An evaluation of the anti-hyperalgesic effects of cannabidiolic acid-methyl ester in a preclinical model of peripheral neuropathic pain

Abstract

Background and purpose: Chronic neuropathic pain (NEP) is associated with growing therapeutic cannabis use. To promote quality of life without psychotropic effects, cannabinoids other than Δ9-tetrahydrocannabidiol, including cannabidiol and its precursor cannabidiolic acid (CBDA), are being evaluated. Due to its instability, CBDA has been understudied, particularly as an anti-nociceptive agent. Adding a methyl ester group (CBDA-ME) significantly enhances its stability, facilitating analyses of its analgesic effects in vivo. This study examines early treatment efficacy of CBDA-ME in a rat model of peripherally induced NEP and evaluates sex as a biological variable.

Experimental approach: After 14 consecutive days of intraperitoneal CBDA-ME administration at 0.01, 0.1 and 1 μg·kg^-1 , commencing 1 day after surgically implanting a sciatic nerve-constricting cuff to induce NEP, the anti-nociceptive efficacy of this cannabinoid was assessed in male and female Sprague-Dawley rats relative to vehicle-treated counterparts. In females, 2 and 4 μg·kg^-1 daily doses of CBDA-ME were also evaluated. Behavioural tests were performed for hind paw mechanical and thermal withdrawal thresholds once a week for 8 weeks. At endpoint, in vivo electrophysiological recordings were obtained to characterize soma threshold changes in primary sensory neurons.

Key results: In males, CBDA-ME elicited a significant concentration-dependent chronic anti-hyperalgesic effect, also influencing both nociceptive and non-nociceptive mechanoreceptors, which were not observed in females at any of the concentrations tested.

Conclusion and implications: Initiating treatment of a peripheral nerve injury with CBDA-ME at an early stage post-surgery provides anti-nociception in males, warranting further investigation into potential sexual dimorphisms underlying this response.

Figure 1

Experimental timeline and action potential parameters. (a) Timeline of experimental procedures. Model induction of peripheral neuropathic pain (NEP) occurred on Day 0. Before model induction, behavioural baseline tests were performed, with weekly testing carried out for 8 weeks. One day after model induction, daily drug administration was initiated and continued for 2 weeks. At the 8‐week experimental endpoint, electrophysiological procedures were performed. (b) Action potential (AP) configuration analysis. Representative intracellular somatic action potentials of sensory neurons were evoked by electrical stimulation of the dorsal root. Upper left: an evoked AP classified as a C‐fibre on the basis of conduction velocity, which was 0.35 mm·ms⁻¹. Upper right: an Aß‐fibre classified based on conduction velocity, which was 12.65 mm·ms⁻¹. Lower left: differentiated derivative trace of a C‐fibre AP. A plateau was identified on the repolarisation branch of the AP in the differentiated recording, which is indicative of a nociceptive neuron. Lower right: differentiated derivative trace of an Aß‐fibre, without inflection on the falling phase in the differentiated recording, which is considered a non‐nociceptive neuron

Figure 2

A comparison of mechanical withdrawal thresholds between naïve, sham, and male and female rats with a sciatic nerve cuff (neuropathy) using the von Frey behavioural test. Mechanical withdrawal thresholds were evaluated by von Frey testing once a week for a total of 4 weeks. Data are expressed as the mean ± SEM for different groups. Two‐way ANOVA with a Bonferroni post hoc test was used for comparisons. A symbol (X) above the graphs indicates significant differences between the naïve and neuropathy groups for each sex. No significant differences were obtained between the naïve and sham groups

