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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Anesthesiology. 2011 Dec;115(6):1239–1250. doi: 10.1097/ALN.0b013e318238c051

Metabotropic Glutamate Receptor 5 Antagonism with Fenobam: Examination of Analgesic Tolerance and Side Effect Profile in Mice

Michael C Montana 1, Beth A Conrardy 1, Laura F Cavallone 1, Benedict J Kolber 1, Leslie K Rao 1, Suellen C Greco 1, Robert W Gereau IV 1
PMCID: PMC3226928  NIHMSID: NIHMS333821  PMID: 22037639

Abstract

Background

The metabotropic glutamate receptor 5 noncompetitive antagonist fenobam is analgesic in rodents. Future development of fenobam as an analgesic in humans will require a favorable long-term treatment profile and a lack of significant deleterious side effects. This study aimed to determine if tolerance to fenobam’s analgesic effects developed over 14 days and to assess for side effects in mice.

Methods

Mouse models of pain, locomotor behavior, and coordination were used. Fenobam or vehicle (n = 8 or 11 per group) was administered for 14 days and analgesic tolerance to fenobam was assessed using the formalin test. Histopathology examination and serum chemistry analysis post-14-day fenobam administration were also assessed (n = 12 or 9). The effects of fenobam on locomotor activity were assessed in the open field and elevated zero maze (n = 8 or 7). Coordination was assessed using ledge crossing and vertical pole descent tasks (n = 11 or 10).

Results

Tolerance to fenobam’s analgesic effect did not develop after 14 days. Chronic fenobam administration resulted in statistically significantly less weight gain compared to vehicle controls, but did not cause any physiologically or statistically significant hematological abnormalities, altered organ function, or abnormal histopathology of the liver, brain, or testes. Fenobam administration resulted in a metabotropic glutamate receptor 5-dependent increase in exploratory behavior but does not impair motor coordination at analgesic doses.

Conclusions

Analgesic tolerance to repeat fenobam dosing does not develop. Chronic dosing of up to 14-days is well tolerated. Fenobam represents a promising candidate for the treatment of human pain conditions.

Introduction

Glutamate is the primary excitatory neurotransmitter in the mammalian nervous system. Abnormal glutamatergic signaling may play a prominent role in several disease processes, including chronic pain1,2. The G-protein coupled glutamate receptor metabotropic glutamate receptor 5 (mGlu5) is expressed at synapses throughout the nervous system and in particular the pain neuraxis3, and agents that modulate mGlu5 may have therapeutic potential in the treatment of pain. Significant inroads into the use of pharmacological agents acting at mGluRs in human patients have been made recently4,5 and findings that activation of mGlu5 is pronociceptive6–8 while its inhibition is antinociceptive9–11 have led to the suggestion that antagonism of mGlu5 may have analgesic efficacy in humans. However, new pharmacological agents that target mGlu5 will also have to possess acceptable safety profiles along with clinical efficacy. This is illustrated by the recent discontinuation of clinical trials involving the Addex Pharmaceuticals (Plan-les-Ouates, Switzerland) mGlu5 antagonist ADX-10059, which, despite 4-fold improvement versus placebo in inducing a pain-free state in migraineurs, resulted in unacceptable liver enzyme increases12.

The recent finding that the clinically validated compound fenobam is a potent and selective mGlu5 negative allosteric modulator13 has led to a resurgence in interest in testing the efficacy of fenobam as a treatment for various neurologic conditions. Originally developed in the 1970s as an anxiolytic with a then unknown mechanism of action, fenobam was found to have a favorable safety profile14,15, however initial results regarding its anxiolytic efficacy were mixed16,17. Further human clinical testing was discontinued in the early 1980s. Fenobam has subsequently been demonstrated to be analgesic in rodents10,11 and moderately effective in managing some of the symptoms of Fragile X in humans4. However in these studies fenobam was only administered as a one-time dose, and future treatment strategies involving fenobam or other mGlu5 antagonists in pain conditions will likely require longer-term treatment. It is presently unknown whether tolerance develops to fenobam’s analgesic effects. In addition, while the elevations in liver enzymes seen with ADX-10059 may be compound-specific, the effects of chronic mGlu5 antagonist administration on liver function and other vital systems are not well characterized in the literature.

Here, we performed a series of experiments to determine whether tolerance develops to the analgesic effects of fenobam and to assess for deleterious side effects. We test whether tolerance to the analgesic effects of fenobam develops following repeated dosing of up to 14 days. In addition, we assessed for alterations in liver function, gross liver histopathology, and hematological abnormalities following chronic fenobam treatment. Sedation and impairment of motor coordination are common dose-limiting factors for some classes of analgesic drugs. In fact, fenobam may act as a stimulant10,16, but it is unknown whether this stimulant effect is due to specific activity at mGlu5. We therefore also assessed the locomotor activity of mGlu5 knockout mice treated with fenobam. Finally, mGlu5 has been shown to play a role in weight gain and energy balance18,19. We sought to expand these results by determining if fenobam mediates any effects on appetitive behavior.

Materials and Methods

Materials

Subjects

Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Washington University School of Medicine (St. Louis, Missouri). Male Swiss-Webster mice (5 weeks old) were purchased from Taconic and group housed five animals to a cage for 1 week prior to starting any behavioral experiments. For experiments involving mice lacking mGlu5 (mGlu5 knockout; 6–9 weeks old), animals were bred in house on a C57BL/6 background and compared to wild type (WT) littermates 20. All C57BL/6 WT mice used in behavioral experiments were also derived from this colony. For experiments involving knockout animals the experimenter was blinded to genotype. Genotyping of mice bred in house was performed using standard polymerase chain reaction techniques. All mice were group housed on a 12/12-light/dark schedule with ad libitum access to food and water, except as described below during food deprivation experiments.

