Themed Section: Cannabinoids 2012
anticonvulsant in mouse
AJ Hill1,2, MS Mercier1*, TDM Hill1, SE Glyn1, NA Jones1,2,
Y Yamasaki1,2,3, T Futamura3, M Duncan4, CG Stott4, GJ Stephens1,
CM Williams2and BJ Whalley1
1Reading School of Pharmacy,University of Reading,Whiteknights, Reading, UK, 2School of
Psychology and Clinical Language Sciences,University of Reading,Reading, UK, 3Otsuka
Pharmaceutical, Co. Ltd,Tokushima, Japan, and 4GW Pharmaceuticals plc,Porton Down
Science Park,Salisbury, Wiltshire, UK
Andrew Hill, Reading School
of Pharmacy and School of
Psychology and Clinical
Language Sciences, University
of Reading, Whiteknights,
Reading, RG6 6AJ, UK. E-mail:
*Present address: MRC Centre
for Synaptic Plasticity, University
of Bristol, Medical Sciences
Building, University Walk,
Bristol, BS8 1TD, UK.
cannabidivarin; seizure; side
23 April 2012
17 August 2012
28 August 2012
BACKGROUND AND PURPOSE
Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and
several exert notable anticonvulsant effects. For the ﬁrst time, we investigated the anticonvulsant proﬁle of the
phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models.
The effect of CBDV (1–100 mM) on epileptiform local ﬁeld potentials (LFPs) induced in rat hippocampal brain slices by
4-aminopyridine (4-AP) application or Mg2+-free conditions was assessed by in vitro multi-electrode array recordings.
Additionally, the anticonvulsant proﬁle of CBDV (50–200 mg·kg-1)in vivo was investigated in four rodent seizure models:
maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures
in rats. The effects of CBDV in combination with commonly used antiepileptic drugs on rat seizures were investigated. Finally,
the motor side effect proﬁle of CBDV was investigated using static beam and grip strength assays.
CBDV signiﬁcantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg2+-free conditions. CBDV
had signiﬁcant anticonvulsant effects on the mES (ⱖ100 mg·kg-1), audiogenic (ⱖ50 mg·kg-1) and PTZ-induced seizures
(ⱖ100 mg·kg-1). CBDV (200 mg·kg-1) alone had no effect against pilocarpine-induced seizures, but signiﬁcantly attenuated
these seizures when administered with valproate or phenobarbital at this dose. CBDV had no effect on motor function.
CONCLUSIONS AND IMPLICATIONS
These results indicate that CBDV is an effective anticonvulsant in a broad range of seizure models. Also it did not signiﬁcantly
affect normal motor function and, therefore, merits further investigation as a novel anti-epileptic in chronic epilepsy models.
This article is part of a themed section on Cannabinoids. To view the other articles in this section visit
AED, antiepileptic drugs; 4-AP, 4-aminopyridine; CBD, cannabidiol; CBDV, cannabidivarin; DG, dentate gyrus;
ESM, ethosuximide; LFP, local ﬁeld potential; MEA, multi-electrode array; mES, maximal electroshock; PTZ,
pentylenetetrazole; D9-THC, D9-tetrahydrocannabinol; TRP, transient receptor potential; VPA, valproate
BJP British Journal of
British Journal of Pharmacology (2012) 167 1629–1642 1629
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society
Epilepsy is a CNS disorder affecting ~1% of the global popula-
tion, and is symptomatically characterized by chronic, recur-
rent seizures. A range of treatments are available, although
there is still a need for more effective and better-tolerated
antiepileptic drugs (AEDs) as illustrated by the pharmacologi-
cal intractability of ~30% of cases and the poor side effect
proﬁle of currently available AEDs (Kwan and Brodie, 2007).
Cannabis sativa has a long history of use for the control of hu-
man seizures (O’Shaughnessy, 1843; Mechoulam, 1986), and is
legally used for this in some countries (Sirven and Berg, 2004).
There are >100 phytocannabinoids present in C. sativa,of
which D9-tetrahydrocannabinol (D9-THC) is the most abun-
dant (Elsohly and Slade, 2005; Mehmedic et al., 2010) and,
via partial agonism of the CB1cannabinoid receptor, is
responsible for the classical psychoactive effects of cannabis
(Pertwee, 2008). Although CB1cannabinoid receptor agonism
can exert anticonvulsant effects in in vitro and in vivo models
(Chesher and Jackson, 1974; Wallace et al., 2001; 2003; Desh-
pande et al., 2007), the most promising non-psychoactive
anticonvulsant phytocannabinoid investigated to date is can-
nabidiol (CBD), which exerts anticonvulsant actions via an,
as yet unknown, non-CB1cannabinoid receptor mecha-
nism(s) in animal models in vitro,in vivo and in humans
(Cunha et al., 1980; Consroe et al., 1982; Wallace et al., 2001;
Jones et al., 2010); CBD’s notable anticonvulsant properties
led us to investigate the anticonvulsant potential of its propyl
analogue, cannabidivarin (CBDV).
CBDV was ﬁrst isolated in 1969 (Vollner et al., 1969). At
present, little is known about the pharmacological properties
of CBDV (Izzo et al., 2009), although Scutt and Williamson
reported that CBDV acts via CB2cannabinoid receptor-
dependent mechanisms (Scutt and Williamson, 2007). More
recently, De Petrocellis and co-workers reported differential
CBDV effects at transient receptor potential (TRP) channels in
vitro, where it acted as a human TRPA1, TRPV1 and TRPV2
agonist (EC50 values: 0.42, 3.6 and 7.3 mM, respectively) and
a TRPM8 antagonist (IC50: 0.90 mM) (De Petrocellis et al.,
2011a,b). Additionally, CBDV has been shown to inhibit
the primary synthetic enzyme of the endocannabinoid,
2-arachidonoylglycerol (Bisogno et al., 2003), diacylglycerol
lipase a(IC50 16.6 mM) in vitro (De Petrocellis et al., 2011a).
While the pharmacological relevance of these effects has not
been conﬁrmed in vivo, they further illustrate the diversity of
non-D9-THC phytocannabinoid pharmacology and support
the emergent role of multiple non-CB receptor targets
(Pertwee, 2010; Hill et al., 2012).
Here, we identiﬁed anticonvulsant effects of CBDV for the
ﬁrst time; CBDV suppressed in vitro epileptiform activity in
brain slices and acted as an anticonvulsant in vivo. However,
normal motor function was not signiﬁcantly affected by
CBDV, therefore, further investigations into the clinical
development of CBDV as a novel AED are warranted.
In vitro electrophysiology
Tissue preparation. All studies involving animals are reported
in accordance with the ARRIVE guidelines for reporting
experiments involving animals (Kilkenny et al., 2010;
McGrath et al., 2010) and all experiments were carried out in
accordance with Home Ofﬁce regulations [Animals (Scientiﬁc
Procedures) Act, 1986]. Transverse hippocampal slices
(~450-mm thick) for multi-electrode array (MEA) recordings
were prepared from female and male adult Wistar Kyoto rats
(P>21; Harlan, Bicester, UK) using a Vibroslice 725 M
(Campden Instruments Ltd., Loughborough, UK) as previ-
ously described (Jones et al., 2010).
