Molecular Targets of Cannabidiol in Neurological Disorders

Abstract

Cannabis has a long history of anecdotal medicinal use and limited licensed medicinal use. Until recently, alleged clinical effects from anecdotal reports and the use of licensed cannabinoid medicines are most likely mediated by tetrahydrocannabinol by virtue of: 1) this cannabinoid being present in the most significant quantities in these preparations; and b) the proportion:potency relationship between tetrahydrocannabinol and other plant cannabinoids derived from cannabis. However, there has recently been considerable interest in the therapeutic potential for the plant cannabinoid, cannabidiol (CBD), in neurological disorders but the current evidence suggests that CBD does not directly interact with the endocannabinoid system except in vitro at supraphysiological concentrations. Thus, as further evidence for CBD's beneficial effects in neurological disease emerges, there remains an urgent need to establish the molecular targets through which it exerts its therapeutic effects. Here, we conducted a systematic search of the extant literature for original articles describing the molecular pharmacology of CBD. We critically appraised the results for the validity of the molecular targets proposed. Thereafter, we considered whether the molecular targets of CBD identified hold therapeutic potential in relevant neurological diseases. The molecular targets identified include numerous classical ion channels, receptors, transporters, and enzymes. Some CBD effects at these targets in in vitro assays only manifest at high concentrations, which may be difficult to achieve in vivo, particularly given CBD's relatively poor bioavailability. Moreover, several targets were asserted through experimental designs that demonstrate only correlation with a given target rather than a causal proof. When the molecular targets of CBD that were physiologically plausible were considered for their potential for exploitation in neurological therapeutics, the results were variable. In some cases, the targets identified had little or no established link to the diseases considered. In others, molecular targets of CBD were entirely consistent with those already actively exploited in relevant, clinically used, neurological treatments. Finally, CBD was found to act upon a number of targets that are linked to neurological therapeutics but that its actions were not consistent withmodulation of such targets that would derive a therapeutically beneficial outcome. Overall, we find that while >65 discrete molecular targets have been reported in the literature for CBD, a relatively limited number represent plausible targets for the drug's action in neurological disorders when judged by the criteria we set. We conclude that CBD is very unlikely to exert effects in neurological diseases through modulation of the endocannabinoid system. Moreover, a number of other molecular targets of CBD reported in the literature are unlikely to be of relevance owing to effects only being observed at supraphysiological concentrations. Of interest and after excluding unlikely and implausible targets, the remaining molecular targets of CBD with plausible evidence for involvement in therapeutic effects in neurological disorders (e.g., voltage-dependent anion channel 1, G protein-coupled receptor 55, CaV3.x, etc.) are associated with either the regulation of, or responses to changes in, intracellular calcium levels. While no causal proof yet exists for CBD's effects at these targets, they represent the most probable for such investigations and should be prioritized in further studies of CBD's therapeutic mechanism of action.

Full text

1 23
Neurotherapeutics
The Journal of the American Society for
Experimental NeuroTherapeutics
ISSN 1933-7213
Neurotherapeutics
DOI 10.1007/s13311-015-0377-3
Molecular Targets of Cannabidiol in
Neurological Disorders
Clementino Ibeas Bih, Tong Chen,
Alistair V.W.Nunn, Michaël Bazelot,
Mark Dallas & Benjamin J.Whalley

1 23
Your article is protected by copyright and all
rights are held exclusively by The American
Society for Experimental NeuroTherapeutics,
Inc.. This e-offprint is for personal use only
and shall not be self-archived in electronic
repositories. If you wish to self-archive your
article, please use the accepted manuscript
version for posting on your own website. You
may further deposit the accepted manuscript
version in any repository, provided it is only
made publicly available 12 months after
official publication or later and provided
acknowledgement is given to the original
source of publication and a link is inserted
to the published article on Springer's
website. The link must be accompanied by
the following text: "The final publication is
available at link.springer.com”.

REVIEW
Molecular Targets of Cannabidiol in Neurological Disorders
Clementino Ibeas Bih
1
&Tong Chen
1
&Alistair V. W. Nunn
2
&Michaël Bazelot
1,3
&
Mark Dallas
1
&Benjamin J. Whalley
1
#The American Society for Experimental NeuroTherapeutics, Inc. 2015
Abstract Cannabis has a long history of anecdotal medicinal use
and limited licensed medicinal use. Until recently, alleged clinical
effects from anecdotal reports and the use of licensed cannabinoid
medicines are most likely mediated by tetrahydrocannabinol by
virtue of: 1) this cannabinoid being present in the most significant
quantities in these preparations; and b) the proportion:potency
relationship between tetrahydrocannabinol and other plant canna-
binoids derived from cannabis. However, there has recently been
considerable interest in the therapeutic potential for the plant can-
nabinoid, cannabidiol (CBD), in neurological disorders but the
current evidence suggests that CBD does not directly interact with
the endocannabinoid system except in vitro at supraphysiological
concentrations. Thus, as further evidence for CBD’s beneficial
effects in neurological disease emerges, there remains an urgent
need to establish the molecular targets through which it exerts its
therapeutic effects. Here, we conducted a systematic search of the
extant literature for original articles describing the molecular phar-
macology of CBD. We critically appraised the results for the
validity of the molecular targets proposed. Thereafter, we consid-
ered whether the molecular targets of CBD identified hold thera-
peutic potential in relevant neurological diseases. The molecular
targets identified include numerous classical ion channels, recep-
tors, transporters, and enzymes. Some CBD effects at these targets
in in vitro assays only manifest at high concentrations, which may
be difficult to achieve in vivo, particularly given CBD’s relatively
poor bioavailability. Moreover, several targets were asserted
through experimental designs that demonstrate only correlation
with a given target rather than a causal proof. When the molecular
targets of CBD that were physiologically plausible were consid-
ered for their potential for exploitation in neurological therapeu-
tics, the results were variable. In some cases, the targets identified
had little or no established link to the diseases considered. In
others, molecular targets of CBD were entirely consistent with
those already actively exploited in relevant, clinically used, neu-
rological treatments. Finally, CBD was found to act upon a num-
ber of targets that are linked to neurological therapeutics but that
its actions were not consistent withmodulation of such targets that
would derive a therapeutically beneficial outcome. Overall, we
find that while >65 discrete molecular targets have been reported
in the literature for CBD, a relatively limited number represent
plausible targets for the drug’s action in neurological disorders
when judged by the criteria we set. We conclude that CBD is very
unlikely to exert effects in neurological diseases through modula-
tion of the endocannabinoid system. Moreover, a number of other
molecular targets of CBD reported in the literature are unlikely to
be of relevance owing to effects only being observed at
supraphysiological concentrations. Of interest and after excluding
unlikely and implausible targets, the remaining molecular targets
of CBD with plausible evidence for involvement in therapeutic
effects in neurological disorders (e.g., voltage-dependent anion
channel 1, G protein-coupled receptor 55, CaV3.x, etc.) are asso-
ciated with either the regulation of, or responses to changes in,
intracellular calcium levels. While no causal proof yet exists for
CBD’s effects at these targets, they represent the most probable
for such investigations and should be prioritized in further studies
of CBD’s therapeutic mechanism of action.
Key Words: Cannabidiol .neurological disorders .
mechanism of action .cannabinoid .pharmacology.
*Benjamin J. Whalley
b.j.whalley@reading.ac.uk
1
School of Chemistry, Food and Nutritional Sciences, and Pharmacy,
University of Reading, Whiteknights, Reading RG6 6AP, UK
2
Broadmind Science Ltd, 47 Bow Field, Hook, Hants RG27 9SA, UK
3
GW Pharmaceuticals Ltd, Sovereign House, Vision Park, Chivers
Way, Histon, Cambridge CB24 9BZ, UK
Neurotherapeutics
DOI 10.1007/s13311-015-0377-3
Author's personal copy

Introduction
For millennia, mankind has associated the use of Cannabis
sativa and its ~100 constituent phytocannabinoids (plant can-
nabinoids) with therapeutic usefulness, including for neuro-
logical disorders such as convulsions and pain [1–9].
However, such traditional use does not constitute valid evi-
dence for the modern medical use of the plant, its extracts or
components, and licensed clinical use of cannabis-based med-
icines remains limited to a small number of disorders such as
pain in multiple sclerosis (MS), appetite stimulation in HIV/
AIDS, and cancer chemotherapy [10–12]. Moreover, the ther-
apeutic effects of currently licensed cannabis-based treatments
rely for the most part upon the pharmacological effects of the
principal psychoactive component derived from the C. sativa
plant, Δ
9
-tetrahydrocannabinol (Δ
9
-THC) [13].
More recently, the therapeutic potential of the typically
second most abundant phytocannabinoid, cannabidiol
(CBD), has been investigated in preclinical animal models
and, together with anecdotal and often ambiguous reports of
crude cannabis extracts containing high proportions of CBD
exerting beneficial effects in treatment-resistant pediatric epi-
lepsies [14], has led to formal human clinical trials of CBD in
a number of epilepsies that will report results in late 2015 [15].
However, despite an extensive preclinical evidence base sug-
gesting therapeutic utility for CBD in several neurological
disorders (reviewed in this issue), a proven lack of cannabi-
noid type 1 receptor (CB
1
R)-mediated psychoactivity [16],
and good tolerability, the specific molecular target(s) through
which CBD exerts its reported therapeutic effects remains
undetermined.
Here, we summarize and assess the current evidence for
CBD exerting plausible pharmacological effects via specific
molecular targets (Part 1) before considering separate evi-
dence of the extent to which these targets may be involved
in mediating therapeutic effects in a variety of neurological
disorders (Part 2) that were selected to complement the re-
views also presented in this issue.
Methods
In order to identify molecular targets of CBD within the
existing literature, a PubMed search, using filters that exclud-
ed review articles, for the term Bcannabidiol^was performed.
Results were thenmanually reviewed to determine whether or
not original results describing CBD effects in molecular target
specific assays were presented. Thus, only peer-reviewed,
original publications that included results from assays specific
to a given molecular target were included in this review.
Part 1 also considers the plausibility of a given molecular
target as having the potential to play a role in CBD’s therapeutic
effects by virtue of potency and efficacy information where
available. With regard to potency, as most studies reviewed
relied upon evidence derived from preclinical animal models
of disease, we selected a specific concentration beyond which
effects reported by in vitro studies cannot realistically be
achieved in vivo. This guideline was based upon data derived
from a detailed report of CBD’s plasma and brain pharmacoki-
netic profile following administration via a number of routes
(orally and intraperitoneally) in both mouse and rat [17]. Thus,
in vitro effects reported in Part 1 that require CBD concentra-
tions ≥10–20 μM were considered supraphysiological and so
such molecular targets were not considered in Part 2.
Part 1: Molecular Targets of CBD
Receptor Targets
Our review of the current literature revealed 11 investigations
of the effect of CBD upon 10 specific receptor targets, which
account for 15% of the known molecular targets of CBD
(Fig. 1, Table 1).
Cannabinoid Receptors
In contrast to Δ
9
-THC, CBD has a very low affinity and
shows little agonist activity at the G protein-coupled
endocannabinoid system (ECS) receptors, CB
1
RandCB
2
R
[29]. However, despite this micromolar affinity, some of the
literature reports CBD as having an antagonistic profile
against CB
1
R/CB
2
R agonists with a nanomolar K
B
[30].
More recently, a statistical meta-analysis of all extant data
describing direct effects of CBD at CB
1
RandCB
2
Rconclud-
ed that there is no direct CBD–CB
1
R interaction that can ac-
count for the reported changes in endocannabinoid signaling
[16]. Indeed, the pharmacology of CBD at cannabinoid recep-
tors is not only complex and highly variable [31–33], but also
typically occurs at supraphysiological concentrations in vitro,
so rendering any contribution to behavioral effects unlikely.
These clear discrepancies between in vitro and in vivo studies
are a warning to pharmacologists that predicted pharmacology
from molecular assay systems is not always replicated at a
system level.
Adenosine
CBD not only elicits effects within the central nervous system
(CNS), but also within the cardiovascular system. Adenosine
receptors have been implicated in regulating coronary blood
flow and oxygen consumption by cardiac muscle and are
present in the brain, most notably in the forebrain [34,35].
CBD (50 μg/kg) inhibits the subsequent ventricular tachycar-
dia following coronary artery occlusion in rats—an effect
abolished by 8-cyclopentyl-1,3-dipropylxanthine, an
Bih et al.
Author's personal copy

adenosine A
1
receptor antagonist (Table 1). These results
demonstrate that CBD can exert an antiarrhythmic effect, pos-
sibly mediated by the adenosine A
1
receptor [36]. In addition
to effects on the A
1
receptor, A
2
receptor-mediated effects of
CBD have also been reported and claimed to mediate anti-
inflammatory effects of CBD [10,31,37,38]. The effects of
CBD on inflammatory signaling cascades have also been ex-
amined (Table 1)[37]. Here, retinal tumor necrosis factor-α
secretion following lipopolysaccharide injection was sup-
pressed by CBD (1 mg/kg), and the pharmacology of this
response was further dissected using ZM 241385, an adeno-
sine A
2
antagonist. The authors also suggested that the ob-
served CBD effects could arise though modulation of adeno-
sine transport (see BTransporters^). Using in vitro and in vivo
models of MS demonstrated that an adenosine A
2
receptor-
mediated component of the inflammatory response was sup-
pressed by CBD (in vivo:5mg/kg;in vitro:1μM) [10].
However, it was noted that the adenosine A
2
-mediated re-
sponse was only partially responsible for the anti-
inflammatory observations, again through use of the adeno-
sine A
2
antagonist ZM241385. Studies have suggested that
neuroprotective effects of CBD are mediated via adenosine
A
2
receptor modulation [31]. However, this has also been
contested where species differences and animal developmen-
tal stages in the methodology of these conflicting studies may
underlie the different conclusions drawn [38]. Therefore, a
clear link between the reported neuroprotective effects of
CBD and adenosine A
2
receptors has not yet been shown.
Glycine Receptors
Another molecular target of CBD has been revealed by 2
comprehensive studies investigating glycine-mediated
synaptic transmission [21,22]. Pentameric glycine ionotropic
receptors (GlyR) mediate neuropathic pain and inflammation
through Cl
–
flux where, at present, 4 αsubunit (α
1–4
)isoforms
and 1 isoform of the βsubunit have been described [39].
Using in vitro electrophysiological assays, CBD (1–300
μM) modulated strychnine-sensitive α1andα1βGlyR. At
concentrations > 100 μM, direct activation of these GlyR
was observed [21], although the physiological pertinence of
an effect at such a high concentration remains unknown.
Subsequently, it was shown that in HEK293 cells expressing
α3GlyRs,1μM CBD increased glycine-induced current am-
plitude almost 5-fold in comparison with glycine alone
(Table 1)[22]. Of interest was the observation that
dehydroxyl-CBD (1μM) was more efficacious than CBD as
it initiated a 9-fold increase in current amplitude. This obser-
vation led to the more recent study highlighting a role for
dehydroxyl-CBD in modulating presynaptic GlyR and sug-
gested a homomeric conformational bias for dehydroxyl-
CBD effects [40].
Opioid Receptors
Opioid receptors (ORs) are G
i/o
protein-coupled receptors that
bind opiates [41]. CBD (0.1–100.0 μM) may serve as an al-
losteric modulator at μand δORs (Table 1)[28]. In rat cortical
membranes, disassociation of OR agonist [D-Ala
2
,N-
MePhe
4
,Gly-ol]-encephalinfromμORs was seen following
the application of CBD. A similar effect was observed for δ
ORs. For both receptor isoforms, half maximal inhibition was
produced at approximately 10 μM(Table1). However, the
inhibitory constant varied between subtypes [18.4 μM(δiso-
form) and 31.6 μM(μisoform)] [28].
Fig. 1 Pie chart showing the
proportions of different molecular
targets for cannabidiol described
in the reviewed literature. Chart
shows percentage proportions
from a total of 65 targets. Targets
counted were unique and not
counted per literature report
Molecular Targets of Cannabidiol
Author's personal copy

Serotonin Receptors
CBD may also act via serotonin (5-HT) receptors [26,42–45].
The 5-HT
1A
receptor is coupled to G protein G
i/o,
where it is
thought to mediate inhibitory neurotransmission [46]. CBD
binds to the 5-HT
1A
receptor within a range of 8–32 μM
(Table 1)[26]. In cultured Chinese hamster ovary cells, CBD
displaced radiolabelled 8-OH-DPAT [7-(dipropylamino)-5,6,7,
8-tetrahydronaphthalen-1-ol] from 5-HT
1A
receptors. An in-
crease in guanosine 5’O-[gamma-thio]triphosphate binding to
G
i/o
and a fall in cyclic adenosine monophosphate was seen,
which suggests that CBD can act as an agonist at the 5-HT
1A
receptor. In addition to 5-HT
1A
agonism, the same study pre-
sented evidence for CBD binding to the 5-HT
2A
receptor. In
NIH/3T3 cells, CBD (8–32 μM) served as a partial agonist of
the 5-HT
2A
receptor with weaker efficacy than its action at the
5-HT
1A
receptor [26]. 5-HT receptors are also implicated in
autonomic control [47], and again here CBD has been shown
to modulate 5-HT
1A
-mediated responses in vivo [42]. CBD (1–
20 mg/kg i.p.) attenuated the stress response in male rats, as
evidenced by cardiovascular parameters, and this attenuation
was masked by co-administration of WAY-1000635, a 5-
HT
1A
antagonist (although with nonspecific effects at dopa-
mine D
4
receptors) [48]. Further studies examining central con-
trol of cardiovascular function have also shown CBD and 5-
HT
1A
interactions [49,50]. Further work using WAY-100635
has highlighted that CBD activation of 5-HT
1A
receptors, lo-
calized to the dorsal periaqueductal gray, mediates reported
panicolytic effects [43]. It is clear that CBD can modulate func-
tions involving 5-HT receptor function, although to what extent
such effects are direct remains unclear, particularly as several
in vivo studies did not include 5-HT receptor antagonist-only
Table 1 Receptor targets of cannabidiol
Target Concentration
range (μM)
EC
50
/IC
50
(μM)
K
i
(μM) Preparation or tissue Assay type Reference
CB
1
3; NSC ND ND A549/human Viability [18]
3; NSC ND ND H460/human Viability
NSC >10 ND ND ND [19]
NSC >30 ND HEK293 membrane/human GTPγS[20]
CB
2
3; NSC ND ND A549/human Viability [18]
3; NSC ND ND H460/human Viability
NSC >10 ND ND ND [19]
NSC >30 ND HEK293 membrane/human GTPγS[20]
Glycine receptor
α1 subunit
1–300; (+) 12.3 ND HEK293/ND Patch clamp/current with glycine [21]
1–300; (+) 132.4 ND HEK293/ND Patch clamp/current without glycine
Glycine receptor
α1βsubunit
1–300; (+) 18.1 ND HEK293/ND Patch clamp/current with glycine
1–300; (+) 144.3 ND HEK293/ND Patch clamp/current without glycine
Glycine receptor
α3 subunit
0.01–50.00; (+) 3* ND HEK293/ND Patch clamp/current without glycine [22]
GPR18 10
–4
–100; (+) 51.1 ND HEK293/ND p44/42
MAPK activation
[23]
GPR55 10
–3
–1; (–) 0.45 ND Human osteoclasts Rho and ERK1/2 activation [24]
10
–3
–10
–2
;(–) ND ND Colon Contraction [25]
(–) 0.445 ND HEK293 membrane/human GTPγS[20]
5-HT
1A
8–32; (–) ND ND CHO membrane/human [3H]-8-OH-DPAT ligand binding [26]
16; (–) ND ND CHO membrane/human [35S]-GTPγSassay
16; (–) ND ND CHO/human Forskolin
5-HT
2A
8–32; (+) ND ND NIH 3T3 membrane/rat [3H]-Ketanserin
nAChR α-7 0.1–100.0; (–) 11.3 ND Xenopus oocyte/human Patch clamp/current/acetylcholine [27]
Opioid (δ)0.1–100.0; (–)* 10.7* 18.4 Cerebral cortex membrane/rat [3H]-NTI binding assay [28]
Opioid (μ)0.1–100.0; (-)* 10* 31.6 Cerebral cortex membrane/rat [3H]-DAMGO binding assay
PPARγ3; (+) ND ND A549/human mRNA RT-PCR/Western blot [18]
3; (+) ND ND H460/human mRNA RT-PCR/Western blot
CB
1
= cannabinoid type 1; CB
2
= cannabinoid type 2; GPR = G protein-coupled receptor; 5-HT = serotonin; nAchR = nicotinic acetylcholine receptor;
PPAR = peroxisome proliferator-activated receptor; NSC = no significant change; ND = not described; HEK = human embryonic kidney; CHO =
Chinese hamster ovary; GTPγS=guanosine5’-O-[gamma-thio]triphosphate; MAPK = mitogen-activatedprotein kinase; ERK = extracellular regulated
kinase; [3H]-3-OH-DPAT = 7-(dipropylamino)-5,6,7,8-tetrahydronaphthalen-1-ol; [3H]-NTI = naltrindole; [3H]-DAMGO = D-Ala
2
, N-MePhe
4
,Gly-
ol; RT-PCR = reverse transcription polymerase chain reaction; (+) = stimulation; (-) = inhibition
*Estimated from plots in cited paper
Bih et al.
Author's personal copy

