat mM LiCl). When present, antagonists were incubated for 5 min brain cDNA library. before agonist addition. After 45-min incubation with agonist, the HEK-293 cells were transfected as described previously. Forty- assay was terminated by the aspiration of the assay buffer and the eight hours post-transfection, the cells were harvested and washed addition of 100 ␮ three times with ice-cold PBS and frozen at Ϫ80°C. The pellet was l of 20 mM formic acid to the cells. After 20-min ␮ ␮ suspended in ice-cold 50 mM Tris-HCl buffer containing 10 mM incubation, a 20- l aliquot was mixed with 80 l of yttrium silicate EDTA, pH 7.4, and homogenized with a Polytron (Kinematica, Basel, beads that bind to the inositol phosphates (but not inositol). The Switzerland) for 10 s at 10,000 rpm. After centrifugation at 48,000g plates were counted on a TopCount, and data were expressed as a for 30 min at 4°C, the pellet was resuspended in ice-cold 50 mM percentage of the basal signal, i.e., cells in the absence of added Tris-HCl buffer containing 0.1 mM EDTA, pH 7.4, homogenized, and drugs. recentrifuged as described above. This pellet was further resus- (Intracellular Ca2؉ ([Ca2؉] ) Mobilization Assay (FLIPR pended in a smaller volume of ice-cold 50 mM Tris-HCl buffer con- i taining 0.1 mM EDTA, pH 7.4. After homogenization for 10 s at HEK-293-hmGlu5 stable cell line was seeded at 6 ϫ 104 cells/well Downloaded from 10,000 rpm, the protein content was measured using the bicincho- in the poly-D-lysine-treated, 96-well, black/clear-bottomed plates ninic acid method (Pierce Chemical, Socochim, Lausanne, Switzer- (BD Biosciences, Palo Alto, CA). After 24 h, the cells were loaded for land) with bovine serum albumin as the standard. The membrane 1 h at 37°C with 4 ␮M Flou-4AM (Molecular Probes, Eugene, OR) in homogenate was frozen at Ϫ80°C before use. loading buffer (1ϫ Hanks’ balanced salt solution and 20 mM HEPES). The cells were washed five times with loading buffer to 3 3 2ϩ [ H]Fenobam and [ H]MPEP Bindings remove excess dye, and [Ca ]i mobilization was measured using

mGlu5 receptor binding experiments were performed as described FLIPR (Molecular Devices, Menlo Park, CA) as described previously jpet.aspetjournals.org previously, with similar conditions for [3H]fenobam as for [3H]MPEP (Malherbe et al., 2003). All antagonists were dissolved in 100% ϫ (Malherbe et al., 2003). Briefly, after thawing, the membrane homoge- DMSO, and diluted in assay buffer to a 5 stock (2.5% DMSO). This nates resuspended and polytronized in 15 mM Tris-HCl, 120 mM NaCl, stock was then applied to the cells at a final DMSO concentration of 0.5%. The antagonist potency of various compounds on the hmGlu5 100 mM KCl, 25 mM CaCl2, and 25 mM MgCl2 binding buffer at pH 7.4 to a final assay concentration of 20 and 30 ␮g protein/well for [3H]M- receptors was determined using 10 to 20 nM quisqualate as agonist PEP and [3H]fenobam binding, respectively. Saturation isotherms were (a concentration that gave 60 to 80% of maximum agonist response, determined by addition of 12 radioligand concentrations (0.04–100 nM) determined daily). The antagonists were applied 5 min before the at ASPET Journals on January 24, 2020 to these membranes (in a total volume of 200 ␮l)for1hat4°C([3H]M- application of the agonist. Responses were measured as peak in- PEP binding) and for1hatRT([3H]fenobam binding). At the end of the crease in fluorescence minus basal, normalized to the maximal stim- incubation, membranes were filtered onto unifilter (96-well white mi- ulatory effect induced by 10 nM quisqualate. Inhibition curves were ϭ ϩ nH croplate with bonded GF/C filter preincubated1hin0.1% polyethyl- fitted according to the Hill equation: y 100/(1 (x/IC50) ), where enimine in wash buffer; PerkinElmer Life and Analytical Sciences, nH is slope factor using prism 3.0 (GraphPad Software Inc.). Boston, MA) with a Filtermate 196 harvester (PerkinElmer Life and [35S]GTP␥S Binding Assay Analytical Sciences) and washed three times with ice-cold 50 mM Tris- HCl, pH 7.4, buffer. Nonspecific binding was measured in the presence This assay was performed for selectivity test of fenobam toward ␮ of 10 M MPEP for both radioligands. At the Kd values, the nonspecific mGlu4a and 7a receptors using membranes from Chinese hamster binding for [3H]MPEP and [3H]fenobam was approximately 5 and 35%, ovary (CHO) cells expressing transiently mGlu4a and stably mGlu7a respectively, of total bound radioactivity for both radioligands. The receptors. After thawing, the membranes were washed once and radioactivity on the filter was counted (3 min) on a TopCount microplate resuspended in ice-cold 20 mM HEPES-NaOH buffer containing 10 ␮ scintillation counter with quenching correction after addition of 45 lof mM MgCl2, 2 mM EGTA, and 100 mM NaCl, pH 8.0. Wheat germ Microscint 40 (Canberra Packard S.A., Zu¨rich, Switzerland) and shak- agglutinin SPA beads (RPNQ0001; GE Healthcare) were suspended ing for 20 min. Saturation experiments were analyzed by Prism 3.0 in the same buffer (40 mg of beads/ml). Membranes and beads were (GraphPad Software Inc., San Diego, CA) using the rectangular hyper- mixed (beads, 13 mg/ml; membranes, 200 ␮g protein/ml) and incu- bolic equation derived from the equation of a bimolecular reaction and bated with 10 ␮M GDP at RT for 1 h, with mild agitation. ϭ ϫ ϩ 35 ␥ the law of mass action, B (Bmax [F])/(Kd [F]), where B is the [ S]GTP S binding was performed in 96-well microplates (Pico- amount of ligand bound at equilibrium, Bmax is the maximum number plate; PerkinElmer Life and Analytical Sciences) in a total volume of ␮ ␮ ␮ of binding sites, [F] is the concentration of free ligand, and Kd is the 180 l with 30 g (mGlu4a) and 5 g (mGlu7a) membrane proteins ligand dissociation constant. For inhibition experiments, membranes and 0.3 nM [35S]GTP␥S. Nonspecific binding was measured in the were incubated with 2 nM [3H]MPEP or 20 nM [3H]fenobam and 10 presence of 10 ␮M ice-cold GTP␥S. For mGlu4a selectivity test, 35 ␥ concentrations of the inhibitory compound. IC50 values were derived [ S]GTP S binding was stimulated with various concentrations of ␮ Ϯ ␮ from the inhibition curve and Ki values were calculated according to the L-AP4 (3 nM–30 M) 10 M fenobam. For mGlu7a selectivity test, ϭ ϩ ␮ equation Ki IC50/(1 [L]/Kd), where [L] is the concentration of fenobam concentration response curves (3 nM–100 M) were per- radioligand and Kd is its dissociation constant at the receptor, derived formed in the presence of 0.1 mM (EC20) and 0.7 mM (EC80) L-AP4. from the saturation isotherm. Plates were sealed and agitated at RT for 3 h. The beads were then allowed to settle and the plate counted in a TopCount (PerkinElmer [3H]Inositol Phosphates Accumulation Assay Life and Analytical Sciences) using quench correction. [3H]Inositol phosphates accumulation was measured using the cAMP Accumulation Assay methodology of Brandish et al. (2003) with the following modifica- tions. HEK-293 cells stably expressing hmGlu5 receptors were trans- For selectivity test of fenobam toward mGlu2, measurement of fected with the high-affinity glutamate transporter EAAC1, plated cAMP accumulation assay was performed with a stable CHO cell line at 70,000 cells/well in a polylysine-coated 96-well plate with the expressing mGlu2 receptor using the radioimmunoassay (RIA) kit 714 Porter et al.

