Metabotropic glutamate receptor 5 antagonism with fenobam: examination of analgesic tolerance and side effect profile in mice

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 whether 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. Histopathologic examination, hematology, and clinical chemistry analysis after 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 with vehicle control subjects, but did not cause any physiologically or statistically significant hematologic 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.

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

(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.

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

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.

Figure 3. Fenobam Does Not Impair Motor Coordination

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.

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

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.

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

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.

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

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.

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

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.