Cannabichromene, Related Phytocannabinoids, and 5-Fluoro-cannabichromene Have Anticonvulsant Properties in a Mouse Model of Dravet Syndrome

Article abstract of DOI:10.1021/acschemneuro.0c00677

Cannabis-based products are increasingly being used to treat refractory childhood epilepsies such as Dravet syndrome. Cannabis contains at least 140 terpenophenolic compounds known as phytocannabinoids. These include the known anticonvulsant compound cannabidiol (CBD) and several molecules showing emergent anticonvulsant properties in animal models. Cannabichromene (CBC) is a phytocannabinoid frequently detected in artisanal cannabis oils used in the community by childhood epilepsy patients. Here we examined the brain and plasma pharmacokinetic profiles of CBC, cannabichromenic acid (CBCA), cannabichromevarin (CBCV), and cannabichromevarinic acid (CBCVA) following intraperitoneal administration in mice. The anticonvulsant potential of each was then tested against hyperthermia-induced seizures in the Scn1a+/- mouse model of Dravet syndrome. All phytocannabinoids within the CBC series were readily absorbed and showed substantial brain penetration (brain-plasma ratios ranging from 0.2 to 5.8). Anticonvulsant efficacy was evident with CBC, CBCA, and CBCVA, each significantly increasing the temperature threshold at which Scn1a+/- mice had a generalized tonic-clonic seizure. We synthesized a fluorinated derivative of CBC (5-fluoro-CBC), which showed improved brain penetration relative to the parent CBC molecule but not any greater anticonvulsant effect. Since CBC and derivatives are anticonvulsant in a model of intractable pediatric epilepsy, they may constitute part of the mechanism through which artisanal cannabis oils are anticonvulsant in patients.

Full text of DOI:10.1021/acschemneuro.0c00677

pubs.acs.org/chemneuro
Research Article
Cannabichromene, Related Phytocannabinoids, and 5‑Fluoro-
cannabichromene Have Anticonvulsant Properties in a Mouse
Model of Dravet Syndrome
Lyndsey L. Anderson, Adam Ametovski, Jia Lin Luo, Declan Everett-Morgan, Iain S. McGregor,
Samuel D. Banister, and Jonathon C. Arnold*
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ABSTRACT: Cannabis-based products are increasingly being
used to treat refractory childhood epilepsies such as Dravet
syndrome. Cannabis contains at least 140 terpenophenolic
compounds known as phytocannabinoids. These include the
known anticonvulsant compound cannabidiol (CBD) and several
molecules showing emergent anticonvulsant properties in animal
models. Cannabichromene (CBC) is a phytocannabinoid
frequently detected in artisanal cannabis oils used in the
community by childhood epilepsy patients. Here we examined
the brain and plasma pharmacokinetic profiles of CBC,
cannabichromenic acid (CBCA), cannabichromevarin (CBCV),
and cannabichromevarinic acid (CBCVA) following intraperito-
neal administration in mice. The anticonvulsant potential of each was then tested against hyperthermia-induced seizures in the
Scn1a+/− mouse model of Dravet syndrome. All phytocannabinoids within the CBC series were readily absorbed and showed
substantial brain penetration (brain−plasma ratios ranging from 0.2 to 5.8). Anticonvulsant efficacy was evident with CBC, CBCA,
and CBCVA, each significantly increasing the temperature threshold at which Scn1a+/− mice had a generalized tonic−clonic seizure.
We synthesized a fluorinated derivative of CBC (5-fluoro-CBC), which showed improved brain penetration relative to the parent
CBC molecule but not any greater anticonvulsant effect. Since CBC and derivatives are anticonvulsant in a model of intractable
pediatric epilepsy, they may constitute part of the mechanism through which artisanal cannabis oils are anticonvulsant in patients.
KEYWORDS: Cannabichromene, CBC, cannabichromenic acid, CBCA, cannabichromevarin, CBCV, cannabichromevarinic acid,
CBCVA, Dravet syndrome
availability and affordability.5−8 The alternative artisanal or
commercially manufactured products often contain an array of
INTRODUCTION
■
Epilepsy is a common neurological disease that affects
phytocannabinoids, and it remains conceivable that com-
approximately 1% of the population worldwide.1−3 While
pounds in these formulations other than CBD might
current anticonvulsant drugs are effective in most epilepsy
contribute to their reported anticonvulsant effects.7 The
patients, 30% of patients are refractory to available treatments.4
CBD doses administered in artisanal cannabis-based products
are typically much lower than those shown to be effective in
reducing seizures in clinical trials with Epidiolex, suggesting
The high prevalence of treatment-resistant epilepsy under-
scores the need for novel treatment options. The recent
interest in cannabis-based medicines for epilepsy has arisen
involvement of other phytocannabinoids. Moreover, some of
from numerous media stories reporting remarkable improve-
the phytocannabinoids found in these products exhibit
ments in treatment-resistant childhood epilepsy patients using
anticonvulsant properties in animal models. These include
medicinal cannabis. Additionally, the US Food and Drug
Administration (FDA), the European Medicines Agency
cannabidiolic acid (CBDA, 2), cannabidivarin (CBDV, 3), Δ9-
(EMA), and more recently the Therapeutic Goods Admin-
Received: October 19, 2020
Accepted: December 14, 2020
Published: January 4, 2021
istration (TGA) in Australia, approved the purified preparation
of cannabidiol (CBD, 1; Figure 1), Epidiolex, for the treatment
of Dravet syndrome and Lennox−Gastaut syndrome.