study groups. Therefore, a clear demonstration of in vivo CBD–
5-HT receptor interaction is required.
G Protein-coupled Receptor 55 (An Orphan G
Protein-coupled Receptor)
More recently, attention has turned to interactions between
CBD and non-endocannabinoid G protein-coupled receptors
(GPCRs) [23,24,51]. The orphan GPCR 55 (GPR55) shares
structural similarities in transmembrane domains 1, 2, and 3
when compared with the cannabinoid receptors, which may
indicate a binding site for cannabinoids [24,52]. A
radiolabelled synthetic analogue of Δ
9
-THC has been shown
to bind GPR55 with an EC
50
of 5 nM. Further studies with
CBD (0.001–1.000 μM) have shown an inhibitory effect on
the agonist activity of CP 55,940 at GPR55, with an IC
50
of
445 nM [24]. Functional consequences of this inhibition have
been demonstrated in rat hippocampal preparations [51].
Here, CBD (1 μM) suppressed physiological activation of
the GPR55 receptors, which restricted excitatory output from
pyramidal cells. While the pharmacology and localization of
GPR55 merits further investigation [20,53], CBD has demon-
strable antagonistic effects within rat brain with clear physio-
logical relevance.
Nicotinic Acetylcholine Receptor
Evidence thatCBD modulates nicotinic acetylcholine receptor
(nAchRs) function comes from a study by Mahgoub et al.
[27]. Electrophysiological recordings from Xenopus oocytes
expressing the α-7-nAChR reveal a concentration-dependent
inhibition in the presence of CBD (0.1–100.0 μM; Table 1).
Complementary biochemical evidence highlighted a noncom-
petitive binding to the α-7-nAChR. Furthermore, CBD (1–30
μM) inhibited acetylcholine-induced ion currents recorded in
rat hippocampal slices (IC
50
=12.7μM) [27].
Peroxisome Proliferator-activated Receptors
The peroxisome proliferator-activated receptor (PPAR) γ,oth-
erwise known as the glitazone receptor, is thought to be re-
sponsible for lipid storage and glucose metabolism [18], and
some anticancer effects of CBD are thought to by mediated
through interaction with PPARγ. In human A549 and A460
cancer cell lines, a time-dependent increase in PPARγmRNA
was observed following the application of CBD (1–3μM).
CBD has also been shown to inhibit tumor cell viability; cell
death assays revealed IC
50
values of 3.47 μM (A549) and
2.80 μM (A460). A complete loss of tumor cell viability
was observed at 8 μM (A549) and 7 μM (A460) [18].
Interestingly, these effects on tumor cells were not prevented
by pharmacological tools acting at CB
1
R, CB
2
R, or transient
receptor potential (TRP) vanilloid-type 1 (TRPV1). These
results do not confirm a direct effect of CBD upon PPARγ
but the extant data warrants its inclusion for completeness.
Furthermore, CBD was shown to displace a fluorescent ligand
of PPARγ(Fluormone PPAR Green, Life Technologies,
Paisley, UK) at a relatively low IC
50
(5 μM) in a fluorescence
polarization assay, indicating that CBD may bind to this pro-
tein [54]. In addition, CBD may be directly involved in
PPARγactivation as 10 μM CBD stimulates activity of the
luciferase reporter gene in HEK293 cells transiently overex-
pressing retinoid X receptor and PPARγ[54].
Enzyme Targets
Our review of the current literature revealed 19 investigations
of the effect of CBD upon 32 specific enzyme targets, which
accounts for 49% of the known molecular targets of CBD
(Fig. 1).
Enzymes Involved in Xenobiotic Metabolism
Cytochrome P450
As would be expected for a xenobiotic, CBD modulates sev-
eral cytochrome P450 (CYP450) enzymes. Although early
work did not show any statistically significant effect of CBD
upon CYP450 function [55], more recent studies (see below)
have reported several CYP450s as potential molecular targets.
The CYP450 enzyme system includes P450 and its nicotin-
amide adenine dinucleotide (phosphate) oxidase-linked reduc-
tase and catalyzes a monoxygenation reaction. In eukaryotes,
it is mainly found in the endoplasmic reticulum and mitochon-
dria where it maintains homeostatic control of lipophilic en-
dogenous compounds and the detoxification of lipophilic xe-
nobiotic compounds by oxidation, making them more water
soluble, and is thus the main component of phase 1 metabo-
lism. It is highly diverse, with many different isoforms [56].
CBD inhibits human recombinant CYP2C19 using a
mixed inhibition mechanism (Ki = 0.793 μM) [57].
Additionally, CBD is a potent inhibitor of this enzyme using
either high-affinity substrates such as omeprazole (IC
50
=1.55
μM) or 2-O-methylfluorescein (IC
50
=1.79μM), or a low-
affinity substrate, (S)-mephenytoin (IC
50
=2.51μM), to acti-
vate the reaction. Irrespective of substrate, CBD fully inhibits
the reaction at 10 μM, indicating that it is a potent inhibitor. It
is unclear whether this is involved in the therapeutic effect of
CBD in CNS disorders as evidence of CYP2C19 expression
in brain remains conflicted [58,59]. Two other CYPs,
CYP2C9 and CYP2D6, are also potential targets for CBD as
CBD also completely inhibits their function at 10 μM[60,
61]. However, CBD exerts a more potent effect upon
CYP2C9 (IC
50
=2.7μM) than CYP2D6 (IC
50
=6.01–6.52
μM). An additional study of the CYP1 family revealed that
CBD acts as a more potent inhibitor of CYP1A1 (IC
50
=0.537
Molecular Targets of Cannabidiol
Author's personal copy

μM) than CYP1A2 (IC
50
~3.5 μM) and CYP1B1 (IC
50
~5μM) [62]. With regard to potency, 90% of CYP1A1’sac-
tivity was inhibited by 2.5 μM CBD, while 10 μM CBD
inhibited approximately 65% and 75% of CYP1A2 and
CYP1B1 activity, respectively.
Finally, it has been reported that CBD may inhibit members
of the CYP3 family [63]. Although, CYP3A4, CYP3A5, and
CYP3A7 have been identified as molecular targets for CBD
(Table 2), it appears that the compound is a more potent in-
hibitorofCYP3A5(IC
50
= 1.65 μM) compared with
CYP3A4 (IC
50
= 11.7 μM) and CYP3A7 (IC
50
=24.7μM).
More importantly, around 90% of CYP3A5’s activity was
abolished by 10 μM CBD, whereas CYP3A4 and CYP3A7
required up to 50μM—a physiologically implausible concen-
tration [17]: CBD to achieve ~90 and 72% inhibition, respec-
tively. The mode of inhibition determined in this study was
competitive for CYP3A4 and CYP3A5 but mixed for
CYP3A7.
Other Enzymes Involved in Xenobiotic Metabolism
The involvement of CYP450 is only part of the metabolism of
CBD [77], which has been linked to many other enzymes
involved in the control of redox. Oxidation of xenobiotics
can potentially produce highly reactive intermediates and is
why phase I and II metabolism is integrated with electrophilic
sensors [e.g., the nuclear factor (erythroid-derived 2)-like 2
(NRF2)–Kelch-like ECH-associated protein 1 pathway),
which can upregulate cytoprotective genes and pathways
[78]. In this regard, CBD can activate NRF2 [79].
Several enzymes implicated in the regulation of redox have
been identified as potential molecular target of CBD [55].
Here, 100 μM CBD applied to mouse hepatic 105,000 g su-
pernatant reduced superoxide dismutase and catalase activity
by approximately 76% and 24%, respectively, and nicotin-
amide adenine dinucleotide (NAD)- and NAD phosphate-
dependent NAD(P)H quinone reductase by 80% and 81%,
respectively, when compared with controls. Moreover, CBD,
at the same concentration, also significantly stimulated gluta-
thione peroxidase and glutathione reductase by 24% and 40%,
respectively. However, it is notable that the concentration of
CBD used in this study was far in excess of that which can be
achieved physiologically [17].
Enzymes Involve in Cholesterol Metabolism
Several studies have reported the action of CBD upon en-
zymes involved in cholesterol metabolism. Although some
of the efficacious concentrations reported are achievable
in vivo, several are not and so can be excluded from
consideration.
Acyl-cholesterol acyltransferases (ACATs) catalyze the
formation of cholesterylester from long-chain fatty acyl-
coenzme A (CoA) and cholesterol. CBD may antagonize the
overall activity of ACAT (without distinguishing the individ-
ual activity of each subtype) in human fibroblast cells [64].
The potency of CBD on cholesteryl ester formation in the
presence of 0.1 mM [
14
C]-oleate-CoA and 5 μg/ml 25-OH-
cholesterol could be estimated to < 10 μM. Furthermore,
30 μM CBD appears to reduce 90% of the formation of this
product.
The synthesis of mevalonate from 3-hydroxy-3-
methylglutaryl-CoA is catalyzed by 3-hydroxy-3-
methylglutaryl-CoA reductase, which has been shown to be
stimulated by CBD [64]. Although an EC
50
for CBD was not
determined, the in vitro production of mevalonate was in-
creased 4.5-fold in human fibroblasts exposed to 30 μM
CBD when compared with cells exposed to 1 μMCBD.The
exact mechanism by which CBD exerts its stimulatory effect
on the enzyme is still unclear.
CBD has also been reported to inhibit progesterone 17α-
hydroxylase, testosterone 6α-hydroxylase, and testosterone
16β-hydroxylase [75]. However, CBD exhibits only weak
efficacy as 10 mM CBD is required to inhibit ~20%, 49%,
and 67% of progesterone 17α-hydroxylase, testosterone 6α-
hydroxylase, and testosterone 16β-hydroxylase, respectively.
Thus, it is unrealistic to propose that these enzymes are mean-
ingfully involved in the therapeutic effect of CBD in neuro-
logical disorders.
Effects on Enzymes Controlling Ceramide
Ceramide is a secondary messenger with a range of ef-
fects, including induction of differentiation to apoptosis. It
can be viewed as part of a stress signaling pathway and
can be produced either via sphingomyelinase-dependent
hydrolysis or by de novo synthesis. It also plays an im-
portant role in membrane rafts and the control of ion
channels [80,81].
Over 3 decades ago, CBD was shown to activate the
breakdown of sphingomyelin in fibroblasts taken from a
patient with Niemen–Pick disease [76]. Prior to CBD
treatment, these cells exhibited < 3% normal
sphingomyelinase activity [82]. Although no assay was
performed that showed a direct effect of CBD on
sphingomyelinase, the authors demonstrated that 16 μM
CBD reduced sphingomyelin levels by 77%. In contrast,
when the same in vitro assay was performed on normal
fibroblasts (WI-38 human lung fibroblasts) CBD exerted
no statistically significant effect on sphingomyelin hydro-
lysis. Furthermore, the antitumor effect of CBD has been
shown to be independent on the production of ceramide in
human glioma cells (but was associated with a fall in
mitochondrial potential; see below) [83]andthusitre-
mains unclear as to whether or not CBD acts directly on
the ceramide pathway.
Bih et al.
Author's personal copy

Tab le 2 Enzyme targets of cannabidiol
Target Concentration range
(μM); (effect)
EC
50
/IC
50
(μM)
K
i
/K
m
(μM)
Preparation or tissue Assay type Reference
ACAT 1–30; (–) <10* ND Fibroblast/human [64]
Arylalkylamine N-acetyltransferase 0.1–10.0; (–) ND ND Pineal gland/rat Radiometric assay [65]
Catalase 10; (–) ND ND Hepatic/mouse [55]
Complex I 2–10
2
;(–)* 8.2 ND Pig cortex High-resolution respirometry in malate
and pyruvate
[66]
Complex II 2–10
2
(–)* 19.1 ND Pig cortex High-resolution respirometry in succinate
and rotenone
Complex IV 2–10
2
;(–)* 18.8 ND Pig cortex High-resolution respirometry in antimycin,
ascorbate and TMPD
Complex I 15–60 mg/kg, single
dose (+)
ND ND Prefrontal/cerebral cortex [67]
Complex II ND ND Prefrontal/cerebral cortex/hippocampus
Complex II–III ND ND Prefrontal/cerebral cortex/hippocampus
Complex IV ND ND Prefrontal/cerebral cortex/striatum/
hippocampus
Complex I 15–60 mg/kg/day for
14 days (+)
ND ND Prefrontal/cortex/striatum/hippocampus
Complex II ND ND Prefrontal/cortex/striatum/hippocampus
Complex II–III ND ND Prefrontal/cortex
Complex IV ND ND Prefrontal/cerebral cortex/striatum
COX1 10–10
3
;(–) ND ND Recombinant/ovine Polarography [68]
318; (NSC)* ND ND Recombinant/ovine Scintillation [69]
COX2 10–10
3
;(–) ND ND Recombinant/human Polarography [68]
318; (NSC)* ND ND Recombinant/ovine Scintillation [69]
0.5mg/mouse/day
for 23 days; (+)
ND ND Tumor/ CD-1 nude mice Polarography
CYP2C19 10
–2
–10; (–) 2.51 0.793/50.800 Recombinant/ human HPLC/4'-OH-mephenytoin [57]
10
–2
–10; (–) 1.55 ND/1.26 Recombinant/human HPLC/5-OH-Omeprazole
10
–2
–10; (–) 1.79 ND/2.32 Recombinant/human FLUOstar OPTIMA
‡
/ fluorescein, BMG Labtech,
Offenburg, Germany
CYP2D6 1–40; (–) 6.52 1.16 Recombinant/human FLUOstar OPTIMA
‡
/O-demethylated
metabolite of AMMC
[60]
1–40; (–) 6.01 2.69 Recombinant/human Measuring dextrome-thorphan
O-demethylation
CYP3A4 0.1–50.0; (–) 11.7 1 Baculovirus-infected insect cells/human HPLC for N-desmethyl-diltiazem detection [63]
CYP3A5 0.1–50.0; (–) 1.65 0.195 Baculovirus-infected insect cells/human HPLC for N-desmethyl-diltiazem detection
CYP3A7 0.1–50.0; (–) 24.7 12.3 Baculovirus-infected insect cells/human HPLC for N-desmethyl-diltiazem detection
CYP2C9 0.1–10; (–)* 2.7 2.31 Recombinant/human HPLC/ 4-hydroxy-diclofenac [61]
0.1–10; (–)* 2.67 0.964 Recombinant/human HPLC/ 7-hydroxy-warfarin
CYP1A1 0.1–2.5; (–) 0.537 0.155 Recombinant/human FLUOstar OPTIMA for resorufin detection
‡
[62,70]
CYP1A2 1–10; (–) ~3.5* 2.69 Recombinant/human FLUOstar OPTIMA for resorufin detection
‡
[62]
CYP1B1 1-–10; (–) ~5* 3.63 Recombinant/human FLUOstar OPTIMA for resorufin detection
‡
DAGL-α1–10
2
; (NSC) ND ND COS-7 cells membrane/human Scintillation counting [14C]-oleic acid [71]
Molecular Targets of Cannabidiol
Author's personal copy

Tab le 2 (continued)
Target Concentration range
(μM); (effect)
EC
50
/IC
50
(μM)
K
i
/K
m
(μM)
Preparation or tissue Assay type Reference
FAA H 1–10
2
;(–)* 27.5 ND N18TG2 cell membrane/mouse Scintillation counting [14C]-ethanolamine [19]
1–50; (–) 15.2 ND Brain tissue membrane/rat Scintillation counting [14C]-ethanolamine [71]
0.5 mg/mouse/day
for 23 days; (+)
ND ND Tumor/ CD-1 nude mice [3H]-Arachidonic acid detection/RP-HPLC [68]
10–16; (+) ND ND U87 glioma cells [3H]-Arachidonic acid detection/RP-HPLC
Glutathione reductase 10
2
; (+) ND ND Hepatic tissue/mouse [55]
GSH peroxidase 10
2
; (+) ND ND Hepatic tissue/mouse
HMG-CoA Reductase 1–30; (+) ND ND Fibroblast/human [64]
IDO 0.1–10.0; (–) ND ND LPS stimulated-THP-1 cells/human HPLC/kynurenine–tryptophan [72]
LOX-5 2–200; (–) 73.73 ND LOX inhibitor screening assay kit
(Cayman Chemical)
†
Hydroperoxides treated with chromogen
spectrophotometry
[73]
10–10
3
; (NSC) ND ND Purified from barley Spectropho-tometric detection of
hydroperoxides
[68]
0.5mg/mouse/day
for 23 days; (–)
ND ND Tumor/ CD-1 nude mice Detection of arachidonic acid
detection/RP-HPLC
LOX-15 0.2–10.0; (–) 2.56 ND LOX inhibitor screening assay kit
(Cayman Chemical)
†
Hydroperoxides treated with chromogen
spectropho-tometry
[73]
10–10
3
; (NSC) ND ND Purified from soybean Spectropho-tometric detection of
hydroperoxi-des
[68]
MAGL 1–10
2
; (NSC) ND ND COS-7 cells homogenate/human Scintillation counting [3H]-glycerol [19]
N-acylethanolamine acid amide
hydrolase
1–10
2
; (-) >100 ND HEK293 cells membrane/human Scintillation counting [14C]-ethanolamine
NAD(P)H quinone reductase 10
2
;(–) ND ND Hepatic tissue/mouse NADH-dependent reaction [55]
10
2
;(–) ND ND Hepatic tissue/mouse NADPH-dependent reaction
NAPE-PLD 0.5 mg/mouse/day
for 23 days;
(NSC)
ND ND Tumor/ CD-1 nude mice Assaying [3H]-anandamide formation [68]
Phosholipase A2 31.9–159.5; (+, –) 6.4/134.0 ND Naja naja venom Spectroscopy [74]
Progesterone 17α-hydroxylase 10–10
3
;(–) ND ND Testis microsome/rat HPLC [75]
SOD 10
2
;(–) ND ND Hepatic tissue/mouse [55]
Sphingomyelinase 16.00–63.60; (+)* ND ND Nieman pick Fibroblast/human HPLC, kratos detector and rainin Microsorb
§
[76]
Testosterone 6α-hydroxylase 10–10
3
;(–) ND ND Hepatic microsome/rat [75]
Testosterone 16β-hydroxylase 10–10
3
;(–) ND ND Hepatic microsome/rat
ACAT = acyl-cholesterol acyltransferase; COX = cyclooxygenase; CYP = cytochrome P; DAGL = diacylglycerol lipase; FAAH = fatty acid amide hydrolase; GSH = Glutathione; HMG-CoA = 3-hydroxy-
3-methylglutaryl-coenzyme A; IDO = indoleamine-2,3-dioxygenase; LOX = lipoxygenase; MAGL = monoacylglycerol lipase; NAPE-PLD = N-acyl-phosphatidyl-ethanolamine-selective phospholipase
D; SOD = superoxide dismutase; NSC = no significant change; ND = not described; LPS = lipopolysaccharide; HEK = human embryonic kidney; TMPD = trimethyl pentanediol; HPLC = high-
performance liquid chromatography; AMMC = 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin; RP-HPLC = reverse phase HPLC; NADH = nicotinamide adenine dinu-
cleotide; NADPH = nicotinamide adenine dinucleotide phosphate; (+) = stimulation; (–) = inhibition
*Estimated from plots in cited paper
†
Ann Arbor, MI, USA
§
Agilent, Santa Clara, CA, USA
Bih et al.
Author's personal copy