(RPA559; GE Healthcare). The cells were plated at 40,000 cells/well (i.e., not tongue movements). At the end of the third 24-h period, they in a 96-well plate 24 h before assay. On the day of assay, cells were were administered the test compound p.o. and isolated in a holding washed in PBS and incubated at RT for 30 min in a total volume of chamber until testing 60 min later. During the 10-min test, they 200 ␮l with PBS buffer containing 1 mM 3-isobutyl-1-methylxan- were allowed to drink freely for a cumulative time of 5 s, after which thine (Fluka, Buchs, Switzerland), 1-aminocyclopentane-1,3-dicar- drinking was punished. That is, an electrical shock between the grid ␮ Ϯ ␮ boxylic acid (10 nM–30 M) 10 M fenobam, and the water-soluble floor and the drinking spout (0.5 mA; 250 ms) was triggered every ␥ forskolin analog 7-deacetyl-7-(O-N-methylpiperazino)- -butyryl-for- second of cumulative drinking time. This shock level strongly sup- ␮ skolin dihydrochloride (10 M) (Calbiochem, Lucerne, Switzerland). presses drinking to approximately 10% of normal levels. Fenobam The amount of cAMP formed was quantified by RIA following the was tested at 3, 10, and 30 mg/kg p.o. (n ϭ 12/dose). cAMP SPA Biotrak direct screening assay system protocol from GE Geller-Seifter Conflict. For further details regarding the train- Healthcare: at the end of the incubation period, the plate was cen- ing schedule and criteria for baseline performance, see Martin et al. trifuged and the supernatant was removed, and then 50 ␮l of lysis (2002). This procedure was divided into three stages (one lever was reagent (containing 10% dodecyltrimethylammonium bromide) was extended throughout the whole session): 1) unpunished responding added to each well. After 5 min of shaking, 150 ␮l of immunoreagent solution (containing rabbit anti-succinyl cAMP serum, donkey anti- (5 min) during which the house light was illuminated and animals rabbit IgG second antibodies coated at the surface of SPA beads, and received food reward to reinforce lever pressing on a schedule of adenosine 3Ј,5Ј-cyclic phosphoric acid 2Ј-O-succinyl-3-[125I]iodoty- variable interval of 30 s (VI30); 2) nonrewarded responding (time-out rosine methyl ester as a tracer) was added to each sample. The plates period of 2 min) during which the house light was extinguished and were shaking for 18 h, allowed to settle for3hatRT,andthen animals were able to press the lever, but they did not receive a food counted in a TopCount. The quantization of the cAMP formed was reward; and 3) punished responding (8 min) during which the house Downloaded from performed with the aid of an appropriate RIA cAMP standard curve. light remained off and a cue light above the lever was illuminated. In the latter stage, a fixed ratio of 10 (FR10) was used so that, on every Behavioral Profiling of Fenobam tenth lever press, the rat received simultaneously a food reward and Animals and Drug Treatment. Adult male Sprague-Dawley a foot shock (0.6 mA; 0.5 s). These three stages were repeated in the rats supplied from Charles River (Margate, Kent, UK) and NMRI same order so that the total daily session duration would be 30 min. mice (stress-induced hyperthermia) supplied from Iffa Credo Eight trained rats were included in the experiment to evaluate jpet.aspetjournals.org (L’Arbresele, France) were used. All rats were group housed and fenobam and were tested using a Latin-squares design, twice weekly mice were singly housed in separate holding rooms at controlled with at least a 2-day interval between test sessions. Rats were temperature (20–22°C) and 12-h light/dark cycle (lights on 6:00 AM). trained between test days to maintain baseline performance. Feno- All animals were allowed ad libitum access to food and water, with bam was tested at 10, 30, and 100 mg/kg p.o. the exception of those used in the operant-conditioning tests [Geller- CER. For further details regarding the training schedule and criteria Seifter, conditioned emotional response (CER)], where food was lim- for baseline performance, see Martin et al. (2002). The 60-min session ited to that earned in the test session and 12 to 15 g/rat at the end of consisted of a VI60 schedule with two randomized presentations of a