Although Epidiolex now has regulatory approval for use in
treatment-resistant epilepsy, many patients continue to use
unregistered cannabis-based products, often reflecting issues of
https://dx.doi.org/10.1021/acschemneuro.0c00677
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Figure 1. Chemical structures of natural phytocannabinoids and a synthetic derivative.
tetrahydrocannabinol (Δ9-THC, 4), Δ9-tetrahydrocannabi-
nolic acid (Δ9-THCA, 5), and Δ9-tetrahydrocannabivarin
(Δ9-THCV, 6).9−16 As such, our laboratory has aimed to
systematically mine the cannabis plant for additional,
potentially more potent, anticonvulsant phytocannabinoids,
with cannabichromene (CBC, 7) identified as a likely
candidate.
In a recent Australian study, CBC was one of the most
abundant phytocannabinoids present in the artisanal oils used
to treat childhood epilepsy in the community, with only the
major phytocannabinoids CBD, CBDA, Δ9-THC, and Δ9-
THCA found to be more abundant.7 Notably, in one patient in
that study with reported anticonvulsant effects, CBC was
administered at a dose as high as 4.7 mg/kg. Further, a recent
open-label trial successfully utilized a cannabis herbal extract
formulation containing CBC (4%) to treat children with
intractable epilepsy.5 Although the plasma concentrations of
CBC were lower than those obtained with CBD in these
patients, they were much higher than plasma Δ9-THC
concentrations. Other support for anticonvulsant effects of
CBC comes from animal models. In a study using the maximal
electroshock (MES) seizure model, CBC displayed a modest
anticonvulsant effect by reducing the duration of hindlimb
extension at a 25 mg/kg dose.17 However, in another study
using the MES model, CBC at doses up to 500 mg/kg failed to
protect mice from seizures.18
Conventional induced acute seizure models, such as MES,
often fail to identify therapies for treatment-resistant epilepsies,
particularly the genetic epileptic encephalopathies of childhood
such as Dravet syndrome. Genetic epilepsy models that
recapitulate spontaneous epilepsy in humans are etiologically
relevant models that can be used in drug discovery. Over 80%
of Dravet syndrome patients have a de novo loss-of-function
mutation in SCN1A, the gene that encodes voltage-gated
sodium channel Nav1.1.19 Dravet syndrome typically presents
as febrile seizures that progress to spontaneous afebrile
seizures.20 Accordingly, heterozygous deletion of Scn1a
(Scn1a+/−) in mice recapitulates the hallmark features of
Dravet syndrome, including susceptibility to thermally induced
seizures.21
The present study assessed the anticonvulsant potential of
CBC against hyperthermia-induced seizures in the Scn1a+/−
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Figure 2. CBC is anticonvulsant against hyperthermia-induced seizures. Concentration−time curves for CBC in (A) plasma and (B) brain
following a 10 mg/kg ip injection. Concentrations are depicted as both mass concentrations (left y-axis) and molar concentrations (right y-axis).
Data are expressed as means SEM, with n = 4−5 per time point. (A, inset) Chemical structure of cannabichromene. (C) Dose−effect screening
of CBC against hyperthermia-induced seizures in Scn1a+/− mice. Threshold temperatures of individual mice for generalized tonic−clonic seizure
(GTCS) induced by hyperthermia following acute treatment with vehicle or varying doses of CBC. CBC (100 mg/kg) significantly increased the
temperature threshold for GTCS, an anticonvulsant effect (green, open symbols). The average temperature of GTCS is depicted by the bar. Error
bars represent SEM, with n = 17−19 per group (***p < 0.0005, log-rank Mantel−Cox).
Table 1. Pharmacokinetic Parameters of Cannabichromene Analogues in Mouse Plasma and Brain
a
a
a
CBC
5-fluoro-CBC
CBCA
plasma
brain
plasma
brain
plasma
brain
b
b
b
C (ng/mL)
tmax (min)
t1/2 (min)
AUC (μg·min/mL)
brain−plasma ratio
271
30
98
38
0.83
9
93 13
120
535 76
843 156
8.4 1.4 μg/mL
30
35
606
0.53
CBCVA
1.4 0.1 μg/mL
60
15
53
41
2.7
45
83
193
136
322
b
b
b
32
111
a
a
CBCV
plasma
brain
plasma
brain
b
b
C (ng/mL)
tmax (min)
t1/2 (min)
AUC (μg·min/mL)
479 52
687 81
30
20.2 1.7 μg/mL
15
34
840
0.20
2.5 0.5 μg/mL
30
58
172
15
40
26
5.8
139
b
149
brain−plasma ratio
a
b
Dose 10 mg/kg. Brain concentrations (ng/mL) converted from measured concentrations (ng/mg brain) assuming density of 1 g/mL.
mouse model of Dravet syndrome. An important first step was
to detail the pharmacokinetic profile of CBC to guide
subsequent assessment of its anticonvulsant potential. In
addition to CBC, we examined the basic pharmacokinetic
profiles and anticonvulsant activity of other CBC-related
phytocannabinoids: cannabichromenic acid (CBCA, 8),
cannabichromevarin (CBCV, 9), cannabichromevarinic acid
(CBCVA, 10), and a terminally fluorinated synthetic analogue
of CBC (5-fluoro-CBC, 11). CBCA and CBCVA are formed
enzymatically in the cannabis plant; therefore, they are
relatively abundant in raw cannabis material and cold-extracted
cannabis products.22 The neutral phytocannabinoids, CBC and
CBCV, are formed via decarboxylation of the respective acid
precursor as a result of light and heat exposure.
(t1/2 = 193 min) in brain than in plasma. Total exposure of
CBC in brain tissue was nearly equivalent to that of plasma as
determined by AUC values (brain−plasma ratio 0.83).
