Antinociceptive Effects of Cannabichromene (CBC) in Mice: Insights from von Frey, Tail-Flick, Formalin, and Acetone Tests

Abstract

Cannabis sativa contains minor cannabinoids that have potential therapeutic value in pain management. However, detailed experimental evidence for the antinociceptive effects of many of these minor cannabinoids remains lacking. Here, we employed artificial intelligence (AI) to perform compound-protein interaction estimates with cannabichromene (CBC) and receptors involved in nociceptive signaling. Based on our findings, we investigated the antinociceptive properties of CBC in naïve or neuropathic C57BL/6 male and female mice using von Frey (mechanical allodynia), tail-flick (noxious radiant heat), formalin (acute and persistent inflammatory pain), and acetone (cold thermal) tests. For von Frey assessments, CBC dose (0-20 mg/kg, i.p.) and time (0-6 h) responses were measured in male and female neuropathic mice. For tail-flick, formalin, and acetone assays, CBC (20 mg/kg, i.p.) was administered to naïve male and female mice 1 h prior to testing. The results show that CBC (10 and 20 mg/kg, i.p.) significantly reduced mechanical allodynia in neuropathic male and female mice 1-2 h after treatment. Additionally, CBC treatment caused significant reductions in nociceptive behaviors in the tail-flick assay and in both phase 1 and phase 2 of the formalin test. Finally, we found a significant interaction in neuropathic male mice in the acetone test. In conclusion, our results suggest that CBC targets receptors involved in nociceptive signaling and imparts antinociceptive properties that may benefit males and females afflicted with diverse forms of acute or chronic/persistent pain.

Keywords: cannabichromene; cannabinoids; cannabis; inflammatory pain; neuropathic pain; pain.

Figure 1

DRIFT predicts CBC’s receptor binding profile. (A) Pearson correlation analysis of six phytocannabinoids examined by DRIFT. (B) DRIFT prediction of CBC targets. Compounds are ranked from highest (red) to lowest (green) confidence of prediction. CBN (cannabinol), THC (Δ⁹-tetrahydrocannabinol), CBG (cannabigerol), CBD (cannabidiol), THCV (Δ⁹-tetrahydrocannabivarin), CBC (cannabichromene).

Figure 2

CBC reduces mechanical sensitivity in neuropathic male and female mice. (A) Timeline of experimental procedure for von Frey experiments. (B) Summary graph showing the force required to evoke a paw withdraw response in neuropathic male mice 1 h after treatment with vehicle, varying doses of CBC (5, 10, 20 mg/kg, i.p.), or indomethacin (10 mg/kg, i.p.) (F_(4,51) = 23.76, p < 0.0001; one-way ANOVA with Bonferroni post-test). (C) Summary graph as described in (B) in neuropathic female mice (F_(4,51) = 26.70, p < 0.0001; one-way ANOVA with Bonferroni post-test). (D) Summary graph comparing the effects of 20 mg/kg CBC in neuropathic male and female mice (F_(3,60) = 73.89, p < 0.0001; one-way ANOVA with Bonferroni post-test). (E) Summary graph showing the force required to evoke a paw withdraw response in neuropathic male mice before cisplatin treatment (baseline), after cisplatin treatment (post-cisp.), and at varying time points after treatment with vehicle or CBC (20 mg/kg, i.p.) (F_(6,84) = 3.797, p = 0.0021; two-way repeated-measures ANOVA with Bonferroni post-test). (F) Summary graph described in (E) in neuropathic female mice (F_(6,84) = 15.85, p < 0.0001; two-way repeated-measures ANOVA with Bonferroni post-test). ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

CBC produces analgesia-like effects in male mice in the formalin test. (A) Summary of the composite pain score 0–60 min after formalin injection in male mice treated with vehicle or CBC (20 mg/kg, i.p.) (F_(12,204) = 1.731, p = 0.0624; two-way repeated-measures ANOVA with Bonferroni post-test). (B) Summary graph showing the area under the curve (AUC) during phase 1 of the formalin assay in male mice treated with vehicle or CBC (t₍₁₇₎ = 3.344, p = 0.0038; unpaired Student’s t-test). (C) Summary graph showing the area under the curve (AUC) during phase 2 of the formalin assay in male mice treated with vehicle or CBC (t₍₁₇₎ = 2.768, p = 0.0132; unpaired Student’s t-test). * p < 0.05, ** p < 0.01.

Figure 4

CBC produces analgesia-like effects in female mice in the formalin test. (A) Summary of the composite pain score from 0–60 min after formalin injection in female mice treated with vehicle or CBC (20 mg/kg, i.p.) (F_(12,216) = 2.607, p = 0.0029; two-way repeated-measures ANOVA with Bonferroni post-test). (B) Summary graph showing the area under the curve (AUC) during phase 1 of the formalin assay in female mice treated with vehicle or CBC (t₍₁₈₎ = 4.416, p = 0.0003; unpaired Student’s t-test). (C) Summary graph showing the area under the curve (AUC) during phase 2 of the formalin assay in female mice treated with vehicle or CBC (t₍₁₈₎ = 6.604, p < 0.0001; unpaired Student’s t-test). * p < 0.05, *** p < 0.001.

Figure 5

CBC produces analgesia-like effects in naïve male and female mice in the tail-flick test. (A) Experimental timeline for the tail-flick test. (B) Summary graph showing the percentage maximum possible effect (%MPE) in male mice 1 h after treatment with vehicle, CBC (20 mg/kg, i.p.) or indomethacin (10 mg/kg, i.p.) (F_(2,28) = 14.05, p < 0.0001; one-way ANOVA with Bonferroni post-test). (C) Summary graph as described in (B) in female mice (F_(2,28) = 10.44, p = 0.0004; one-way ANOVA with Bonferroni post-test). ns = not significant, ** p < 0.01, *** p < 0.001.

Figure 6

CBC effects in the acetone test in both male and female mice. (A) Summary graph showing the time to evoke a behavioral response following acetone exposure to the hind paw in naïve male mice 1 h following vehicle, CBC (20 mg/kg, i.p.), or indomethacin (10 mg/kg, i.p.) (F_(2,28) = 13.24, p < 0.0001; one-way ANOVA with Bonferroni post-test). (B) Summary graph of the same procedure outlined in (A) in naïve female mice (F_(2,28) = 6.321, p = 0.0054; one-way ANOVA with Bonferroni post-test). (C) Summary graph showing the time to evoke a behavioral response following acetone exposure to the hind paw in neuropathic male mice at varying time points (30 min, 1, 2, 4, 6 h) following vehicle or CBC (20 mg/kg, i.p.) injection (F_(4,56) = 2.596, p = 0.0459; two-way repeated-measures ANOVA with Bonferroni post-test). (D) Summary graph of the same procedure outlined in (C) in neuropathic female mice (F_(4,56) = 1.974, p = 0.1110; two-way repeated-measures ANOVA with Bonferroni post-test). ** p < 0.01, *** p < 0.001.