the day. The experimental procedures used in the present investiga- at ASPET Journals on January 24, 2020 conditioned stimulus (CS) for 2 min. The CS consisted of a 2.9-kHz tone tion received prior approval from the City of Basel Cantonal Animal delivered from a Sonalert system and a cue light above the lever. Protection Committee based on adherence to federal and local regu- During the final 0.5 s of the CS, animals received a foot shock (0.6–1.0 lations. All compounds were prepared immediately before use in vehicle, mA; 0.5 s). The number of lever presses during the CS period and 0.3% Tween 80 (v/v) saline. The volume of administration was 1 during the 2 min before this period were recorded and used to calculate ml/kg (Geller-Seifter, CER) or 5 ml/kg (Vogel) body weight for rats suppression ratios (SRs): number of lever presses during CS/total num- and 10 ml/kg for mice. Fenobam or vehicle was administered p.o. 60 ber of lever presses before and during CS. An SR value of 0 indicates min before testing. All doses were calculated as weight of base. that animals have suppressed responding during the tone presentation Stress-Induced Hyperthermia in Mice. A detailed description due to a conditioned fear response. An SR value of 0.5 indicates that the of the stress-induced hyperthermia (SIH) test procedure can be animals are responding equivalently during the conditioning period and found in Spooren et al. (2002). Briefly, drug naive individually 2 min immediately before this period. housed (macrolon cage, 26 ϫ 21 ϫ 14 cm) male NMRI mice were Twelve trained rats were included in the experiment to evaluate used. Body temperature was measured in each mouse twice at t ϭ 0 fenobam and were tested using a Latin-squares design, twice weekly (T1) and t ϭϩ15 min (T2). T1 served as the handling stressor. The with at least a 2-day interval between test sessions. During a test difference in temperature (T2 Ϫ T1) was considered to reflect stress- session, the animals did not receive a foot shock after the condition- induced hyperthermia. Mice received treatment with fenobam (3, 10, ing periods. However, animals always received foot shock during or 30 mg/kg p.o.) or vehicle 60 min before T1 and subsequently intervening baseline days. Rats were trained between test days to underwent the SIH procedure. maintain baseline performance. Fenobam was tested at 10, 30, and Operant Conditioning Tests. The operant conditioning boxes 100 mg/kg p.o. were obtained from MED Associates (St. Albans, VT), and the pro- Behavior Data Analysis. SIH data were statistically evalu- tocols were run by the Kestrel Control System from Conclusive ated using a Dunnett multiple comparison test (two-tailed) after a Marketing Ltd. (Harlow, UK) operating on an IBM-compatible com- one-way ANOVA. Geller-Seifter data were analyzed using a re- puter. peated measures ANOVA followed by a paired t test. For the CER Vogel Conflict Drinking. Male Sprague-Dawley rats (around test, the SR data were analyzed using nonparametric statistics 200 g) were water-restricted for three consecutive 24-h periods. At the end of the first 24-h period, animals were allowed access to water because this is a ratio with a cut-off value of 0.5; i.e., a Friedman’s in their home cage for 60 min. At the end of the second 24-h period, test followed by a Wilcoxon rank sum test. The total number of they were placed into the test chamber containing a drinking bottle lever presses made during the 1-h session was analyzed using and were allowed to drink freely for 15 min, after which they received repeated measures ANOVA followed by paired t tests. For the drinking water in their home cage for another 60 min. The drinking Vogel test, drinking time was analyzed by Kruskal-Wallis test, time in the test chamber was used to randomize the animals over the because the data are not normally distributed, followed by a different treatments. The drinking time (in seconds) was determined Mann-Whitney U test. In all studies, the accepted level of signif- as the time spent by the rat at the drinking spout. An optical sensor icance was p Ͻ 0.05. For uniformity, all of the data are expressed (MED Associates) was used to detect the presence of the rat’s snout as mean Ϯ S.E.M. in the figures. Fenobam: A Noncompetitive Antagonist of mGlu5 Receptors 715 Results Discovery of Fenobam as HTS Screening Hit against mGlu5 Receptor A series of urea derivatives were previously patented within F. Hoffmann-La Roche as anxiolytics (Hunkeler and

Kyburz, 1980) that did not act via GABAA receptors but via a molecular target, which was unknown. During an HTS func- tional (FLIPR) screening of F. Hoffmann-La Roche random small-molecule library against rat mGlu5 receptors, we iden- tified urea derivatives, including fenobam, as HTS-hits. Fenobam has a chemical structure that is different from the benzodiazepines (diazepam), competitive group I mGlu an- tagonist (S-4-CPG), and noncompetitive mGlu5 receptor an- tagonists (MPEP and its analog MTEP) (Fig. 1).