CBC Is Anticonvulsant against Hyperthermia-In-
duced Seizures in Scn1a+/− Mice. CBC was evaluated for
efficacy against thermally induced seizures in Scn1a+/− mice.
Between postnatal day 14 and 16, Scn1a+/− mice were treated
with a single ip injection of vehicle or CBC and challenged
with a thermal event. CBC was anticonvulsant against
hyperthermia-induced seizures (Figure 2C, Table 2). A
significant increase in GTCS temperature threshold was
observed with 100 mg/kg CBC (p = 0.0002). By comparison,
the effective anticonvulsant dose of CBD against hyperthermia-
induced seizures is also 100 mg/kg.10,23,24
Fluorination of CBC Achieves Greater Brain Pene-
tration than CBC. We hypothesized that the anticonvulsant
efficacy of CBC might be improved by enhanced brain
penetration. To this end, a fluorinated derivative of CBC was
synthesized, 5-fluoro-CBC, that was predicted to have reduced
lipophilicity and better brain penetration. The synthesis of
CBC and 5-fluoro-CBC is shown in Figure 3. Citral (12) was
condensed with either olivetol (13) or 5-(5-fluoropentyl)-
RESULTS AND DISCUSSION
■
Pharmacokinetic Profile of CBC in Mouse Plasma and
Brain. Intraperitoneal (ip) administration of CBC resulted in
rapid absorption (plasma tmax 30 min) and a relatively long
half-life (t1/2 = 98 min; Figure 2, Table 1). Distribution into
brain tissue was delayed relative to plasma, not reaching tmax
until 120 min (Figure 2B). CBC exhibited a longer half-life
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knowing the anticonvulsant mode of action(s), it is difficult to
reach any firm conclusions.
Table 2. Plasma Cannabinoid Concentrations in
Experimental Scn1a+/− Mice
Pharmacokinetic Profiles of Other CBC-Related
Phytocannabinoids: CBCA, CBCV, and CBCVA. The
pharmacokinetic profiles of three CBC-related phytocannabi-
noids (CBCA, CBCV, and CBCVA) were then characterized
in mouse plasma and brain (Figure 5A).
cannabinoid
CBC
dose (mg/kg)
plasma concentration (μg/mL)
10
30
100
10
394 128 ng/mL
4.1 0.2
12.4 0.8
15.8 1.2
53.7 6.3
CBCA
For CBCA, a high maximal plasma concentration (Cmax
=
30
8.4 1.4 μg/mL; Figure 5B, Table 1) was observed 30 min
after ip injection of 10 mg/kg. Distribution into brain tissue
was delayed, reaching tmax at 60 min (Figure 5C). CBCA
exhibited a long half-life (t1/2 = 136 min) in brain compared to
35 min in plasma. Although overall drug exposure of CBCA in
brain tissue was lower than that in plasma, CBCA had
appreciable brain penetration (brain−plasma ratio 0.53). This
substantial brain penetration of CBCA observed here is in
contrast to our previous study where CBCA was not detectable
in brain tissue following a 5 mg/kg ip injection.9 An important
difference is that in the previous study vegetable oil was used as
a vehicle, while here an ethanol−Tween80−saline (ratio
1:1:18) vehicle was used. A similar result was observed for
CBDA where its brain−plasma ratio was significantly increased
in a Tween-based vehicle compared to an oil vehicle.9
For CBCV, rapid absorption was observed with a tmax of 15
min in plasma and 30 min in brain (Figure 5D,E; Table 1).
Elimination from plasma was rapid (t1/2 = 40 min); however, a
long half-life (t1/2 = 139 min) was observed in brain, which led
to a greater AUC value for brain compared to plasma. Total
exposure of CBCV in brain tissue was nearly six times that of
plasma (brain−plasma ratio 5.8).
Similarly, CBCVA was rapidly absorbed with a plasma tmax of
15 min and a half-life of 34 min (Figure 5F, Table 1). CBCVA
achieved high concentrations in the brain (Cmax = 2.5 0.5
ng/mg brain); however, its plasma Cmax (20.2 1.7 μg/mL)
was significantly higher (Figure 5G). The brain−plasma ratio
of CBCVA was 0.20.
CBCA and CBCVA Are Anticonvulsant against Hyper-
thermia-Induced Seizures in Scn1a+/− Mice. Finally, the
anticonvulsant efficacy of CBCA, CBCV, and CBCVA was
determined against thermally induced seizures in Scn1a+/−
mice. Compared to vehicle, a significant elevation in GTCS
temperature threshold was observed with 100 mg/kg CBCA (p
= 0.0001; Figure 6A). CBCV had no effect on hyperthermia-
induced seizures at any dose tested despite its substantial brain
uptake (Figure 6B). Its acid form, CBCVA, however, was
anticonvulsant against thermally induced seizures with 100
mg/kg significantly elevating the temperature threshold for
GTCS compared to vehicle (p = 0.0365; Figure 6C).
100
10
30
100
10
30
100
10
30
95.7 18.4
1013 52 ng/mL
2.6 0.4
8.9 1.3
24.1 4.8
102.3 11.2
157.7 16.1
714 79 ng/mL
1.9 0.4
CBCV
CBCVA
5-fluoro-CBC
100
8.2 0.9
resorcinol (14) in the presence of catalytic ethylenediamine
diacetate to produce CBC or 5-fluoro-CBC, respectively.
5-fluoro-CBC was rapidly absorbed in plasma (tmax = 15
min; Figure 4A, Table 1) and brain (tmax = 45 min; Figure 4B).