Chemicals and Reagents

Fenobam was purchased from Tocris (Ellisville, MO) or was custom synthesized for our use by Scynexis (Research Triangle Park, NC) and dissolved in dimethyl sulfoxide (DMSO - Sigma-Aldrich, St. Louis, MO) on the day of experiment. All intraperitonial injection volumes were 20 microliters. Throughout all experiments the investigator was blinded to pharmacological treatment. All other reagents were high-performance liquid chromatography grade and purchased from Sigma-Aldrich Co.

Behavioral Assays

Open Field Locomotor Test

Locomotor activity was measured in an open field chamber (42L x 42W x 30H cm) attached to the VersaMax Animal Activity Monitoring System (AccuScan Instruments, Columbus, OH). For experiments involving drug naive C57BL/6 WT and mGlu5 knockout littermate mice, animals were brought to the testing room in their home cage and acclimated for at least 2 h prior to testing. Locomotor activity was assessed individually by recording photobeam breaks for 60 min. Total distance traveled, time spent moving, and the number of beam breaks (horizontal activity), were calculated for the entire chamber. For experiments designed to assess the effects of fenobam on locomotor behavior in mGlu5 knockout mice, animals were habituated in their home cages for at least 2 h, and then injected intraperitoneally with either 30 mg/kg fenobam or vehicle (DMSO) immediately prior to placement in the chamber. Animals were allowed to explore the chamber for 90 min and locomotor activity was measured individually as described above and the total distance traveled was calculated for the entire chamber.

Elevated zero maze

Locomotor activity was measured in low light conditions using a zero maze (Stoelting Co., Wood Dale, IL) placed 70 cm off of the ground and consisting of two closed sections (wall height, 30 cm) and two open sections (wall height, 1.3cm) on a circular track (diameter of track, 60 cm). C57BL/6 WT mice were habituated to testing room for 1 h prior to injections, and then injected intraperitoneally with fenobam (30mg/kg) or vehicle (DMSO). Fifteen minutes after injection, mice were placed individually in the closed area of the zero maze for a 600-s trial. Movement during the trial was recorded using two digital video cameras (Logitech 9000 Pro webcam, Romanel-sur-Morges, Switzerland). Total distance travelled, number of entries into open sections, and time spent in the open sections was scored off-line using AnyMaze video tracking software (Stoelting Co.).

Motor Sensory Assessment

A series of three tests was performed on drug naive C57BL/6 WT mice to assess the effects of fenobam on gross motor behavior and coordination 21,22. All tests were performed two times.

Ledge Crossing Task

Each mouse was tested to see how long it could maintain its balance on a 0.75 cm wide plexiglass ledge without falling (60 s maximum). A score of 60 s was also assigned if the mouse traversed the entire 51 cm length of the ledge and returned to the starting point in less than 60 s without falling.

Vertical Pole Descent Task

Mice were placed head upwards at the top of a vertical metal rod (8 mm diameter, 55 cm height). The rod was finely textured with a file to provide a gripping surface. Mice were given a maximum of 120 s to turn 180 decrees and climb down to reach the bottom of the pole and place all four paws on the tabletop. Mice were required to reverse direction and actively climb down. If a mouse slid down the pole without reversing direction or fell down the pole it was given a score of 120 s.

Inverted Screen Hang

Mice were placed on a wire mesh screen (16 squares per 10 cm, 47 cm high × 18 cm wide) oriented at 60 degrees. The screen was then inverted to 180 degrees so that mice were upside down. The time mice spent hanging on the screen was measured for two minutes. A maximum score of 120 s was given to an animal that did not fall.

Spontaneous Formalin-induced Nocifensive Behavior following Chronic Fenobam Treatment

Male Swiss Webster mice were injected intraperitoneally once per day chronically for 5 days with 30 mg/kg of fenobam or vehicle (DMSO). On the sixth day mice were placed in transparent plexiglas boxes (10L × 10W × 15H cm, assembled by the Washington University School of Medicine Instrument Machine Shop) on a glass surface and acclimated for 2 h prior to any drug injection. Animals were then pretreated by intraperitoneal injection with vehicle or fenobam (30 mg/kg). Ten microliters of dilute formalin solution (5% dissolve in normal saline, Sigma, St. Louis, MO) was injected subcutaneously into the plantar surface of the right hind paw. Some mice from the chronic-vehicle group were injected with fenobam, while others received vehicle, followed 5 min later by intraplantar formalin injection. Thus, three separate groups were analyzed, 1) chronic vehicle / acute vehicle; 2) chronic vehicle, acute fenobam; 3) chronic fenobam, acute fenobam; with chronic administration defined as 5 days. Spontaneous behavior was recording using a digital video camera (Logitech 9000 Pro webcam) placed underneath the glass platform. Video recordings were scored after the collection of all data by an observer blinded to treatment (both chronic and acute) and the time spent in nocifensive behavior defined as licking, lifting, or flicking of the injected paw, was scored in 5-min intervals for 45 min after paw injection. A separate set of experiments was performed exactly as described above, except animals were injected with fenobam (30 mg/kg) or vehicle for 14 days instead of 5, with formalin testing performed on the 15th day. Animals from this longer time period of injections were killed immediately after the cessation of the formalin test and tissue obtained from them was used in the serum chemistry analysis and tissue histology described below. All animals were weighed daily immediately prior to intraperitoneal injection.