MEA recordings. MEA recordings and analyses were con-
ducted as described in Hill et al. (2010). Once established [by
addition of either 100 mM 4-aminopyridine (4-AP) or omission
of MgSO4.7H2O without substitution], epileptiform activity
was permitted to continue for 30 min (control bursting) before
sequential addition of 1, 10 and 100 mM CBDV (30 min each).
Epileptiform activity was characterized by spontaneous local
ﬁeld potentials (LFPs) recorded simultaneously from 59 elec-
trodes covering the majority of the hippocampal slice prepa-
ration. The amplitude and duration of epileptiform LFPs were
analysed for each electrode. Data from individual electrodes,
based on their position in each hippocampal subregion, were
pooled to provide mean results for each subregion across nⱖ
5 slices from nⱖ5 animals per model. Matlab 6.5 and 7.0.4
(Mathworks, Natick, MA, USA), Microsoft Excel (Microsoft,
Redmond, WA, USA), MC_DataTool and MC_Rack (Multi
Channel Systems GmbH, Reutlingen, Germany) were used to
process and present data as described in Hill et al. (2010).
Inherent changes in LFP amplitude and frequency were cor-
rected for, as described previously (Hill et al., 2010). For refer-
ence, the extent of amplitude rundown correction applied is
illustrated in Figure 1C and D. LFP frequency was calculated
per slice (nⱖ5 for each model) and represents the number of
LFP bursts per unit time. Examples of single bursts from each
model can be seen in Figure 1A and B. Drug-induced changes
in burst duration, amplitude and frequency are expressed as
normalized proportions of control values ⫾SEM, corrected
where necessary, and were analysed by Wilcoxon’s paired test
with Holm’s sequential Bonferroni correction.
In vivo seizure models
Animals. In all cases before seizure induction, animals were
maintained on a 12 h light/dark cycle with free access to food
and water (with the exception of rats that received oral CBDV,
see later). Audiogenic seizure experiments with dilute, brown,
non-Agouti (DBA/2) mice (3–4 weeks old; Elevage Janvier, Le
Genest-Saint-Isle, France) were performed at Porsolt Research
Laboratory (Le Genest-Saint-Isle, France) in accordance with
French legislation and under licence from the French Minis-
try for Agriculture and Fisheries. mES experiments with
ICR (CD-1) mice (5 weeks old; SLC Japan Inc., Shizuoka,
Japan) were performed at Otsuka Pharmaceuticals Co, Ltd.
(Tokushima, Japan) in accordance with the guidelines of the
Physiological Society of Japan. In total, 80 mice were used.
Seizure studies in male Wistar Kyoto rats (Harlan, 3–4 weeks
old; in total, 640 rats were used) were performed at the Uni-
versity of Reading, UK; all experiments were carried out in
accordance with UK Home Ofﬁce regulations [Animals (Sci-
entiﬁc Procedures) Act 1986].
CBDV administration. CBDV (50, 100 or 200 mg·kg-1;GW
Pharmaceuticals Ltd., Salisbury, UK) in an ethanol : Cremo-
BJP AJ Hill et al.
1630 British Journal of Pharmacology (2012) 167 1629–1642
phor: saline (0.9% w v-1NaCl vehicle; 2:1:17; all Sigma, Poole,
UK) was administered by an i.p. injection 1 h before seizure
induction in all the models, with the exception of mES where
it was administered 30 min before seizure induction. All
experiments included a control group, which received
volume-matched vehicle, against which other groups were
assessed. In mice experiments, n=10 per group and in rat,
n=15 per group. In experiments where CBDV was adminis-
tered p.o. (gavage), 400 mg·kg-1CBDV or volume-matched
vehicle [20% solutol (Sigma) in 0.9% w v-1NaCl] was admin-
istered after the animals had been deprived of food for 13.5 h
and 3.5 h before i.p. administration of pentylenetetrazole
(PTZ), n=15 for both groups (see Supporting Information
Appendix S1 for details on oral dose levels).
Seizure induction. mES seizures were induced in mice by a
stimulator (Ugo Basile ECT, Comerio, Italy) via earlap
clamps at a current of 30 mA delivered at 100 Hz for
200 ms. DBA/2 mice were placed in a Plexiglas jar 1 h after
CBDV/vehicle administration. A mounted bell (110–
120 dB) was activated until occurrence of a tonic audiogenic
seizure or for a maximum of 60 s. To induce generalized
seizures in rats, 85 mg·kg-1PTZ was injected i.p. Status epi-
lepticus with a temporal lobe focus was induced in rats by
injecting pilocarpine hydrochloride (Sigma; in 0.9% w v-1
NaCl) 380 mg·kg-1i.p., 45 min after pretreating the rats
with methylscopolamine (Sigma; in 0.9% w v-1NaCl)
1 mg·kg-1i.p., which blocks the peripheral effects of
Seizure analysis. In mES experiments, mice were observed for
10 s during electroshock, tonic hindlimb extension occur-
rence was noted and expressed as a percentage of the total
number of animals for each group. Audiogenic seizure behav-
iour was observed visually, while rat seizures were video
recorded (Farrimond et al., 2009). For audiogenic seizures, the
incidence (as a percentage) of the most severe (tonic–clonic)
seizures, mortality and seizure-free animals were calculated
for each group. These parameters, as well as seizure duration
and severity, were also determined for rat seizures. Rat behav-
iour was coded blind ofﬂine using The Observer Pro software
(Noldus, Wageningen, The Netherlands) and seizure severity
scales appropriate to each seizure type (Table 1). Values are
expressed as mean ⫾SEM throughout.
Co-administration experiments. The effect of co-
administration of clinically-used AEDs with 200 mg·kg-1
CBDV on PTZ- and pilocarpine-induced seizures was investi-
gated. For details, see Supporting Information Appendix S1.
Brieﬂy, in each experiment, an AED was administered i.p., at
either ~20, ~40 or ~70% maximal effective dose, in the
absence or presence of 200 mg·kg-1CBDV (n=15 per group,
120 per experiment); the convulsant (PTZ or pilocarpine) was
administered 1 h after CBDV or its vehicle. The experimental
design is illustrated and summarized in Table 2. In the PTZ
model, CBDV was co-administered with valproate (VPA) or
ethosuximide (ESM) before PTZ, and with VPA or phenobar-
bital (PB) before pilocarpine. These AEDs were chosen based
on their clinical proﬁle and their reported efﬁcacy in the
models used here, with VPA suppressing both seizure types
and ESM and phenobarbital suppressing PTZ and pilocarpine
respectively (Loscher et al., 1991; Soﬁa et al., 1993; Shantilal
et al., 1999; Lindekens et al., 2000; Loscher, 2011). In co-
administration experiments, seven (2.9%) rats exhibited a
fatal reaction to CBDV administration. Behaviourally, this
manifested as rapid development (within 300 s) of lethargic
convulsive movements followed by death. Overall, across all
PTZ and pilocarpine experiments, this effect was seen in 2.6%
of all rats that received 200 mg·kg-1CBDV, but not at all in
side effect tests. No adverse effects of other CBDV doses were
observed in rats, and none at any dose in mice. The animals
that died were omitted from all analyses.