The simple substitution of fluorine caused striking improve-
ments in plasma and brain absorption compared to those of
CBC, which had tmax values in plasma and brain of 30 and 120
min, respectively. The Cmax (843 156 ng/mL) achieved in
the brain was significantly greater than the plasma Cmax (543
76 ng/mL). Total exposure of 5-fluoro-CBC in brain tissue
was nearly three times that of the plasma (brain−plasma ratio
2.7), confirming the addition of the fluorine increased the
brain penetration as predicted. These results are consistent
with reports that an appropriately placed single fluorine
substitution can dramatically improve absorption, distribution,
metabolism, and excretion (ADME) properties of drugs.25,26
The effect of 5-fluoro-CBC on hyperthermia-induced
seizures in Scn1a+/− mice was then evaluated. The 100 mg/
kg dose of 5-fluoro-CBC was found to be anticonvulsant,
significantly elevating the GTCS temperature threshold
compared to vehicle (p = 0.0376, Figure 4C). Despite 5-
fluoro-CBC exhibiting substantially increased brain penetration
compared to CBC, it did not show greater anticonvulsant
efficacy. Both CBC and 5-fluoro-CBC were anticonvulsant
against hyperthermia-induced seizures at 100 mg/kg. Since the
brain−plasma ratio for 5-fluoro-CBC was nearly three times
that of CBC, it was hypothesized that it would be more potent
with perhaps a 30 mg/kg dose having anticonvulsant effects. It
is possible that the introduction of the fluorine disrupted
engagement with the molecular target(s) responsible for the
anticonvulsant effect of CBC. However, in the absence of
Brain and Plasma Pharmacokinetic Parameters of
CBC-Related Compounds in Mice. The current study
presents the first pharmacokinetic data for CBC, 5-fluoro-CBC,
CBCV, and CBCVA in mice. Pharmacokinetic parameters have
Figure 3. Synthesis of CBC (7) and 5-fluoro-CBC (11). Reagents and conditions: ethylenediamine diacetate (25 mol %), toluene, 110 °C, 6 h;
yield 35% (7) and 31% (11).
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Figure 4. 5-fluoro-CBC exhibits substantial brain penetration. Concentration−time curves for 5-fluoro-CBC in (A) plasma and (B) brain following
a 10 mg/kg ip injection. Concentrations are depicted as both mass concentrations (left y-axis) and molar concentrations (right y-axis). Data are
expressed as means SEM, with n = 4 per time point. (A, inset) Chemical structure of 5-fluoro-CBC. (C) Dose−effect screening of 5-fluoro-CBC
against hyperthermia-induced seizures in Scn1a+/− mice. Threshold temperatures of individual mice for generalized tonic−clonic seizure (GTCS)
induced by hyperthermia following acute treatment with vehicle or varying doses of 5-fluoro-CBC. 5-Fluoro-CBC (100 mg/kg) significantly
increased the temperature threshold for GTCS, indicating an anticonvulsant effect (green, open symbols). The average temperature of GTCS is
depicted by the bar. Error bars represent SEM, with n = 15−16 per group (*p < 0.05, log-rank Mantel−Cox).
been characterized previously for CBCA; however, here a
Tween-based vehicle was used. In general, the phytocannabi-
noids within this CBC series were rapidly absorbed in plasma
following ip injection with plasma tmax values ≤30 min.
Absorption into brain tissue tended to be slightly delayed
compared to plasma, with tmax values between 30 and 120 min.
These absorption rates are consistent with those of other
phytocannabinoids.9,27 Interestingly, while the half-lives of
each in plasma were relatively short (34−98 min), brain half-
lives were substantially longer. The half-lives in brain ranged
from 1.5 to 3.5 times longer than the plasma half-lives. This is
likely due to the lipophilic nature of these compounds, which
tend to store in lipid-rich tissues like the brain.
Overall, there was good brain penetration within the CBC
series, with CBC exhibiting a brain−plasma ratio of 0.83. By
addition of a fluorine to CBC (5-fluoro-CBC), the brain−
plasma ratio nearly tripled (2.7). The increased permeability is
not surprising as fluorine substitutions are a common strategy
employed in drug discovery and development to increase
blood−brain barrier (BBB) penetration.28 Surprisingly, CBCA
and CBCVA had fair brain penetration (brain−plasma ratios of
0.53 and 0.20, respectively) despite containing a carboxylic
acid functional group. Drugs with a carboxylic acid group are
negatively charged at physiological pH, which hampers passive
diffusion across the BBB. Nonsedative antihistamines purpose-
fully contain a carboxylic acid moiety to restrict them
peripherally.29
In a previous study, CBCA was not detectable in brain tissue
when administered in a vegetable oil vehicle.9 Here, when
CBCA was administered in a Tween-based vehicle, its brain
penetration increased substantially. Increased brain perme-
ability with a Tween-based vehicle is not unusual. Several
reports in the literature describe increased biomembrane
permeability and altered pharmacokinetic profiles of drugs
when administered with nonionic surfactants, including
Tween80.30−33 In fact, the brain−plasma ratio of CBDA was
nearly 50 times greater in a Tween-based vehicle compared to
a vegetable oil vehicle (brain−plasma ratios of 1.9 and 0.04,
respectively).9 Nonionic surfactants have been shown to
inhibit P-glycoprotein (P-gp)-mediated transport, and since
some phytocannabinoids are known P-gp substrates, it is
possible that CBCA is also a substrate.34−37 In this case,
Tween80 could inhibit its efflux from the brain; future studies
could investigate this possibility.