Effects of Fenobam on Food Intake and Weight Gain

Food deprivation studies were performed in a manner similar to that previously described 18. For experiments designed to assess the acute effects of fenobam on food intake mice were weighed and housed individually in their home cages 3 days prior to food deprivation. Cage bedding was replaced with a raised wire mesh bottom. On the third day mice were weighed again and food deprived for 24 h starting at one hour after lights on until 1 h after lights on the next day. Mice were then weighed again and injected intraperitoneal with either 30 mg/kg fenobam or vehicle and preweighed food was placed onto the cage bottom. Food intake was measured for each mouse at 15, 30, 60, 90, and 180 min, as well as at 3 h prior to lights off (i.e., 8 h later). In order to compensate for variation of body weight, mouse food intake was normalized to body weight. Mice were weighed to the nearest 0.1 g and food was weighed to the nearest 0.01 g.

Serum Chemistry Analysis and Complete Blood Count with Differential

Plasma and Tissue Collection

All mice from the 14 day chronic fenobam injection study (see above) were administered an overdose of sodium pentobarbital (75 mg/kg) following the cessation of the formalin test. Whole blood was obtained by transcutaneous cardiac puncture and placed into plasma separator tubes with lithium heparin for blood chemistries or EDTA tubes for complete blood counts (BD Microtainer, Franklin Lakes, NJ). Serum chemistry analysis and necropsy was performed immediately posttissue collection.

General Instrumentation

Complete blood counts were analyzed using the Hemavet 1700 (Drew Scientific, Waterbury, CT). White blood cell differentials were determined by direct examination of blood smears. Serum chemistries were analyzed using the Vitros DT60 system dry reagent chemistry analyzer (Ortho Clinical Diagnostics, Rochester, NY).

Histological Analysis

Tissues were formalin-fixed, paraffin-embedded, and sectioned at 5 microns. Sections were prepared according to standard histologic techniques and stained with Hematoxylin and Eosin.

Statistical Analysis

Behavioral Data Analysis

Statistical analysis of behavioral data was performed using Prism 5.0 (GraphPad Software, Inc., La Jolla, CA). All data collected over multiple time points from the open field assay, the spontaneous formalin test, and the food deprivation studies were statistically analyzed using a Bonferroni multiple comparison test after a nonrepeated measures two-way ANOVA. The factors used for these analyses were genotype/treatment and time. Summed data from these tests were analyzed using a two-tailed Student’s t-test when comparisons were made between two groups or a Bonferroni multiple comparisons tests after a one-way ANOVA when comparisons were made between more than two groups. In all studies, the accepted level of significance was p < 0.05. In all figures data are reported as mean +/− SEM. 95% confidence intervals (CIs) of the difference between means are also listed in the text.

Serum Chemistry and Cell Count Data Analysis

Statistical analysis of data was performed using Prism 5.0 (GraphPad Software, Inc.). All data collected from chronically injected fenobam versus vehicle-injected animals were statistically analyzed using an unpaired t-test. In all studies, the accepted level of significance was p < 0.05. In all figures data are reported as mean +/− SEM.

Results

Effects of Genetic and Pharmacological Disruption of mGlu5 in the Open Field Task

Previous work from our lab suggests that mGlu5 inhibition via fenobam may affect locomotor activity 10. We expanded upon previous findings to determine whether altered locomotor behavior following fenobam administration is mediated via inhibition of mGlu5 or through an off-target effect of the drug. The open field task was used to assess the effects of mGlu5 deletion and inhibition on locomotor activity. When the effects of genetic deletion of mGlu5 were examined in the open field, we found that drug naive mGlu5 knockout mice traveled a statistically significant farther distance in the open field over a 60-min time period as compared to their drug naive WT littermates (7365 ± 876.7 cm vs. 12171 ± 1111 cm; 95% CI −7843 to −1770 cm; p < 0.001; fig. 1 A, B). To assess whether the increased locomotor activity following fenobam administration was due to inhibition of mGlu5, knockout mice and WT littermates were injected with fenobam (30 mg/kg) or vehicle (DMSO) and then immediately placed in the open field for 90 min. There was a statistically significant increase in the total distance travelled in the fenobam-treated WT mice and both the vehicle and fenobam treated mGlu5 knockout as compared to vehicle-treated WT mice (95% CI between vehicle treated WT mice and fenobam treated WT, vehicle treated KO, and fenobam treated KO −11735 to −2044 cm, −9924 to −541.8 cm, and −13329 to −3946 cm respectively; p < 0.001; fig. 1 C, D). No differences were noted between vehicle- or fenobam-treated mGlu5 knockout animals.

Figure 1. The Effects of Fenobam and mGlu5 Disruption on Open Field Locomotor Behavior.

Figure 1

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(A) Drug-naive mGlu5 knockout mice traveled significantly farther compared to their WT littermates at multiple time points (2-Way ANOVA Main Effect p < 0.0001; Bonferroni Post Test *,**,*** p < 0.05, 0.01, 0.001) and (B) as a sum total of distance traveled in 60 min (Unpaired t-test p = 0.0044) n = 8 per group. (C–D) WT mice traveled significantly less than all other groups at multiple time points (c, 2-Way ANOVA Main Effect p < 0.0001; Bonferroni Post Test * p < 0.05) and as a sum total of distance traveled in 90 min (d, 1-Way ANOVA p = 0.0001; Bonferroni Post Test *,**,*** p <0.05, 0.01, 0.001) Fenobam did not affect the total distance traveled in mGlu5 knockout mice as compared to vehicle-treated mGlu5 knockouts (Bonferroni Post Test p > 0.05), (n = 7 per group in WT-vehicle and WT-fenobam and n = 8 per group in KO-vehicle and KO-fenobam groups respectively).

cm = centimeter; KO = knockout; WT = wild type.