Statistics. In experiments where i.p. CBDV alone was admin-
istered, the effects of CBDV on seizure severity, onset latency
and seizure duration were assessed by one-way ANOVA with
post hoc Tukey’s tests as appropriate. Chi-squared tests fol-
lowed by post hoc Fisher’s exact tests were used where appro-
Seizure behaviour scoring scales for PTZ and pilocarpine-induced seizures
Score PTZ-induced seizures Pilocarpine-induced seizures
0 Normal behaviour Normal behaviour
1 Isolated myoclonic jerks Mouth clonus
2 Atypical clonic seizure Unilateral forelimb clonus
3 Fully developed bilateral forelimb clonus Bilateral forelimb clonus
3.5 Forelimb clonus with tonic component and body twist NA
4 Tonic–clonic seizure with suppressed tonic phase* Bilateral forelimb clonus with rearing and falling
4.5 NA Tonic–clonic seizure with postural control retained
5 Fully developed tonic–clonic seizure* Tonic–clonic seizure*
Seizure severity scoring scales are shown for each model, although no equivalency of severity should be assumed between scales for different
*Indicates a loss of righting reﬂex.
NA =not applicable.
Cannabidivarin as an anticonvulsant
British Journal of Pharmacology (2012) 167 1629–1642 1631
priate to assess differences in incidence parameters. Where
CBDV was co-administered with an AED, two-way ANOVA or
log-linear modelling was used to analyse the effects of CBDV
and AEDs. Log-linear modelling was used to model the inter-
actions between drug co-administration and incidence
parameters (e.g. mortality, % seizure-free). If the model indi-
cated a signiﬁcant effect of drug treatment, further analysis to
determine the contribution of CBDV, the relevant AED and
any drug ¥drug interaction was performed; these analyses are
given in the text and in Supporting Information Tables S1
Motor function assays
The effects of CBDV (50, 100 and 200 mg·kg-1) and VPA (125,
250 and 350 mg·kg-1) on normal rat motor function were
assessed on a 1 m raised static beam and by a grip strength
test (see Supporting Information Appendix S1 for details).
All receptor and ion channel nomenclature conforms to
BJP’s Guide to Receptors and Channels (Alexander et al., 2011).
Effects of pure CBDV in the Mg2+-free and
4-AP in vitro models of epileptiform activity
The effects of CBDV (1–100 mM) on epileptiform activity,
induced by Mg2+-free aCSF (Figure 1A) or 100 mM 4-AP
(Figure 1B), in rat acute hippocampal slices were examined.
CBDV signiﬁcantly decreased the amplitude and duration of
epileptiform LFPs induced by Mg2+-free aCSF (Figure 1C and
D; Pⱕ0.05); signiﬁcant effects were seen at ⱖ10 mM, and the
CA3 region was more resistant to the effects of CBDV than
the dentate gyrus (DG) or CA1 (Figure 1C and D). Conversely,
CBDV signiﬁcantly increased Mg2+-free-induced LFP fre-
quency (ⱖ10 mM; Figure 1E; Pⱕ0.05).
An anti-epileptiform effect of 100 mM CBDV on the
amplitude of 4-AP-induced epileptiform LFPs was observed in
the CA1 region alone (Figure 1F; Pⱕ0.05), whereas LFP
duration was signiﬁcantly lowered in all hippocampal regions
by ⱖ10 mM CBDV (Figure 1G) and, by contrast to the Mg2+-
free model, 4-AP-induced LFP frequency was signiﬁcantly
decreased by all CBDV concentrations tested (Figure 1H;
Pⱕ0.05). Thus, CBDV attenuated the duration of amplitude
of LFPs in both models, and had differential effects on
Effects of CBDV on maximal electroshock
(mES) and audiogenic seizures in mice
The effects of CBDV (50–200 mg·kg-1) on mES convulsions
and audiogenic seizures in mice were investigated. CBDV had
a signiﬁcant anticonvulsant effect on animals displaying
tonic hindlimb extension after mES [c2(3) =15.000; Pⱕ
0.001; Figure 2A]; signiﬁcantly fewer animals that received
100 or 200 mg·kg-1CBDV exhibited hindlimb extension
(both groups 30%) than those that received vehicle (90%,
Figure 2A; Pⱕ0.001 vs. vehicle-treated group). Audiogenic
seizures were also signiﬁcantly attenuated by CBDV
(Figure 2B–D). The incidence of tonic convulsions was signiﬁ-
cantly lower after CBDV administration [c2(3) =19.436, Pⱕ
0.001; Figure 2B]; 80% of vehicle-treated animals developed
tonic convulsions compared with only 20% (50 mg·kg-1
CBDV), 10% (100 mg·kg-1CBDV) and 0% (200 mg·kg-1
CBDV) after drug treatment (each Pⱕ0.001 vs vehicle). The
percentage of animals that remained seizure-free was signiﬁ-
cantly higher after administration of 200 mg·kg-1CBDV
(90%) than vehicle [0%; c2(3) =27.461, Pⱕ0.001; Figure 2C].
Finally, a statistical trend was observed for the mortality rate
[c2(3) =6.667, Pⱕ0.1], with lower mortality after 100 and
200 mg·kg-1CBDV treatment than vehicle (0% vs 30%,
respectively; Figure 2D). Thus, CBDV exhibits strong and sig-
niﬁcant anticonvulsant effects in two broad-screen mouse
Experimental design and time course of co-administration experiments
CBDV/vehicle treatment (i.p.)
(min) AED treatment (i.p.)
200 mg·kg-1CBDV (n=60) 30 VPA vehicle, 50, 100, 250 mg·kg-1VPA (n=15 each) 30 85 mg·kg-1PTZ
CBDV vehicle (n=60) VPA vehicle, 50, 100, 250 mg·kg-1VPA (n=15 each)
200 mg·kg-1CBDV (n=60) 30 ESM vehicle, 60, 120, 175 mg·kg-1ESM (n=15 each) 30
CBDV vehicle (n=60) ESM vehicle, 60, 120, 175 mg·kg-1ESM (n=15 each)
200 mg·kg-1CBDV (n=60) 15 VPA vehicle, 62.5, 125, 250 mg·kg-1VPA (n=15 each) 45 380 mg·kg-1
CBDV vehicle (n=60) VPA vehicle, 62.5, 125, 250 mg·kg-1VPA (n=15 each)
200 mg·kg-1CBDV (n=60) 15 PB vehicle, 10, 20, 40 mg·kg-1PB (n=15 each) 45
CBDV vehicle (n=60) PB vehicle, 10, 20, 40 mg·kg-1PB (n=15 each)
‘Time A’ column: time between CBDV/CBDV vehicle and AED administration. ‘Time B’ column: time between AED/vehicle and convulsant.
The duration of the seizure recording is indicated in the ﬁnal column. PB, phenobarbital, VPA, valproate, ESM, ethosuximide.
BJP AJ Hill et al.