Electron Transport Chain
The mitochondrial electron transport chain (ETC) comprises a
group of 4 enzymatic complexes (known as, complex I, II, III,
and IV) involved in mitochondrial bioenergetics. Recent stud-
ies suggest that CBD may alter mitochondrial bioenergetics
via modulation of ETC activity [67]. Moreover, mitochondrial
bioenergetics may also be modified by CBD via interaction
with mitochondrial voltage-dependent anion channel 1
(VDAC1; see BIon Channels^)[84], which may affect the
production of adenosine triphosphate (ATP). VDAC1 is in-
volved in the transport of ions (e.g., calcium) and multiple
metabolites, including adenosine diphosphate [85], the sub-
strate of ATP.
There are still some discrepancies surrounding the nature of
CBD’s action on the complexes comprising the ETC. Acute or
chronic in vivo administration (Table 2) of CBD (60 mg/kg,
i.p.) increased the activity of complex I, II/III, and IV in tissue
obtained from cerebral and the prefrontal cortex in rat [67],
while in vitro application of CBD to isolated mitochondria
from pig brain antagonized the activity of complex I (IC
50
=
8.2 μM), II (IC
50
=19.2μM), and IV (IC
50
=18.8μM) [66];
apart from complex I, the IC
50
values presented are inconsis-
tent with the concentrations likely to be achieved by the
in vivo study to which we have compared [17]. Moreover, this
latter study determined that CBD exerted a greater effect on
complexes I and II than on complex IVas 100 μM CBD fully
inhibited complexes I and II, while complex IVretained ~75%
activity. These results were confirmed by a recent in vitro
study which showed that CBD inhibits directly complexes I,
II/III, and IVonly at higher concentrations (50 μM) [86].
In summary, it would seem that CBD acts differently on
mitochondrial complexes depending on the route of adminis-
tration. While the number of studies in this area remains small,
they appear to suggest that CBD effects on ETC complexes
may involve indirect mechanisms (e.g., increase ETC com-
plex expression or mitochondrial biogenesis) that ultimately
result in the bioenergetic increases reported by Valvassori
et al. [67], although a direct interaction between CBD and
complex I may inhibit this enzyme [66].
Arylalkylamine N-Acetyltransferase
Arylalkylamine N-acetyltransferase (AANAT) plays a role in
melatonin synthesis, which controls circadian rhythm, orches-
trated by the pineal gland, although melatonin performs other
functions,including control of neuroexcitation, immune mod-
ulation, and protection against oxidative stress [87–89]. The
effect of CBD on norepinephrine-induced activation of
AANAT in the rat pineal gland and in cell-free lysates has
been studied where 10 μM CBD significantly attenuated nor-
epinephrine induction of melatonin synthesis by 35% com-
pared with controls in isolated pineal glands, while 1 μM
inhibited AANAT activity by 35% [65]. This effect was shown
to be independent of CB
1
RorCB
2
R and due to direct
AANAT inhibition demonstrated using cell-free lysates of
the pineal gland where 10 μM CBD inhibited AANAT by
40% [65].
Effect of CBD on Phospholipases
Phospholipases are responsible for the breakdown of complex
lipids and play a key role in lipid signaling. In particular,
phospholipase A2 (PLA2) may play a key role in brain in-
flammation and thus neurodegeneration [90]. CBD has been
reported to act upon Naja naja venom PLA2, while it has no
effect on N-acylphosphatidyl-ethanolamines-hydrolyzing
phospholipase D, monoacylglycerol lipase, or diacylglycerol
lipase-α(Table 2). Using an in vitro assay, the effect of CBD
upon N. naja venom PLA
2
was reportedly biphasic where low
concentration CBD stimulated activity (EC
50
=6.4μM; max-
imum induction = 262% induction at 39.1 μM), while at
higher concentrations, activity was inhibited (IC
50
=134μM;
maximum inhibition ~60 % inhibition at 159.5 μM) [74].
While the reported EC
50
is physiologically achievable
in vivo, it is unclear whether CBD exerts the same effects upon
mammalian PLA2s as they are likely to be structurally and
functionally different from N. naja venom PLA2 [91].
While studying the effect of CBD on phospholipases in-
volved in regulation of endocannabinoids, repeated adminis-
tration of CBD (0.5 mg/mouse) in female CD-1 nude mice for
23 days was shown not to change the in vivo activity of N-
acylphosphatidyl-ethanolamine–phospholipase D. Similarly,
the same authors showed that CBD does not act upon the
human monoacylglycerol lipase and diacylglycerol lipase-α
expressed in COS-7 cells [68].
Effect on Fatty Acid Amide Hydrolase
Fatty acid amide hydrolase (FAAH) is an enzyme involved in
the catabolism of fatty acid amides (e.g.,the endocannabinoid,
anandamide) and reports of CBD effects at this target are
conflicted throughout the literature. Two studies that investi-
gated the effects of CBD on membrane extracted FAAH
in vitro reported different IC
50
values (27.5 μMand15.2
μM, respectively) [19,71]; marginal differences that could
conceivably arise via tissue and/or species differences
(Table 2). These results suggest that higher CBD concentra-
tions may exert inhibitory effects on FAAH. In contrast, an
in vitro study in U87 cells suggested that 16 μM CBD stimu-
lated FAAH by ~2-fold when compared with untreated cells
[68]. Furthermore, the samestudy found that mice treated with
0.5 mg CBD peritumorally for 23 days exhibited increased
FAAH activity via a possible post-translational modification
as the protein content did not differ from untreated mice [68].
Molecular Targets of Cannabidiol
Author's personal copy

Consequently, based on the limited and conflicting evi-
dence, it appears that CBD’s effects upon FAAH may depend
upon the physiological environment in which it is studied as
cellular or systemic systems reveal CBD-induced FAAH acti-
vation, while membrane extract assays reveal CBD-induced
inhibition. Such an environment-dependent effect suggests
that more than one mechanism may be involved—short-term
direct effects and longer-term, post-translational modifications
are probably both at play [68]. It is also notable that reported
effects upon FAAH only occur at relatively high micromolar
concentrations.
Cyclooxygenases and Lipoxygenases
Cyclooxygenases (COXs) and lipoxygenases (LOXs) metab-
olize free arachidonic acid into 2-series prostaglandins and 4-
series leukotrienes, respectively [92]. Here, COX1 and COX2
activity was partially inhibited (50% and 40%, respectively)
by CBD at very high concentrations (500 μM) in an in vitro
assay (Table 2)[68]. Although IC
50
values were not deter-
mined in this study, inhibitory effects of CBD were reported
from 50 μM and 100 μM for COX1 and COX2, respective-
ly—concentrations hard to achieve in vivo [17]. Conversely, a
different study showed that 318 μM CBD had no effect upon
the in vitro activities ofCOX1 and COX2 (Table 2)[69], while
in vivo administration of CBD had no effect upon COX2
activity in tumor tissue [68] (Table 2). However, a further
study suggested that CBD may modulate COX2 activity by
stimulating transcription and/or translation in some subtypes
of cells [18]. While the concentrations described above are
unlikely to be physiologically achievable, a more recent study
suggested that 10 μM CBD increased arterial vasorelaxation
in diabetic Zucker rats, which was associated with COX1 and
COX2 stimulation via an allosteric mechanism [93].
CBD has also been reported to act as an inhibitor of 15-
LOX and 5-LOX. However, CBD exerts a more potent effect
on 15-LOX (IC
50
=2.56μM) than 5-LOX (IC
50
=73.73μM)
in vitro [73]. Furthermore, 200 μM CBD cannot fully abolish
5-LOX activity, while 15-LOX activity can be fully blocked
by 10 μM CBD, indicating that the efficacy of CBD is higher
upon 15-LOX. However, these results are in contradiction
with previous work which showed that CBD exerted no effect
upon 5- and 15-LOX in vitro,despitein vivo administration
inhibiting 5-LOX activity in tumor tissue [68].
Indoleamine-2,3-dioxygenase
Indolamine-2,3-dioxygenase (IDO) is activated by inflamma-
tion, in particular by interferon-γand is thought to be involved
in inflammatory-associated depression (cytokine-induced
sickness behavior). Its overactivation may be important in
neurodegeneration as it can result in oxidative stress. It
catalyzes the degradation of ring-containing compounds, in
particular tryptophan to kynurenine [94].
CBD exerts biphasic effects on the kynuenine/tryptophan
ratio in human monocytic cells stimulated with concavlin A or
phytohemagglutinin (PHA), suggesting modulation of IDO
where 0.03–0.3 μM CBD stimulated them, whereas the IC
50
was about 8.9 μM. The highest concentration tested (16 μM)
almost completely inhibited IDO mRNA expression induced
by PHA. CBD was also shown to inhibit LPS stimulation of
THP-1 cell IDO activity (IC
50
=0.9μM). Given the bidirec-
tional nature of CBD effects upon IDO, it is most likely that
CBD concentrations typically achieved in in vivo studies in
animal models of disease will inhibit IDO [17].
Ion Channel Targets
The available literature revealed 10 investigationsof the effect
of CBD upon 10 specific ion channel targets, which accounted
for 15% of the known molecular targets of CBD (Fig. 1).
TRP Channels
CBD has been reported to act as an agonist at human TRPV1
channel expressed in HEK293 cells when assessed using a
fluorescence-based, high-throughput assay with an efficacy
of ~70% that of a saturating concentration of the positive
control, ionomycin (4 μM) (Table 3)[19]. TRP channels are
present in the plasma membrane of a variety of cells in many
tissues and act as ligand-gated, nonselective cation channels
permeable to sodium, calcium, and magnesium ions [102].
Here, CBD-induced increases in intracellular calcium
([Ca
2+
]
i
) were reportedly abolished by the TRPV1 antagonist
capsazepine (10 μM), suggesting a TRPV1-specific effect of
CBD. The authors also asserted that CBD did not affect
[Ca
2+
]
i
in non-TRPV1-expressing HEK293 cells but did not
present supporting data. Notably, other molecular targets of
CBD are present in HEK293 cells (e.g., VDAC1; see Tables 1,
2,3and 4) but their expression on intracellular membranes is
likely to prevent CBD-mediated effects from being identified
by an assay such as this. This group used the same approach in
a later study that examined anticancer effects of CBD [99],
where CBD effects at human TRPV1 expressed in HEK293
cells were again assessed and reported in the same way as
above. While specific EC
50
and efficacy results for were not
formally presented, an estimate from the concentration–re-
sponse curves published in suggests that CBD was both less
efficacious (~50% vs 70%) and less potent (~6.3 μMvs 3.5
μM) than previously reported [99]. The same group used the
same approach to assess CBD effects at human TRPV1
expressed in HEK293 cells for a third and final time [71].
Again, some disparity between the results presented and those
previously published was evident as CBD’s efficacy was con-
sistent with that reported by Ligresti et al. [99] but not that
Bih et al.
Author's personal copy

reported by Bisogno et al. [19]; however, potency differed
from both preceding papers (EC
50:
1.0 μMvs 6.3μM([71]
estimated from Ligresti et al. [99]vs 3.5 μM[19]). Taken
together, these results suggest that CBD can submaximally
activate TRPV1 receptors, although the potency of CBD’s
effects remains less welldefined. Analysis of the above results
yields an approximate mean EC
50
value of 3.5 μMwithan
SEM of 1.5 μM, which suggests that the 95% confidence
interval is greater than that values measured and could be a
consequence of variability in TRPV1 in the expression sys-
tems used (V. DiMarzo, personal communication) [110].
In contrast to the above studies, comparable fluorescence-
based Ca
2+
imaging in HEK293 cells expressing rat TRPV1
receptors revealed that CBD (100 μM) produced a response
that was only 21% of that produced by the positive control,
capsaicin (500 nM), which was insufficient to determine
reliablyanEC
50
value [96]. Furthermore, a recent study used
patch clamp techniques to assess the effect of CBD (3–30 μM)
on the bidirectional current recorded in HEK293 cells overex-
pressing rat TRPV1 receptors that can be evoked by application
of the TRPV1 agonist, capsaicin (1–10 μM) [98]. Although a
complete concentration–response relationship for CBD was not
determined, detectable currents were evoked by 10 and 30 μM
CBD but not by 3 μM. While species-specific differences in
TRPV1 responsiveness could account for efficacy and potency
differences in the preceding studies, the use of rat TRPV1 here
and by Qin et al. [96] justifies the target’s consideration. It is
notable that the highest CBD concentration (30 μM) studied by
Iannotti et al. [98] elicited a peak response of ~30% of that
produced by capsaicin (c.f., 21%). While it remains unknown
whether the response to 30 μM CBD represents a saturating
response, it is notable that CBD concentrations > 20 μMare
Table 3 Ion channel targets of cannabidiol
Target Concentration
range (μM)
EC
50
/IC
50
(μM)
K
i
(μM) Preparation or tissue Assay type Reference
Cav3.1 T-type 10
–2
–10; (–) 0.82 ND HEK293 cells/human Patch clamp/ current clamp [95]
Cav3.2 T-type 10
–2
–10; (–) 0.78 ND HEK293 cells/Hhuman
Cav3.3 T-type 10
–2
–30; (–) 3.7 ND HEK293 cells/human
TRPA1 ND-100; (–) 81.4 ND HEK 293 cells/rat Calcium mobilization assays [96]
10
–3
–10; (–)* 0.096 ND HEK 293 cells/rat Calcium assay/Fluo4-AM [97]
3–30; (+) ND ND HEK 293 cells/rat Patch clamp/current [98]
10
–3
*–25; (–) 0.11 ND HEK 293 cells/rat Calcium assay/Fluo4-AM [71]
TRPM8 10
–3
–10; (–)* 0.14 ND HEK 293 cells/rat Calcium assay/Fluo4-AM [97]
10
–3
–25*; (–) 0.06 ND HEK 293 cells/rat [71]
TRPV1 0.1–10.0; (+) 3.5 ND HEK 293 cells/human Calcium assay/Fluo-3 [19]
10
–3
–25; (+)* 1 ND HEK 293 cells/human Calcium assay/Fluo4-AM [71]
ND-100; (NSC) ND ND HEK 293 cells/rat Calcium mobilization assays [96]
3–30; (+) ND ND HEK 293 cells/rat Patch clamp/current [98]
3; (NSC) ND ND A549/human Viability [18]
3; (NSC) ND ND A549/human DNA fragmentation
10
–3
–10; (+)* 0.7* ND MDA-MB-231 cells Calcium assay/Fluo4 [99]
TRPV2 0.05–10
3
; (+)* 31.7 ND HEK 293 cells/human Calcium mobilization assay [96]
0.1–200.0; (+)* 22.2 ND U87MG glioma cells/rat Calcium mobilization assay [100]
10
–3
–25*; (+) 1.25 ND HEK 293 cells/rat Calcium assay/Fluo4-AM [71]
0.05–500.00; (+)* 3.7 ND HEK 293 cells/rat Calcium mobilization assay [96]
3–30; (+) ND ND HEK293 cells/rat Patch clamp/current [98]
3; (NSC) ND ND H460/human Viability [18]
3; (NSC) ND ND H460/human DNA fragmentation
TRPV3 10
–1
–10
3
; (+)* 3.7 ND HEK 293 cells/rat Calcium assay/Fluo4-AM [101]
TRPV4 10
–2
–10
2
; (+)* 0.8 ND HEK 293 cells/rat Calcium assay/Fluo4-AM
VDAC1 20; (–) ND ND Liver VDAC1 channel in
planar lipid bilayer/sheep
Bilayer Clamp BC-525B
amplifier/current
[84]
0.1–75.0; (+)* ND Kd; 11.2 Liver VDAC1 channel in
planar lipid bilayer/sheep
Thermophoretic analysis/
CBD–VDAC1 interaction
TRP = transient receptor potential; TRPA1 = TRP ankyrin type 1; TRPM8 = TRP subfamily M; TRPV1 = TRP vanilloid-type 1; VDAC = voltage-dependent
anion channel; NSC = no significant change; ND = not described; HEK = human embryonic kidney; CBD = cannabidiol; (+) = stimulation; (–): inhibition
*Estimated from plots in cited paper
Molecular Targets of Cannabidiol
Author's personal copy

unlikely to be reached in vivo andsoareunlikelytobephysi-
ologically meaningful [17].
In addition to reports of TRPV1 receptor agonism, CBD
has also been reported to interact with the TRPV2 receptor. In
the first study reporting such effects [96], CBD agonized hu-
man TRPV2 receptors overexpressed in HEK293 cells when
assessed using a fluorescence-based assay, a finding subse-
quently confirmed using patch clamp electrophysiology and
extended to reveal similar effects upon rat TRPV2
overexpressed in the same cell line, although with markedly
different potency [EC
50
=3.7μM(human)vs 31.7 μM(rat)]
(Table 3). CBD effects upon rat TRPV2 receptors were also
separately investigated in the same expression system and
using patch clamp electrophysiology [98]. Here, similar re-
sults were obtained where CBD activated TRPV2 at concen-
trations ≥10 μM, although a complete concentration–re-
sponse relationship was not. Subsequently, TRPV2-mediated
effects of CBD upon the chemosensitivity of a glioblastoma
multiforme cell line to conventional cytotoxic agents were
reported [100]. Interestingly, in addition to activating
TRPV2, CBD also stimulated TRPV2 expression in this cell
line. Finally, such TRPV2-dependent effects of CBD in ma-
lignant cell lines were extended by a similar demonstration of
CBD-induced increases in chemosensitivity in a multiple my-
eloma cell line [111]. In both cases, TRPV2-mediated effects
of CBD in malignant cells required moderate micromolar con-
centrations (≥20 μM) to achieve significant effects.
A single report has also described CBD effects at rat
TRPV3 and TRPV4 receptors expressed in HEK293 cells
where it acted as a potent agonist in both cases (Table 3),
although with variable efficacy when compared with positive
controls (TRPV3: 50%; TRPV4: 16%) [71].
Several reports have also described CBD interactions with
the transient receptor potential ankyrin type 1 (TRPA1) recep-
tor. A first report stated that CBD acts as a potent and effica-
cious agonist at rat TRPA1 receptors overexpressed in
HEK293 cells (Table 3), as assessed using a fluorescence-
based assay, but went on to report that high concentrations
(≥100 μM) of CBD failed to elicit [Ca
2+
]
i
increases in mustard
oil-sensitive dorsal root ganglion neurons [97]. Thereafter,
CBD was reported to activate fully rat TRPA1 expressed in
HEK293 cells when assessed in a similar fluorescence-based
assay, but with much lower potency (EC
50
= 81.4 μM;
Table 3)[96]. Subsequently, the first report was recapitulated
by the same group and reported similar effects of CBD at rat
TRPA1 in HEK293 cells but extended the study to show that
CBD can also act as a potent desensitizer (IC
50
=0.16μM) of
this receptor [97]. Finally, CBD effects at TRPA1 receptors
expressed in HEK293 cells were assessed using patch clamp
electrophysiology where CBD (≥10 μM) evoked a ruthenium
Table 4 Transporter targets of cannabidiol
Target Concentration
range (μM);
(effect)
EC
50
/IC
50
(μM)
K
i
(μM)
Preparation or tissue Assay type Reference
ABCC1 0.08–50.00; (–) 5.5 ND SF9/ human ATPase/N-ethyl-maleimide-
glutathione
[103]
ABCG2 0.08–50.00; (–) 7.3 ND ATPase/ sulfasalazine [104]
Adenosine uptake 10
–3
–10
2
;(–) 3.5 ND Striatal tissue synaptosome/rat Dual-label counting
[3H]-adenosine
[105]
Adenosine uptake 10
–2
–1; (–) 0.12 ND EOC-20 microglia/ mouse Scintillation counting/
[3H]-adenosine
[106]
Anandamide
uptake (AMT)
5–50; (–)* 22 ND RBL –2H3 cells/rat Scintillation counting/
[14C]-anandamide
[19]
1–25; (–)25.3ND [71]
Dopamine uptake 0.5–100.0; (–)* 16.2 ND Striatal tissue synaptosome/ rat Dual-label counting/
[3H]-dopamine
[105]
0.005–10.000;(-)* ND ND Hippocampus/corpus striatum/
rat
[3H]-dopamine
Glutamate uptake 1–10
2
;(–) 43.8 ND Striatal tissue synaptosome/rat Dual-label counting/
[3H]-glutamate
Mg
2+
-ATPase 1–10
3
;(–) ND ND Brain cortical vesicules/ rat Phosphate release [107]
NA uptake 0.005–10.000;(–)* ND ND Hippocampus/corpus striatum/
rat
Method: Coyle and Snyder
(1969)/ [3H]-NA
[108]
Thymidine uptake 10
–2
–1; (–) 0.19 ND EOC-20 microglia/ mouse Scintillation counting/
[3H]-thymidine
[106]
Choline uptake 0.24–240.00; (–) 15.9 ND Hippocampus synaptosome/rat Method: Whittaker et al. (1964) [109]
AMT = anandamide membrane transporter; Mg
2+
-ATPase = magnesium-activated adenosine triphosphatase; NA = noradrenaline; ND = not described;
(+) = stimulation; (–) = inhibition
*Estimated from plots in cited paper
Bih et al.
Author's personal copy