Effects of Fenobam on Performance in an Elevated Zero Maze

Fenobam (30 mg/kg) injected mice travelled a statistically significant farther distance during a ten minute period in an elevated zero maze when compared to vehicle-injected mice (41.44 ± 3.099 meters vs. 15.59 ± 2.651; 95% CI −35.30 to −16.40 meters; p = 0.0002). In addition fenobam-injected mice more frequently entered the open sections of the zero maze (p = 0.001; fig. 2A), spent a statistically significant longer amount of time in the open section of the zero maze (p < 0.01, fig. 2B), and travelled a statistically significant farther distance as a percentage of total distance travelled in the open sections of the zero maze (i.e., distance in open Arms divided by Total Distance) (p < 0.01; fig. 2C) as compared to vehicle-injected controls.

Figure 2. Fenobam Increases the Time Spent in the Open Sections of an Elevated Zero Maze.

Figure 2

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Drug naive C57 WT mice were injected with either vehicle or fenobam (30 mg/kg). Fenobam injected mice (A) entered the open sections significantly more than their vehicle injected littermates, (B) spent significantly more time in the open sections, and (c) travelled significantly farther as a percentage of total distance travelled in the open sections (unpaired t-test ** p < 0.01) over a 10-min period. (n = 5 in the vehicle group and 6 in the fenobam per group). sec = seconds; WT = wild type.

When knockout mice were compared to WT controls no differences were noted in the total distance travelled (WT n = 8, 19.04 ± 1.633 meters vs. knockout n = 7, 21.49 ± 2.770 meters; 95% CI −9.344 to 4.452 meters; p > 0.05), the number of entries into the open sections of the zero maze (WT 13.75 ± 1.916 vs. knockout 21.14 ± 4.512; 95% CI −15.70 to 4.450 entries; p > 0.05), the distance travelled as a percentage of total distance travelled in the open arms (WT 21.41 ± 3.300 percent vs. knockout 18.99 ± 4.310 percent; 95% CI −9.223 to 14.06 percent; p > 0.05), or the amount of time spent in the open sections of the maze (WT 86.19 ± 13.83 s vs. knockout 140.8 ± 26.19 s; 95% CI −118.1 to 8.933 s; p > 0.05).

Effects of Fenobam on Motor Coordination

As compared to vehicle, fenobam injection resulted in no physiologically or statistically significant differences in performance on three separate tasks designed to assess motor coordination. Fenobam-injected mice (30 mg/kg) performed equivalent to vehicle-injected mice when required to hang from an inverted screen for 120 s (95% CI for trial 1 −52.87 to 6.106, for trial 2 −33.56 to 25.41; fig. 3A), descend from a thin (8 mm diameter, 55 cm height) vertically-oriented metal pole (95% CI for trial 1 −48.85 to 49.06, for trial 2 −46.52 to 51.39; fig. 3B), and cross a thin (0.75 cm wide, 51 cm long) horizontally oriented ledge (fig. 3C). No physiologically or statistically significant difference between vehicle-injected and fenobam-injected C57 WT mice was observed over two consecutive trials.

Figure 3. Fenobam Does Not Impair Motor Coordination.

Figure 3

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Drug naive C57 WT mice were injected with either vehicle or fenobam (30 mg/kg). Within 20 min their performance was assayed twice on three separate tasks designed to assess motor coordination (A) the inverted screen hang time, (B) the vertical pole descent task, and (C) the ledge crossing task. No differences were noted between fenobam- and vehicle-injected mice in any task. (n = 10 in the vehicle group and 11 in the fenobam group).

s = seconds; Veh = vehicle; WT = wild type.

The Effects of Repeat Dosing of Fenobam on Analgesic Efficacy

To test whether tolerance to the analgesic effects of fenobam develops over time, Swiss Webster WT mice were injected with either vehicle or fenobam (30mg/kg) for 5 days. On the sixth day mice from the chronic-vehicle group were injected with either fenobam or vehicle, and mice from the chronic-fenobam group were injected with fenobam 5 min prior to performing a formalin test. Regardless of whether they had been treated chronically with fenobam or vehicle, mice acutely treated with fenobam demonstrated statistically significantly less spontaneous nocifensive behavior postformalin injection when compared to chronic-vehicle mice that were injected acutely with vehicle (p < 0.001; fig. 4A). Acute pretreatment with fenobam (30 mg/kg intraperitoneal) statistically significantly reduced the time mice spent licking or lifting the formalin injected paw during the first phase (95% CI of the chronic vehicle/acute vehicle treated mice compared to chronic vehicle/acute fenobam and chronic fenobam/acute fenobam 14.57 to 179.0 and 30.16 to 200.8 s respectively; p = 0.0097; fig. 4B) and second phase compared to 5 day Veh / Veh mice, regardless of fenobam pretreatment (95% CI of the chronic vehicle/acute vehicle treated mice compared to chronic vehicle/acute fenobam and chronic fenobam/acute fenobam 124.2 to 740.5 and 81.56 to 721.1 s respectively; p = 0.0069; fig. 4C).