1632 British Journal of Pharmacology (2012) 167 1629–1642
seizure models. Next, we investigated the anticonvulsant
potential of CBDV in two further models of seizure in rat that
emulate more speciﬁc seizure types.
Effects of CBDV on PTZ- and
pilocarpine-induced seizures in rats
CBDV signiﬁcantly decreased PTZ seizure severity (F3,58 =
4.423, Pⱕ0.05; Figure 3A); the median seizure severity after
vehicle administration was tonic–clonic convulsion score 5,
but after 200 mg·kg-1CBDV administration seizure severity
was signiﬁcantly lowered to a median severity of bilateral
clonic convulsion score 3 (Pⱕ0.05). CBDV also signiﬁcantly
reduced mortality (c2(3) =10.356, Pⱕ0.05; Figure 3B) at 100
and 200 mg·kg-1CBDV (Pⱕ0.01). The percentage of animals
that remained seizure-free was signiﬁcantly increased by
CBDV administration [c2(3) =7.809, Pⱕ0.05; Figure 3C];
33.3% of animals that received 200 mg·kg-1CBDV exhibited
no signs of seizure compared with only 6.7% of animals that
received vehicle (Pⱕ0.01). Furthermore, seizure onset was
signiﬁcantly delayed by CBDV treatment (F3,50 =2.971, Pⱕ
0.05; Figure 3D); mean onset latency was signiﬁcantly longer
after administration of 200 mg·kg-1CBDV than vehicle (65 ⫾
11 s and 40 ⫾4 s, respectively; Pⱕ0.05). Thus, CBDV,
administered alone, exhibited strong and signiﬁcant anticon-
vulsant effects on PTZ seizures at 200 mg·kg-1(Figure 3A–D)
with more limited, but signiﬁcant, effects at 100 mg·kg-1
Effects of CBDV on hippocampal epileptiform activity. (A and B) Example traces showing effects of 100 mM CBDV on epileptiform LFPs induced
by Mg2+-free conditions (A) or 100 mM 4-AP (B) in the CA1 region. The black bar represents amplitude as corrected for inherent rundown (see
Methods); the dotted line below represents control burst duration. Scale in (A): 100 mV/200 ms; (B): 150 mV/200 ms. (C–H) Effects of CBDV on
amplitude (C and F), duration (D and G) and frequency (E and H) of epileptiform LFPs induced by Mg2+-free conditions (C–E) or 100 mM 4-AP
(F–H). Data are presented as mean ⫾SEM normalized to control (pre-drug) conditions and corrected for background changes where appropriate
(see Methods). LFP amplitude and duration values are expressed for each hippocampal region as in the key. n=9–12. *Pⱕ0.05, **Pⱕ0.01 and
Cannabidivarin as an anticonvulsant
British Journal of Pharmacology (2012) 167 1629–1642 1633
We extended our studies to investigate the effects of
CBDV (50–200 mg·kg-1) on the convulsions associated with
pilocarpine-induced status epilepticus (380 mg·kg-1). CBDV
(50–200 mg·kg-1) had no signiﬁcant effect on the severity
(F3,59 =0.049, P>0.1; Figure 3E) or resultant mortality of
pilocarpine convulsions [c2(3) =1.779, P>0.1; Figure 3F].
Similarly, CBDV did not signiﬁcantly affect the percentage of
animals that remained seizure-free [c2(3) =0.110, P>0.1;
Figure 3G] or the latency to the onset of convulsions (F3,53 =
0.404, P>0.1; Figure 3H).
Effect of co-administration of CBDV and
AEDs on PTZ- and pilocarpine-induced
seizures in rats
We investigated the effects of CBDV when co-administered
with AEDs before PTZ or pilocarpine treatment. The effects of
combined drug treatment (CBDV +AED) on seizure param-
eters are illustrated in Figures 4 and 5, as is the contribution
of CBDV to these effects. The contribution of AEDs is illus-
trated in Figures 4 and 5 while statistical analyses of AED
effects and any interaction between CBDV and AEDs are
shown in Supporting Information Tables S1 and S2.
CBDV 200 mg·kg-1was co-administered with VPA (50–
250 mg·kg-1) or ESM (60–175 mg·kg-1). In the CBDV +VPA
experiments, drug co-administration had signiﬁcant anti-
convulsant effects on all seizure parameters except the per-
centage of animals remaining seizure-free. CBDV and VPA
co-administration signiﬁcantly decreased seizure severity
(F7,112 =10.449, Pⱕ0.001; Figure 4A). When modelled
by log-linear analyses, our data indicated that drug
co-administration decreased mortality (Figure 4B) and the
incidence of the most severe (tonic–clonic) seizures
(Figure 4C). Seizure onset was signiﬁcantly delayed by drug
co-administration (F7,109 =13.285, Pⱕ0.001; Figure 4D) and
the mean duration of seizures was increased (F7,103 =5.250,
Pⱕ0.001). VPA contributed signiﬁcantly to all these effects
(Figure 4A–D, Supporting Information Table S1). CBDV sig-
niﬁcantly contributed to the overall decrease in severity
(F1,112 =5.748, Pⱕ0.05; Figure 4A) and mortality [c2(1) =
6.639, Pⱕ0.01; Figure 4B] and the increase in onset latency
(F1,109 =7.393, Pⱕ0.01; Figure 4C). CBDV did not signiﬁ-
cantly affect tonic–clonic seizure incidence (Figure 4D) or
seizure duration (P>0.1). No effect of drug treatment on the
number of seizure-free animals was observed [X2(14) =8.930,
P>0.1] and no signiﬁcant positive or negative interactions
between the effects of 200 mg·kg-1CBDV and VPA were
observed (Supporting Information Tables S1, P>0.1).
Co-administration of 200 mg·kg-1CBDV and ESM (60–
175 mg·kg-1) had signiﬁcant anticonvulsant effects on all
parameters of PTZ-induced seizures: CBDV and ESM
co-administration signiﬁcantly decreased seizure severity
(F7,110 =12.556, Pⱕ0.001; Figure 4E), when modelled with
log-linear analysis, our data indicated that co-administration
also decreased mortality (Figure 4F) and the incidence of the
most severe seizures (Figure 4G). Seizure onset latency was
signiﬁcantly increased (F7,76 =7.885, Pⱕ0.001; Figure 4H), as
was the percentage of animals that remained seizure-free (log-
linear model; Figure 4I); seizure duration was also signiﬁ-
cantly decreased (F7,102 =6.934, Pⱕ0.001). ESM signiﬁcantly
Effects of CBDV on mES and audiogenic seizures in mice. (A) The effect of CBDV on the percentage of animals that exhibited tonic hindlimb
extension in response to mES. (B–D) The effect of CBDV (50–200 mg·kg-1) on the percentage of animals that displayed tonic convulsions (B),
remained seizure-free (C) or suffered mortality (D) as a result of audiogenic seizure induction. n=10 in all cases, ***Pⱕ0.001.
BJP AJ Hill et al.