[3H]MPEP and [3H]Fenobam Binding and Displacement Studies Downloaded from To characterize the in vitro binding of [3H]MPEP and [3H]fenobam, saturation binding analyses were performed at binding equilibrium (1-h incubation at 4°C and room temper- ature, respectively), as outlined under Materials and Meth- ods on membranes isolated from the HEK-293 transiently

transfected with the rat and human mGlu5 receptors. The jpet.aspetjournals.org saturation isotherm was monophasic ([3H]MPEP concentra- tions from 0.04 to 100 nM and [3H]fenobam from 0.04 to 400 nM) and best fitted to a one-site model for both radioligands

(Fig. 2). The dissociation constants (Kd) and the maximum number of binding sites (Bmax) derived from the saturation isotherms are given in Table 1. [3H]Fenobam bound to re-

combinantly expressed human and rat mGlu5 receptors with at ASPET Journals on January 24, 2020 Ϯ Ϯ a Kd of 31.14 4.10 nM and 53.60 5.58, respectively. The 3 Ki values for [ H]fenobam displacement studies with various noncompetitive mGlu5 antagonists, glutamate, and quis- qualate using membranes containing recombinant human and rat mGlu5 receptors are given in Table 2. For com- Fig. 2. Saturation bindings of [3H]MPEP and [3H]fenobam to membranes parison, the data for in vitro binding and displacement of from HEK-293 cell transfected transiently with hmGlu5 receptor. Each Ϯ [3H]MPEP are also given in Tables 1 and 2. Although feno- data point is mean S.E. (bars) of three individual experiments per- 3 formed in triplicate. The data were analyzed by nonlinear regression bam is less potent than [ H]MPEP (Kd of 1.5 nM at rat and analysis using Prism 3.0 software and a single-site binding model. human receptors), the pharmacological profile of standard 2ϩ mGlu5 ligands was similar at displacing either fenobam or creases in [Ca ]i by noncompetitive (SIB-1757, SIB-1893, MPEP (Table 2). We have also investigated the displacement and MPEP) and competitive (S-4-CPG) hmGlu5 antagonists; 3 of [ H]fenobam binding to recombinant rmGlu5a membrane their derived IC50 values are shown in Fig. 3 and Table 3. by the compounds that are phenylglycine (PG) derivatives To characterize the inhibition mode of fenobam, the con- (S-4C3HPG, S-DHPG, S-3HPG, 2Me4CPG, -4-CPG, (ϩ)- centration-response curves for [3H]IP formation stimulated MCPG, and CHPG) or rigidified analogs of glutamate, in- by quisqualate in the presence of 0, 100 nM, 300 nM, and 1 cluding 2-(carboxycyclopropyl)glycine, (2S,2ЈR3ЈRЈ)-2-(2Ј,3Ј- ␮M fenobam in HEK-293-hmGlu5 stable cell line are shown dicarboxycyclopropyl)glycine, 1-aminocyclopentane-1,3-di- in Fig. 4. As seen, fenobam behaves as a noncompetitive carboxyclic acid, ␣-amino-3-hydroxy-5-methylisoxazole-4- antagonist shifting the agonist concentration-response curve propionic acid, L-AP4, kainate, and 1-aminoindan-1,5-dicar- to the right with a concomitant decrease in maximal response boxylic acid. All of these compounds were inactive (Ki of consistent with a noncompetitive mode of action. Ͼ10,000 ␮M). Furthermore, the noncompetitive mGlu1 an- MPEP has previously been shown to inhibit the mGlu5 tagonists 7-hydroxyiminocyclopropan[b]chromen-1a-carbox- constitutive activity (Pagano et al., 2000). Similarly, we have ylic acid ethyl ester and NPS 2390 inhibited poorly [3H]feno- tested the inverse agonist activity of fenobam using IP accu- bam binding to recombinant rmGlu5a membrane with Ki of mulation assay. To optimize the condition for detection of 50 Ϯ 5.5 and 3.66 Ϯ 1.4 ␮M, respectively. mGlu5 constitutive activity, HEK-293-hmGlu5 stable cell Fenobam Is a Potent and Noncompetitive Antago- line expressing a high level of recombinant receptor was nist of mGlu5 Receptor with Inverse Agonist Activity. transiently transfected with a plasmid containing high-affin- In HEK-293 cells stably expressing human mGlu5 receptors, ity glutamate transporter EAAC1 cDNA and the basal level 2ϩ 3 fenobam inhibits quisqualate-evoked [Ca ]i response with of [ H]IP formation was then measured in the presence of ϭ Ϯ ϭ Ϯ IC50 58 2 nM, nH 1.7 0.1. For comparison, the glutamate pyruvate transaminase (a glutamate-degrading concentration-dependent inhibition of quisqualate-evoked in- enzyme). As seen in Fig. 5, fenobam inhibited the basal 716 Porter et al.

