Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy

Abstract

Background and purpose: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties.

Experimental approach: We used the Scn1a^+/- mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a^+/- mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets.

Key results: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a^+/- mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABA_A receptors.

Conclusion and implications: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class.

Keywords: CBGA; Dravet syndrome; cannabinoids; epilepsy.

FIGURE 1

Phytocannabinoid screen against thermally induced seizures in Scn1a ^+/− mice yields compounds with novel anticonvulsant activity. Change in threshold temperature for generalized tonic–clonic seizure (GTCS) induced by hyperthermia compared with vehicle‐treated controls following acute intraperitoneal (mg·kg⁻¹) administration of varying doses of compounds. Cannabidivarin (CBDV; 30 mg·kg⁻¹), cannabidivarinic acid (CBDVA; 100 mg·kg⁻¹), cannabigerolic acid (CBGA; 30 and 100 mg·kg⁻¹) and cannabigerovarinic acid (CBGVA; 100 mg·kg⁻¹) treatments were anticonvulsant resulting in a significantly improved response to thermal seizure induction (green bars), whereas Δ⁹‐tetrahydrocannabivarin (Δ⁹‐THCV) (3 mg·kg⁻¹) was proconvulsant and significantly lowered the thermal threshold (red bar). Error bars represent SEM, with n = 12–18 per group (^* P < 0.05; log‐rank Mantel–Cox). Treatments where mice were protected from GTCS were as follows: ‐ 0.3‐mg·kg⁻¹ cannabigerol (CBG), four mice seizure free; 10‐mg·kg⁻¹ CBG, one mouse seizure free. All vehicle‐treated mice had seizures. Temperatures of GTCS induction for individual mice, including vehicle‐treated mice, are found in Figure S2

FIGURE 2

Cannabigerolic acid (CBGA) worsens spontaneous seizure frequency but not survival in Scn1a ^+/− mice. (a) Chemical structure of CBGA. (b) Average CBGA plasma concentrations in experimental Scn1a ^+/− mice treated with acute (solid bars) or subchronic (hatched bars) CBGA. Acute treatment of CBGA was administered as a single intraperitoneal injection for hyperthermia‐induced seizure experiments. Subchronic CBGA treatment was administered orally through supplementation in chow for spontaneous seizure and survival experiments. Error bars represent SEM, with n = 4–8 per group. CBGA concentrations are depicted as both mass concentrations (left Y axis) and molar concentrations (right Y axis). (c) Spontaneous generalized tonic–clonic seizure (GTCS) frequency of individual untreated and CBGA‐treated mice. Treatment began after the induction of a single hyperthermia‐induced seizure and spontaneous GTCS frequency was subsequently quantified over a 60‐h recording period. Treatment with the higher dose of CBGA (2500‐mg·kg⁻¹ chow) significantly increased seizure frequency, with n = 15–19 per treatment (^* P < 0.05; one‐way ANOVA followed by Dunnett's post hoc). (d) Survival curves comparing untreated and CBGA‐treated mice. Treatment with CBGA had no effect on survival of Scn1a ^+/− mice, with n = 15–19 per treatment (log‐rank Mantel–Cox, P > 0.05)