1634 British Journal of Pharmacology (2012) 167 1629–1642
contributed to all anticonvulsant effects (Figure 4E-I; Sup-
porting Information Table S1). CBDV contributed signiﬁ-
cantly to the overall decreases in seizure severity (F1,112 =
7.474, Pⱕ0.01; Figure 4E) and mortality [c2(1) =5.174, Pⱕ
0.05; Figure 4F]; the contribution of CBDV to the increase in
onset latency showed a statistical trend (F1,76 =2.791, Pⱕ0.1;
Figure 4H). CBDV did not signiﬁcantly contribute to the
effects on seizure duration, the proportion of animals that
remained seizure-free (both P>0.1) or the incidence of the
most severe seizures (P>0.1; Figure 4G). No signiﬁcant
positive or negative interactions between the effects of
200 mg·kg-1CBDV and ESM were observed (Supporting Infor-
mation Tables S1, P>0.1).
We next investigated whether 200 mg·kg-1CBDV affected
the anticonvulsant actions of VPA or phenobarbital
on pilocarpine-induced convulsions. Interestingly, these
co-administration experiments highlighted signiﬁcant anti-
convulsant effects of 200 mg·kg-1CBDV not previously
observed when CBDV was administered alone. Co-
administration of VPA (50–250 mg·kg-1) with 200 mg·kg-1
CBDV had signiﬁcant anticonvulsant effects on all the
parameters except the percentage of animals that remained
convulsion-free: CBDV and VPA co-administration signiﬁ-
cantly decreased severity (F7,100 =16.477, Pⱕ0.001;
Figure 5A); when modelled by log-linear analysis, our data
indicated that mortality (Figure 5B) and the incidence of the
most severe (tonic–clonic) convulsions (Figure 5C) were also
decreased by drug co-administration; onset latency was
signiﬁcantly increased (F7,105 =8.649, Pⱕ0.001; Figure 5D).
VPA contributed signiﬁcantly to all anticonvulsant effects
(Figure 5A-D, Supporting Information Table S2) with the
interesting exception of mortality. Mortality was higher (but
not signiﬁcantly so) when 62.5 and 125 mg·kg-1VPA were
co-administered with vehicle (Figure 5B); however, CBDV
had an anticonvulsant effect, signiﬁcantly decreasing mortal-
ity compared with administration of its vehicle [c2(1) =4.010,
Pⱕ0.05; Figure 5D]. CBDV also signiﬁcantly contributed to
the overall anticonvulsant effects of treatment on severity
(F1,110 =22.711, Pⱕ0.001; Figure 5A) and the incidence of
tonic–clonic convulsions [c2(1) =4.010, Pⱕ0.01; Figure 5C],
although it had no signiﬁcant effect on onset latency
(P>0.1; Figure 5D). The percentage of animals that remained
convulsion-free [c2(6) =1.564, P>0.1] was unaffected by
treatment. No signiﬁcant interactions between CBDV and
VPA effects were observed (Supporting Information Tables S2,
Co-administration of 200 mg·kg-1CBDV and phenobarbi-
tal (10–40 mg·kg-1) had signiﬁcant anticonvulsant effects on
the severity of pilocarpine-induced convulsions (F7,108 =
19.352, Pⱕ0.001; Figure 5E). When modelled with log-linear
analysis, our data indicated that there was no effect of treat-
ment on mortality (Figure 5F), whereas the percentage of
animals that developed tonic–clonic convulsions was signiﬁ-
cantly decreased (Figure 5G). No effect of drug treatment
Effects of CBDV on PTZ- and pilocarpine-induced seizures in rats. (A–D) The effect of CBDV on PTZ-induced seizures: seizure severity (A), mortality
(B), the proportion of animals remaining seizure-free (C) and the onset latency (D). (E–H) The effect of CBDV on pilocarpine-induced convulsions:
severity (E), mortality (F), the percentage of animals remaining seizure-free (G) and the onset latency (H). In (D and H), onset latency is presented
as mean ⫾SEM In (A and E), median severity is represented by a thick horizontal line, the 25th and the 75th percentiles by the box and maxima
and minima are represented by ‘whiskers’. n=15 in all cases. *Pⱕ0.05, **Pⱕ0.01 and ***Pⱕ0.001.
Cannabidivarin as an anticonvulsant
British Journal of Pharmacology (2012) 167 1629–1642 1635
was observed on seizure onset latency (P>0.1; Figure 5H);
however, when modelled with log-linear analysis, our data
indicated that the percentage of animals that remained
convulsion-free was signiﬁcantly increased (Figure 5I). Phe-
nobarbital signiﬁcantly contributed to all anticonvulsant
effects (Figure 5E–I; Supporting Information Table S2). CBDV
signiﬁcantly contributed to the overall decrease seen in sever-
ity (F1,108 =4.480, Pⱕ0.05), and the effects of CBDV and
phenobarbital interacted signiﬁcantly due to a convergence
of the severity observed in the absence and presence of CBDV
(Figure 5F, Supporting Information Table S2; F3,108 =3.105,
Pⱕ0.05), no further signiﬁcant interactions between the
effects of CBDV and phenobarbital were observed (P>0.1;
Supporting Information Table S2).
Data from the co-administration experiments demon-
strate that the AEDs strongly suppress PTZ-induced seizures
and pilocarpine-induced convulsions in a dose-dependent
manner (Figures 4 and 5). From several, but not all, of the
parameters examined, 200 mg·kg-1CBDV signiﬁcantly con-
tributed to the anticonvulsant effects observed in these
experiments. To more precisely assess the effect of CBDV on
AED actions in these studies, we performed pairwise compari-
sons at each dose of AED between groups that received CBDV
vehicle and groups that received 200 mg·kg-1CBDV; these
analyses were only performed if two-way ANOVA or log-
linear analysis results indicated an overall effect of CBDV
upon a given parameter. Based on these analyses and
Figure 5F–I, the effect of CBDV on the actions of phenobar-
bital in the pilocarpine model appears limited and is not
signiﬁcant. Similarly, the effect of CBDV on the actions of
VPA in the PTZ model was limited (Figure 4A–D); the primary
effect of CBDV is on delaying seizure onset, as 200 mg·kg-1
CBDV signiﬁcantly improved the effect of 50 mg·kg-1VPA
(Pⱕ0.05; Figure 4D) and showed a statistical trend towards
the same effect with 100 mg·kg-1VPA (P<0.1). More notably,
CBDV signiﬁcantly improved the effect of 60 mg·kg-1ESM on
PTZ-induced seizure severity and onset latency (Pⱕ0.05;
Figure 4E and H) and also showed a statistical trend to
improvement of the 120 mg·kg-1ESM effect for both these
measures (P<0.1). Furthermore, when 200 mg·kg-1CBDV
was administered together with VPA before pilocarpine
administration, it signiﬁcantly improved the effects of VPA
on severity (62.5 and 250 mg·kg-1;Pⱕ0.05), mortality (62.5
and 125 mg·kg-1;Pⱕ0.05) and the percentage of animals
that experienced the most severe seizures (all doses, Pⱕ0.01;
Thus, CBDV is well-tolerated when co-administered with
AEDs and does not interact antagonistically with any of the
AEDs studied in either seizure model. Furthermore, CBDV has
signiﬁcant anticonvulsant effects when co-administered with
ESM in the PTZ model and even greater effects when
co-administered with VPA in the pilocarpine model, where
beneﬁcial effects were generally observed at low and medium
AED doses. CBDV did not affect the effects of phenobarbital
Effects of co-administration of CBDV and AEDs on PTZ-induced seizures in rats. The effects of CBDV co-administration with VPA (A–D) or ESM (E–I)
on PTZ-induced seizures: severity (A and E), mortality (B and F), the incidence of tonic–clonic seizures (C and G), onset latency (D and H) and (for
CBDV +ESM only) the percentage of animals that remained seizure-free. In (D and H), onset latency is presented as mean ⫾SEM. In (A and E),
median severity is represented by a thick horizontal line, the 25th and 75th percentiles by the box and maxima and minima are represented by
‘whiskers’. Signiﬁcance of CBDV treatment is given in text. n=15 in all cases. *Pⱕ0.05, **Pⱕ0.01 and ***Pⱕ0.001 for AED effects.