The Discovery of CBC-Related Phytocannabinoids
with Novel Anticonvulsant Activity in a Mouse Model
of Intractable Childhood Epilepsy. In addition to
characterizing the pharmacokinetic parameters of the phyto-
cannabinoids within the CBC series, we examined the effect of
each on hyperthermia-induced seizures in the Scn1a+/− mouse
model of Dravet syndrome. CBC was anticonvulsant and
significantly increased the temperature threshold of GTCS.
This is in contrast to two previous studies in mice that
reported CBC lacked anticonvulsant efficacy in the conven-
tional MES seizure model, as CBC (up to 500 mg/kg) did not
protect against generalized seizures.18 Although, one study
reported hints of anticonvulsant activity, with CBC modestly
reducing the duration of MES-induced hindlimb extension,
albeit not in a dose-dependent manner.17 Taken together with
the present findings, this might imply that CBC has
preferential anticonvulsant potential against childhood epi-
leptic encephalopathies.
The fluorination of CBC, generating 5-fluoro-CBC,
increased the brain−plasma ratio by nearly 3-fold compared
to CBC itself. Despite significantly increasing the brain−
plasma permeability, 5-fluoro-CBC was no more potent than
CBC against hyperthermia-induced seizures. The observations
that CBC and its fluorinated derivative were anticonvulsant at
the same dose as CBD against thermally induced seizures
suggest some promise in pursuing these compounds
clinically.10,23,24 Additionally, we identified two novel anti-
convulsant phytocannabinoids, CBCA and CBCVA, that were
also both effective at 100 mg/kg.
Future studies might usefully probe the anticonvulsant
mode(s) of action of this CBC series. Knowledge on the
pharmacological activity is extremely limited for CBC and
virtually nonexistent for the other phytocannabinoids such as
CBCA, CBCV, and CBCVA. Recently, CBC has been
identified as a CB2 receptor agonist, which could be relevant
to the anticonvulsant effects observed here.38 In a pentylenete-
trazole (PTZ)-induced seizure model, genetic deletion of CB2
receptors increased seizure susceptibility.39 Furthermore, CB2
receptor expression appears to be increased in lymphocytes of
Dravet syndrome patients.40 Importantly, CBC has little
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Figure 5. Pharmacokinetic analysis of CBCA, CBCV, and CBCVA in mouse plasma and brain samples. (A) Chemical structures of the
phytocannabinoids cannabichromevarin (CBCV), cannabichromenic acid (CBCA), and cannabichromevarinic acid (CBCVA). Concentration−
time curves for CBCV in (B) plasma and (C) brain, CBCA in (D) plasma and (E) brain, and CBCVA in (F) plasma and (G) brain following 10
mg/kg ip injections. Concentrations are depicted as both mass concentrations (left y-axis) and molar concentrations (right y-axis). Data are
expressed as means SEM with n = 4 per time point.
activity at CB1 receptors so is unlikely to produce THC-like
psychotropic effects.38 CBC has also been reported to be a
potent agonist at the transient receptor potential ankyrin 1
(TRPA1) channel (EC50 = 60 nM).41 However, long-term
activation of TRPA1 channels exacerbated seizure activity in
PTZ-induced kindled rats.42
explored in future studies. Epilepsy-relevant targets might
include voltage-gated sodium and calcium channels, transient
receptor potential vanilloid type 1 (TRPV1), and GABAA
channels, as well as GPR55 and 5-HT1A receptors.24,43−48
Moreover, CBC metabolites, which have been characterized in
several species including mice, could be examined to observe
whether they contribute to the anticonvulsant effects of
CBC.49
Other molecular targets of the more extensively studied
phytocannabinoid CBD might also apply to CBC and could be
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Figure 6. Dose−effect screening of CBCA, CBCV, and CBCVA using hyperthermia-induced seizures in Scn1a+/− mice. Threshold temperatures of
individual mice for generalized tonic−clonic seizure (GTCS) induced by hyperthermia following acute treatment with varying doses of (A) CBCA,
(B) CBCV, or (C) CBCVA. CBCA (100 mg/kg) and CBCVA (100 mg/kg) significantly increased the temperature threshold for GTCS, indicating
anticonvulsant effects (green, open symbols). CBCV had no effect on thermally induced seizures. The average temperature of GTCS is depicted by
the bar. Error bars represent SEM with n = 15−18 per group (*p < 0.05 and ***p < 0.0005, log-rank Mantel−Cox).
Sample Preparation. Plasma samples (50 μL, 5 μL for CBCA
and CBCVA) were spiked with diazepam (2 μg/mL) as an internal
standard, and protein precipitation was achieved by vortex-mixing
CONCLUSION
■
Here, the brain and plasma pharmacokinetic profiles of CBC
and related phytocannabinoids were determined and could be
used to guide future in vivo experiments with these
compounds. CBC, 5-fluoro-CBC, CBCA, and CBCVA all
exhibited anticonvulsant properties against hyperthermia-
induced seizures in the Scn1a+/− mouse model of Dravet
syndrome. Further studies could explore whether these
cannabinoids are also anticonvulsant against spontaneous
seizures or prolong the lifespan of Scn1a+/− mice.
with 200 μL of acetonitrile. The organic layer was isolated by
centrifugation (4000g for 10 min) and evaporated to dryness with N2.
Samples were reconstituted in acetonitrile (90 μL) and 0.1% formic
acid in water (300 μL) for supported-liquid extraction (SLE) with
methyl tert-butyl ether (MTBE) using Biotage Isolute SLE+ columns
(Uppsala, SWE). Samples were evaporated to dryness with N2 and
reconstituted in acetonitrile and 0.1% formic acid in water (1:1, v/v)
for analysis. Brains were prepared as described previously.10 Briefly,
each half brain was homogenized in 5× volume of acetonitrile.