TABLE 1 ͓3H͔fenobam and ͓3H͔MPEP binding properties at human and rat mGlu5 Saturation binding isotherms of ͓3H͔fenobam and ͓3H͔MPEP were performed on membrane preparations from HEK-293 cells transiently transfected with the human and Ϯ rat mGlu5 receptors as described under Materials and Methods. Values are mean S.E.M. of the Kd and Bmax values, calculated from three independent experiments (each performed in triplicate).

͓3H͔MPEP Binding ͓3H͔Fenobam Binding

Human Rat Human Rat Ϯ Ϯ Ϯ Ϯ Kd (nM) 1.46 0.21 1.49 0.03 31.14 4.10 53.60 5.58 Ϯ Ϯ Ϯ Ϯ Bmax (fmol/mg protein) 3300 341 4608 265 2951 219 5301 348

TABLE 2 ͓3 ͔ ͓3 ͔ Ki values for inhibition of H MPEP (2 nM) and H fenobam (10 nM) bindings to human and rat mGlu5 receptors transfected HEK-293 cell membranes by various compounds Ϯ Values are mean S.E.M. of the Ki calculated from three independent experiments, each performed in duplicate. ͓3H͔MPEP Binding ͓3H͔Fenobam Binding

Human Rat Human Rat

nM

MPEP 3.3 Ϯ 0.3 3.1 Ϯ 0.2 6.7 Ϯ 0.7 8.7 Ϯ 1.0 Downloaded from MTEP 5.3 Ϯ 0.3 5.4 Ϯ 0.4 14.1 Ϯ 2.0 17.8 Ϯ 1.8 Fenobam 53.0 Ϯ 3.7 50.8 Ϯ 1.8 40.8 Ϯ 2.7 56.3 Ϯ 3.3 SIB-1757 328.4 Ϯ 31.6 346.4 Ϯ 13.4 357.3 Ϯ 40.0 486.4 Ϯ 23.6 SIB-1893 219.4 Ϯ 15.7 242.9 Ϯ 13.4 333.7 Ϯ 21.5 423.0 Ϯ 53.0 Glutamate Ͼ10,000 Ͼ10,000 Ͼ10,000 Ͼ10,000 Quisqualate Ͼ10,000 Ͼ10,000 Ͼ10,000 Ͼ10,000 jpet.aspetjournals.org at ASPET Journals on January 24, 2020

Fig. 3. Comparison of group I mGlu antagonism on quisqualate-evoked Fig. 4. Schild analysis showing that the noncompetitive mode of antag- 2ϩ onism by fenobam at mGlu5 receptors. Quisqualate concentration re- [Ca ]i in the HEK-293 cells stably expressing mGlu5 receptor. Concen- 2ϩ sponses in the absence (control) and presence of various concentrations of tration-dependent inhibition quisqualate-stimulated increases in [Ca ]i by various antagonists as assayed using the Ca2ϩ-sensitive dye Flou-4 fenobam were generated using the IP accumulation assay and HEK-293- and a fluorometric imaging plate reader. Responses are normalized to the hmGlu5 stable cells that transiently express the high-affinity glutamate Ϯ first control response. Each curve represents mean Ϯ S.E. (bars) of the transporter EAAC1. Each data point is mean S.E. (bars) of three three to six dose-response measurements (each performed in triplicate). individual experiments performed in quadruplet.

TABLE 3 fenobam at 10 ␮M, respectively (Fig. 5). Note that under Potencies of various mGlu5 antagonists on inhibition of quisqualate- identical experimental conditions, the competitive antago- induced ͓Ca2ϩ͔ response i nist S-4-CPG did not exhibit any inverse agonist activity (A. IC50 and Hill coefficient (nH) values for the inhibition of quisqualate (20 nM)-evoked ͓ 2ϩ͔ Mu¨hlemann, data not shown). Ca i response in the HEK-293 cells stably expressing the hmGlu5a receptor. Data are means Ϯ S.E. of the three to six dose-response measurements (each performed in Selectivity. To evaluate the selectivity of fenobam rela- triplicate). tive to other mGlu receptors, functional assays evaluating

Compound IC50 nH both potential antagonist and agonist/enhancer activity ␮M against mGlu1a transiently expressed in HEK-293 using SIB-1757 1.1 Ϯ 0.06 2.3 Ϯ 0.3 FLIPR assay (Fig. 6, A and B), mGlu2 stably expressed in SIB-1893 0.8 Ϯ 0.09 1.4 Ϯ 0.1 CHO using cAMP accumulation assay (Fig. 6C), mGlu4a Ϯ Ϯ MPEP 0.028 0.003 1.9 0.2 transiently expressed in CHO using [35S]GTP␥S binding as- Fenobam 0.058 Ϯ 0.002 1.7 Ϯ 0.1 S-4-CPG 1971 Ϯ 205 1.3 Ϯ 0.2 say (Fig. 6D), mGlu7a stably expressed in CHO using [35S]GTP␥S binding assay (Fig. 6E), and mGlu8a stably ex- pressed in CHO-G␣15 using FLIPR assay (Fig. 6F) were ϭ Ϯ ϭ ␮ activity dose dependently with an IC50 84 13 nM, nH established. When tested at concentrations up to 10 M, Ϯ ϭ Ϯ ϭ Ϯ 1 0.2, this is compared with IC50 21 3 nM, nH 1 fenobam was not active in assay conditions that did detect 0.1, for MPEP under identical conditions. The basal levels of known modulators. Furthermore, the pharmacological spec- [3H]IP formation were reduced to 32 and 34% by MPEP and ificity of fenobam was confirmed by testing in the diversity Fenobam: A Noncompetitive Antagonist of mGlu5 Receptors 717