red-sensitive bidirectional conductance, although a full con-
centration–response relationship was not presented. The
highest concentration of CBD tested (30 μM) was only able
to induce a TRPA1-mediated current that was ~20% of that
evoked by the positive control TRPA1 agonist, mustard oil,
and was rapidly desensitized by continued exposure to CBD
[98].
Finally, CBD has been reported to act as a potent antagonist
(Table 3) at the rat TRP subfamily M (TRPM8) receptor
expressed in HEK293 cells [71,97].
VDAC1 (Mitochondrial Porin)
CBD has also been reported to act upon sheep liver VDAC1.
VDAC1 is a promiscuous ion channel, also known as mito-
chondrial porin, and is most often located on the outer mem-
brane of mitochondria, although has also been reported to be
present on cell plasma membranes [112]. VDAC1 plays a
complex and important role in a variety of cell functions such
as ATP rationing, Ca
2+
homeostasis, apoptosis, and protection
against oxidative stress. The effect of CBD upon VDAC1
channels reconstituted into an asolectin-based planar lipid bi-
layer has been examined using patch clamp electrophysiology
[84]. Here, CBD (20 μM) inhibited VDAC1-mediated cur-
rents to varying extents at different voltage steps (–60 mV to
+60 mV). This functional finding was further confirmed via a
thermophoretic analysis, which demonstrated an interaction
between CBD and VDAC1 with a K
d
of 11.2 μM(Table3).
Voltage-gated Calcium Channels (VGCCs)
CBD has been reported to inhibit human T-type voltage-gated
calcium channels (VGCCs) encoded by the Ca
V
3genefamily
when expressed in HEK293 cells and assessed using patch
clamp electrophysiology [95]. CBD was able to abolish fully
conductances via Ca
V
3.1, 3.2 and 3.3 T-type channels with
comparable potency for Ca
V
3.1, 3.2 but lower potency for
Ca
V
3.3 (Table 3). This finding was extended to investigation
of CBD effects upon native T-type conductances in mouse
trigeminal ganglion neurons where similar inhibitory effects
were observed, although a complete concentration response
relationship was not described.
Voltage-gated Sodium Channels
CBD can block voltage-gated sodium channels (VGSCs) in a
number of in vitro assays but, interestingly, this VGSC block-
ade per se does not confer anticonvulsant effects in whole-
animal models of seizure [113]. Here, the effects of CBD
(1–10 μM) were examined in rat brain slices, cultured mouse
cortical neurons, and human SH-SY5Y cells using patch
clamp electrophysiology and in Chinese hamster ovary cells
expressing human Na
V
1.1 and Na
V
1.2 VGSC subtypes using
a fluorescence-based assay. In all in vitro tests, CBD inhibited
VGSC conductances at micromolar concentrations, although
the block was sudden with no apparent conventional concen-
tration–response relationship. This was response is suggestive
of a nonspecific effect (e.g., disruption of the lipid membrane
in which the channels are located) and was also seen for the
structurally similar plant cannabinoid, cannabigerol. When
compared in a model of generalized seizure, CBD was strong-
ly anticonvulsant but cannabigerol exerted no effect, suggest-
ing that the apparent in vitro block of VGSC by CBD and
cannabigerol are artefactual.
Transporter Targets
Our review of the current literature revealed 9 investigations
of the effect of CBD upon 13 specific transporter targets,
which accounts for 20% of the known molecular targets of
CBD (Fig. 1). These are further illustrated in Table 4.
Neurotransmitter Transporters
The uptake of neurotransmitters is vital to homeostasis in the
brain and prevents overstimulation of receptors, which can
result in cell death [114]. A major route for neurotransmitter
uptake is through transporter proteins, which shuttle specific
transmitters. Work in the 1970s utilized rat brain synapto-
somes to determine the effect of CBD on the activity of var-
ious transporters (Table 4)[115]. Here, CBD (1–100 μM)
showed a concentration-dependent effect, significant at 50
μM, on 5-HT, noradrenaline (NA), and γ-aminobutyric acid
(GABA) uptake, with greater potency for inhibition of 5-HT
and NA uptake (50 μM CBD; 78% and 81% inhibition, re-
spectively). No significant changes were recorded for 1 μM
CBD, which draws on concerns regarding the physiological
relevance of these data. A more recent study employed rat
hippocampal and striatal synaptosomes to investigate the roles
of CBD on neurotransmitter uptake [116]. By measuring
radiolabelled uptake of NA and dopamine, significant inhibi-
tion of NA and dopamine was observed by 1 μM CBD in both
brain regions investigated.
With regard to dopamine transport, further studies showed
a dose-dependent reduction in dopamine uptake following
application of CBD (0.5–100.0 μM; IC
50
=16.2μM) in rat
striatal synaptosomes, consistent with a previous study
(Table 4)[105]. Glutamate uptake was again inhibited in a
dose-dependent manner following application of CBD (1–
100 μM) to rat striatal synaptosomes. However, unlike
CBD’s effect on dopamine uptake, half maximal inhibition
was achieved at the higher concentration of 43.8 μM[105].
These studies indicate that CBD can inhibit uptake of adeno-
sine, dopamine, and glutamate in vitro, but with variable po-
tency. While examining the anticonvulsant properties of CBD
some investigation of the cholinergic system was undertaken
Molecular Targets of Cannabidiol
Author's personal copy

but did not reveal significant changes in choline uptake across
several brain regions following 60 mg/kg intraperitoneally
administered CBD [117]. This finding was in contrast to
in vitro observations where an IC
50
of 15.9 μM was reported
for CBD upon choline uptake in a rat hippocampal prepara-
tion. This study highlights the need for caution in interpreting
in vitro assays and extrapolating and/or making assumptions
regarding the in vivo effects of CBD.
Microglia act as macrophages of the CNS, and are
thought to serve as the first and main form of active im-
mune defense. In previous studies, CBD has been shown
to exert anti-inflammatory effects, but a distinct mecha-
nism of action for this effect remains unresolved [106],
despite hypotheses that CBD exerts anti-inflammatory ef-
fects by facilitating adenosine transmission [10,106]. To
test this hypothesis, the effect of CBD on the uptake of
[
3
H]-adenosine was investigated (see Table 4). In vitro
studies on EOC 20 rat microglia demonstrated that CBD
(1–1000 nM) inhibited adenosine uptake in a dose-
dependent manner. A similar effect was observed in
RAW264.7 macrophages where a K
i
of 225nM was re-
ported. Further studies, which examined the affinity of
CBD for the equilibrative nucleoside transporter 1
(ENT1) showed that CBD displaced radiolabelled 6-
S-[(4-Nitrophenyl)methyl]-6-thioinosine ([
3
H]-NMBPR)
with a K
i
value of 237 nM, consistent with the result
obtained from RAW264.7 macrophages. These findings
indicate that CBD can inhibit adenosine uptake by bind-
ing ENT1 [106]. Thus, while it is clear that CBD can
modulate adenosine signaling at both the receptor (see
BReceptors^) and transporter levels, the contribution of
these effects to the in vivo pharmacology of CBD still
requires definitive study.
Anandamide Transporters
CBD has been implicated in modulating the metabolism of the
endocannabinoid, arachidonoylethanolamide (AEA; also
‘anandamide’)in vitro [118,119]. Transporter proteins are
involved in regulating AEA-mediated cellular signaling and
have also been reported to be targets for CBD [19,71].
CBD inhibits the anandamide membrane transporter,
which is thought to be responsible for the reuptake of AEA
into cells prior to degradation [110]. Scintillation counting of
[
3
H]-AEA revealed that CBD inhibits AEA uptake in a
concentration-dependent manner within a range of 5 to
50 μM(IC
50
=22μM) in rat basophilic leukemia cells
(RBL-2H3) [19]. Using the same techniqueand cellline, these
findings were replicated by De Petrocellis et al. [71], where
the IC
50
obtained was 25.3 μM, although the CBD concentra-
tion range tested was 1–25 μM[71]. Previous studies under-
taken by Di Marzo et al. [120] have shown that AEA exerts
anti-inflammatory effects. Therefore, the anti-inflammatory
effects also exerted by CBD may arise from facilitation of
AEA transmission [19].
Overall, while the concentrations at which effects on AEA
transporters have been reported are relatively high, it remains
possible that incomplete inhibition by CBD could underlie
some effects in vivo.
Multidrug Resistance Transporters
ABCC1 or multidrug resistance-associated protein 1, is a
transporter expressed in various tissues and displays a
wide substrate specificity, including important therapeu-
tics. Investigating intracellular substrate retention, using
Fluo3 and vincristine, CBD (IC
50
=128.3 μM and 30.9
μM, respectively) was reported to be a Bpotent^inhibitor
of ABCC1-meditated transport in vitro [103]. The use of
the word potent in this regard is questionable given the
reported IC
50.
Previous work by this group [104]using
flow cytometric analysis of substrate accumulation and
ATPase activity assays also highlighted the ABCG2 trans-
porter as a molecular target for CBD. Again, inhibition
was demonstrated in the presence of 10 and 50 μM
CBD (Table 4). The ATP-dependent transporter P-
glycoprotein has also been implicated in CBD effects
[121]. ATPase assays revealed a CBD concentration-
dependent inhibition of transport activity mediated by P-
glycoprotein (IC
50
=39.6μM). All these studies highlight
in vitro evidence that adds these substrate transporters to
the list of molecular targets; however, in vivo evidence is
still lacking.
Mg-ATPase Transporter
Magnesium-activated ATP (Mg
2+
-ATPase) is a transporter
that is expressed in erythrocytes, where Mg
2+
flows through
the plasma membrane to drive ATP hydrolysis [122]. In ves-
icles obtained from the rat cortex, CBD was reported to be a
potent inhibitor of Mg
2+
-ATPase within a range of 1–
1000 μM[107].
Part 2: The Relevance of CBD Molecular Targets
in Neurological Disorders
Part 1 considered the available pharmacological evidence de-
scribing CBD’s effects at specific molecular targets, regardless
of any a priori evidence of their involvementin a given disease
or disorder. Here, in part 2, we consider whether: 1) there is a
role for each of CBD’s plausible pharmacological targets in
neurological disease; and whether 2) CBD’s modulation of a
given target would be beneficial or detrimental.
Bih et al.
Author's personal copy

Epilepsy
Epilepsy is a highly heterogenous neurological disease char-
acterized by the manifestation of spontaneous recurrent sei-
zures and has a significant unmet clinical need whereby >
30% of people with epilepsy do not gain full control of their
seizures from currently available treatments [123]. Cannabis
has a long anecdotal history of use for the treatment of epilep-
sy, although, confusingly, significant reports of proconvulsant
effects are also present in the literature (for a detailed review
see [124]). CBD has a more consistent record and has been
repeatedly shown to exert anticonvulsant effects in a variety of
preclinical models and small, but flawed, clinical trials but the
mechanism through whichthese effects are exerted remains to
be determined [124]. Here, we consider the potential role that
the pharmacological targets identified in part 1 can play in
epilepsy and its symptoms.
CBD Ion Channel Targets in Epilepsy
While CBD’s effects at TRPV channel subtypes have been
investigated with some rigor, the involvement of this subtype
of TRP channel in epilepsy remains unclear. In humans, 2
reports describe increases in TRPV1 mRNA and protein ex-
pression in small numbers of patients with mesial temporal lobe
epilepsy, tuberous sclerosis, and focal cortical dysplasia type
IIb, suggesting that TRPV1 may be involved in epilepsy [125].
However, it remains unclear whether the increased TRPV1
expression described reflects an uninvolved and downstream
consequence of disease or an integral part of the process. The
preclinical literature describing TRPV1 involvement in epilep-
sy is conflicted where studies suggest no involvement [126,
127], a proconvulsant effect, or an anticonvulsant effect of
TRPV1 activation [128–134]. Overall, a larger number of stud-
ies suggest that the consequences of TRPV1 activation in epi-
lepsy are detrimental which, given the large number of validat-
ed studies asserting overwhelmingly anticonvulsant effects of
CBD in these animal models, suggests that TRPV1 activation
by CBD is not part of its antiepileptic mechanism of action. To
our knowledge, neither TRPV2 nor TRPV3 have been reported
to have any involvement in epilepsy, while only a single study
to date has suggested the involvement of TRPV4 in epilepsy
[135]. Here, TRPV4 blockade was proposed to reduce febrile
seizures in larval zebrafish [136], and so, again, the ability of
CBD to activate TRPV4 would be in direct contrast to its clear
anticonvulsant effects.
With regard to other TRP family members, only a single
preclinical report associates TRPA1 expression changes with
epilepsy without insight into any causal relationship, and no
reports linking TRPM8 and epilepsy have been made [137].
Finally, the potential conflict between TRP agonist-
mediated increases in [Ca
2+
]
i
and anticonvulsant effects of
CBD should be considered alongside the propensity for many
TRP agonists to elicit Ca
2+
-dependent desensitization, includ-
ing CBD in some cases [98,137]. However, it remains to be
seen whether a hypothetical CBD-induced TRP channel de-
sensitization achieved from steady-state CBD dosing could
contribute to the antiepileptic effects reported. In conclusion,
based on the existing evidence, the TRP channels affected by
CBD are unlikely to account for the antiepileptic effect seen.
Although the literature makes few direct links between
VDAC1 and epilepsy, and such that exist remain limited to
the hypothetical, mitochondrial dysfunction that plays a sig-
nificant role in some epilepsies [138,139]. Therefore, while
VDAC1 remains a plausible molecular target for CBD, it is
too early to assert confidently any involvement of VDAC1 in
the mechanism of action underlying CBD’s antiepileptic
mechanism but should be investigated further. Related to
VDAC1 as a mitochondrial target is the effect of CBD via
PPARγ, a nuclear receptor type expressed in brain with a
significant role in regulating metabolism, immune response,
and development. However, while such a physiological role
could have profound implications for epilepsy, no specific link
between PPARγand epilepsy has yet been made, and direct
effects of CBD at (c.f., via) this target have yet to be demon-
strated [140].
While VGCCs are long-established molecular targets for
antiepileptic drugs (AEDs) such as lamotrigine,
eslicarbazepine, felbamate, levetiracetam, gabapentin, and
pregabalin, most target N-, P/Q-, and L-type VGCCs and
not the low voltage-activated T-type VGCCs which CBD
can block [141]. T-type channels are, however, blocked by a
smaller number of existing AEDs such as ethosuximide,
zonisamide, and possibly valproate, where this action is
thought to inhibit synchronized depolarization of neuronal
populations which can lead to generalized seizures [141].
Such drugs have found particular utility in the treatment of
absence seizures, while T-type VGCC mutations have been
associated with the pathogenesis of some absence epilepsies
[142]. Thus, while a definitive role for blockade of T-type
Ca
2+
channels in the antiepileptic mechanism of CBD has
not been established, both CBD’s potency at this target and
the target’s validity in this disease make it a plausible target for
further investigation. It is also notable that CBD’seffectsatT-
type channels produces a hyperpolarizing shift of the activa-
tion potential, while changes in steady-state activation and
activity, even at strongly hyperpolarizing potentials, suggest
the ability to induce a block when the channel is in either the
open or closed state. Interestingly, while ethosuximide in-
duces a similar hyperpolarizing shift, it is only able to block
channels when in the open state [143].
CBD Receptor Targets in Epilepsy
CBD exhibits reasonable affinity at plausible concentration
for 5-HT
1A
and 5-HT
2A
receptors, and 5-HT
2A
receptors act
Molecular Targets of Cannabidiol
Author's personal copy

as a target for fenfluramine, a drug for which some evidence
exists supporting efficacy in drug-resistant epilepsies such as
Dravet syndrome [144]. A very limited number of studies
have reported changes in 5-HT receptor expression and func-
tion in people with epilepsy, although it remains unclear
whether this is a consequence of the disease or a component
of pathogenesis. Thus, while 5-HT involvement in pathogen-
esis remains uncertain, some 5-HT receptor subtypes may
represent a valid therapeutic target in epilepsy through which
CBD could be acting [145,146].
GlyR are predominantly expressed in the CNS in neuronal
cells in the brainstem and spinal cord and, as such, there is
much less evidence of their role in disorders of the cerebrum,
such as epilepsy. However, recent research in rodent species
has shown significant, functional GlyR expression in cortex
and hippocampus at least up to postnatal day 14 where they
serve to modulate neuronal network function [147], and
emerging evidence suggests a role in hyperexcitability disor-
ders [148]. These findings suggests that investigation of GlyR
function in healthy and epileptic, mature human cortex iswar-
ranted in order to lend further credence to GlyR-mediated
antiepileptic effects of CBD.
Links between μand δOR function and a variety of brain
disorders, including epilepsy, have been extensively investi-
gated [149,150]. However, OR agonism and antagonism have
each been confusingly associated with both pro- and anticon-
vulsant effects, and so make any antiepileptic therapeutic strat-
egy involving OR targets uncertain. Moreover, the relatively
high micromolar concentrations at which CBD exerts a block
of μand δOR in highly refined and optimized systems (see
Part 1) further argues against this mechanism underlying the
drug’s well established antiepileptic effects [151].
GPR55 is an orphan receptor for which the endogenous
ligand lysophosphatidylinositol has been proposed where
GPR55activationtriggersreleaseofCa
2+
from intracel-
lular stores to facilitate neurotransmission in hippocampus
[51]. However, while this functional role for GPR55 in
modulating synaptic transmission has been demonstrated,
its validity as a target for modulation of hyperexcitability
disorders such as epilepsy remains unknown. CBD shows
good affinity for GPR55 and the antagonistic effects re-
ported would be consistent with an attenuation of synaptic
transmission which could be antiepileptic. However, de-
finitive evidence to support this antiepileptic mechanism
of action is required.
nAChRs have been directly implicated in a rare, familial
form of epilepsy, autosomal dominant nocturnal frontal lobe
epilepsy where mutant forms of the α4β2* subtype are widely
expressed in cortex and, more generally, α4 subunit mutations
are associated with febrile seizures [152]. However, the role of
these mutant nAChRs during brain development to produce
this epilepsy or influence febrile seizure susceptibility remains
unclear [153].Thus, while nAChRs are a plausible target for
CBD, their relevance in ictogenesis and hence the therapeutic
effect of the drug remains unknown.
CBD has also been shown to mediate effects via both A
1
and A
2
adenosine receptors in studies of peripheral tissues.
However, both receptor subtypes are present in brain, and
adenosinergic modulation of neuronal activity serves to pro-
tect against cellular damage resulting from excessive metabol-
ic demand (e.g., during seizures) in addition to many agonists
at these receptors exerting significant anticonvulsant effects in
a wide range of animal models [154]. However, exploitation
of adenosine agonists as epilepsy treatments has been unsuc-
cessful owing to the very narrow therapeutic window that can
result in significant adverse events. In particular, those affect-
ing the cardiovascular system in humans and in animal
models, which suggests that direct agonism of A
1
/A
2
recep-
tors by CBD is unlikely and, as such, indirect elevation of
local adenosine levels is not only more plausible but could
represent a viable mechanism underlying CBD’santiconvul-
sant effects [124,155].
CBD Enzyme Targets in Epilepsy
While CYP enzymes are present in the endoplasmic reticulum
of CNS cells, their expression is typically only 0.5% that seen
in hepatic tissues [156]. Moreover, while CNS CYP450 have
been shown to modulate GABA and cholesterol levels, it is
unclear whether a drug effect on CNS CYP450 would confer a
functional change in CNS function through such pathways
[156].
AANAT has not been implicated in epilepsy and so the
pertinence of this target is unknown. Moreover, while neuro-
inflammation has long been established as a hallmark conse-
quence of epilepsy and PLA2 levels are reportedly increased
in some animal models and human studies of epilepsy, the
relevance of this enzyme as a therapeutic drug target remains
uncertain [157]. Similarly, reported effects upon COX and
IDO enzymes could also be argued to exert effects upon epi-
lepsy via involvement in inflammatory processes. However,
conflicting results and the very high CBD concentrations re-
quired for such effects casts doubt over the relevance of these
targets for CBD’s effects in epilepsy.
Conversely, FAAH inhibition has been shown to affect
seizure states in animal models and so represents a putative
antiepileptic target [158,159]. However, as CBD has been
claimed to inhibit and activate FAAH, conclusive elucidation
of CBD’s effects at this target are required. Furthermore, as
antiepileptic effects of FAAH result in CB
1
R-mediated anti-
convulsant effects and such effects are prone to tolerance,
investigation of the persistence of FAAH-mediated interven-
tions in epilepsy is also required.
Finally, while not exerting a direct therapeutic effect, it
should be noted that CBD-induced inhibition of enzymes such
as CYP3A4, which is responsible for the hepatic metabolism
Bih et al.
Author's personal copy