Homogenates were centrifuged (20000g for 30 min, 4 °C), and brain
supernatants were spiked with diazepam (10 μg/mL). Extraction was
achieved by vortex-mixing with 3× volume of ice-cold acetonitrile for
10 min. The organic layer was isolated by centrifugation (20000g for
15 min, 4 °C). Supernatants were filtered through Amicon Ultracel-
3K (Merck-Millipore; Burlington, USA) filtration devices. Filtrates
were evaporated to dryness with N2 and underwent SLE extraction
with MTBE and reconstitution for analysis as described above.
Analytical Methods. Samples were assayed by LC-MS/MS using
a Shimadzu Nexera ultra-HPLD coupled to a Shimadzu 8030 triple
quadrupole mass spectrometer (Shimadzu Corp.; Kyoto, JPN) as
previously described.9,10 The mass spectrometer was operated in
positive electrospray ionization mode (negative mode for CBCVA)
with multiple reaction monitoring. Details regarding MS conditions
for each cannabinoid are provided in Table 3. Quantification of
cannabinoids in plasma and brain samples was performed by
comparing samples to standards prepared with known amounts of
drug.
Pharmacokinetic Calculations. Plasma and brain cannabinoid
concentrations at each time point were averaged, and pharmacoki-
netic parameters were calculated by noncompartmental analysis.
Elimination rate constants were determined by linear regression of the
terminal component of the concentration−time curve using Graph-
Pad Prism 7.2 (La Jolla, USA). The log−linear trapezoidal method
was used to calculated total drug exposure (area under concen-
tration−time curve) using equations described previously.21
Hyperthermia-Induced Seizures. Hyperthermia-induced seiz-
ure experiments were conducted on male and female Scn1a+/− mice at
P14−16 as previously described with n = 15−19 per group (each sex
accounted for approximately one-half of each group).21,23 Briefly,
mice were pretreated with vehicle or cannabinoid by a single ip
injection. Fifteen minutes prior to the target experimental (postdose)
time point for each drug, the rectal probe was inserted, mice
acclimated for 5 min, and the hyperthermia protocol was initiated.
Following the hyperthermia-induced seizure protocol, plasma samples
were isolated and stored at −80 °C until assayed. Seizure threshold
METHODS
■
Chemicals and Reagents. All reagents, reactants, and solvents
were purchased from Sigma-Aldrich (St. Louis, USA) or WuXi
AppTec (Shanghai, CHN) and used as purchased. CBCV was
purchased from Epichem (Bentley, AUS). CBC and 5-fluoro-CBC
were synthesized as described below. CBCA and CBCVA were
generously provided by Professor Michael Kassiou at the University of
Sydney. The purity of all the cannabinoids used were CBC (>95%),
CBCA (>99%), CBCV (>96%), and CBCVA (>95%). When
available, analytical standards were purchased from Novachem Pty
Ltd. (Heidelberg West, AUS).
Animals. All animal care and experimental procedures were
approved by the University of Sydney Animal Ethics Committee, and
all procedures were in accordance with the Australian Code of
Practice for the Care and Use of Animals for Scientific Purposes.
Scn1a+/− mice were purchased from The Jackson Laboratory (stock
37107-JAX; Bar Harbor, USA) and maintained as a congenic line on
the 129S6/SvEvTac background (129.Scn1a+/−). For experiments, F1
mice were generated by breeding heterozygous 129.Scn1a+/− mice
with wild-type C57BL/6J (Jackson Laboratory stock 000664; Animal
Resources Centre; Canning Vale, AUS). The Scn1a genotype was
determined as previously described.50
Pharmacokinetic Studies. Drugs were prepared fresh on the day
of the experiment as solutions in ethanol/Tween-80/saline (1:1:18).
CBCA (100 mg/kg) was a suspension, with CBCV (100 mg/kg) near
its solubility limit. Drugs were administered as an intraperitoneal (ip)
injection in a volume of 10 mL/kg. Wild-type male and female mice
(postnatal day 21−28, P21−28) received a single ip injection of 10
mg/kg CBC, 5-fluoro-CBC, CBCA, CBCV, or CBCVA. At selected
time points (15−120 min), mice were anesthetized with isoflurane
and whole blood was collected by cardiac puncture. Plasma was
isolated by centrifugation (9000g for 10 min, 4 °C) and stored at −80
°C until assayed. Whole brain was also collected and stored at −80
°C. Each time point (n = 4−5) contained at least one male and one
female per group.