understanding of physiological role played by this receptor in central nervous system processes. In numerous preclin- ical studies, MPEP and MTEP are active in animal models of anxiety and depression (Spooren et al., 2000; Tatarczyn- ska et al., 2001; Pilc et al., 2002; Wieronska et al., 2002, 2004; Busse et al., 2004; Ballard et al., 2005), Parkinson’s disease (Breysse et al., 2002), pain (Zhu et al., 2004), and addiction (McGeehan et al., 2004). The discovery in the present studies that mGlu5 is the molecular target of fenobam, a clinically validated nonbenzodiazepine anxio- lytic, demonstrates further the pivotal function of mGlu5 in mood and anxiety. SIB-1757 and SIB-1893 were the first novel nonamino acid, selective, and noncompetitive antagonists of mGlu5 re- ␮ ceptor (IC50 values of 3.1 and 2.3 M at hmGlu5, respec- tively, in IP accumulation assay) to be reported (Varney et Fig. 5. Inhibition of mGlu5 basal activity by fenobam and MPEP. The al., 1999). MPEP, a novel derivative of SIB-1757, was the basal activity was measured using the IP accumulation assay and HEK-

ϭ Downloaded from 293-hmGlu5 stable cells that transiently expressing the high-affinity first highly potent mGlu5-selective antagonist (IC50 36 nM glutamate transporter EAAC1. Each data point is mean Ϯ S.E. (bars) of at hmGlu5a in the IP accumulation assay) to be described three individual experiments performed in quadruplet. (Gasparini et al., 1999). Pagano et al. (2000) elucidated the site of action of [3H]M-MPEP, which binds within the 7TM profile at Cerep (Paris, France) (www.cerep.fr). Fenobam was domain of hmGlu5 in close contact with the residues Ala810 considered inactive (Ͻ50% activity at 10 ␮M) at all targets in TM7 and Pro655 and Ser658 in TM3. Furthermore, the tested with the exception of the adenosine A receptor, where 3 homology modeling of 7TM helices of mGlu5 receptors re- jpet.aspetjournals.org it caused a 65% displacement of specific binding at 10 ␮M sulted in identification of additional residues in the TM3, 5, (Table 4). 6, and 7 regions that are important molecular determinants of the high-affinity binding site of [3H]MPEP (Malherbe et Behavioral Profiling of Fenobam al., 2003). The structure-activity relationship analysis of Effect of Fenobam on SIH in Mice. Fenobam (3, 10, or MPEP has later led to MTEP (Cosford et al., 2003) and 30 mg/kg p.o.) at doses 10 (p Ͻ 0.001) and 30 mg/kg (p Ͻ similar close analogs.

0.001) p.o. significantly reversed SIH (Fig. 7). Fenobam had Fenobam was discovered as an mGlu5 receptor antagonist at ASPET Journals on January 24, 2020 no effect on T1 (data not shown). serendipitously in a screening campaign. Interestingly, the The Vogel Test as a Measure of Conflict Behavior in structure of fenobam belongs to a chemical class different Rats. Fenobam (3, 10, and 30 mg/kg p.o.) increased the from MPEP and its analogs. [3H]Fenobam binds to a single drinking time in the Vogel conflict test (Kruskal-Wallis, p Ͻ saturable site on recombinantly expressed human and rat

0.01; Fig. 8). Although there was no significant effect at the mGlu5 receptors (Bmax of 2951 and 5301 fmol/mg protein, two lower doses (p Ͼ 0.1 versus vehicle, as determined by the respectively) with high affinity (Kd of 31.1 and 53.6 nM, Mann-Whitney U test), at the dose of 30 mg/kg, the effect of respectively). Binding of [3H]fenobam was temperature-de- fenobam in increasing drinking time was highly significant pendent and increased substantially at 25°C. Interestingly, (p Ͻ 0.001). fenobam did consistently exhibit a more potent profile at Geller-Seifter Conflict Test in Rats. Fenobam (10, 30, human than at rat receptors. The noncompetitive mGlu5 and 100 mg/kg p.o.) dose dependently increased punished antagonists displaced [3H]fenobam binding with the follow- responding [F(3,27) ϭ 3.7; p Ͻ 0.05] in the rat conflict test ing rank order of potency: MPEP Ͼ MTEP Ͼ Fenobam Ͼ with an MED of 10 mg/kg p.o. (Fig. 9C). Unpunished re- SIB-1893 Ͼ SIB-1757, which is consistent with the rank sponding was decreased [F(3,27) ϭ 8.9; p Ͻ 0.001], with a order potency of these ligands in inhibiting quisqualate- 2ϩ significant reduction at 100 mg/kg compared with vehicle evoked [Ca ]i response at mGlu5 receptors. Moreover, glu- (Fig. 9A). None of the doses affected unrewarded responding tamate, quisqualate, ␣-amino-3-hydroxy-5-methyl-4-isox- (Fig. 9B). azolepropionic acid, kainate, and a series of phenylglycine Conditioned Emotional Response Test in Rats. Feno- derivatives or rigidified analogs of glutamate (acting as com- bam produced a significant dose-dependent increase in the petitive agonist or antagonists) were unable to displace the SR (Friedman test, p Ͻ 0.01), which reached statistical sig- [3H]fenobam binding to rmGlu5a receptors expressed in nificance at 30 and 100 mg/kg p.o. (Fig. 10A). Fenobam at 30 HEK-293 cell membranes. Because MPEP and its analog 3 and 100 mg/kg significantly reduced the total number of lever MTEP strongly inhibited the [ H]fenobam binding with Ki presses made during the 1-h session (Fig. 10B). values of 6.7 Ϯ 0.7 and 14.1 Ϯ 2.0 nM, respectively, we conclude that fenobam shares a common binding pocket in Discussion the transmembrane region of the receptor, at least overlap- ping with that of MPEP. The G protein-coupled metabotropic In functional studies, fenobam was able to inhibit quis- 2ϩ mGlu5 is important in the modulation of neuronal func- qualate-induced [Ca ]i response in a HEK-293-hmGlu5 sta- tion. The identification of MPEP, as a highly potent, sys- ble cell line with an IC50 value of 58 nM (similar in potency temically active, and allosteric antagonist of mGlu5 recep- to MPEP). The investigation of the antagonism mechanism tor (Gasparini et al., 1999), has greatly contributed to our by fenobam revealed that it behaved as a noncompetitive 718 Porter et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on January 24, 2020