FIGURE 3

Cannabigerolic acid (CBGA) enhanced the anticonvulsant effect of clobazam against hyperthermia‐induced seizure in Scn1a ^+/− mice. (a) Temperature threshold for generalized tonic–clonic seizure (GTCS) induced by hyperthermia in individual mice following acute intraperitoneal treatment with CBGA (30 mg·kg⁻¹, blue bar) or clobazam (1 mg·kg⁻¹, light blue bar) administered individually or in combination (dark blue bar). All treatments resulted in a significantly improved response to thermal seizure induction compared with vehicle (P < 0.05; log‐rank Mantel–Cox). Combination CBGA and clobazam treatment was significantly more effective than either treatment alone, with n = 13–16 per group (^* P < 0.05; log‐rank Mantel–Cox). (b) Average CBGA plasma concentrations in Scn1a ^+/− mice from hyperthermia‐induced seizure and spontaneous seizure experiments. CBGA was administered acutely (solid bars) or subchronically (hatched bars) in the presence (blue) and absence (dark blue) of clobazam, with n = 5 per treatment. Significantly higher CBGA plasma levels were observed following acute combination treatment with clobazam (^* P < 0.05; Student's t‐test). CBGA concentrations are depicted as both mass concentrations (left Y axis) and molar concentrations (right Y axis). (c) Spontaneous GTCS frequency of individual untreated and drug‐treated Scn1a ^+/− mice. Treatments were administered orally via supplementation in chow and began at P18 following a hyperthermia‐induced seizure. Clobazam in both the absence and presence of CBGA significantly reduced the frequency of GTCS, with n = 17–19 per group (^* P < 0.05; one‐way ANOVA followed by Dunnett's post hoc). Combination clobazam and CBGA treatment also significantly reduced the proportion of mice experiencing GTCS (^# P < 0.05, Fisher's exact test). Untreated data are replotted from Figure 3c for clarity. (d) Survival curves comparing untreated and drug‐treated mice. Clobazam treatment alone or in combination with CBGA had no effect on survival, with n = 17–19 per group (log‐rank Mantel–Cox). Untreated data are replotted from Figure 2d for clarity. Additional data presented in Figure S3

FIGURE 4

Cannabigerolic acid (CBGA) has divergent anticonvulsant and proconvulsant effects in conventional seizure models. (a) Dose–response of CBGA in the maximal electroshock (MES) acute seizure model. CBGA (30 mg·kg⁻¹) was anticonvulsant and significantly increased the critical current (CC₅₀) at which 50% of the mice exhibit a seizure with maximal hindlimb extension (green bar). Data are expressed as mean ± SEM, with n = 12 per treatment (^* P < 0.05; one‐way ANOVA followed by Dunnett's post hoc). By comparison, 100% of mice treated with the positive control, valproic acid, were protected from hindlimb extension, with n = 12. (b) Dose–response of CBGA in the 6‐Hz seizure model. CBGA was proconvulsant with 10‐ and 30‐mg·kg⁻¹ doses significantly reducing the CC₅₀ for mice to have a psychomotor seizure (red bars). Data are expressed as mean + SEM, with n = 12 per treatment (^* P < 0.05; one‐way ANOVA followed by Dunnett's post hoc). By comparison, 100% of mice treated with valproic acid were protected from psychomotor seizures, with n = 12

FIGURE 5

Cannabigerolic acid (CBGA) affects various epilepsy‐relevant drug targets in vitro. (a) Concentration–response for CBGA inhibition of GPR55 activation measured by ERK phosphorylation (pERK). GPR55 activation was measured following treatment with either LPI (black symbols) or ML‐186 (magenta symbols) in the presence of varying concentrations of CBGA and expressed as a percentage of responses to ligand alone. Data are expressed as mean ± SEM, with n = 6–7 per group. Curves represent fit to a three‐parameter log function and IC₅₀ values are listed. (b) Concentration–response curves for activation of GPR55 by ML‐186 across varying concentrations of CBGA. Data are expressed as mean ± SEM, with n = 6 per group. Curve represents fit to the Hill equation. (c) Concentration–response for CBGA inhibition of TRPV1 determined by a FLIPR calcium influx assay to measure channel activity in cells expressing human TRPV1 channels. TRPV1 activity was measured following treatment with capsaicin (10 μM) in the presence of varying concentrations of CBGA and expressed as a percentage of capsaicin response. Data are expressed as mean ± SEM, with n = 4–8 per group. Curve represents fit to a three‐parameter log function. (d) Concentration–response curve for CBGA at GABA_A receptors expressed in oocytes. CBGA modulates currents evoked by 15 μM GABA. Data are shown as mean ± SEM fit to the Hill equation, with n = 5 per group. (e) Representative trace currents evoked by GABA (1 mM) with (red) and without (black) preincubation of CBGA (10 μM) with varying preincubation times. (f) Time course of CBGA preincubation on GABA_A receptor activity. Currents evoked by 1 mM GABA were measured following varying preincubation times with 10 μM CBGA (red symbols) and without (black symbols) and expressed as a percentage of GABA response. Dotted line represents no change in current. Data are shown as mean ± SEM, with n = 5 per group (^* P < 0.05, Student's t‐test), and curve represents fit to a one‐phase decay. Additional data presented in Figures S4 and S5