336
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ACS Chem. Neurosci. 2021, 12, 330−339

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
7-(5-Fluoropentyl)-2-methyl-2-(4-methylpent-3-en-1-yl)-2H-
chromen-5-ol (5-Fluoro-CBC, 11). Subjecting 5-(5-fluoropentyl)-
benzene-1,3-diol (14, 327 mg, 1.65 mmol) to the general procedure
gave, following purification by flash column chromatography, the title
compound (158 mg, 31%) as a yellow oil. Rf = 0.76 (hexanes/EtOAc,
Table 3. Parameters for LC-MS/MS Detection of
Cannabinoids
plasma
a
a
molecular parent > daughter
LOQ
brain LOQ
(ng/mg brain)
1
compound
CBC
weight
ions (m/z)
(μg/mL)
80:20); H NMR (400 MHz, CDCl3) δ 6.61 (d, J = 10.0 Hz, 1H),
314.5
315.20 > 193.25
315.20 > 81.25
333.45 > 277.35
0.01−20
0.03−0.25
6.25 (s, 1H), 6.11 (s, 1H), 5.50 (d, J = 10.0 Hz, 1H), 5.12−5.07 (m,
1H), 4.72 (brs, 1H), 4.43 (dt, J = 47.3, 6.2 Hz, 2H), 2.49−2.45 (m,
2H), 2.16−2.05 (m, 2H), 1.77−1.67 (m, 3H), 1.67 (s, 3H), 1.66−
1.58 (m, 3H), 1.57 (s, 3H), 1.46−1.39 (m, 2H), 1.38 (s, 3H) ppm;
13C NMR (100 MHz, CDCl3) δ 154.3, 151.2, 144.3, 131.8, 127.5,
5-fluoro-
CBC
333.2
0.01−10
0.05−1
333.45 > 211.10
359.10 > 341.25
359.10 > 316.00
287.00 > 165.15
287.00 > 231.20
287.00 > 81.15
329.35 > 163.25
329.35 > 311.30
329.35 > 285.20
1
124.3, 116.9, 109.3, 107.8, 107.2, 84.2 (d, JC−F = 164.1 Hz), 78.4,
CBCA
CBCV
358.4
286.4
0.005- 10
0.05−4
0.05−1
41.2, 35.9, 30.7, 30.4 (d, 2JC−F = 19.5 Hz), 26.4, 25.8, 25.0 (d, 3JC−F
=
5.5 Hz), 22.9, 17.8 ppm; IR νmax 3140, 2965, 2929, 2858, 1622, 1575,
1429, 1575, 1429, 1081, 830, 774 cm−1; LCMS (ESI+): 333.2 ([M +
H]+, 100%), 355.3 ([M + Na]+, 5%).
0.01−10
CBCVA
330.4
0.01−20
0.05−4
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00677.
a
LOQ, Limit of Quantification. Range from lower to upper limits.
1H and 13C NMR spectra and LCMS trace for 5-fluoro-
CBC (PDF)
temperatures were compared using Mantel−Cox log-rank test, and p
< 0.05 was considered statistically significant. No significant sex
differences were observed, so groups were combined across sex.
Experimental time points were based on determined time-to-peak
plasma and brain concentrations from our pharmacokinetic studies.
Experimental time points used were as follows: 45 min, 5-fluoro-CBC;
30 min, CBC and CBCA; 15 min, CBCV and CBCVA, which were
administered after the 5 min acclimation period.
AUTHOR INFORMATION
Corresponding Author
■
Jonathon C. Arnold − Brain and Mind Centre, Discipline of
Pharmacology, Faculty of Medicine and Health, and Lambert
Initiative for Cannabinoid Therapeutics, The University of
Sydney, Sydney, NSW 2050, Australia;
Synthesis of Cannabichromene (CBC) and 5-Fluoro-canna-
bichrome (5-fluoro-CBC). All reactions were performed under an
atmosphere of nitrogen unless otherwise specified. Analytical thin-
layer chromatography was performed using Merck aluminum-backed
silica gel 60 F254 (0.2 mm) plates (Merck; Darmstadt, GER), which
were visualized using shortwave (254 nm) UV fluorescence. Flash
chromatography was performed using a Biotage Isolera Spektra One
and Biotage SNAP KP-Sil silica cartridges, with gradient elution
terminating at the solvent combination indicated for each compound.
Nuclear magnetic resonance spectra (NMR) were recorded at 298 K
using an Agilent 400 MHz spectrometer. The data are reported as
chemical shift (δ ppm) relative to the residual protonated solvent
resonance, multiplicity (s = singlet, br s = broad singlet, d = doublet,
br d = broad doublet, t = triplet, q = quartet, app quin. = apparent
quintet, m = multiplet), coupling constants (J Hz), relative integral,
and assignment. Assignment of carbon signals for the parent
compounds was assisted by correlation spectroscopy (COSY),
distortionless enhancement by polarization transfer (DEPT),
heteronuclear single quantum coherence (HSQC), and heteronuclear
multiple-bond correlation (HMBC) experiments where necessary.
General Procedure for the Synthesis of CBC and 5-fluoro-
CBC. Using a modified procedure, a flame-dried reaction vessel was
charged with the appropriate resorcinol (5 mmol, 1 equiv), citral (1,
1.09 mL, 6 mmol, 1.2 equiv), ethylenediamine diacetate (225 mg,
1.25 mmol, 0.25 equiv), and toluene (50 mL).51 The solution was
heated at reflux for 6 h before being cooled to ambient temperature,
and the solvent was removed under reduced pressure. The resulting
crude oils were purified via flash column chromatography.