Fig. 6. Fenobam displayed no effect toward rat mGlu1a, 2, 4a, 7a, and 8a receptors. A, fenobam (1 nM–10 ␮M) was not able to inhibit 100 nM quisqualate in HEK-293 cell transiently expressing mGlu1a. B, quisqualate concentration response in absence and presence of 10 ␮M fenobam. C, 1-aminocyclopentane-1,3-dicarboxylic acid dose-response curves in absence and presence of 10 ␮M fenobam in CHO-mGlu2 cell line. Effect of 10 ␮M fenobam on L-AP4 concentration responses in CHO cell transiently expressing mGlu4a (D) and in CHO-G␣15 cell stably expressing mGlu8a (F). The fenobam concentration responses in the presence 0.1 mM (EC20) and 0.7 mM (EC80) in CHO-mGlu7a stable line are shown in E. Each data point is mean Ϯ S.E. (bars) of two to four experiments performed in triplicate or quadruplet. antagonist, very similar to MPEP, shifting the quisqualate cell line, fenobam was found to be an inverse agonist compa- dose-response curves to the right in the presence of increas- rable with MPEP. Moreover, fenobam displayed no activity ing fenobam concentrations with a concomitant decrease in toward rat mGlu1a, 2, 4a, 7a, and 8a receptors in a functional the maximal effect of quisqualate using IP accumulation assay at concentration up to 10 ␮M. With the exception of the assay. In the present study, using a high receptor-expressing A3 receptor where it displaced 65% of specific binding at 10 Fenobam: A Noncompetitive Antagonist of mGlu5 Receptors 719

TABLE 4 Broad Cerep screen undertaken to determine the pharmacological activity of fenobam

% Control % Control Target (10 ␮M) Target (10 ␮M)

A1 (h) 89.9 Opiate (nonsel) 114.8 A2A (h) 96.7 ORL1 (h) 101.4 A3 (h) 35.3 PCP 95.3 ␣ 1 (nonsel) 103.3 P2X 108.5 ␣ 2 (nonsel) 102.1 P2Y 95.8 ␤ 1 (h) 95.9 5-HT (nonsel) 94.1 ␤ ␴ 2 (h) 79.0 1 88.2 ␴ AT1 (h) 91.2 2 79.2 Fig. 8. Increase in drinking time in the Vogel conflict test in rats. Male AT2 (h) 109.6 Glucocorticoid (h) 96.7 naive Sprague-Dawley rats received fenobam at doses of 3, 10, and 30 GABAA-BZD 97.6 Estrogen (h) 100.2 mg/kg p.o. (1-h pretreatment time) and were tested in the Vogel conflict Ϯ B1 108.5 Progesterone (h) 105.8 test. Data are mean S.E.M. based on 12 animals per group. Vehicle, Ͻ ءءء Ͻ ءء Ͻ ء B2 (h) 102.1 Androgen (h) 108.0 0.3% Tween 80 in 0.9% NaCl. , p 0.05; , p 0.01; , p 0.001 CB1 (h) 95.0 TRH 90.6 versus vehicle, as determined by the Mann-Whitney U test. CB2 (h) 100.5 V1 103.7 CCKA (h) 112.8 V2 (h) 109.4 2ϩ ␮M, it did not bind to a battery of GPCRs in a broad CEREP CCKB (h) 84.8 Ca L channel (DHP) 96.7 ϩ 2 profiling. Downloaded from CRF1 106.9 Ca L channel (dilt) 104.6 2ϩ D1 (h) 100.0 Ca L channel (verap) 150.9 The behavioral data indicate that fenobam exhibits anxio- ϩ D2 (h) 95.3 K ATP channel 136 ϩ lytic-like activity in all four anxiety tests included in the D3 (h) 100.6 K V channel 103.6 ϩ 2ϩ present study; three rat conflict tests (Geller-Seifter, CER, D4.4 (h) 86.7 SK Ca channel 97.9 ϩ ETA (h) 93.3 Na channel 88.9 and Vogel conflict) and a mouse model of autonomic hyper- Ϫ ETB (h) 85.1 Cl channel 103.7 reactivity in anxiety/stress. Fenobam was active in a compa- GABA (nonsel) 107.5 ADO transporter 99.6 AMPA 93.1 NE transporter (h) 99.9 rable dose range after oral administration in all tests (MED jpet.aspetjournals.org Kainate 103.2 DA transporter (h) 98.2 10–30 mg/kg p.o.). These data confirm the previously re- NMDA 91.9 GABA transporter 104.7 ported preclinical studies with fenobam (i.e., rat conflict Glycine (strych-sens) 126.2 Choline transporter 81.4 tasks including Geller-Seifter and Vogel) (Patel et al., 1982; Glycine (strych-insens) 104.1 5-HT transporter (h) 101.2 Goldberg et al., 1983). The data are also in line with previous H1 89.9 PDE I 100.3 H2 97.6 PDE II (h) 99.1 findings with selective mGlu5 receptor antagonists, particu- H3 89.8 PDE III (h) 95.9 larly the prototypical mGlu5 receptor antagonist MPEP (for I1 83.2 PDE IV (h) 93.4