BJP AJ Hill et al.
1636 British Journal of Pharmacology (2012) 167 1629–1642
in the pilocarpine model and had only very limited effects on
the onset of seizures when co-administered with VPA before
CBDV motor side effect proﬁle and
anticonvulsant efﬁcacy when
To further determine the suitability of CBDV as a clinical
candidate, we assessed both its motor side effect proﬁle and
whether it could suppress seizures when administered p.o.
before PTZ treatment. Many currently used AEDs have sig-
niﬁcant side effects at clinically effective doses, particularly
on motor function (Schachter, 2007). Additionally, a prereq-
uisite for human epilepsy treatment is that a drug is effective
after oral administration.
We used two motor tasks to investigate the side effect
proﬁle of CBDV (50–200 mg·kg-1): a static beam test to assess
motor coordination (Stanley et al., 2005; Roberts et al., 2006)
and a grip strength test to assess drug-induced muscle relaxa-
tion and functional neurotoxicity (Nevins et al., 1993;
Crofton et al., 1996). CBDV had no signiﬁcant effects on
motor performance at any dose compared with vehicle treat-
ment (Figure 6A–D). In the static beam assay, the pass rate
[c2(3) =4.053; P>0.1; Figure 6A] and mean distance travelled
(F3,79 =1.335; P>0.1; data not shown) were both unaffected
by CBDV. CBDV had no signiﬁcant overall effect on the mean
number of foot slips (F3,79 =0.858; P>0.1; Figure 6B),
although we did note a non-signiﬁcant increase in foot slips
in animals treated with 200 mg·kg-1CBDV (0.70 ⫾0.25 slips,
compared with 0.30 ⫾0.11 slips after vehicle treatment).
CBDV had no effect on grip strength (F3,79 =0.465; P>0.1,
Figure 6C). To validate the tests’ ability to detect AED-
induced motor deﬁcits, a second group of animals received
VPA (125–350 mg·kg-1) or saline vehicle. VPA signiﬁcantly
affected the percentage of animals that successfully com-
pleted the static beam test [c2(3) =35.084; Pⱕ0.001;
Figure 6A], with doses ⱖ250 mg·kg-1signiﬁcantly decreasing
the pass rate (Pⱕ0.01). Similarly, both the number of foot
slips (F3,78 =9.140; Pⱕ0.001; Figure 6B) and the mean dis-
tance travelled (F3,78 =15.561; Pⱕ0.001; data not shown)
were signiﬁcantly, negatively and dose-dependently affected
by treatment with ⱖ250 mg·kg-1VPA (Pⱕ0.01). VPA also
signiﬁcantly affected the grip strength of animals (F3,79 =
3.175; Pⱕ0.05; Figure 6C), with a small, but signiﬁcant
decrease in mean strength induced by 350 mg·kg-1VPA
Finally, we investigated the ability of 400 mg·kg-1CBDV
administered p.o. (see Supporting Information Appendix S1
for dose details) to suppress PTZ seizures (90 mg·kg-1);
Effects of co-administration of CBDV and AEDs on pilocarpine-induced convulsions in rats. The effects of CBDV co-administration with VPA (A–D)
or phenobarbital (E–I) on pilocarpine-induced convulsions: severity (A and E), mortality (B and F), the incidence of tonic–clonic convulsions (C and
G), onset latency (D and H) and (for CBDV +phenobarbital only) the percentage of animals that remained seizure-free. In (D and H), onset latency
is presented as mean ⫾SEM In (A and E), median severity is represented by a thick horizontal line, the 25th and 75th percentiles by the box and
maxima and minima are represented by ‘whiskers’. Signiﬁcance of CBDV treatment is given in text. n=15 in all cases. *Pⱕ0.05, **Pⱕ0.01 and
***Pⱕ0.001 for AED effects.
Cannabidivarin as an anticonvulsant
British Journal of Pharmacology (2012) 167 1629–1642 1637
400 mg·kg-1CBDV signiﬁcantly lowered the severity of PTZ-
induced seizures (Figure 6D, Pⱕ0.05) from 5 to 3.5. There
were no signiﬁcant effects of CBDV on seizure onset latency
(vehicle 58.6 ⫾3.7 s; CBDV 61.8 ⫾5.2 s; P>0.1), percentage
mortality (vehicle 26.7%; CBDV 20%; P>0.1) or develop-
ment of tonic–clonic seizures (vehicle 53.3; CBDV 33.3; P>
0.1). Overall, we demonstrated that the anticonvulsant
effects of CBDV in rat are due to genuine anticonvulsant
properties and not motor suppression, and that CBDV is
anticonvulsant when administered p.o. as well as i.p. in the
This study demonstrates, for the ﬁrst time, that CBDV has
anticonvulsant properties, and, to date, is the only study that
has investigated the effects of CBDV in whole animals. Our
main ﬁnding is that CBDV suppresses seizures in four in vivo
seizure models at doses ⱖ50 mg·kg-1. CBDV also did not affect
normal motor function and was well-tolerated when
co-administered with AEDs. Moreover, CBDV suppressed epi-
leptiform activity in vitro.
In vitro effects of CBDV
In both in vitro models of epileptiform activity, LFP duration
and amplitude were signiﬁcantly decreased by CBDV, with
efﬁcacy varying between hippocampal subregions and
models. The CA3 region was most resistant to CBDV effects,
potentially due to its role as the epileptiform focus (Perreault
and Avoli, 1992; Hill et al., 2010). It has also been reported
that a smaller proportion of neurons in the CA1 contribute to
burst activity than in the CA3 (Perreault and Avoli, 1992),
potentially rendering the CA1 region more sensitive to the
effects of anti-epileptiform drugs. CBDV effects on LFP fre-
quency in the two models were opposite; CBDV increased
Mg2+-free-induced LFP frequency, but decreased 4-AP-induced
LFP frequency. This may be due to a genuine, model-
dependent CBDV effect on LFP frequency; however, the
response of frequency in the Mg2+-free model is in direct
contrast to all other ﬁndings across both models, where
varying degrees of anti-epileptiform effects were observed. In
addition, we have observed that LFPs in the Mg2+-free model
exhibit greater variation in frequency than the 4-AP model;
sporadic bursts of LFPs occur with periods of relative quies-
cence between them (see Hill et al., 2010). Thus, while the
frequency of LFPs in this Mg2+-free model was corrected to
allow for inherent increases, it may be that the unpredictabil-
ity of LFP incidence limits the accuracy of this process.