Email: jonathon.arnold@sydney.edu.au
Authors
Lyndsey L. Anderson − Brain and Mind Centre, Discipline of
Pharmacology, Faculty of Medicine and Health, and Lambert
Initiative for Cannabinoid Therapeutics, The University of
Sydney, Sydney, NSW 2050, Australia; orcid.org/0000-
0002-0037-0891
Adam Ametovski − Brain and Mind Centre, Lambert
Initiative for Cannabinoid Therapeutics, and School of
Chemistry, Faculty of Science, The University of Sydney,
Sydney, NSW 2050, Australia
Jia Lin Luo − Brain and Mind Centre, Lambert Initiative for
Cannabinoid Therapeutics, and School of Psychology, Faculty
of Science, The University of Sydney, Sydney, NSW 2050,
Australia
Declan Everett-Morgan − Lambert Initiative for Cannabinoid
Therapeutics, The University of Sydney, Sydney, NSW 2050,
Australia
Iain S. McGregor − Brain and Mind Centre, Discipline of
Pharmacology, Faculty of Medicine and Health, and School
of Psychology, Faculty of Science, The University of Sydney,
Sydney, NSW 2050, Australia
Samuel D. Banister − Brain and Mind Centre, Lambert
Initiative for Cannabinoid Therapeutics, and School of
Chemistry, Faculty of Science, The University of Sydney,
Sydney, NSW 2050, Australia; orcid.org/0000-0002-
4690-4318
2-Methyl-2-(4-methylpent-3-en-1-yl)-7-pentyl-2H-chromen-5-ol
(CBC, 7). Subjecting olivetol (13, 900 mg, 5 mmol) to the general
procedure gave, following purification by flash column chromatog-
raphy, the title compound (556 mg, 35%) as a yellow oil. Rf = 0.74
(hexanes/EtOAc, 80:20); NMR (400 MHz, CD3OD) δ 6.65 (dd, J =
10.1 Hz, 1H), 6.17 (d, J = 1.5 Hz, 1H), 6.10 (d, J = 1.5 Hz, 1H), 5.47
(d, J = 10.1 Hz, 1H), 5.15−5.11 (m, 1H), 92.47−2.41 (m, 2H), 2.17−
2.06 (m, 2H), 1.67 (s, 3H), 1.67−1.63 (m, 2H), 1.66−1.53 (m, 2H),
1.58 (s, 6H), 1.39−1.29 (m, 4H), 1.35 (s, 3H), 0.92 (t, J = 7.1 Hz,
3H) ppm. All spectral data is consistent with that previously reported
(Figure S1, S2).52
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00677
Author Contributions
L.L.A., J.C.A., S.D.B., and I.S.M. contributed to the conception
and design of the study. L.L.A., A.A., J.L.L., and D.E.M.
337
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ACS Chem. Neurosci. 2021, 12, 330−339

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(2018) Composition and Use of Cannabis Extracts for Childhood
Epilepsy in the Australian Community. Sci. Rep. 8 (1), 10154.
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dravet syndrome. Ann. Clin. Transl. Neurol. 5 (9), 1077−1088.
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Arnold, J. C. (2019) Pharmacokinetics of Phytocannabinoid Acids
and Anticonvulsant Effect of Cannabidiolic Acid in a Mouse Model of
Dravet Syndrome. J. Nat. Prod. 82 (11), 3047−3055.
contributed to the acquisition and analysis of data. All authors
contributed to drafting the manuscript or figures.
Funding
This study was supported by the Lambert Initiative for
Cannabinoid Therapeutics, a philanthropically funded center
for medicinal cannabis research at the University of Sydney.
Additional funding was provided by the National Health and
Medical Research Council of Australia (NHMRC) to J.C.A.,
S.D.B., and I.S.M. (APP1161571).
(10) Anderson, L. L., Low, I. K., McGregor, I. S., and Arnold, J. C.
(2020) Interactions between cannabidiol and Δ 9 -tetrahydrocanna-
binol in modulating seizure susceptibility and survival in a mouse
model of Dravet syndrome. Br. J. Pharmacol. 177 (18), 4261−4274.
(11) Amada, N., Yamasaki, Y., Williams, C. M., and Whalley, B. J.
(2013) Cannabidivarin (CBDV) suppresses pentylenetetrazole
(PTZ)-induced increases in epilepsy-related gene expression. PeerJ
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Tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo
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Notes
No unexpected, new, or significant hazards or risks to report.
The authors declare the following competing financial
interest(s): Associate Professor Jonathon Arnold is Deputy
Academic Director of the Lambert Initiative. He has served as
an expert witness in various medicolegal cases involving
cannabis and cannabinoids and served as a temporary advisor
to the World Health Organization on their review of cannabis
and cannabinoids. Iain McGregor is Academic Director of the
Lambert Initiative for Cannabinoid Therapeutics. He has
served as an expert witness in various medicolegal cases
involving cannabis, has received honoraria from Janssen, is
currently a consultant to Kinoxis Therapeutics, and has
received research funding and fellowship support from the
Lambert Initiative, NHMRC, and Australian Research Council.
He currently sits on medical advisory board of BOD Australia
and holds a variety of patents for non-cannabinoid
therapeutics.
ACKNOWLEDGMENTS
■
(16) Benson, M. J., Anderson, L. L., Low, I. K., Luo, J. L., Kevin, R.
C., Zhou, C, McGregor, I. S., and Arnold, J. C. (2020) Evaluation of
the Possible Anticonvulsant Effect of Δ 9 -Tetrahydrocannabinolic
Acid in Murine Seizure Models. Cannabis Cannabinoid Res.,
DOI: 10.1089/can.2020.0073.
The authors gratefully acknowledge Barry and Joy Lambert for
their continued support of the Lambert Initiative for
Cannabinoid Therapeutics. In addition, we thank Katelyn
Lambert for inspiring our work on novel cannabinoid therapies
for childhood epilepsy. We also thank Rebecca Vogel for
technical assistance.
(17) Davis, W.M., and Hatoum, N. S. (1983) Neurobehavioral
actions of cannabichromene and interactions with Δ9-tetrahydrocan-
nabinol. Gen. Pharmacol. 14 (2), 247−252.
(18) Karler, R, and Turkanis, S. A. (1979) Cannabis and Epilepsy.
Marihuana Biological Effects 22−23, 619−641.
ABBREVIATIONS
■
(19) Marini, C., Scheffer, I. E., Nabbout, R., Suls, A., De Jonghe, P,
Zara, F., and Guerrini, R. (2011) The genetics of Dravet syndrome.
Epilepsia 52, 24−29.
CBC, cannabichromene; 5-fluoro-CBC, 5-fluoro-cannabichro-
mene; CBCA, cannabichromenic acid; CBCV, cannabichro-
mevarin; CBCVA, cannabichromevarinic acid
(20) Dravet, C., and Oguni, H. (2013) Dravet syndrome (severe
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