of a variety of AEDs and can be induced by others [160],
could exert an indirect effect by altering circulating levels of
concurrent medications. The clinical relevance for such indi-
rect effects of CBD on circulating AED levels has recently
been highlighted in the case of clobazam co-administration
[161].
CBD Transporter Targets in Epilepsy
While CBD’s reported inhibition of glutamate reuptake is en-
tirely inconsistent with is well establishedantiepileptic effects,
the inhibition of dopamine and adenosine reuptake could play
a role as increased levels of both transmitters have been asso-
ciated with anticonvulsant effects [6,162]. Nevertheless, the
high micromolar concentrations of CBD required to elicit the-
se effects for some targets suggests that further investigation
of physiologically pertinent concentrations at defined molec-
ular targets is required to clarify their involvement, if any.
CBD reportedly inhibits AEA uptake, although the specific
molecular transporter responsible for this role remains to be
identified. Moreover, in the CNS, AEA is less abundant than
the other major endocannabinoid, 2-arachidonoyl glycerol,
which renders a functional role for AEA uptake inhibition in
the antiepileptic effects of CBD uncertain [163]. Furthermore,
AEA uptake inhibition would lead to increased CB
1
Ractiva-
tion that could conceivably produce psychoactive effects that
have never been reported for CBD. Moreover, as describe
above (BCBD Enzyme Targets in Epilepsy^), CB
1
R-mediated
modulation of seizure states is prone to treatment tolerance,
which does not appear to occur with prolonged CBD treat-
ment in animal models.
Movement Disorders
Movement disorders cover a wide range of diseases that hin-
der the ability to initiate and control movement. CBD has been
shown to alleviate the symptoms of dystonia, dyskinesia, and
catalepsy resulting from antipsychotic treatment or neurode-
generative diseases [164,165].
CBD Ion Channel Targets in Movement Disorders
It is known that CBD acts on multiple subtypes of TRPV
channels, and TRPV1 agonists, such as capsaicin, have been
shown alleviate hyperkinesia in rodent models of
Huntingdon’s disease (HD) by restoring the neurochemical
deficits [164,165]. CBD also desensitizes the TRPV1 recep-
tor, so it is not certain if TRPV1 serves as a plausible target. At
present, neither TRPV2 nor TRPV3 receptors have been re-
ported to play a role in movement disorders. One study has
shown that TRPV4 R269H mutations are responsible for es-
sential tremors in Charcot–Marie–Tooth disease. However,
TRPV4 does not serve as a suitable target for CBD, as high
micromolar concentrations (100 μM) are required for channel
blockade [71]. Regarding other TRP subtypes, no studies
linking TRPA1 or TRPM8 to movement disorders have been
made. Based on the data so far, it is unlikely that interactions
between CBD and TRP channels account for its
antihyperkinetic effects.
Current evidence proves that CBD changes the conduc-
tance of VDAC1 [84]. VDAC1 plays a significant role in
Parkinson’s disease (PD), where it is required for the degra-
dation of mitochondria. This causes the loss of dopaminergic
neurons, leading to the movement disorders seen in PD [166].
Thus, VDAC1 may be involved in the effect of CBD in move-
ment disorders.
Activation of PPARγby pioglitazone has been reported to
reverse oral dyskinesia in rodent models of schizophrenia, but
adverse effects on memory retention have been observed
[167]. CBD has been shown to upregulate and activate
PPARγ[18,54], and therefore it may be able to replicate the
effects of pioglitazone.
CBD acts as a potent inhibitor of T-type VGCCS [95]. T-
type VGCC α1G (CACNA1G) subunit expression is signifi-
cantly increased in Purkinje cells in rodent models of ataxia,
which results in the abnormal regulation of motor function
[168]. Numerous studies on rodent models have also shown
that palatal, Parkinsonian, and essential tremors are all depen-
dent on T-type VGCC conductance [169,170]. Tremors were
abolished upon channel blockade by zonisamide, a selective
T-type VGCC antagonist [171]. T-type VGCCs are blocked
by CBD at low doses, and therefore CBD may be effective in
treating the diseases mentioned.
CBD Receptor Targets in Movement Disorders
CBD activates 5-HT
1A
and 5-HT
2A
receptor isoforms with
reasonable potency [26]. Activation of the 5-HT
1A
subtype
by buspirone has been shown to alleviate catalepsy in rodent
models of PD [172]. Similar results were observed following
CBD treatment, where the effect of CBD was abolished by 5-
HT
1A
receptor blockade, but it is not clear if CBD directly
interacted with 5-HT
1A
to exert its anticataleptic effect [50].
Despite this, the 5-HT
1A
subtype seems to be a suitable ther-
apeutic target for the anticataleptic action of CBD.
GlyRs are expressed in the CNS, where they mediate in-
hibitory neurotransmission. Disorders in glycine transmission
have been implicated in spastic disorders, for example re-
search in the 1980s showed a loss of glycine synthesis in the
spinal cord of spastic animals [173]. High micromolar con-
centrations of CBD are required to activate GlyRs; therefore,
GlyRs are not appropriate targets for CBD.
Present research shows that blockade of δORs and μORs
abolishes the extrapyramidal effects of levodopa treatments in
humans [174]. However, high micromolar concentrations of
CBD are required to antagonize either receptor subtype, so
Molecular Targets of Cannabidiol
Author's personal copy

μORs and δORs are not plausible targets for CBD [28]. There
is no evidence linking the GPR55 receptor with movement
disorders.
Α4β2andα7 nAchRs are potently inhibited by CBD [27].
As rapid desensitization of either subtype reduces dyskinesia
[174], it is possible that the blockade of these receptors by
CBD is responsible for its antidyskinetic effect.
CBD has been reported to modulate adenosinergic activity
in vitro. However, no current studies that link the A
1
receptor
with movement disorders have been undertaken. Adenosine
A
2A
receptors are highly expressed in GABAergic neurons
within the indirect pathway of the basal ganglia, where their
activation exerts an inhibitory effect on movement. Blockade
of the A
2A
receptor in Parkinsonian rats abolished the motor
deficits. The side effect profile was favorable owing to low
A
2A
receptor expression in other regions ofthe brain [111]. As
CBD activates A
2A
receptors, this interaction is unlikely to
have any beneficial effect in PD.
CBD Enzyme Targets in Movement Disorders
CBD is a potent inhibitor of CYP2D6 [60]. Following treat-
ment with various antipsychotic agents, tardive dyskinesia
appeared to be greater in patients with the poor metabolizer
phenotype ofCYP2D6, but the difference was not statistically
significant [175]. It is not clear if the inhibition of CYP2D6 by
CBD will be beneficial in preventing antipsychotic-induced
tardive dyskinesia. No other CYP450 subtypes have been in-
vestigated in movement disorders. AANAT and COX2 have
not been implicated in movement disorders; high CBD con-
centrations are required to an interaction with either enzyme,
thus they are not suitable targets. Studies investigating the use
of FAAH inhibitors totreat restless leg syndrome and periodic
limb movement disorder are ongoing, but no publications
have been released so far.
CBD Transporter Targets in Movement Disorders
CBD inhibits the uptake of glutamate, dopamine, and adeno-
sine, but only at high micromolar concentrations [105].
Impaired glutamate uptake has been reported in transgenic
rodent models of HD, but the effect of this on motor function
has not been determined [176]. Dopamine transporter avail-
ability is significantly lower in patients exhibiting symptoms
of PD [177]. Adenosine uptake has not been implicated in this
disease. Based on the data so far, inhibition of glutamate or
dopamine uptake will not be beneficial in the treatment of
movement disorder.
CBD binds an unknown AEA transporter to inhibit AEA
uptake [19,71]. Blockade of AEA uptake by UCM707
abolished hyperkinesia in rodent models of HD and reduced
spasticity in MS rats [178]. However, the elevation in AEA
could produce psychotropic effects and tolerance upon CB
1
R
activation [179], which has not been seen as a side effect of
CBD [180]. Therefore, the binding of CBD to the AEA trans-
porter will not provide a therapeutic effect in movement
disorders.
Neurodegenerative Diseases
Several preclinical studies have shown that CBD can
exert benefit in mouse models of neurodegenerative dis-
ease [181,182]. A common finding among neurodegen-
erative diseases is chronic neuroinflammation and initi-
ation of positive feedback between failure of calcium
homeostasis, mitochondrial dysfunction, and excessive
cell death, where age and oxidative stress are strong
correlates [183–185]. Dementia is caused by many dif-
ferent disorders, resulting in deterioration of memory,
mental functions, and behavior. It was estimated that,
globally, in 2010, 35.6 million people were living with
it—and it was predicted that this number would double
every year: Alzheimer’s disease (AD) is generally con-
sidered to be the most common subtype, resulting in
60–80% of cases [186]. As it is associated with the
excessive build-up of β-amyloid (Aβ)and
hyperphosphorylated forms of the microtubule-
associated protein, tau, this led to the proposal of the
Bamyloid cascade hypothesis^as the underlying patho-
genic basis for this disease [187]. However, manifesta-
tion of AD can be highly heterogeneous with clear
gene–environment interactions [188], and precipitating
are factors highly variable, that is, any insult leading
to an excessive and chronic neuroinflammatory response
(e.g., traumatic brain injury) or energy failure [189],
which is often seen in early-onset neurodegenerative
diseases, such as Leigh syndrome [190]. Genome-wide
analysis has highlighted several risk genes, many of
which alter clearance of misfolded proteins and modu-
late inflammation, while environment also influences ex-
pression via systemic inflammation (e.g., obesity),
which may make targeting risk factors and the immune
system important treatment strategies [191].
CBD Receptor Targets in Neurodegeneration
It has been suggested that the ECS may be hypofunctional,
and/or dysfunctional in neurodegenerative diseases, suggest-
ing circumstance-dependent protective or potentially damag-
ing effects (reviewed in [192,193]) and consistent with its
largely adaptive role in mammalian physiology.
In relation to CBD, McPartland et al. [16] conclude that
CBD does not act directly at CB
1
R but may act indirectly at
potentially supraphysiological concentrations by enhancing
ECS tone. However, as discussed in the transporter section
(see below), CBD may also bind tofatty acid binding proteins
Bih et al.
Author's personal copy

(FABPs), which could partly explain its apparent indirect ef-
fects on the ECS among others [194].
As indicated in Table 1, CBD exerts effects via 5-HT re-
ceptors and the serotonergic system, including the 5-HT
1A
receptor. While implicated in neurodegeneration, serotonergic
involvement is complex as both increases and decreases in
receptor density across various regions of the brain have been
associated with cognitive decline [195]. Although not strictly
a neurodegenerative disorder, CBD has been shown to be
effective in a mouse model of hepatic encephalopathy in-
duced by bile duct ligation—a condition that has a large ele-
ment of neuroinflammation. In this study, mice received 5
mg/kg of CBD or vehicle for 5 weeks after ligation. Treated
mice were significantly protected, but the effect was largely
negated by co-administration of a 5-HT
1A
receptor antagonist
[196]. However, as there was no bile duct ligation control
group treated with the antagonist only, it is not possible to
determine if the antagonist was blocking CBD’s effects di-
rectly, as it could simply be a behavioral effect. Similarly,
Pazos et al. [197] showed that in a pig model of hypoxic
ischemic brain injury, CBD, at a final concentration in brain
tissue of ~0.2 μM, could be protective when given after the
insult. Critically, the protective effects were blocked when a
5-HT
1A
antagonist was co-administered with the CBD [197].
However, since no antagonist-only group was included in the
study, a clear demonstration of CBD effects via 5HT1A re-
ceptors was achieved. There is thus some evidence in vivo
that 5-HT may play in a role in CBD’s actions, but it is not
possible to determine if this effect is directly mediated via this
receptor.
CBDcanbindtoPPARγ,andPPARγactivation is becom-
ing an important target in neurodegeneration, as it is anti-
inflammatory and its activation improves mitochondrial func-
tion and resistance to oxidative stress [54,140]. Here, the pro-
tective effects of CBD in an Aβ-induced neuroinflammatory
mouse model were abolished by a PPARγantagonist, but no
evidence was given as to whether this was a direct or indirect
effect [198]. CBD can also positively modulate COX via in-
creased mRNA and protein expression of PPARγand COX2,
which also suggests that a COX2-generated increase in PGD
2
and 15d-PGJ
2
could underlie PPARγactivation in addition to
any direct activation of PPARγby CBD [18,54,93]. A possi-
ble mechanism for this is suggested by the observation that
NRF2 can increase PPARγexpression as CBD can activate
NRF2 [79,199,200].
CBD can antagonize the GPR55 receptor [20,51]. This
receptor, when activated by its proposed endogenous ligand,
lysophosphatidylinositol, appears to be neuroprotective.
Activation of GPR55 by a variety of ligands, including
lysophosphatidylinositol, can release calcium from intracellu-
lar stores [201],and can boost the release of neurotransmitters,
such as glutamate at synapses [51]. Critically, activation of
GPR55 can stimulate the calcineurin–nuclear factor of
activated T cells (NFAT) pathway—a well-known target of
calcium signaling [202]. The NFAT pathway is a master reg-
ulator of the immune response and is inhibited by ciclosporin,
but can also display antiapoptotic actions in neurons [203,
204]. However, Aβ-induced neuritic damage, in an in vitro
model, mimics the increased intracellular calcium often seen
in neurodegenerative disorders and is associated with upregu-
lation of the calcineurin–NFAT pathway, inhibition of this
pathway, both in vivo and in vitro, can reverse markers of
neurodegeneration [205]. Indeed, in the brains of patients with
AD, increased nuclear NFAT is a common finding—and cog-
nitive decline in this disease is associated with selective
changes calcineurin/NFAT signaling. This suggests that al-
though GPR55 activation of NFAT can be antiapoptotic in
some circumstances, blocking GPR55 with CBD could be
beneficial in neuroinflammation, as it might suppresses the
calcineurin–NFAT pathway [206].
CBD antagonizes nAChRs and although α7-nAChR ago-
nists have limited use in AD to improve cognition, in Down’s
syndrome increased Aβ
1–42
binding to nAChRs accelerates
Aβ
1–42
internalization that could worsen neuroinflammation
[207]. Thus, there are putative benefits and disadvantages to
agonism and antagonism of this receptor that are dependent on
the prevailing physiological conditions; both antagonists and
agonists can have benefit but also exhibit biphasic effects
[207,208]. At this juncture, it is therefore unclear whether
or not this is an important target for CBD.
CBD can act as a positive allosteric modulator of ORs [28,
209]. The opioid system has many functions that could be
important in neuroprotection, ranging from control of cellular
proliferation to immunomodulation. Although opposing ef-
fects can be seen in some circumstances, activation of ORs,
in particular, the δOR, is considered neuroprotective, espe-
cially in hypoxic/ischemic injury, and tends to be immunosup-
pressive. Certainly, in some animal models of MS and PD,
opioid agonists may be effective. However, there are cases of
movement disorders occurring in patients after anesthesia and
use of opioid drugs, which can be reversed by the opioid
antagonist naloxone and so suggests that the role of the opioid
system in neuroprotection might be more complex [210].
Thus, the potential role of CBD in modulating neuroprotec-
tion via the opioid system remains unclear.
CBD Enzyme Targets in Neurodegeneration
CBD can directly inhibit enzymes in the ETC in isolated mi-
tochondria [66]. However, brain extracts obtained from rats
acutely and repeatedly treated with CBD revealed significant-
ly increased ETC activity [67]. In neurodegenerative disease,
the ETC is often impaired [211]. This might therefore suggest
that CBD, like many other compounds that can inhibit mito-
chondrial function, can induce an adaptive upregulation of
mitochondrial function and resistance to oxidative stress, a
Molecular Targets of Cannabidiol
Author's personal copy

well-described process involving reactive oxygen species
[212,213].
CBD can exert biphasic effects on the kynurenine/
tryptophan pathways, causing activation at < 1 μM but inhi-
bition at higher concentrations [72]. A key enzyme in this
pathway is IDO, which, when activated by inflammatory me-
diators, might not only increase tryptophan degradation, and
thus 5-HT depletion, but also the production of metabolites
that could induce oxidative stress [94,214]. Thus, although
there is no direct evidence for inhibition of IDO by CBD,
suppression of this pathway could be an attractive approach
to reduce neuroinflammation.
As previously mentioned, CBD can positively allosterical-
ly modulate COX, and increased COX1 and COX2 activities
are thought to play an important role in the development of
neuroinflammation in several neurodegenerative disorders
[93,215]. However, interpretation is complicated because
COX enzymes can also metabolize docosahexaenoic acid/
eicosapentaenoic acid to the anti-inflammatory resolvins
[216]. In this regard, it has been suggested that low-dose as-
pirin, in combination with fish oil, is strongly anti-
inflammatory because it can allosterically activate COX2,
and in the presence of docosahexaenoic acid be protective in
AD [217]. As CBD can upregulate PPARγandCOX2atlow
micromolar doses, it is possible that CBD could have anti-
inflammatory affects both via PPARγand resolvins [18].
Finally, one property of CBD that has long thought to be
important in its mode of action is its ability to act as an anti-
oxidant and was originally considered to arise from its ability
to directly scavenge ROS [218,219]. However, the protective
effect of CBD has also been associated with an upregulation
of Cu,Zn superoxide dismutase mRNA, an antioxidant en-
zyme [220]. Indeed, CBD can activate the redox-sensing fac-
tor NRF2, which is key in detecting electrophilic xenobiotics
[79,200,221]. Mazur et al. [222]havesuggestedCBDisa
good candidate for phase 1 metabolism, as it is very lipophilic,
so would be oxidized and thus result in activation of phase II
metabolism. It therefore appears that CBD may be both a
direct and indirect antioxidant, with the latter effect arising
from activation of the xenobiotic system. Because of its anti-
oxidant properties, and its well-described anti-inflammatory
effects, it is under investigation for its potential use against
neurodegeneration [223].
CBD Ion Channels Targets in Neurodegeneration
CBD binds to and modulates mitochondrial VDAC1 [84].
VDAC1 levels are elevated in the brains of patients with AD
and the protein may interact with Aβand phosphorylated tau
which supports the idea that both affect mitochondrial func-
tion [224]. In contrast, reduced VDAC1 expression appears to
protect against AD [225]. VDAC1 plays a key role in control-
ling calcium flux into mitochondria, and intracellular calcium
can increase VDAC1 expression and oligomerization, which
is associated with increased apoptosis; in particular, it is
thought that VDAC1 selectively transfers apoptotic calcium
signals to the mitochondrion [226,227]. Two other groups
have also shown that CBD could apparently control intracel-
lular calcium flux in a mitochondrially dependent way, which,
overall, suggests that CBD, via a VDAC1-related mechanism,
could play an important role in controlling calcium flux in
neurodegeneration [228,229].
TRP channels have also been extensively investigated as
CBD targets where it acts largely as an agonist. Overactivation
of TRPV1 has been implicated in neurodegeneration via cal-
cium overload of mitochondria, although the rapid desensiti-
zation of TRPV1 following agonist stimulation suggests a
plausible longer-term mechanism by which agonists could
be neuroprotective [230]. In fact, TRPV1-mediated modula-
tion of presynaptic neurotransmitter release is partly depen-
dent on mitochondrial calcium signaling, and TRPV1 activa-
tion can enhance mitochondrial function [231,232]. That
TRPV1 can both enhance and inhibit mitochondrial function
can be reconciled by the fact that calcium tightly controls
mitochondrial function and biogenesis such that a small
amount can stimulate function but too much can induce apo-
ptosis [212,233]. Finally, vanillin-induced TRPV1 activation
reduced markers of oxidative stress in model of HD and im-
proved motor function, suggesting further validity for this
target in relation to CBD [234].
CBD Transporter Targets in Neurodegeneration
CBD can inhibit cellular uptake of adenosine via inhibition of
the ENT1 carrier at nanomolar concentrations, and it is now
thought that adenosinergic signaling could play a role in neu-
roprotection [10,106,228,235]. Although possible evidence
that CBD might act, in part, via this system was suggested by
the observation that in brain slices taken from a newborn
mouse hypoxia model that A
2A
and A
1A
receptor antagonists
inhibited a protective CBD effect, the most significant effect
was only observed at 100 μM—a physiologically implausible
concentration [31]. In contrast, Mecha et al. [10], using a
virally induced mouse inflammatory model of MS, demon-
strated that anantagonist of the A
2A
receptor partially blocked
the in vivo ability of CBD to suppress inflammation and im-
mune cell infiltration in the brains of infected mice; the ad-
ministration of the antagonist on its own had no effect. In cell
lines, the antagonist could also block the anti-inflammatory
effects of 1 μM CBD in a dose-responsive manner [10]. Thus,
there is some evidence that CBD might partially work through
increasing adenosine levels, but it does require further
research.
Computational analysis and ligand displacement assays
suggest that CBD is transported by FABPs, which also trans-
port AEA and so could explain the ability of CBD to increase
Bih et al.
Author's personal copy