Figure 4. The Effects of Chronic Fenobam Injection on Spontaneous Formalin Behavior.

Figure 4

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Mice that were administered either fenobam or vehicle for five days (A–C) prior to the formalin test demonstrated significantly decreased time spent licking or lifting the injected paw when administered fenobam (30 mg/kg) 5 min prior to intraplantar formalin injection as compared to mice treated with vehicle for five days and vehicle on the day of the experiment (2-Way ANOVA Main Effect of Treatment p < 0.0001; Bonferroni Post Test ** = p < 0.01 compared to 5 Day Veh / Veh). Both the first phase (b) and the second phase (c) were reduced (1-Way ANOVA Main Effect of Treatment p = 0.0097, p = 0.0069 respectively; Dunnett’s Post Test *, ** = p < 0.05, 0.01, respectively as compared to 5 Day Veh / Veh mice, n = 6 in Veh/Veh per group and n = 7 in Veh/Fen and Fen/Fen groups respectively). Mice that were administered either fenobam or vehicle for 14 days (D–F) prior to the formalin test demonstrated significantly decreased time spent licking or lifting the injected paw when administered fenobam (30 mg/kg) 5 min prior to intraplantar formalin injection as compared to mice treated with vehicle for 14 days and vehicle on the day of the experiment (2-Way ANOVA Main Effect of Treatment p < 0.0001; Bonferroni Post Test ** = p < 0.01 compared to 14 Day Veh/Veh). The second phase (F) was reduced in both groups compared to Veh / Veh mice (1-Way ANOVA Main Effect of Treatment p = 0.0154; Dunnett’s Post Test * = p < 0.05) as compared to 14 Day Veh / Veh mice. The first phase (e) was significantly reduced in vehicle / fenobam mice when compared to Veh / Veh mice (1-Way ANOVA Main Effect of Treatment p = 0.0258; Dunnett’s Post Test * = p < 0.05 as compared to 14 Day Veh / Veh mice). (n = 8 in the Veh/Veh and n = 11 in the Veh/Fen and Fen/Fen groups respectively).

Fen = fenobam; sec = seconds; Veh = vehicle.

This experiment was then repeated with a second group of Swiss Webster WT mice, with the one alteration that fenobam or vehicle was injected for 14 days prior to performing a formalin test on the 15th day. Similar to the five day experiment above, mice acutely treated with fenobam demonstrated statistically significantly less spontaneous nocifensive behaviors post formalin injection when compared 14 day Veh / Veh mice (p < 0.001; fig. 4D). Mice that were chronically treated with vehicle and then pretreated with fenobam exhibited statistically significantly reduced time spent licking or lifting in the first phase when compared to 14 day Veh / Veh mice (95% CI 16.02 to 156.7 s; p = 0.0258; fig. 4E). Acute pretreatment with fenobam (30 mg/kg intraperitoneal) also statistically significantly reduced the time mice spent licking or lifting the formalin injected paw during the second phase compared to 5 day Veh / Veh mice, regardless of fenobam pretreatment (95% CI of the chronic and acutely vehicle treated mice compared to chronic vehicle/acute fenobam and chronic fenobam/acute fenobam 34.97 to 575.3 and 65.50 to 583.9 s respectively; p = 0.0154; fig. 4F).

The Effects of Chronic Fenobam Injection on Histopathology, Serum Chemistries, and Complete Blood Count

Serum was collected from mice chronically injected with either vehicle or fenobam for 14 days. Serum chemistry values for cholesterol, total bilirubin, triglycerides, creatinine, total protein, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), lactate dehydrogenase (LDH), and amylase were measured. No differences in any values measured were seen between fenobam- (30 mg/kg intraperitoneal for 14 days) and vehicle-injected (vehicle intraperitoneal for 14 days) mice (fig. 5A–I; 95% CIs: cholesterol: −23.87 to 10.17 mg/dl, total bilirubin: −0.07 to 0.10 mg/dl, triglycerides: −37.00 to 9.46 mg/dl, creatine: −0.043 to 0.021 mg/dl, total protein: −0.42 to 0.13 g/dl, ALT: −83.38 to 289.5 units, AST: −32.61 to 116.4 units, LDH: −1310 to 3167 units, and amylase: −1361 to 2788 units; p > 0.05 for all comparisons).

Figure 5. The Effects of Chronic Fenobam Injection on Serum Chemistries.

Figure 5

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No differences were found between mice injected daily with fenobam (30 mg/kg intraperitoneal) or vehicle in serum (A) cholesterol, (B) total bilirubin, (C) triglycerides, (D) creatinine, (E) total protein, (F) alanine-aminotransferase (ALT), (G) aspartate-aminotransferase (AST), (H) lactate dehydrogenase (LDH), (I) or amylase, (Unpaired t-test p > 0.05 vehicle compared to fenobam). (n = 13 for vehicle and 20 for fenobam).

L = liters; U = units.

Whole blood was also obtained from mice chronically injected with either vehicle or fenobam for 14 days and assayed for counts of white blood cells (WBC), erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), or platelets. No differences were found between mice injected daily with fenobam (30 mg/kg intraperitoneal for 14 days) or vehicle (vehicle intraperitoneal for 14 days). (fig. 6A–H; 95% CIs: WBC −0.8969 to 1.052 K/cu mm, erythrocytes −3.187 to 0.8982 M/cu mm, hemoglobin −4.845 to 1.005 g/dl, hematocrit −18.84 to 4.038 percent, MCV −7.191 to 2.258 cu microns, MCH −2.358 to 0.4581 picograms, MCHC −9.907 to 4.767 percent, and platelets −342.6 to 330.8 K/cu mm; p > 0.05 for all comparisons).