Figure 3

A comparison of mechanical withdrawal thresholds in the behavioural study between vehicle and CBDA‐ME‐treated male and female rats. Mechanical withdrawal thresholds of sciatic cuff‐implanted rats were evaluated by von Frey testing once a week to determine the effect of CBDA‐ME treatment, which was assessed via dose–response analyses. Data are expressed as the mean ± SEM for each treatment group. Two‐way ANOVA with a Bonferroni post hoc test was used for comparisons. A symbol (X) above the left‐hand graph indicates significant differences between vehicle and CBDA‐ME‐1, CBDA‐ME‐0.1 and CBDA‐ME‐0.01, represented by an upper, middle, or lower row of this symbol, respectively, during the fourth fifth, sixth, and eighth weeks. Asterisks (*) across the two graphs indicate significant differences between males and females receiving the CBDA‐ME‐1 treatment, with these differences occurring during weeks 4 and 8. CBDA‐ME‐1, CBDA‐ME‐0.1, and CBDA‐ME‐0.01 represent different concentrations, corresponding to 1, 0.1 and 0.01 μg·kg⁻¹ respectively. In addition, in females, two higher concentrations, CBDA‐ME‐2 and CBDA‐ME‐4, were tested, corresponding to 2 and 4 μg·kg⁻¹, respectively

Figure 4

Comparison of thermal withdrawal thresholds in the behavioural study between vehicle and CBDA‐ME‐treated male and female rats. Thermal withdrawal thresholds of sciatic cuff‐implanted rats were measured using the Hargreaves' test once a week to determine the effect of CBDA‐ME treatment, assessed via a dose–response analysis. Data are expressed as the mean ± SEM for each treatment group. Two‐way ANOVA with a Bonferroni post hoc test was used for comparisons. The absence of a symbol or an asterisk indicates lack of a statistically significant difference. CBDA‐ME‐1, CBDA‐ME‐0.1 and CBDA‐ME‐0.01 represent different concentrations, corresponding to 1, 0.1, and 0.01 μg·kg⁻¹ respectively. In addition, in females, two higher concentrations, CBDA‐ME‐2 and CBDA‐ME‐4, were tested, corresponding to 2 and 4 μg·kg⁻¹, respectively

Figure 5

Comparison of the activation threshold of mechanical sensory neurons in response to intracellular current injection in vehicle and CBDA‐ME‐treated male and female rats. The current threshold was defined as the minimum current required to evoke an action potential (AP) by intracellular current injection. Data are expressed as the mean ± SEM for each treatment group. The comparison between vehicle and CBDE‐ME‐1 treatment, corresponding to 1 μg·kg⁻¹ for each sex, was analysed by Mann–Whitney analysis. A symbol (X) above the graphs indicates significant differences between the vehicle and CBDA‐ME‐1 treatment. Two‐way ANOVA with a Dunn's post hoc test was then applied for statistical comparisons between sexes with vehicle and CBDA‐ME‐1 treatment. Asterisks (*) between the graphs indicate a significant difference between males and females in the vehicle and CBDA‐ME‐1 treatment groups. AβHTM, Aβ‐fibre high threshold mechanoreceptor; cutaneous (CUT), Aβ‐fibre cutaneous neuron; CHTM, C‐fibre high threshold mechanoreceptor; CLTM, C‐fibre low threshold mechanoreceptor; MS, Aβ‐fibre muscle spindle neuron

Figure 6

Representative raw recordings in response to intracellular current injection in vehicle and CBDA‐ME‐treated male and female rats. Discharge was evoked by injecting a series of depolarizing current pulses into DRG soma through the recording electrode [X‐axis: time (ms); Y‐axis: voltage (mV)]. Representative raw recordings show the threshold and repetitive charges of action potentials (APs) evoked by intracellular current injection in different types of mechanoreceptor neurons. APs were evoked by current pulses of 0.5 to 4 nA, in increments of 0.5 nA. Neurons from males and females treated with either CBDA‐ME at the 1 μg·kg⁻¹ concentration or vehicle were tested. AβHTM: Aβ‐fibre high threshold mechanoreceptor; CHTM, C‐fibre high threshold mechanoreceptor; CLTM, C‐fibre low threshold mechanoreceptor; MS, Aβ‐fibre muscle spindle neuron; CUT: Aβ‐fibre cutaneous neuron