AEA levels; the K
i
of CBD determined by displacement as-
says was in the region of 1.5–1.9 μMfor3FABPs—which is
comparable to AEA in the same assay (0.8–3.1 μM) [194].
FABPs are involved in intracellular trafficking of hydrophobic
ligands within cells and are thus important in the transport of
endogenouslipid and xenobiotics [236]. Although evidence is
limited, FABP levels appear to be increased in neurodegener-
ative states and may be a potential biomarker [237].
Moreover, increased FABP3 expression accentuates arachi-
donic acid-induced α-synuclein oligomerization and cell
death [238]. As already discussed, the ECS is thought to be
important in neurodegeneration, so the ability of CBD to bind
competitively to FABP could indicate that it is an important
target.
CBD inhibits ABCG2 (IC
50
=21–30 μM) and ABCC1
(IC
50
=31–128 μM) [103,104]. ATP-binding cassette trans-
porters, as well as being involved in lipid metabolism, choles-
terol efflux, and drug transport, are important in removing
intracellular Aβand are dysfunctional in AD; as they are
ATP dependent, it has been suggested that reduced energy
production could lead to a build-up of Aβ[239].
Specifically, both ABCG2 and ABCC1 have been found to
be involved in Aβtransport [240,241]. Whether or not CBD
may beneficially act through these transporters is difficult to
gauge, as not only might they inhibit ABC-mediated transport
of Aβ, but the reported IC
50
may also be too high to be rele-
vant physiologically.
CBD can inhibit both dopamine and glutamate uptake
[105]. The dopaminergic system is involved in some stages
of AD, for instance its degeneration can result in apathy and
although its precise role is still debated, agonists can be of
some benefit [242]. Glutamate, as the major excitatory neuro-
transmitter, has long been associated with neurodegeneration
but its ultimate effect is very much dependent upon the gluta-
mate receptor subtype mediating its effects; some can be pro-
tective while others exacerbate damage [243]. Thus, CBD
might have some beneficial effect on dopamine levels, but
the higher concentrations required for effects on glutamate
suggest that this is less likely to be an important mechanism.
Finally, CBD inhibits low-density lipoprotein-induced
cholesteryl ester formation in cultured human fibroblasts, not
via direct inhibition of ACAT but possibly via increased cho-
lesterol transport out of the cell—an effect that was apparent
from 10 to 30 μMincellculture[64]. This suggests that it
might modulate cholesterol metabolism—in particular, re-
verse cholesterol transport. Apolipoprotein E (APOE) is the
major apolipoprotein in the CNS, and in combination with
high-density lipoprotein is mainly responsible for transporting
cholesterol and amyloid around the brain. The APOE4 allele
is associated with decreased APOE levels and is associated
with AD. APOE is mostly synthesized by glial cells and can
bind to the low-density lipoprotein receptor on neurons and
thus be taken up. It has been suggested that APOE4 can result
in excessive amyloid build up, interference with Tau metabo-
lism, and/or disrupt lipid homeostasis and reduced neuronal
plasticity; upregulating APOE levels might thus be protective.
There is thus a close relationship between cholesterol trans-
port and amyloid deposition—although the precise inter-
relationship needs to be clarified [244,245]. Although an
interesting effect, the possible influence of CBD on cholester-
ol efflux has not been further verified, and the precise molec-
ular targets are not known;the concentrationsrequired are also
quite high.
Pain
The International Association for the Study of Pain defines
pain as Ban unpleasant sensory and emotional experience as-
sociated with actual or potential tissue damage, or described in
terms of such damage^. In many diseases such as MS or
cancer, pain no longer serves its protective role, which is to
avoid further tissue damage. Instead, because it is prolonged,
it turns out to deteriorate the quality of life of individuals
affected by these diseases. Although such chronic pain can
be caused by either injury in the peripheral tissue or lesion
in the CNS or in nerves, the common factor in both cases is
inflammation. Current treatments are partially effective and
are accompanied with side effects that can be more or less
severe [246,247]; therefore, new options of treatment involv-
ing cannabinoids (e.g., CBD) are now been pursued. In the
present review, we are focusing on the mechanism of action
underlying the therapeutic action of CBD only.
Although the effect of CBD on acute or chronic pain is still
not well understood, a few studies using animal models of pain
do suggest that CBD can be used to control inflammatory and
neuropathic pain. It has been shown that a per os administration
of CBD exerts antihyperalgesic effects on rat models of neuro-
pathic and inflammatory pain [248,249]. Microinjections of 3
nM CBD in the ventrolateral periaqueductal gray appears to
reduce the frequency of spikes generated by both ON cells
and OFF cells (localized in the rostral ventromedial medulla
(RVM), controlling the descending pathway of antinociception
[250]. Furthermore, CBD appears to control allodynia and neu-
ropathic pain caused by paclitaxel in mice [251,252].
CBD Ion Channel Targets in Pain
TRP channels are currently seen as promising targets for pain
management. A recent study suggests that each of them (e.g.,
TRPA1 and TRPV1) play distinct roles depending on the type
of pain in rodent models [253]. So far, only TRPA1 and TRPV1
have been involved in the therapeutic action of CBD regarding
the management of pain [249,251]. Capsazepine (intraperito-
neally), a TRPV1 antagonist, abolished the therapeutic action of
CBD in different rat models of pain such as carrageenan-
induced thermal hyperalgesia, chronic constriction injury of
Molecular Targets of Cannabidiol
Author's personal copy

the sciatic nerve, and the Freund's adjuvant model [248,249].
As CBD may inhibit the cellular uptake of AEA, although at
arguably supraphysiological concentrations, one could argue
that TRPV1 is activated simultaneously by the excess of extra-
cellular AEA and CBD—although such effects may only occur
at supraphysiological concentrations. Interestingly, AM404, a
blocker of AEA uptake, also exhibits analgesic characteristics
in a rat model of neuropathic pain [254]. However, its beneficial
effect is not prevented by capsazepine [249], indicating that the
action of AEA upon TRPV1 is also involved in the manage-
ment of neuropathic pain. Conversely, the action of CBD upon
TRPV1 does not seem to regulate the descending pathway of
antinociception. It appears that the reduction spikes frequency
in both ON cells and OFF cells in the RVM caused by CBD is
not reversed with 5′-iodo-resiniferatoxin, which antagonises
TRPV1 [250]. On the contrary, it appears that the mechanism
of action of CBD upon this descending pathway of
antinociception may be mediated by TRPA1 [250]. Finally,
although the inhibition of TRPM8 does not seem to be involved
in the alleviation of inflammatory and neuropathic pain [255], it
does appear that it attenuates cold hyperalgesia and tactile
allodynia [253].
VGCCs present at nerve endings, such as Cav2.2, are in-
volved in pain and appear to be important targets in the treat-
ment of inflamed and neuropathic pain. A blockade of Cav2.2
using ω-conotoxin MVIIA has been shown to alleviate both
inflamed and neuropathic pain [256,257]. However, regard-
ing other VGCCs such as Cav3.1, Cav3.2, and Cav3.3, which
are targets for CBD [95], it is still not known whether they are
channels that are important targets in the treatment of pain.
To date there has been no association between VDAC1 and
pain. Therefore, it is not known whether the effect of CBD
upon this protein is relevant to treat pain.
CB
1
R represent a useful target in the treatment of pain,
although both the alleged antagonistic profile of CBD at
CB
1
R at supraphysiological concentrations in vitro and ab-
sence of CB
1
R-mediated effects in vivo indicate that any role
of CBD in pain relief does not involve direct interaction with
CB
1
RandCB
2
R[22,249,250,252].
CBD Receptor Targets in Pain
With regard to other receptor types, only 1 study has demon-
strated that the mode of action of CBD upon the descending
pathway of antinociception is dependent on the stimulation of
A
1
adenosine receptor [250]. 1, 3-Dipropyl-8-
cyclopentylxanthine, an A
1
receptor antagonist, abolishes the
effect of CBD on the spikes frequency generated by ON cells
and OFF cells in the RVM, while blockade of this receptor
reverses the beneficial effect of norisoboldine, which attenu-
ates inflammatory pain [258]. To date, there is no evidence
demonstrating a direct implication ofadenosine A
2
receptor in
the therapeutic action of CBD in pain relief. Nevertheless, as
CBD suppresses the inflammatory response caused by aden-
osine A
2
receptor, we can hypothesize that the inactivation of
A
2
adenosine receptor would promote pain. Interestingly, it
has been shown that the knockout of adenosine A
2A
receptor
in male and female mice protects against carrageenan-induced
mechanical hyperalgesia [259].
The analgesic effect of CBD in a mice model of chronic
pain is dependent on α3GlyR[22], as, in a model of chronic
pain, mice that do not express these GlyR were insensitive to
the analgesic effect of CBD (50 mg/kg i.p.) [22].
It is still not clear whether CBD is able to manage
paclitaxel-induced neuropathic pain through the activation of
5-HT
1A
receptor or through the inhibition of D
4
dopamine
receptor [251,252], as studies that often investigate the in-
volvement of 5-HT
1A
receptor in therapeutic action of CBD
employ WAY-100635, which not only acts as an antagonist of
5-HT
1A
receptor [260], but is also a potent agonist at the D
4
dopamine receptor [48].
GPR55 has been implicated in inflammatory pain [261]. In
fact, mice that do not express GPR55 do not exhibit inflam-
matory mechanical hyperalgesia for up to 14 days after the
administration of Freund's complete adjuvant, although
whether or not CBD mediates its action via GPR55 in vivo
is unknown.
Although the activation of α-7-nACh receptor has been re-
ported as anti-inflammatory and protective against nociception
in neuropathic pain [262–265], it is unlikely that the analgesic
proprieties of CBD are mediated by this receptor as CBD tends
to antagonize (IC
50
=12.7μM) its action in vitro [27].
ORs exert analgesic effects [266]. However, their stimula-
tion can be associated with several side effects, including ad-
diction [267]. CBD has been described as a modulator of μ
and δORs [28]; there are, however, no studies demonstrating
this behaviorally. This might indicate that CBD can modulate
the undesirable effect caused by opioids, such as addiction.
CBD Enzyme Targets in Pain
In addition, it is possible that CBD affects the metabolism of
opioid analgesics by inhibiting CYP2D6, because polymor-
phism of CYP2D6 has been linked to distinct pain sensitivity
and difference of response to opioid analgesics [268].
Among the targets of CBD, it appears that the TPRV1,
TRPA1, and TRPM8 may be of greater significance regarding
the treatment of pain.
Psychosis and Anxiety
CBD Ion Channel Targets in Psychosis
CBD activates and rapidly desensitizes TRPV1 receptors at
low micromolar concentrations. TRPV1 activation has been
shown to cause anxiety-like behavior in rats. Current evidence
Bih et al.
Author's personal copy

indicates that CBD acts as an anxiolytic; therefore, CBD ag-
onist activity at TRPV1 channels is not responsible for its
anxiolytic effects [269]. A recent study indicates that the acti-
vation TRPV3 channels in rats reduced anxiety [270]. The
TRPV3 receptor is not a plausible target for CBD, as high
micromolar concentrations (100 μM) are required for channel
activation [71]. At present, neither TRPV2 nor TRPV4 have
been reported to have any involvement in anxiety disorder or
psychosis. Regarding other TRPV isoforms, no publications
linking TRPA1 or TRPM8 to either disease have been made.
CBD also binds VDAC1 and PPARγ, but their roles in anx-
iety and psychosis have not been established.
At present there are no antianxiety drugs that target
VGCCs, but CBD has been proven to act as a blocker of
Cav3.1 and Cav3.2 T-type channels at low micromolar con-
centrations [271]. However, studies on mice indicate that
Cav3.2 knockouts exhibit greater anxiety-like behavior
[272]. Thus, blockade of Cav3.2 cannot explain the anxiolytic
effects of CBD.
CBD Receptor Targets in Psychosis
CBD is a potent activator of the 5-HT
1A
and 5-HT
2A
receptors
[26]. Buspirone activates the 5-HT
1A
receptor to treat gener-
alized anxiety disorder. Work undertaken recently shows that
anxiety is greater in 5-HT
1A
knockout animals. However, 5-
HT
2A
knockouts produced the opposite outcome [169]. Based
on the evidence so far, the 5-HT
1A
receptor represents a good
target for CBD in anxiety. It has been proposed that 5-HT
2A
activation leads to psychotic symptoms. It is unlikely that
CBD exerts its therapeutic effects through 5-HT
2A
receptor
activation, as CBD has never been shown to exert psychotro-
pic effects. Thus, involvement of 5-HT
2A
in the antipsychotic
effect of CBD is unlikely [273].
As mentioned previously, GlyRs are mainly expressed in
the brainstem and spinal cord, where they mediate inhibitory
transmission. The activation of strychnine-sensitive GlyRs by
taurine reduced anxiety in rodent models [274]. High micro-
molar concentrations of CBD are required to activate GlyRs
and therefore GlyRs are not appropriate targets for CBD.
The roles of the μand δORs has been studied in rodent
models of anxiety. μOR activation has been associated with
anxiogenic effects, while δOR activation reduced anxiety-like
behavior [275]. Although their role in anxiety has been
established, the high micromolar concentrations of CBD re-
quired for OR activation are not achievable in vivo [28].
Therefore, it is unlikely that CBD effects at ORs mediate
anxiety, and no reports linking opioid transmission to psycho-
sishavebeenmadesofar.
The GPR55 receptor is expressed in the hippocampus,
where they trigger Ca
2+
release from the sarcoplasmic reticu-
lum upon activation [276]. It has been demonstrated that
GPR55 activation produces anxiolytic effects in rodent
models; therefore, the antagonistic properties of CBD at
GPR55 are not consistent with its antianxiety response. It is
not known if GPR55 is involved in psychosis.
In vitro studies have shown that A
1
and A
2
receptors are
potently activated by CBD. In zebrafish, the A
1
receptor sub-
type has been implicated in anxiety, where its blockade caused
anxiety-like behaviors. Current pharmacological data do not
support the involvement of A
2
receptors in anxiety [277], or
either receptor isoform in psychosis, but blockade of adeno-
sine clearance produced antipsychotic effects in rats [278].
Adenosine agonists cannot be used as treatments, as they re-
duce cardiac output [170]. This effect was not observed when
CBD was administered to either humans or animals; therefore,
it is likely that CBD causes an indirect elevation of adenosine
release as opposed to receptor activation.
CBD Enzyme Targets in Psychosis
Although CYP enzymes are present in the CNS, it is not
known if they are involved in anxiety or psychosis. AANAT
has not been implicated in either disease. An increased expres-
sion of COX2 has been reported in patients with schizophre-
nia [279]. A very high concentration of CBD is required to
interact with these enzymes, so it is unlikely they are involved
in the anxiolytic or antipsychotic effect of CBD.
An elevation in FAAH activity has been reported following
chronic restraint stressin animal models, and alsoanxiety-like
behaviors [280]. FAAH inhibition also alleviates psychotic
symptoms in patients with schizophrenia [281]. CBD is a
potent inhibitor of FAAH; therefore, it is worth investigating
if this will provide a therapeutic effect.
CBD Transporter Targets in Psychosis
The inhibition of glutamate uptake by CBD is not consistent
with its anxiolytic effects, as blockade of the glutamate trans-
porter has been shown to create anxiety-like behaviors in ro-
dents. It has been proposed that patients with reduced levels of
glutamate in the thalamus are at greater risk of psychosis, thus
blockade of glutamate uptake will not treat psychosis in pa-
tients [167]. Elevated levels of adenosine lead to greater acti-
vation of the A
1
receptor, which has been linked to anxiolytic
effects. Thus, inhibition of ENT1, responsible for adenosine
uptake, may provide a therapeutic effect. Dopamine uptake
has not been implicated in anxiety or psychosis [282].
CBD inhibits AEA uptake at likely supraphysiological
concentrations, possibly by binding an unknown AEA trans-
porter [19,71]. Although the inhibition of AEA uptake has
produced anxiolytic responses in animal models, previous
studies have shown that CB
1
R activation leads to psychotro-
pic effects; this has never been observed following CBD treat-
ment [279]. Furthermore, prolonged CB
1
R activation leads to
tolerance, which has not been reported in CBD-treated
Molecular Targets of Cannabidiol
Author's personal copy

animals [180]. Thus, it is unlikely that the AEA transporter is
involved in the anxiolytic or antipsychotic effect of CBD.
Addiction
The therapeutic effect of CBD on addiction has not yet been
well established. So far, only 2 studies have approached this
question directly [283,284]. Preliminary studies performed on
rats trained to self-administer heroin for 6 sessions showed
that CBD (5 mg/kg and 20 mg/kg i.p.) did not reduce heroin
self-administration behavior. However, when CBD is admin-
istered 24 h before the test, it attenuates conditioned cue-
induced heroin-seeking behavior after a 2-week period of ab-
stinence [284]. Conversely, the same treatment (24 h prior to
test) did not significantly reduce drug-seeking behavior after
heroin prime. Furthermore, the authors showed that CBD pro-
motes the extinction of heroin self-administration [284].
Another study recently performed on 24 tobacco smokers
suggested that CBD could reduce (~40%) the number of cig-
arettes smoked [283], but the effect did not persist after treat-
ment nor did it alter craving.
CBD Ion Channel Targets in Addiction
If CBD did exert antiaddictive proprieties, the evidence so far
would suggest that molecular targets involved in the metabo-
lism of drug such as CYP450, the transporter of drugs (e.g.,
ABCG2) and ORs, would be of greatest interest.
Its action upon TRPV1 may not mediate any therapeutic
effects in addiction, AS blocking this channel turns out to be
beneficial to control addictive behavior [285,286]. Equally,
the roles of VDAC1, VGCCs, and the other TRP channels that
are targets for CBD have not yet had their role in addiction
firmly established. Although, it has been shown that opiates
are involvedin the internalization of TRPM8, which can affect
thermosensitization and pain [287], it remains unclear whether
this plays any role in addiction.
CBD Enzyme Targets in Addiction
A reduced activity of CYP2D6 has been associated with pro-
tection against addictive behavior caused by oral opiate [288].
Interestingly, CYP2D6 is highly expressed in the brain of
alcoholics [289]. This may indicate that the activity of
CYP2D6 is of great importance in the pathway of addiction
associated with drugs. For further details about the opioid
system and CBD see the section on pain.
CBD Transporter Targets in Addiction
Finally, it is possible that the effect of CBD upon ABCG2 may
impact drugs transport and drug abuse [290].
Conclusions
We have identified a large number of potential molecular tar-
gets of CBD which are likely to be of direct relevance to many
of the therapeutic effects of this compound reported in large
number of preclinical and smaller numbers of clinical studies.
However, despite such an extensive literature, there is a pau-
city of studies that incontrovertibly demonstrate which specif-
ic molecular targets underlying different therapeutic effects of
CBD. A significant area of confusion in this regard has been
the assumption that, being a cannabinoid, CBD acts through
the ECS; an assumption that has now been largely dismissed.
Moreover, the lipophilic nature of CBD and the membrane-
bound nature of many of the targets implicated means that
many of the effects reported at high micromolar (or higher)
concentrations in vitro are likely to be nonspecific and of
dubious relevance to any therapeutic effect. Thus, consider-
able caution should be exercised when both interpreting the
extant literature and designing new studies to determine CBD
mechanisms of action. While definitive statements regarding
the specific molecular targets underlying CBD’s effects can-
not yet be made, our review of the literature has identified
several targets per therapeutic indication which, on the bal-
ance of evidence, warrant further, focused investigation in
studies that establish causal relationships between target and
effect (Table 5).
Required Author Forms Disclosure forms provided by the authors are
available with the online version of this article.
Tab le 5 Most plausible molecular targets of cannabidiol (CBD)
warranting further investigation in specific disease stated
Disease or disease group Most plausible molecular targets of CBD
Epilepsy VDAC1, CaV3.x, 5-HT
1A
, GlyR, GPR55,
adenosine modulation (ENT1)
Movement disorders CaV3.x, 5-HT
1A
,VDAC1
Neurodegenerative diseases VDAC1, FABP, GPR55, NRF2, ENT1
Pain TRPV1, TRPA1, TRPM8
Psychosis and anxiety 5-HT
1A
, adenosine modulation (ENT1)
Addiction CYP2D6, opioid receptors, ABCG2
VDAC1 = voltage-dependent anion channel 1; 5-HT = serotonin; GlyR=
glycine receptor; GPR55= G protein-coupled receptor 55; ENT1 = equil-
ibrative nucleoside transporter 1; FABP = fatty acid binding protein;
NRF2 = Nuclear factor erythroid 2-related factor 2; TRPV1 = transient
receptor potential vanilloid-type 1; TRPA1 = transient receptor potential
ankyrin type 1; TRPM8 = transient receptor potential subfamily M; CYP
= cytochrome P
Bih et al.
Author's personal copy