Figure 6. The Effects of Chronic Fenobam Injection on Complete Blood Counts.

Figure 6

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No differences were found between mice injected daily with fenobam (30 mg/kg intraperitoneal) or vehicle in serum (A) white blood cell count (WBC), (B) erythrocyte cell count, (C) hemoglobin, (D) hematocrit, (E) mean corpuscular volume (MCV), (F) mean corpuscular hemoglobin (MCH), (G) mean corpuscular hemoglobin concentration (MCHC), or (H) platelets. (Unpaired t-test p > 0.05 vehicle compared to fenobam). (n = 6 for vehicle and 10 for fenobam).

cu = cubic; K = thousand; M = million.

In addition a 100-count white blood cell differential was performed on all mice from which whole blood was obtained. No differences were noted in the number of segmented neutrophils (all values presented as mean number of cells per cubic mm +/− std. dev.) (vehicle = 506.9 +/−140.6; fenobam = 651.8 +/− 428.1, p > 0.05), lymphocytes (vehicle = 1194 +/− 725.2; fenobam = 945.3 +/− 632.5, p > 0.05), or monocytes (vehicle = 2.5 +/− 6.1; fenobam = 5.1 +/− 10.8, p > 0.05). No band forms, eosinophils, or basophils were observed from any mice in either injection group.

Several animals from each group were examined by necropsy. In addition, tissue histopathology was performed on brain, liver, and testes on all animals examined by necropsy. Additional organs that were noted to be abnormal by gross necropsy also underwent tissue histopathology on a case-by-case basis. In all animals, the hair coat and skin were normal, skeletal palpation revealed no evidence of malformation or trauma, there was no nasal or ocular discharge or diarrhea, and hydration and body fat were normal. Whole body and individual organ weights are detailed below in table 1. No physiologically or statistically significant differences were found in total body weight or any organ weight between fenobam- and vehicle-treated mice. The major thoracic and abdominal organs and brain were examined in all animals. No abnormalities were noted in the respiratory system, digestive system, musculoskeletal system, urinary system, genital system, brain, thymus, spleen, lymph nodes, adrenal, thyroid, pituitary, or eye. Gross abnormalities and histopathologic examinations of the brain, liver, testes, and any other organs found to exhibit abnormalities on gross examination are noted in table 2. No physiologically or statistically significant differences were found between the fenobam- and vehicle-treated animals with regard to the type or prevalence of any abnormalities.

Table 1.

The Effects of Chronic Fenobam Injection on Visceral Organ Weights.

Total Animal Std Dev Heart Std Dev Lung Std Dev Liver Std Dev
Vehicle 30.11 2.32 0.16 0.01 0.20 0.02 1.65 0.16
Fenobam 29.83 2.04 0.16 0.05 0.20 0.02 1.59 0.17
Spleen Std Dev Thymus Std Dev Right Kidney Std Dev Right Testicle Std Dev
Vehicle 0.09 0.01 0.08 0.01 0.26 0.03 0.12 0.01
Fenobam 0.10 0.02 0.06 0.02 0.25 0.04 0.11 0.01
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All weights in grams, n = 9 for Vehicle, n = 12 for fenobam. No significant differences were noted for any organs (unpaired t-test, p > 0.05).

Table 2.

The Effects of Chronic Fenobam Injection on Gross Organ Appearance and Histopathology.

Gross Necropsy Examination Vehicle Fenobam
No Gross Abnormalities 5/9 4/12
Heart Enlarged Due to Left Ventricle Dilation 0/9 1/12
Lesions Related to Repeated Intraperitoneal Injection Vehicle Fenobam
Focal Adhesions between Abdominal Fat and Viscera 3/9 5/12
Seminal Vesicles Dark in Color 1/9 2/12
Liver capsule minimally thickened by inflammatory infiltrate 1/9 1/12
Seminal Vesicles Showed Mild Fibrosis and Inflammatory Infiltrate 0/9 2/12
Histopathologic Examination Vehicle Fenobam
No Significant Brain Lesions 9/9 12/12
No Significant Liver Lesions 8/9 11/12
No Significant Testicular Lesions 8/9 11/12
Cardiomyopathy 0/9 1/12
Testes showed moderate seminiferous tubule degeneration affecting a limited number of tubules 0/9 1/12
Testes showed advanced seminiferous tubule degeneration affecting 5 – 40% of tubules 1/9 0/12
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The Effects of Fenobam on Weight gain and Post-Food Deprivation Food Intake

Animals injected with fenobam or vehicle for the 14-day tolerance study described above were weighed daily prior to injection. When the percent change in body weight was calculated as a function of starting weight for each animal, daily fenobam injection was found to have a statistically significant effect on percent change in body weight over a 14-day period, as compared to vehicle (fig. 7A, p < 0.0001). No differences were found in the average starting weights of the animals (Veh = 29.34 ± 0.77g, Fenobam = 29.42 ± 0.65 g, 95% CI −2.112 to 1.946 g; fig. 7A, inset, p > 0.05).

Figure 7. The Effects of mGlu5 Inhibition on Weight Gain and Post-fasting Food Intake. (A).