Overall, the magnitude of the effects of CBDV on LFP ampli-
tude and duration are comparable with those observed with
both CBD and clinically used AEDs (Sagratella, 1998; Hill
et al., 2010; Jones et al., 2010).
In vivo effects of CBDV and
We demonstrated that CBDV has signiﬁcant anticonvulsant
effects in four seizure models with different bases across two
species. CBDV was effective in three models of generalized
seizure – mES and audiogenic in mice and PTZ in rats. In
particular, CBDV (200 mg·kg-1) completely prevented tonic–
clonic convulsions in the audiogenic seizure model and had
robust effects in the mES model, in line with the reported
Effects of CBDV on performance in the static beam and forelimb grip strength assays in rat and as an orally administered anticonvulsant. (A and
B): static beam performance; including the pass rate (A) and foot slips (B). (C) Performance in the grip strength assay. (A) Pass rate is represented
as percentage; (B and C), represented as mean ⫾SEM n=20 for static beam data, 10 for grip strength. (D) Effect of orally administered
400 mg·kg-1CBDV on the severity of PTZ-induced seizures. (A–C): n=20, (D): n=15. *Pⱕ0.05, **Pⱕ0.01 and ***Pⱕ0.001 respectively. (A–C):
V=CBDV vehicle; S =VPA vehicle (saline).
BJP AJ Hill et al.
1638 British Journal of Pharmacology (2012) 167 1629–1642
efﬁcacy of VPA and other AEDs in these models (Gareri et al.,
2004; Luszczki et al., 2011; 2012). Moreover, positive ﬁndings
in the mES model – a primary screen for putative anticonvul-
sants (Loscher, 2011) – are predictive of clinical efﬁcacy
against generalized human seizures (Loscher, 2011). Audio-
genic seizures, although providing limited predictive differ-
entiation of future efﬁcacy against human seizure types
(Loscher, 2011), are also a useful model of generalized seizure
(Pitkanen et al., 2006). Attenuation of PTZ-induced seizures
can be predictive of efﬁcacy against absence seizures, as well
as predicting effective suppression of generalized seizures in
humans (Veliskova, 2006). Hence, CBDV should also be
investigated in non-convulsive seizure models (e.g. WAG/Rjj
rats; Coenen and Van Luijtelaar, 2003). Importantly, p.o.
CBDV (400 mg·kg-1) also suppressed PTZ-induced seizures,
showing that CBDV can exert anticonvulsant effects when
Systemic administration of pilocarpine induces status epi-
lepticus with a temporal lobe focus that subsequently gener-
alizes and is associated with motor convulsions (Curia et al.,
2008). Interestingly, the anticonvulsant effects of CBDV only
became apparent when 200 mg·kg-1CBDV and AEDs were
co-administered. Thus, effects were observed only in higher-
power experiments in which 60, as opposed to 15, animals
received 200 mg·kg-1CBDV. These effects were limited (see
later), suggesting that CBDV is less effective in this model
than in the others studied here. However, our statistical
analyses revealed that the effects of CBDV in these experi-
ments were independent of, and separate from, the actions of
AED. Hence, it would be of interest to characterize the effects
of CBDV on pilocarpine-induced status epilepticus using direct
recordings of brain activity, for example via electroencepha-
lographic or electrocorticographic recordings in this model as
status epilepticus activity can persist in the absence of motor
Many AEDs exert signiﬁcant motor side effects (Schachter,
2007), which can limit patient quality of life. To address this
and conﬁrm that CBDV’s anticonvulsant actions were due to
direct actions on seizures and not motor suppression, we
investigated the effects of CBDV on the performance of rats in
the static beam and grip strength tasks. These tests assess
balance, coordination, muscle relaxation and drug-induced
functional neurotoxicity (Nevins et al., 1993; Crofton et al.,
1996; Stanley et al., 2005; Muller et al., 2008). CBDV did not
affect grip strength, and although the number of foot slips
did increase after 200 mg·kg-1CBDV treatment, this effect
was not signiﬁcant. Our tests were validated by the ﬁnding
that, consistent with previous studies, VPA negatively
affected all motor parameters (Roks et al., 1999).
Our in vivo results showing that CBDV has comparatively
strong anticonvulsant effects in a range of seizure models,
indicate that CBDV has signiﬁcant potential for the treat-
ment of generalized, human seizures and should be further
investigated against temporal lobe seizures. Furthermore,
data from the motor function assays indicate that CBDV does
not have signiﬁcant adverse motor effects at anticonvulsant
doses. In the future, it will be of great interest to investigate
CBDV’s properties in models of chronic epilepsy and hyper-
excitability. The effect of chronic CBDV treatment on behav-
iour in healthy and epileptic animals is also worthy of
Clinical investigation of new anticonvulsants is typically per-
formed using the candidate AED as an adjunctive treatment
to the patient’s current treatment regimen (French et al.,
2001). Therefore, we investigated the effects of CBDV
(200 mg·kg-1) when co-administered with clinically used anti-
convulsants. The three anticonvulsants used were chosen
based on their use as prescribed AEDs and, more pragmati-
cally, reported efﬁcacy in the seizure models used (Loscher
et al., 1991; Soﬁa et al., 1993; Shantilal et al., 1999; Lindekens
et al., 2000; Loscher, 2011). No negative interactions between
CBDV and the AEDs were observed, indicating that CBDV is
well-tolerated when co-administered with the three clinically
used AEDs employed in these studies. The anticonvulsant
effect of CBDV beyond that of these AEDs was variable, in our
study. When administered with ESM before PTZ or VPA
before pilocarpine, CBDV contributed signiﬁcantly to the
effects seen on severity (both cases), mortality (VPA in pilo-
carpine only), latency (ESM only) and the incidence of tonic–
clonic convulsions (VPA in pilocarpine only). The majority of
the signiﬁcant facilitatory effects of CBDV were seen at the
lower two doses; this could be due to the greater potential for
anticonvulsant actions when the AED is not producing a
maximal effect itself. However, 200 mg·kg-1CBDV appeared
to have little effect on pilocarpine-induced convulsions when
administered with phenobarbital at any dose, although it
should be noted that all doses of phenobarbital strongly
suppressed seizure activity, probably limiting CBDV’s effect.
CBDV had limited effects on PTZ-induced seizures when
co-administered with VPA. Thus, CBDV had AED-dependent
effects in these experiments, producing notable improve-
ments over AED treatment alone in two of four experiments.
Based on these data, we postulate that CBDV is well-tolerated
when co-administered with three AEDs used in the clinic for
a variety of epileptic syndromes, but that further investiga-
tion of its anticonvulsant properties in combination with
other drugs is required, for example, using isobolographic
experimental design and analysis (e.g. Luszczki et al., 2010).