References
1. Brunner TF. Marijuana in ancient Greece and Rome? The literary
evidence. Bull Hist Med 1973;47:344-355.
2. Dark P. The environment of Britain in the first millennium AD.
UK: Bloomsbury Academic Press, 2000.
3. Li H-L. An archaeological and historical account of cannabis in
China. Econ Bot 1973;28:437-448.
4. Manniche L. An ancient Egyptian herbal. British Museum Press.
5. Mathre ML. Cannabis in medical practice: a legal, historical and
pharmacological overview of the therapeutic use of marijuana.
McFarland & Co Inc., Jefferson, NC. USA, 1997.
6. Mikuriya TH. Marijuana in medicine: past, present and future.
Calif Med 1969;110:34-40.
7. Morningstar PJ. Thandai and chilam: traditional Hindu beliefs
about the proper uses of Cannabis. J Psychoactive Drugs
1985;17:141-165.
8. Pollington S. Leechcraft: early English charms, plant lore, and
healing. Anglo-Saxon Books. UK, 2000.
9. Touw M. The religious and medicinal uses of Cannabis in China,
India and Tibet. J Psychoactive Drugs 1981;13:23-34.
10. Mecha M, Feliú A, Iñigo PM, Mestre L, Carrillo-Salinas FJ,
Guaza C. Cannabidiol provides long-lasting protection against
the deleterious effects of inflammation in a viral model of multiple
sclerosis: a role for A2A receptors. Neurobiol Dis 2013;59:141-
150.
11. Serpell M, Ratcliffe S, Hovorka J, et al. A double-blind, random-
ized, placebo-controlled, parallel group study of THC/CBD spray
in peripheral neuropathic pain treatment. Eur J Pain 2014;18:999-
1012.
12. Shrivastava A, Kuzontkoski PM, Groopman JE, Prasad A.
Cannabidiol induces programmed cell death in breast cancer cells
by coordinating the cross-talk between apoptosis and autophagy.
Mol Cancer Ther 2011;10:1161-1172.
13. Pertwee R. Handbook of cannabis. Oxford University Press.
14. Porter BE, Jacobson C. Report of a parent survey of cannabidiol-
enriched cannabis use in pediatric treatment-resistant epilepsy.
Epilepsy Behav 2013;29:574-577.
15. GW Pharma. GW Pharmaceuticals provides update on Epidiolex®
program in treatment-resistant childhood epilepsies. Available at:
http://www.gwpharm.com/GW%20Pharmaceuticals%
20Provides%20Update%20on%20Epidiolex%20Program%
20in%20Treatment-Resistant%20Childhood%20Epilepsies.aspx.
Accessed August 4, 2015.
16. McPartland JM, Duncan M, Di Marzo V, Pertwee RG. Are
cannabidiol and Δ(9)-tetrahydrocannabivarin negative modula-
tors of the endocannabinoid system? A systematic review. Br J
Pharmacol 2015;172:737-753.
17. Deiana S, Watanabe A, Yamasaki Y, et al. Plasma and brain phar-
macokinetic profile of cannabidiol (CBD), cannabidivarine
(CBDV), Δ
9
-tetrahydrocannabivarin (THCV) and cannabigerol
(CBG) in rats and mice following oral and intraperitoneal admin-
istration and CBD action on obsessive-compulsive behaviour.
Psychopharmacology (Berl) 2012;219:859-873.
18. Ramer R, Heinemann K, Merkord J, et al. COX-2 and PPAR-γ
confer cannabidiol-induced apoptosis of human lung cancer cells.
Mol Cancer Ther 2013;12:69-82.
19. Bisogno T, Hanus L, De Petrocellis L, et al. Molecular targets for
cannabidiol and its synthetic analogues: effect on vanilloid VR1
receptors and on the cellular uptake and enzymatic hydrolysis of
anandamide. Br J Pharmacol 2001;134:845-852.
20. Ryberg E, Larsson N, Sjögren S, et al. The orphan receptor
GPR55 is a novel cannabinoid receptor. Br J Pharmacol
2007;152:1092-1101.
21. Ahrens J, Demir R, Leuwer M, et al. The nonpsychotropic canna-
binoid cannabidiol modulates and directly activates alpha-1 and
alpha-1-Beta glycine receptor function. Pharmacology 2009;83:
217-222.
22. Xiong W, Cui T, Cheng K, et al. Cannabinoids suppress inflam-
matory and neuropathic pain by targeting α3 glycine receptors. J
Exp Med 2012;209:1121-1134.
23. McHugh D. GPR18 in microglia: implications for the CNS and
endocannabinoid system signalling. Br J Pharmacol 2012;167:
1575-1582.
24. Whyte LS, Ryberg E, Sims NA, et al. The putative cannabinoid
receptor GPR55 affects osteoclast function in vitro and bone mass
in vivo. Proc Natl Acad Sci U S A 2009;106:16511-16516.
25. Li K, Fichna J, Schicho R, et al. A role forO-1602and G protein-
coupled receptor GPR55in the control of colonic motility in mice.
Neuropharmacology 2013;71:255-263.
26. Russo EB, Burnett A, Hall B, Parker KK. Agonistic properties of
cannabidiol at 5-HT1a receptors. Neurochem Res 2005;30:1037-
1043.
27. Mahgoub M, Keun-Hang SY, Sydorenko V, et al. Effects of
cannabidiol on the function of α7-nicotinic acetylcholine recep-
tors. Eur J Pharmacol 2013;720:310-319.
28. Kathmann M, Flau K, Redmer A, Tränkle C, Schlicker E.
Cannabidiol is an allosteric modulator at mu- and delta-opioid
receptors. Naunyn. Schmiedebergs Arch Pharmacol 2006;372:
354-361.
29. Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of
three plant cannabinoids: delta9-tetrahydrocannabinol,
cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol
2008;153:199-215.
30. Thomas A, Baillie GL, Phillips AM, Razdan RK, Ross RA,
Pertwee RG. Cannabidiol displays unexpectedly high potency as
an antagonist of CB1 and CB2 receptor agonists in vitro. Br J
Pharmacol 2007;150:613-623.
31. Castillo A, Tolón MR, Fernández-Ruiz J, Romero J, Martinez-
Orgado J. The neuroprotective effect of cannabidiol in an
in vitro model of newborn hypoxic-ischemic brain damage in mice
is mediated by CB(2) and adenosine receptors. Neurobiol Dis
2010;37:434-440.
32. Járai Z, Wagner JA, Varga K, et al. Cannabinoid-induced mesen-
teric vasodilation through an endothelial site distinct from CB1 or
CB2 receptors. Proc Natl Acad Sci U S A 1999;96:14136-14141.
33. Schmuhl E, Ramer R, Salamon A, Peters K, Hinz B. Increase of
mesenchymal stem cell migration by cannabidiol via activation of
p42/44 MAPK. Biochem Pharmacol 2014;87:489-501.
34. Yang J-N, Chen J-F, Fredholm BB. Physiological roles of A1 and
A2A adenosine receptors in regulating heart rate, body tempera-
ture, and locomotion as revealed using knockout mice and caf-
feine. Am J Physiol Heart Circ Physiol 2009;296:H1141-H1149.
35. Elmenhorst D, Meyer PT, Winz OH, et al. Sleep deprivation in-
creases A1 adenosine receptor binding in the human brain: a pos-
itron emission tomography study. J. Neurosci. Off J Soc Neurosci
2007;27:2410-2415.
36. Gonca E, DarıcıF. The effect of cannabidiol on ischemia/
reperfusion-induced ventricular arrhythmias: the role of adenosine
A1 receptors. J Cardiovasc Pharmacol Ther 2015;20:76-83.
37. Liou GI, Auchampach JA, Hillard CJ, et al. Mediation of
cannabidiol anti-inflammationin the retina by equilibrative nucle-
oside transporter and A2A adenosine receptor. Invest Ophthalmol
Vis Sci 2008;49:5526-5531.
38. Sagredo O, Ramos JA, Decio A, Mechoulam R, Fernández-Ruiz
J. Cannabidiol reduced the striatal atrophy caused 3-nitropropionic
acid in vivo by mechanisms independent of the activation of can-
nabinoid, vanilloid TRPV1 and adenosine A2A receptors. Eur J
Neurosci 2007;26:843-851.
Molecular Targets of Cannabidiol
Author's personal copy

39. Lynch JW. Molecular structure and function of the glycine recep-
tor chloride channel. Physiol Rev 2004;84:1051-1095.
40. Xiong W, Chen S-R, He L, et al. Presynaptic glycine receptors as a
potential therapeutic target for hyperekplexia disease. Nat
Neurosci 2014;17:232-239.
41. Madar I, Lesser RP, Krauss G, et al. Imaging of delta- and mu-
opioid receptors in temporal lobe epilepsy by positron emission
tomography. Ann Neurol 1997;41:358-367.
42. Resstel LBM, Tavares RF, Lisboa SFS, Joca SRL, Corrêa FMA,
Guimarães FS. 5-HT1A receptors are involved in the cannabidiol-
induced attenuation of behavioural and cardiovascular responses
to acute restraint stress in rats. Br J Pharmacol 2009;156:181-188.
43. Soares V de P, Campos AC, Bortoli VC de, Zangrossi H Jr,
Guimarães FS, Zuardi AW. Intra-dorsal periaqueductal gray ad-
ministration of cannabidiol blocks panic-like response by activat-
ing 5-HT1A receptors. Behav Brain Res 2010;213:225-229.
44. Yang K-H, Galadari S, Isaev D, Petroianu G, Shippenberg TS, Oz
M. The nonpsychoactive cannabinoid cannabidiol inhibits 5-
hydroxytryptamine3A receptor-mediated currents in Xenopus
laevis oocytes. J Pharmacol Exp Ther 2010;333:547-554.
45. Zanelati TV, Biojone C, Moreira FA, Guimarães FS, Joca SRL.
Antidepressant-like effects of cannabidiol in mice: possible in-
volvement of 5-HT1A receptors. Br J Pharmacol 2010;159:122-
128.
46. Saigal N, Bajwa AK, Faheem SS, et al. Evaluation of serotonin 5-
HT1A receptors in rodent models using [18F]Mefway PET.
Synapse 2013;67:596-608.
47. Austgen JR, Kline DD. Endocannabinoids blunt the augmentation
of synaptic transmission by serotonin 2A receptors in the nucleus
tractus solitarii (nTS). Brain Res 2013;1537:27-36.
48. Chemel BR, Roth BL, Armbruster B, Watts VJ, Nichols DE.
WAY-100635 is a potent dopamine D4 receptor agonist.
Psychopharmacology (Berl) 2006;188:244-251.
49. Alves FHF, Crestani CC, Gomes FV, Guimarães FS, Correa FMA,
Resstel LBM. Cannabidiol injected into the bed nucleus of the
stria terminalis modulates baroreflex activity through 5-HT1A re-
ceptors. Pharmacol Res 2010;62:228-236.
50. Gomes FV, Alves FHF, Guimarães FS, Correa FMA, Resstel
LBM, Crestani CC. Cannabidiol administration into the bed nu-
cleus of the stria terminalis alters cardiovascular responses in-
duced by acute restraint stress through 5-HT1A receptor. Eur.
Neuropsychopharmacol 2013;23:1096-1104.
51. Sylantyev S, Jensen TP, Ross RA, Rusakov DA. Cannabinoid-
and lysophosphatidylinositol-sensitive receptor GPR55 boosts
neurotransmitter release at central synapses. Proc Natl Acad Sci
U S A 2013;110:5193-5198.
52. Baker D, Pryce G, Davies WL, Hiley CR. In silico patent
searching reveals a new cannabinoid receptor. Trends Pharmacol
Sci 2006;27:1-4.
53. Oka S, Nakajima K, Yamashita A, Kishimoto S, Sugiura T.
Identification of GPR55 as a lysophosphatidylinositol receptor.
Biochem Biophys Res Commun 2007;362:928-934.
54. O’Sullivan SE, Sun Y, Bennett AJ, Randall MD, Kendall DA.
Time-dependent vascular actions of cannabidiol in the rat aorta.
Eur J Pharmacol 2009;612:61-68.
55. Usami N, Yamamoto I, Watanabe K. Generation of reactive oxy-
gen species during mouse hepatic microsomal metabolism of
cannabidiol and cannabidiol hydroxy-quinone. Life Sci 2008;83:
717-724.
56. Zhou S-F, Liu J-P, Chowbay B. Polymorphism of human cyto-
chrome P450 enzymes and its clinical impact. Drug Metab Rev
2009;41:89-295.
57. Jiang R, Yamaori S, Okamoto Y, Yamamoto I, Watanabe K.
Cannabidiol is a potent inhibitor of the catalytic activity of
cytochrome P450 2C19. Drug Metab Pharmacokinet
2013;28:332-338.
58. Booth Depaz IM, Toselli F, Wilce PA, Gillam EMJ. Differential
expression of cytochrome P450 enzymes from the CYP2C sub-
family in the human brain. Drug Metab Dispos Biol Fate Chem
2015;43:353-357.
59. Wu M, Chen S, Wu X. Differences in cytochrome P450 2C19
(CYP2C19) expression in adjacent normal and tumor tissues in
Chinese cancer patients. Med Sci Monit 2006;12:BR174-BR178.
60. Yamaori S, Okamoto Y, Yamamoto I, Watanabe K. Cannabidiol, a
major phytocannabinoid, as a potent atypical inhibitor for
CYP2D6. Drug Metab Dispos 2011;39:2049-2056.
61. Yamaori S, Koeda K, Kushihara M, Hada Y, Yamamoto I,
Watanabe K. Comparison in the in vitro inhibitory effects of major
phytocannabinoids and polycyclic aromatic hydrocarbons
contained in marijuana smoke on cytochrome P450 2C9 activity.
Drug Metab Pharmacokinet 2012;27:294-300.
62. Yamaori S, Kushihara M, Yamamoto I, Watanabe K.
Characterization of major phytocannabinoids, cannabidiol and
cannabinol, as isoform-selective and potent inhibitors of human
CYP1 enzymes. Biochem Pharmacol 2010;79:1691-1698.
63. Yamaori S, Ebisawa J, Okushima Y, Yamamoto I, Watanabe K.
Potent inhibition of human cytochrome P450 3A isoforms by
cannabidiol: role of phenolic hydroxyl groups in the resorcinol
moiety. Life Sci 2011;88:730-736.
64. Cornicelli JA, Gilman SR, Krom BA, Kottke BA. Cannabinoids
impair the formation of cholesteryl ester in cultured human cells.
Arteriosclerosis 1981;1:449-454.
65. Koch M, Dehghani F, Habazettl I, Schomerus C, Korf H-W.
Cannabinoids attenuate norepinephrine-induced melatonin bio-
synthesis in the rat pineal gland by reducing arylalkylamine N-
acetyltransferase activity without involvement of cannabinoid re-
ceptors. J Neurochem 2006;98:267-278.
66. Fišar Z, Singh N, Hroudová J. Cannabinoid-induced changes in
respiration of brain mitochondria. Toxicol Lett 2014;231:62-71.
67. Valvassori SS, Bavaresco DV, Scaini G, et al. Acute and chronic
administration of cannabidiol increases mitochondrial complex
and creatine kinase activity in the rat brain. Rev Bras Psiquiatr
2013;35:380-386.
68. Massi P, Valenti M, Vaccani A, et al. 5-Lipoxygenase and anan-
damide hydrolase (FAAH) mediate the antitumor activity of
cannabidiol, a non-psychoactive cannabinoid. J Neurochem
2008;104:1091-1100.
69. Ruhaak LR, Felth J, Karlsson PC, Rafter JJ, Verpoorte R, Bohlin
L. Evaluation of the cyclooxygenase inhibiting effects of six major
cannabinoids isolated from Cannabis sativa. Biol Pharm Bull
2011;34:774-778.
70. Yamaori S,Okushima Y, Masuda K, et al. Structural requirements
for potent direct inhibition of human cytochrome P450 1A1 by
cannabidiol: role of pentylresorcinol moiety. Biol Pharm Bull
2013;36:1197-1203.
71. De Petrocellis L, Ligresti A, Moriello AS, et al. Effects of canna-
binoids and cannabinoid-enriched Cannabis extracts on TRP
channels and endocannabinoid metabolic enzymes. Br J
Pharmacol 2011;163:1479-1494.
72. Jenny M, Santer E, Pirich E, Schennach H, Fuchs D. Delta9-
tetrahydrocannabinol and cannabidiol modulate mitogen-induced
tryptophan degradation and neopterin formation in peripheral
blood mononuclear cells in vitro. J Neuroimmunol 2009;207:75-
82.
73. Takeda S, Usami N, Yamamoto I, Watanabe K. Cannabidiol-2’,6’-
dimethyl ether, a cannabidiol derivative, is a highly potent and
selective 15-lipoxygenase inhibitor. Drug Metab Dispos Biol
Fate Chem 2009;37:1733-1737.
74. Evans AT, Formukong E, Evans FJ. Activation of phospholipase
A2 by cannabinoids. Lack of correlation with CNS effects. FEBS
Lett 1987;211:119-122.
Bih et al.
Author's personal copy