Figure 7

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The percent change in weight of fenobam injected animals was significantly less than that of vehicle injected animals over a two week period (2-Way ANOVA Main Effect of fenobam *** = p < 0.0001). (A inset) The average starting weights of the animals in the two groups did not differ (unpaired t-test p > 0.05). (n = 13 for the vehicle and 16 for the fenobam group). (B) Fenobam (30 mg/kg) significantly decreased food intake following a 24-h fast as compared to vehicle (2-Way ANOVA Main Effect of Fenobam *** = p < 0.0001, Bonferroni Post Test *, *** = p < 0.05, 0.001). n = 6 per group.

WT = wild type.

The effects of fenobam on food intake following a 24-h food deprivation were assessed in WT Swiss Webster mice (fig. 7B). Fenobam (30 mg/kg) administered at the time of refeeding statistically significantly decreased the amount of food consumed, as compared to vehicle-treated animals (fig. 7B, p < 0.001). Fenobam-treated mice consumed more food 60 min (95% CI −36.42 to −11.72 mg/g body weight), 90 min (95% CI −36.97 to −12.28 mg/g body weight), and 180 min (95% CI −26.17 to −1.470 mg/g body weight) after refeeding compared to vehicle-treated mice. However, by 8 h after the start of refeeding food intake of the fenobam-treated mice was not different from vehicle treated animals (vehicle = 97.3 +/− 8.8 mg/g; fenobam = 87.9 +/− 7.8 mg/g; 95% CI −16.77 to 35.55 mg/g body weight; unpaired T-test p = 0.44). No differences in body weight were noted in animals prior to re-feeding (Veh = 32 +/− 0.82 g, Fenobam = 31.73 +/− 1.25 g; 95% CI −3.052 to 3.585; unpaired T-test p = 0.86).

Discussion

Activation of mGlu5 with the group I agonist (R,S)-3,5-dihydroxyphenylglycine is proalgesic6, while antagonism with fenobam results in decreased nociceptive behaviors10,11. These data provide evidence that mGlu5 may represent a viable therapeutic target for the treatment of pain. However, any future development of mGlu5 antagonists as analgesics in human patients will require both drugs that are selective for mGlu5 and for mGlu5 antagonism at sites outside the pain neuraxis to be devoid of clinically significant deleterious effects. Here we report that drug naive mGlu5 knockout mice exhibited increased locomotor activity at multiple time points when compared to WT littermates, and the level of locomotor activity of mGlu5 knockout mice was unchanged following either vehicle or fenobam injection. In contrast, when fenobam was injected into WT mice before placement in the open field increased locomotor activity was exhibited at multiple time points as compared to vehicle-treated mice. This suggests that the increased locomotor activity is due to antagonism of mGlu5. The fact that fenobam does not increase locomotor activity in mGlu5 knockout mice also suggests that the effect is mediated by mGlu5, although it is possible that ceiling effects are preventing any increased locomotion in mGlu5 knockout mice, since drug naive mGlu5 knockout mice are hypermotile.

Fenobam was originally developed as an anxiolytic15, and when administered to rodents13 and humans15 it is anxiolytic. Increased locomotion in an open-field enclosure could represent an anxiolytic effect of fenobam. To test this possibility we assessed the effects of fenobam on drug-naive mice in an elevated zero maze. Fenobam injected mice entered the open sections of the zero maze significantly more and spent significantly more time in the open sections when compared to vehicle-injected mice. They also traveled significantly farther in the zero maze as a whole and traveled significantly more of this distance in the open sections of the maze when compared to vehicle-injected mice. These findings suggest that the effects of fenobam are both stimulatory and anxiolytic. The fact that these findings were not seen in drug-naive mGlu5 knockout mice compared to WT controls may be due to a compensatory effect due to genetic deletion of mGlu5 since birth in knockout animals.

Increased locomotor activity following mGlu5 antagonism with fenobam is in opposition to results seen with 2-Methyl-6-(phenylethynyl)pyridine (MPEP)9,23 and the related mGlu5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP)9, which have been shown to reduce locomotor activity at analgesic doses. MPEP has also been reported to impair motor coordination at analgesic doses9. While our previous findings10 did not show impaired motor coordination following fenobam injection, the discrepant findings between MPEP and fenobam compelled us to perform further assessment of the effects of fenobam on motor coordination. No differences were seen in fenobam- versus vehicle-injected mice in the ledge crossing test or the vertical pole descent task. Both of these tasks require a mouse to navigate with limited foot purchase and execute complex turning behaviors without falling. A test of strength where mice hung upside down for two minutes also yielded no differences, further suggesting that while mGlu5 antagonism may result in increased locomotive behaviors, motor coordination remains intact. Thus, while fenobam may increase locomotor activity, the potentially more deleterious side effect of altered motor coordination appears to be absent at the tested analgesic dose. Furthermore the stimulant side effects of fenobam could be beneficially exploited in patients that are also suffering from concurrent depression, assuming that any psychostimulant effects are not aversive. No major adverse reactions were reported by three healthy volunteers administered fenobam at doses of 150 mg4. While these findings are encouraging they represent results from a limited number of subjects. Further human clinical testing will be necessary to confirm these data.

A major limitation of certain analgesics, such as opiates, is that tolerance to their analgesic effects occurs over time. In order for mGlu5 antagonists to be useful in humans they would have to avoid this limitation. Seven days of repeated dosing of the mGlu5 antagonist MTEP has been demonstrated to result in analgesic tolerance in the formalin test24. Whether tolerance develops to the analgesic effects of fenobam is not currently known. Therefore we injected groups of mice daily for either 5 or 14 days with an analgesic dose of fenobam (30 mg/kg) or vehicle. On the 6th or 15th day, respectively, mice were injected with either fenobam or vehicle, followed by a formalin test. Mice chronically injected with fenobam did not display tolerance to the analgesic effects of acute fenobam on the test day. Fenobam was analgesic in both the first and second phase of the formalin test. While this is not conclusive evidence that tolerance to fenobam would not develop if the drug was administered over a longer time course, it is nonetheless encouraging evidence that tolerance may not be a limiting factor in the use of fenobam as an analgesic.