Anticonvulsant mechanisms of CBDV
This is the ﬁrst investigation of CBDV effects in any in vivo
model or system; in vitro information on CBDV pharmaco-
logical properties, while growing, is limited (Scutt and Wil-
liamson, 2007; De Petrocellis et al., 2011a,b) and remains of
unknown in vivo or clinical relevance. For example, reported
effects of CBDV at recombinant TRP channels are as yet
unconﬁrmed in native tissue and it is unknown how such
TRP-based mechanisms of action could affect excitability in
epileptogenic areas. While TRPV1 expression in brain areas
including the hippocampus remain controversial (Mezey
et al., 2000; Cavanaugh et al., 2011), the functional expres-
sion of other TRP subtypes in relevant parts of the brain has
yet to be conﬁrmed (Crawford et al., 2009; Hirata and Oku,
2010). CBDV has also been reported to inhibit diacylglycerol
lipase (DAGL) a(De Petrocellis et al., 2011a), the enzyme
responsible for the production of the endocannabinoid
2-arachidonoylglycerol (2-AG; Stella et al., 1997). The effect
of inhibiting 2-AG production is likely to be complex. The
initial effect would be to decrease 2-AG levels and subsequent
activation of CB1cannabinoid receptors. However, the overall
effect of this on seizure activity would depend on propor-
Cannabidivarin as an anticonvulsant
British Journal of Pharmacology (2012) 167 1629–1642 1639
tional CB1cannabinoid receptor expression and localization
on different presynapses (i.e. excitatory or inhibitory), and
the contribution of inhibitory GABAergic circuits in brain
areas crucial to epileptogenesis, as a decrease in 2-AG would
result in less suppression of both excitatory and inhibitory
synapses. Furthermore, over longer time courses, it has been
reported that CB1cannabinoid receptor levels can be affected
by changes in agonist levels, that is higher levels of CB1
cannabinoid receptor agonists can increase internalization of
the receptor (Coutts et al., 2001). Thus, reduced 2-AG levels
could cause increased the number of CB1cannabinoid recep-
tors at the membrane. In addition, in this study the effects of
CBDV were only investigated on acute seizures and CB1can-
nabinoid receptor expression changes during both animal
models (e.g. pilocarpine-induced spontaneous recurrent sei-
zures as a model of temporal lobe epilepsy) of chronic epi-
lepsy and in human epilepsy (Magloczky et al., 2010; Karlocai
et al., 2011), which could affect the consequences of changes
in endocannabinoid levels upon seizure activity. D9-THC has
been reported to have a direct anticonvulsant action via
CB1cannabinoid receptor agonism (Wallace et al., 2001).
However, the effects of CBDV on CB1cannabinoid receptors
have not been characterized. Furthermore, 200 mg·kg-1
CBDV had no signiﬁcant effects in the motor function assays
used here, whereas CB1cannabinoid receptor agonists
produce signiﬁcant motor deﬁcits (Carlini et al., 1974), which
suggests that CBDV does not act via CB1cannabinoid recep-
CBDV is the propyl analogue of CBD and a qualitative
comparison of the effects of CBD and CBDV on PTZ-induced
seizures showed that both compounds improve mortality and
severity. However, CBD produced these effects at 100 mg·kg-1,
a dose at which CBDV did not affect severity. CBD did not
appear to affect onset latency (ⱕ100 mg·kg-1), whereas CBDV
delayed seizure onset in a dose-dependent manner that
reached signiﬁcance at 200 mg·kg-1. The comparison between
CBD and CBDV in the pilocarpine model is less simple as
CBDV at 200 mg·kg-1had wider-ranging anticonvulsant
effects in our co-administration experiments (on severity,
mortality and latency as well as the proportion of animals
that developed tonic–clonic convulsions), but was not effec-
tive in initial experiments at any dose, whereas low-dose CBD
affected tonic–clonic convulsions, but no other measures.
Hence, it would be of interest to perform a direct experimen-
tal comparison both of efﬁcacy and how similarly CBD and
CBDV affect seizures. Although assumptions of pharmaco-
logical similarity between plant cannabinoids on the basis of
structural homology should be made with caution (e.g. the
opposing effects of D9-THC and D9-THCV on CB1cannabinoid
receptors), CBD is anticonvulsant in animals and humans,
and more is known about CBD’s pharmacological properties,
if not its speciﬁc anticonvulsant mechanism(s) of action.
CBD has a wide range of known pharmacological targets,
which are unlikely to include CB1cannabinoid receptors, that
could underlie its anticonvulsant effects (Hill et al., 2012).
These include inhibition of T-type Ca2+channels (Ross et al.,
2008), inhibition of GPR55 in some tissues/preparations
(Ryberg et al., 2007), modulation of mitochondrial calcium
handling in neurons (Ryan et al., 2009) and increased activity
of inhibitory non-cannabinoid GPCRs including 5-HT1A
(direct agonism; Russo et al., 2005) and adenosine A1(via
effects on adenosine uptake; Carrier et al., 2006). Thus, if
CBDV shares some or all of CBD’s pharmacological targets, it
is possible that CBDV also acts via multiple mechanisms to
produce its overall anticonvulsant effect, as opposed to exert-
ing a high-efﬁcacy action at a single target. However, there is
no a priori reason to assume a common target and there is
clearly some divergence between the properties of CBD and
CBDV, for example CBD, but not CBDV, inhibits FAAH (De
Petrocellis et al., 2011a).
In conclusion, our most important ﬁnding is that CBDV
possesses strong anticonvulsant properties in a range of in
vivo seizure models that parallel a variety of human seizure
types and pathologies; anticonvulsant effects were also seen
after oral, as well as i.p., administration. As with many clini-
cally used AEDs, further work is required to determine the
anticonvulsant mechanism of CBDV, but the signiﬁcant anti-
convulsant effects and favourable motor side effect proﬁle
demonstrated in this study identify CBDV as a potential
standalone AED or as a clinically useful adjunctive treatment
alongside other AEDs.
UoR authors thank GW Pharmaceuticals and Otsuka Pharma-
ceuticals for research sponsorship and the provision of CBDV
and thank Simon Marshall for technical assistance.
Conﬂict of interest
The work reported was funded by grants to BJW, CMW & GJS
from GW Pharmaceuticals and Otsuka Pharmaceuticals. BJW,
AJH, NAJ, CMW & GJS were responsible for experimental
design. YY and TF are employees of Otsuka Pharmaceuticals
and hold stocks in this company. MD and CGS are GW
Pharmaceuticals employees, and CGS is a stockholder.
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Additional Supporting Information may be found in the
online version of this article:
Appendix S1 Methods.
Tables S1 and S2 For each seizure parameter that was
affected by CBDV +AED treatment, the analysis of the indi-
vidual AED effect is given (either as ANOVA or Chi-squared).
The directions of signiﬁcant effects are also given by an
upward or downward arrow (irrespective of the parameter, all
signiﬁcant AED effects described are anticonvulsant). Addi-
tionally, the doses at which AEDs were signiﬁcantly anticon-
vulsant are indicated with post hoc p values given after.
Finally, analyses of interactions between CBDV and AED
effects are given.
BJP AJ Hill et al.
1642 British Journal of Pharmacology (2012) 167 1629–1642