review, see Spooren et al., 2001). MPEP and MTEP have at ASPET Journals on January 24, 2020 I2 66.3 PDE V (h) 106.3 LTB4 (h) 99.0 PKC 96.0 previously been shown to be active in the Vogel conflict test LTD4 (h) 106.2 COMT 98.9 (Tatarczynska et al., 2001; E. Prinssen, unpublished obser- MC4 (h) 92.2 GABA transaminase 94.6 M (nonsel) 94.6 Glu. decarboxylase 105.7 vations) and the Geller-Seifter conflict test (Brodkin et al., 2002; Busse et al., 2004). Furthermore, we have recently NK1 (h) 102.2 MAO-A (h) 70.5 NK2 (h) 89.7 PNMT 88.9 shown that MPEP produces a robust anxiolytic-like effect in NK (h) 104.1 TH 122.6 3 all three rat conflict tests (Geller-Seifter, CER, and Vogel) at NPY (nonsel) 102.5 ATPase 97.4 N (neuronal) 119.4 Acetylcholinesterase (h) 98.5 an equivalent dose range after oral administration, with a minimum effective dose of 10 mg/kg p.o. (Ballard et al., 2005). h, human; nonsel, nonselective; NMDA, N-methyl-D-aspartate; AMPA, ␣-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid; PDE, phosphodiesterase; PKC, pro- Moreover, the anxiolytic effect of MPEP was comparable with tein kinase C; PNMT, phenylethanolamine-N-methyltransferase; MAO-A, monoam- that of diazepam, which was used as a positive control in the ineoxidase-A; COMT, catechol-O-methyl transferase; LTB, leukotriene B; LTD, leukotriene D; NPY, neuropeptide Y; ET, endothelin; CRF, corticotrophin-releasing same tests. factor; CCK, cholecystokinin; CB, cannabinoid; 5-HT, serotonin; TRH, thyrotrophin- In the current study, fenobam differs from MPEP and releasing factor; DHP, dihydropyridine; ADO, adenosine; NE, norepinephrine; DA, dopamine; TH, tyrosine hydroxylase; Dilt, diltiazem; Verap, verapamil; PCP, phen- MTEP in that it has a greater effect on nonspecific aspects of cyclidine; P2X, purinergic 2X; P2Y, purinergic 2Y; BZD, benzodiazepine. the task; i.e., significant reduction in lever pressing in CER and in unpunished responding in Geller-Seifter (Busse et al., 2004; Ballard et al., 2005). The greater behavioral disruptive properties of fenobam, in contrast to that observed with MPEP and MTEP in the animal models, may have some consistency with the clinical data reporting of side effects of fenobam and may be due to the very variable interindividual oral bioavailability of fenobam (Itil et al., 1978). Interest- ingly, analogs of fenobam (Hunkeler and Kyburz, 1980) or the more structurally diverse mGlu5 receptor antagonists MPEP and MTEP do not cause the same behavioral disrup- tion as fenobam in animals. Consequently, we cannot rule out an additional site of action of fenobam or possibly the Fig. 7. Reversal of stress-induced hyperthermia in mice. Male NMRI formation of a biologically active metabolite because fenobam mice received fenobam at doses of 3, 10, and 30 mg/kg p.o., 1 h before T1. is extensively metabolized in vivo (Wu et al., 1995). Ϯ Data are mean S.E.M. based on eight animals per group. veh, vehicle Antagonism of mGlu5 receptors by MPEP (Gasparini et al., ,p Ͻ 0.001 versus vehicle ,ءءء ;p Ͻ 0.01 ,ءء .(Tween 80 in NaCl 0.9% 0.3%) Dunnett multiple comparison test (two-tailed) following a one-way 1999) and the anxiolytic-like effects of mGlu5 receptor an- ANOVA. tagonists have been shown to be independent of any benzo- 720 Porter et al.

Fig. 9. Rat Geller-Seifter conflict test. Effect of fenobam (10, 30, and 100 mg/kg p.o.) on responding in the Geller-Seifter conflict test. The data are presented according to each component of the test schedule: unpunished (VI30) period (A), nonrewarded (time-out) period (B), and punished (FR10) Downloaded from period (C). n ϭ 8 male Sprague-Dawley rats were used in this study. Data are expressed as the mean Ϯ S.E.M. number of lever presses per minute. .(p Ͻ 0.05 versus vehicle (V ,ء jpet.aspetjournals.org Fig. 10. Rat CER test. Effect of fenobam (10, 30, and 100 mg/kg p.o.) on responding in CER test. Data are expressed as mean Ϯ S.E.M. A, suppres- sion ratio. B, total number of lever responses re- corded over the test session. n ϭ 12 male Sprague- p Ͻ 0.05 ,ء .Dawley rats were used in this study versus vehicle (V). at ASPET Journals on January 24, 2020

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