Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism

Abstract

Background and purpose: Epilepsy is the most prevalent neurological disease and is characterized by recurrent seizures. Here, we investigate (i) the anticonvulsant profiles of cannabis-derived botanical drug substances (BDSs) rich in cannabidivarin (CBDV) and containing cannabidiol (CBD) in acute in vivo seizure models and (ii) the binding of CBDV BDSs and their components at cannabinoid CB1 receptors.

Experimental approach: The anticonvulsant profiles of two CBDV BDSs (50-422 mg·kg(-1) ) were evaluated in three animal models of acute seizure. Purified CBDV and CBD were also evaluated in an isobolographic study to evaluate potential pharmacological interactions. CBDV BDS effects on motor function were also investigated using static beam and grip strength assays. Binding of CBDV BDSs to cannabinoid CB1 receptors was evaluated using displacement binding assays.

Key results: CBDV BDSs exerted significant anticonvulsant effects in the pentylenetetrazole (≥100 mg·kg(-1) ) and audiogenic seizure models (≥87 mg·kg(-1) ), and suppressed pilocarpine-induced convulsions (≥100 mg·kg(-1) ). The isobolographic study revealed that the anticonvulsant effects of purified CBDV and CBD were linearly additive when co-administered. Some motor effects of CBDV BDSs were observed on static beam performance; no effects on grip strength were found. The Δ(9) -tetrahydrocannabinol and Δ(9) -tetrahydrocannabivarin content of CBDV BDS accounted for its greater affinity for CB1 cannabinoid receptors than purified CBDV.

Conclusions and implications: CBDV BDSs exerted significant anticonvulsant effects in three models of seizure that were not mediated by the CB1 cannabinoid receptor and were of comparable efficacy with purified CBDV. These findings strongly support the further clinical development of CBDV BDSs for the treatment of epilepsy.

Keywords: anticonvulsant; cannabidiol; cannabidivarin; cannabinoid; epilepsy; isobologram; radioligand binding assays; seizure; tolerability.

Figure 1

Effects of CBDV BDS components in the PTZ model of acute convulsion. (A, B) Experiment 1.1: dose response of modified BDS. (C, D) Experiment 1.2: purified CBDV compared with modified BDS. (E, F) Experiment 1.3: comparison of modified CBDV BDS against matching levels of CBDV and CBD. (G, H) Experiment 1.4: BDS-pCB. (A, C, E, G) Maximum observed convulsion severity (median severity in grey, box represents interquartile range, whiskers represent maxima and minima (Kruskal–Wallis test, with a post hoc Mann–Whitney U-tests). (B, D, F, H) Mortality (chi-squared test, with post hoc Fisher exact test); n = 15 for each dose; ^#P ≤ 0.1, *P ≤ 0.05, **P ≤ 0.01. In all panels ‘V’ represents vehicle treatment.

Figure 2

Anticonvulsant and motor effects of modified and unmodified BDSs. (A, B) Experiment 2.1: effect of BDSs on the severity (A) and associated mortality (B) of PTZ-induced convulsion. In (A) median severity is shown in grey, box represents interquartile range and whiskers represent maxima and minima (Kruskal–Wallis test, with a post hoc Mann–Whitney U-tests); in (B), mortality is given as a percentage (chi-squared test, with post hoc Fisher exact test). (C–F) Experiment 2.2: side effect profile of BDSs in motor assays. (C–E) Performance on the static beam assay showing the failure rate (C; chi squared, Fisher exact post hoc test), the mean number of foot slips per metre (D) and the mean distance covered (E) after treatment with BDS or vehicle. (F) Mean forelimb grip strength (kgf). Data in (D–F) presented as mean ± SEM and analysed by anova with Tukey post hoc test. (A, B) n = 15, (C–F) n = 10; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 3

Effects of CBDV BDS modified and unmodified, and isobolographic study of CBDV and CBD. (A, B) Experiments 3.1–3.2: percentage of animals exhibiting each convulsion parameter (WR, wild running; clonic, clonic convulsions; tonic, tonic convulsions; chi-squared test, with post hoc Fisher exact test) for unmodified BDS (A) and modified BDS (B). (C–E) Experiments 3.3–3.4: isobolographic determination of CBDV and CBD interactions. (C) Dose–response relationships of CBD and CBDV. (D) ED₅₀ isobole of CBDV and CBD with theoretical and actual ED₅₀s marked (chi-squared test). (E) Results from co-administration study and predicted results based on individual pCB studies (two-way anova); n = 10; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4

Modified and unmodified CBDV BDS, and purified cannabinoids in the acute pilocarpine model in rat. (A) Experiment 4.1: unmodified CBDV BDS. (B) Experiment 4.2: modified and unmodified CBDV BDS, and matched doses of pure CBDV and CBD. Both panels show maximum observed seizure severity (median severity in grey, box represents interquartile range, whiskers represent maxima and minima; Kruskal–Wallis test, with a post hoc Mann–Whitney U-tests). n = 15; ^#P ≤ 0.1, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 5

Radioligand binding properties of CBDV BDSs. Displacement of [³H]CP55940 by pure CBDV and unmodified CBDV BDS from (A) specific binding sites on MF1 mouse brain membranes and (B) hCB₁-CHO cell membranes. Displacement of [³H]CP55940 by (C) unmodified CBDV BDS and modified CBDV BDS, (D) unmodified CBDV BDS and BDS-pCB plus pure CBDV, (E) unmodified CBDV BDS and BDS-pCB plus purified Δ⁹-THCV, and (F) unmodified CBDV BDS and BDS-pCB plus purified Δ⁹-THC from specific binding sites on MF1 mouse brain membranes. Symbols represent mean values ± SEM. Modified CBDV BDS lacks both Δ⁹-THC and Δ⁹-THCV. n = 4 in all cases.