A fascinating effect of mGlu5 deletion and pharmacological inhibition is a significant effect on weight gain and postfast feeding behavior. mGlu5 knockout mice have been previously reported to weigh less than their WT littermates18,19. Differences in ad libitum food intake were not found18, so this is not believed to account for the differences. However both mGlu5 knockout mice, and mice injected with MPEP, exhibit significantly less refeeding behavior following a 14-h fast, suggesting that mGlu5 does play a role in appetite and feeding behavior18. Here we report that fenobam administration significant l y decreases postfast refeeding. Fenobam’s psychostimulant properties and appetite-suppressive effects should certainly be viewed as separate from its analgesic effects. However, rather than being dose limiting, it might be possible to exploit these off-target effects to beneficial clinical use in certain diseases that have pain as a major symptom. Osteoarthritis is a prime candidate as weight loss has been associated with both a decrease in risk of developing osteoarthritis25 and both weight loss and increased activity result in an improvement of existing osteoarthritis symptoms26. Again, further testing in human patients will be necessary to know for certain.

Future endeavors to bring mGlu5 antagonists to human patients will require that pharmacological agents have acceptable safety profiles. This is underscored by the cessation of clinical trials involving the mGlu5 antagonist ADX-10059 due to unacceptable increases in liver enzymes12. While these changes were felt to be compound-specific27, mGlu5 is expressed in liver tissue28, and thus mGlu5 antagonists as a class could theoretically cause damage to that tissue. Therefore, we sought to determine whether chronic administration of fenobam would also result in unacceptable changes to liver, as well as other organs. Fenobam administration for up to 14 days resulted in no significant gross lesions in multiple organs and systems, including hepatobiliary, respiratory, digestive, urinary, nervous, or endocrine, when compared to vehicle-injected controls, or changes seen by histopathology in brain, liver, or testes. In addition, no major changes were seen in standard blood chemistry measurements or in complete blood counts. While occasional animals did demonstrate cardiomyopathy and testicular degeneration, these lesions can occur spontaneously as animals age29. Given that the animal with the significant testicular lesions was treated with vehicle, this is likely to be an incidental finding. An absence of brain lesions and a lack of altered motor coordination suggests an absence of adverse effects in the spinal cord, however we did not directly verify this histologically. Many other lesions seen, including focal adhesions, liver capsule inflammatory infiltrates, and the darkened color of the seminal vesicles, were seen in both vehicle- and fenobam-injected mice and may simply be due to repeated intraperitoneal injections30.

Finally, while these experiments were designed to test the analgesic tolerance of fenobam and better understand its toxicity profile, it is critical to note that only one dose was tested. This dose was chosen based on previous dose-response studies indicating that 30 mg/kg was an effective analgesic dose in mice10. Furthermore, this dose corresponds to the highest single dose of fenobam so far tested in human trials with different clinical indications4,14,15. Possible toxicity of fenobam at higher doses or administered for longer time periods have not been ruled out. In addition, as mGlu5 has been implicated in learning and memory11, future studies should test the effects of fenobam on these processes.

In conclusion, we report that fenobam, a clinically validated mGlu5 antagonist with analgesic and anxiolytic properties has an acceptable safety profile in rodents following chronic daily dosing of 30mg/kg for up to 14 days. In addition, we report that tolerance to the analgesic effects of fenobam does not develop during this time period. Furthermore, while this study and previous studies suggest that fenobam and other mGlu5 antagonists may have a psychostimulant effect, we continue to find no evidence that fenobam impairs motor coordination. Finally, we suggest that while fenobam may have effects outside of the pain neuraxis, at least some of its nonanalgesic effects of could be used for beneficial clinical effect. Fenobam, and mGlu5 antagonists in general, represent promising candidates for the treatment of human pain conditions.

Summary Statement.

What we already know about this topic

  • Fenobam, an antagonist for the metabotropic glutamate receptor 5, produces acute analgesia in rodents and might be developed for clinical use

  • Whether chronic fenobam treatment results in tolerance has not been studied

What this article tells us that is new

  • In mice, 2 week daily dosing, a duration which produces extreme tolerance to opioids, did not result in tolerance to analgesia from fenobam

Acknowledgments

Disclosure of Funding: This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke, Bethesda, Maryland [Grant NS42595, NS06462601, NS06776101] (to R.W.G., M.C.M., and B.J.K. respectively). Additional support was provided by the National Institutes of Health Neuroscience Blueprint Interdisciplinary Center Core Grant P30 NS057105 (Washington University).

We would like to acknowledge the work of Sherri Vogt, B.S., Research Technician, and Chang Shen Qiu, M.D., Laboratory Manager, Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri, in maintaining our mouse colony. In addition we would like to acknowledge the work of Niecey Hinkle, B.S., Research Technician, Division of Comparative Medicine, Washington University in St. Louis, St. Louis, Missouri, who worked on the hematology and clinical chemistry analyses. Finally, we would like to thank Mena Morales, B.S., PhD Candidate, Program in Neuroscience and Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri, for extensive manuscript editing.

Footnotes

Work Attributable Department: Washington University Pain Center - Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri

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