75. Watanabe K, Motoya E, Matsuzawa N, et al. Marijuana extracts
possess the effects like the endocrine disrupting chemicals.
Toxicology 2005;206:471-478.
76. Burstein S, Hunter SA, Renzulli L. Stimulation of sphingomyelin
hydrolysis by cannabidiol in fibroblasts from a Niemann-Pick pa-
tient. Biochem Biophys Res Commun 1984;121:168-173.
77. Jiang R, Yamaori S, Takeda S, Yamamoto I, Watanabe K.
Identification of cytochrome P450 enzymes responsible for me-
tabolism of cannabidiol by human liver microsomes. Life Sci
2011;89:165-170.
78. Bryan HK, Olayanju A, Goldring CE, Park BK. The Nrf2 cell
defence pathway: Keap1-dependent and -independent mecha-
nisms of regulation. Biochem. Pharmacol 2013;85:705-717.
79. Juknat A, Pietr M, Kozela E, et al. Microarray and pathway anal-
ysis reveal distinct mechanisms underlying cannabinoid-mediated
modulation of LPS-induced activation of BV-2 microglial cells.
PloS One 2013;8:e61462.
80. Bock J, Szabó I, Gamper N, Adams C, Gulbins E. Ceramide
inhibits the potassium channel Kv1.3 by the formation of mem-
brane platforms. Biochem. Biophys Res Commun 2003;305:890-
897.
81. Cremesti AE, Goni FM, Kolesnick R. Role of sphingomyelinase
and ceramide in modulating rafts: do biophysical properties deter-
mine biologic outcome? FEBS Lett 2002;531:47-53.
82. Maziere JC, Maziere C, Mora L, Routier JD, Polonovski J. In situ
degradation of sphingomyelin by cultured normal fibroblasts and
fibroblasts from patients with Niemann-Pick disease type A and C.
Biochem Biophys Res Commun 1982;108:1101-1106.
83. Massi P, Vaccani A, Ceruti S, Colombo A, Abbracchio MP,
Parolaro D. Antitumor effects of cannabidiol, a nonpsychoactive
cannabinoid, on human glioma cell lines. J Pharmacol Exp Ther
2004;308:838-845.
84. Rimmerman N, Ben-Hail D, Porat Z, et al. Direct modulation of
the outer mitochondrial membrane channel, voltage-dependent
anion channel 1 (VDAC1) by cannabidiol: a novel mechanism
for cannabinoid-induced cell death. Cell Death Dis 2013;4:e949.
85. Shoshan-Barmatz V, Mizrachi D. VDAC1: from structure to can-
cer therapy. Mol Cell Oncol 2012;2:164.
86. Singh N, Hroudová J, Fišar Z. Cannabinoid-induced changes in
the activity of electron transport chain complexes of brain mito-
chondria. J Mol Neurosci 2015 Mar 29 [Epub ahead of print].
87. Escames G, León J, López LC, Acuña-Castroviejo D.
Mechanisms of N-methyl-D-aspartate receptor inhibition by mel-
atonin in the rat striatum. J Neuroendocrinol 2004;16:929-935.
88. Ozkanlar S, Kara A, Sengul E, Simsek N, Karadeniz A, Kurt N.
Melatonin modulates the immune system response and inflamma-
tion in diabetic rats experimentally-induced by alloxan. Horm
Metab Res 2015 May 4 [Epub ahead of print].
89. Ramis MR, Esteban S, Miralles A, Tan D-X, Reiter RJ. Protective
effects of melatonin and mitochondria-targeted antioxidants
against oxidative stress: a review. Curr Med Chem 2015 Jun 18
[Epub ahead of print].
90. Sun GY, Shelat PB, Jensen MB, He Y, Sun AY, Simonyi A.
Phospholipases A2 and inflammatory responses in the central ner-
vous system. Neuromolecular Med 2010;12:133-148.
91. Arriagada E, Cid H. Search for a Btoxic site^in snake venom
phospholipases A2. Arch Biol Med Exp 1989;22:97-105.
92. Calder PC. Omega-3 fatty acids and inflammatory processes.
Nutrients 2010;2:355-374.
93. Wheal AJ, Cipriano M, Fowler CJ, Randall MD, O’Sullivan SE.
Cannabidiol improves vasorelaxation in Zucker diabetic fatty rats
through cyclooxygenase activation. J Pharmacol Exp Ther
2014;351:457-466.
94. Hoyo-Becerra C, Schlaak JF, Hermann DM. Insights from
interferon-α-related depression for the pathogenesis ofdepression
associated with inflammation. Brain Behav Immun 2014;42:222-
231.
95. Ross HR, Napier I, Connor M. Inhibition of recombinant human
T-type calcium channels by Delta9-tetrahydrocannabinol and
cannabidiol. J Biol Chem 2008;283:16124-16134.
96. Qin N, Neeper MP, Liu Y, Hutchinson TL, Lubin ML, Flores CM.
TRPV2 is activated by cannabidiol and mediates CGRP release in
cultured rat dorsal root ganglion neurons. J Neurosci 2008;28:
6231-6238.
97. De Petrocellis L, Vellani V, Schiano-Moriello A, et al. Plant-
derived cannabinoids modulate the activity of transient receptor
potential channels of ankyrin type-1 and melastatin type-8. J
Pharmacol Exp Ther 2008;325:1007-1015.
98. Iannotti FA, Hill CL, Leo A, et al. Nonpsychotropic plant canna-
binoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate
and desensitize transient receptor potential vanilloid 1 (TRPV1)
channels in vitro: potential for the treatment of neuronal hyperex-
citability. ACS Chem Neurosci 2014;5:1131-1141
99. Ligresti A, Moriello AS, Starowicz K, et al. Antitumor activity of
plant cannabinoids with emphasis on the effect of cannabidiol on
human breast carcinoma. J Pharmacol Exp Ther 2006;318:1375-
1387.
100. Nabissi M, Morelli MB, Santoni M, Santoni G. Triggering of the
TRPV2 channel by cannabidiol sensitizes glioblastoma cells to
cytotoxic chemotherapeutic agents. Carcinogenesis 2013;34:48-
57.
101. De Petrocellis L, Orlando P, Moriello AS, et al. Cannabinoid ac-
tions at TRPV channels: effects on TRPV3 and TRPV4 and their
potential relevance to gastrointestinal inflammation. Acta Physiol
2012;204:255-266.
102. Billeter AT, Hellmann JL, Bhatnagar A, Polk HC. Transient recep-
tor potential ion channels: powerful regulators of cell function.
Ann Surg 2014;259:229-235.
103. Holland ML, Allen JD, Arnold JC. Interaction of plant cannabi-
noids with the multidrug transporter ABCC1 (MRP1). Eur J
Pharmacol 2008;591:128-131.
104. Holland ML, Lau DTT, AllenJD, Arnold JC. The multidrug trans-
porter ABCG2 (BCRP) is inhibited by plant-derived cannabi-
noids. Br J Pharmacol 2007;152:815-824.
105. Pandolfo P, Silveirinha V, Santos-Rodrigues A dos, et al.
Cannabinoids inhibit the synaptic uptake of adenosine and dopa-
mine in the rat and mouse striatum. Eur J Pharmacol 2011;655:38-
45.
106. Carrier EJ, Auchampach JA, Hillard CJ. Inhibition of an equili-
brative nucleoside transporter by cannabidiol: a mechanism of
cannabinoid immunosuppression. Proc Natl Acad Sci U S A
2006;103:7895-7900.
107. Gilbert JC, Pertwee RG, Wyllie MG. Effects of delta9-
tetrahydrocannabinol and cannabidiol on a Mg2+-ATPase of syn-
aptic vesicles prepared from rat cerebral cortex. Br J Pharmacol
1977;59:599-601.
108. Coyle JT, and Snyder SHJ. Catecholamine uptake by synapto-
somes in homogenates of rat brain: Stereospecificity in different
areas. Pharmacol Exp Ther 1969;170:221.
109. Whittaker VP, Michaelson IA, Kirkland RJA. The separation of
synaptic vesicles from nerve-ending particles ('synaptosomes').
Biochem J 1964;90:293-303.
110. Di Marzo V, Fontana A,Cadas H, et al. Formation and inactivation
of endogenous cannabinoid anandamide in central neurons.
Nature 1994;372:686-691.
111. MorelliMB,OffidaniM,AlesianiF,etal.Theeffectsof
cannabidiol and its synergism with bortezomib in multiple mye-
loma cell lines. A role for transient receptor potential vanilloid
type-2. Int J Cancer 2014;134:2534-2546.
Molecular Targets of Cannabidiol
Author's personal copy

112. Shoshan-Barmatz V, De Pinto V, Zweckstetter M, Raviv Z, Keinan
N, Arbel N. VDAC, a multi-functional mitochondrial protein reg-
ulating cell life and death. Mol Aspects Med 2010;31:227-285.
113. Hill AJ, Jones NA, Smith I, et al. Voltage-gated sodium (NaV)
channel blockade by plant cannabinoids does not confer anticon-
vulsant effects per se. Neurosci Lett 2014;566:269-274.
114. Vandenberg RJ, Ryan RM. Mechanisms of glutamate transport.
Physiol Rev 2013;93:1621-1657.
115. Banerjee SP, Snyder SH, Mechoulam R. Cannabinoids: influence
on neurotransmitter uptake in rat brain synaptosomes. J Pharmacol
Exp Ther 1975;194:74-81.
116. Poddar MK, Dewey WL. Effects of cannabinoids on catechol-
amine uptake and release in hypothalamic and striatal synapto-
somes. J Pharmacol Exp Ther 1980;214:63-67.
117. Lindamood C 3rd, Colasanti BK. Effects of delta 9-
tetrahydrocannabinol and cannabidiol on sodium-dependent high
affinity choline uptake in the rat hippocampus. J Pharmacol Exp
Ther 1980;213:216-221.
118. Rakhshan F, Day TA, Blakely RD, Barker EL. Carrier-mediated
uptake of the endogenous cannabinoid anandamide in RBL-2H3
cells. J Pharmacol Exp Ther 2000;292:960-967.
119. Watanabe K, Kayano Y, Matsunaga T, YamamotoI, Yoshimura H.
Inhibition of anandamide amidase activity in mouse brain micro-
somes by cannabinoids. Biol Pharm Bull 1996;19:1109-1111.
120. Di Marzo V, Melck D, De Petrocellis L, Bisogno T.
Cannabimimetic fatty acid derivatives in cancer and inflamma-
tion. Prostaglandins Other Lipid Mediat 2000;61:43-61.
121. Zhu H-J, Wang J-S, Markowitz JS, et al. Characterization of P-
glycoprotein inhibition by major cannabinoids from marijuana. J
Pharmacol Exp Ther 2006;317:850-857.
122. Szemraj J, Sobolewska B, Gulczynska E, Wilczynski J, Zylinska
L. Magnesium sulfate effect on erythrocyte membranes of asphyx-
iated newborns. Clin Biochem 2005;38:457-464.
123. Schmidt D, Schachter SC. Drug treatment of epilepsy in adults.
BMJ 2014;348:g254.
124. Devinsky O, Cilio MR, Cross H, et al. Cannabidiol: pharmacology
and potential therapeutic role in epilepsy and other neuropsychi-
atric disorders. Epilepsia 2014;55:791-802.
125. Shu H-F, Yu S-X, Zhang C-Q, et al. Expression of TRPV1 in
cortical lesions from patients with tuberous sclerosis complex
and focal cortical dysplasia type IIb. Brain Dev 2013;35:252-260.
126. Rüden EL von, Jafari M, Bogdanovic RM, Wotjak CT, Potschka
H. Analysis in conditional cannabinoid 1 receptor-knockout mice
reveals neuronal subpopulation-specific effects on epileptogenesis
in the kindling paradigm. Neurobiol Dis 2015;73:334-347.
127. Samineni VK, Premkumar LS, Faingold CL. Post-ictal analgesia
in genetically epilepsy-prone rats is induced by audiogenic sei-
zures and involves cannabinoid receptors in the periaqueductal
gray. Brain Res 2011;1389:177-182.
128. Bhaskaran MD, Smith BN. Cannabinoid-mediated inhibition of
recurrent excitatory circuitry in the dentate gyrus in a mouse mod-
el of temporal lobe epilepsy. PloS One 2010;5:e10683.
129. Chen C-Y, Li W, Qu K-P, Chen C-R. Piperine exerts anti-seizure
effects via the TRPV1 receptor in mice. Eur J Pharmacol
2013;714:288-294.
130. Ghazizadeh V, Nazıroğlu M. Electromagnetic radiation (Wi-Fi)
and epilepsy induce calcium entry and apoptosis through activa-
tion of TRPV1 channel in hippocampus and dorsal root ganglion
of rats. Metab Brain Dis 2014;29:787-799.
131. Gonzalez-Reyes LE, Ladas TP, Chiang C-C, Durand DM. TRPV1
antagonist capsazepine suppresses 4-AP-induced epileptiform ac-
tivity in vitro and electrographic seizures in vivo. Exp Neurol
2013;250:321-332.
132. Manna SSS, Umathe SN. Involvement of transient receptor poten-
tial vanilloid type 1 channels in the pro-convulsant effect of
anandamide in pentylenetetrazole-induced seizures. Epilepsy Res
2012;100:113-124.
133. Nazıroğlu M, Özkan FF, Hapil SR, Ghazizadeh V, ÇiğB.
Epilepsy but not mobile phone frequency (900 MHz) induces
apoptosis and calcium entry in hippocampus of epileptic rat:
involvement of TRPV1 channels. J Membr Biol 2015;248:
83-91.
134. Shirazi M, Izadi M, Amin M, Rezvani ME, Roohbakhsh A,
Shamsizadeh A. Involvement of central TRPV1 receptors in
pentylenetetrazole and amygdala-induced kindling in male rats.
Neurol Sci 2014;35:1235-1241.
135. Kong W-L, Min J-W, Liu Y-L, Li J-X, He X-H, Peng B-W. Role of
TRPV1 in susceptibility to PTZ-induced seizure following repeat-
ed hyperthermia challenges in neonatal mice. Epilepsy Behav
2014;31:276-280.
136. Hunt RF, Hortopan GA, Gillespie A, Baraban SC. A novel
zebrafish model of hyperthermia-induced seizures reveals a role
for TRPV4 channels and NMDA-type glutamate receptors. Exp
Neurol 2012;237:199-206.
137. Lin Y-W, Hsieh C-L. Auricular electroacupuncture reduced
inflammation-related epilepsy accompanied by altered TRPA1,
pPKCα,pPKCε, and pERk1/2 signaling pathways in kainic
acid-treated rats. Mediators Inflamm 2014;2014:493480.
138. Brinckmann A, Weiss C, Wilbert F, et al. Regionalized pathology
correlates with augmentation of mtDNA copy numbers in a patient
with myoclonic epilepsy with ragged-red fibers (MERRF-syn-
drome). PloS One 2010;5:e13513.
139. Jiang W, Du B,Chi Z, et al. Preliminary explorations of the role of
mitochondrial proteins in refractory epilepsy: some findings from
comparative proteomics. J Neurosci Res 2007;85:3160-3170.
140. Quintanilla RA, Utreras E, Cabezas-Opazo FA. Role of PPAR γin
the differentiation and function of neurons. PPAR Res 2014;2014:
768594.
141. LasońW, Chlebicka M, Rejdak K. Research advances in basic
mechanisms of seizures and antiepileptic drug action. Pharmacol
Rep 2013;65:787-801.
142. Adams PJ, Snutch TP. Calcium channelopathies: voltage-gated
calcium channels. Subcell Biochem 2007;45:215-251.
143. Gomora JC, Daud AN, Weiergräber M, Perez-Reyes E. Block of
cloned human T-type calcium channels by succinimide antiepilep-
tic drugs. Mol Pharmacol 2001;60:1121-1132.
144. Ceulemans B, Boel M, Leyssens K, et al. Successful use of fen-
fluramine as an add-on treatment for Dravet syndrome. Epilepsia
2012;53:1131-1139.
145. Theodore WH, Hasler G, Giovacchini G, et al. Reduced hippo-
campal 5HT1A PET receptor binding and depression in temporal
lobe epilepsy. Epilepsia 2007;48:1526-1530.
146. Theodore WH, Wiggs EA, Martinez AR, et al. Serotonin 1A re-
ceptors, depression, and memory in temporal lobe epilepsy.
Epilepsia 2012;53:129-133.
147. Avila A, Nguyen L, Rigo J-M. Glycine receptors and brain devel-
opment. Front Cell Neurosci 2013;7:184.
148. Harvey RJ, Carta E, Pearce BR, et al. A critical role for glycine
transporters in hyperexcitability disorders. Front Mol Neurosci
2008;1:1.
149. Chu Sin Chung P, Kieffer BL. Delta opioid receptors in brain
function and diseases. Pharmacol Ther 2013;140:112-120.
150. Snead OC. Opiate-induced seizures: a study of mu and delta spe-
cific mechanisms. Exp Neurol 1986;93:348-358.
151. Sagratella S, Scotti de Carolis A. In vivo and in vitro epileptogenic
effects of the enkephalinergic system. Ann Ist Super Sanita
1993;29:413-418.
152. Saghazadeh A, Mastrangelo M, Rezaei N. Genetic background of
febrile seizures. Rev Neurosci 2014;25:129-161.
Bih et al.
Author's personal copy

153. Becchetti A, Aracri P, Meneghini S, Brusco S, Amadeo A. The
role of nicotinic acetylcholine receptors in autosomal dominant
nocturnal frontal lobe epilepsy. Front Physiol 2015;6:22.
154. Dias RB, Rombo DM, Ribeiro JA, Henley JM, Sebastião AM.
Adenosine: setting the stage for plasticity. Trends Neurosci
2013;36:248-257.
155. Świąder MJ, Kotowski J, Łuszczki JJ. Modulation of
adenosinergic system and its application for the treatment of ep-
ilepsy. Pharmacol Rep 2014;66:335-342.
156. Hedlund E, Gustafsson JA, Warner M. Cytochrome P450 in the
brain; a review. Curr Drug Metab 2001;2:245-263.
157. Adibhatla RM, Hatcher JF. Altered lipid metabolism in brain in-
jury and disorders. Subcell Biochem 2008;49:241-268.
158. Coomber B, O’Donoghue MF, Mason R. Inhibition of
endocannabinoid metabolism attenuates enhanced hippocampal
neuronal activity induced by kainic acid. Synapse 2008;62:746-
755.
159. Vilela LR, Medeiros DC, Rezende GHS, de Oliveira ACP, Moraes
MFD, Moreira FA. Effects of cannabinoids and endocannabinoid
hydrolysis inhibition on pentylenetetrazole-induced seizure and
electroencephalographic activity in rats. Epilepsy Res 2013;104:
195-202.
160. Villikka K, Kivistö KT, Mäenpää H, Joensuu H, Neuvonen PJ.
Cytochrome P450-inducing antiepileptics increase the clearance
of vincristine in patients with brain tumors. Clin Pharmacol Ther
1999;66:589-593.
161. Geffrey AL, Pollack SF, Bruno PL, Thiele EA. Drug-drug inter-
action between clobazam and cannabidiol in children with refrac-
tory epilepsy. Epilepsia 2015 Jun 26 [Epub ahead of print].
162. Clinckers R, Smolders I, Meurs A, Ebinger G, Michotte Y.
Anticonvulsant action of hippocampal dopamine and serotonin
isindependentlymediatedbyDand5-HTreceptors.J
Neurochem 2004;89:834-843.
163. Zoerner AA, Gutzki F-M, Batkai S, et al. Quantification of
endocannabinoids in biological systems by chromatography and
mass spectrometry: a comprehensive review from an analytical
and biological perspective. Biochim Biophys Acta 2011;1811:
706-723.
164. Dawbarn D, Harmar AJ, Pycock CJ. Intranigral injection of cap-
saicin enhances motor activity and depletes nigral 5-
hydroxytryptamine but not substance P. Neuropharmacology
1981;20:341-346.
165. Hajós M, Engberg G, Nissbrandt H, Magnusson T, Carlsson
A. Capsaicin-sensitive vasodilatatory mechanisms in the rat
substantia nigra and striatum. J Neural Transm 1988;74:129-
139.
166. Geisler S, Holmström KM, Skujat D, et al. PINK1/Parkin-
mediated mitophagy is dependent on VDAC1 and p62/
SQSTM1. Nat Cell Biol 2010;12:119-131.
167. Stone JM, Day F, Tsagaraki H, et al. Glutamate dysfunction in
people with prodromal symptoms of psychosis: relationship to
gray matter volume. Biol Psychiatry 2009;66:533-539.
168. Sramek JJ, Tansman M, Suri A, et al. Efficacy of buspirone in
generalized anxiety disorder with coexisting mild depressive
symptoms. J Clin Psychiatry 1996;57:287-291.
169. Bourin M, Nic Dhonnchadha BA. 5-HT2 receptors and anxiety.
Drug Dev Res 2005;65:133-140.
170. Bush A, Busst CM, Clarke B, Barnes PJ. Effect of infused aden-
osine on cardiac output and systemic resistance in normal subjects.
Br J Clin Pharmacol 1989;27:165-171.
171. Litvin Y, Phan A, Hill MN, Pfaff DW, McEwen BS. CB1 receptor
signaling regulates social anxiety and memory. Genes Brain
Behav 2013;12:479-489.
172. Mohajjel Nayebi A A, Sheidaei H. Buspirone improves
haloperidol-induced Parkinson disease in mice through 5-HT1A
receptors. Daru 2010;18:41-45.
173. Becker CM, Hermans-Borgmeyer I, Schmitt B, Betz H. The gly-
cine receptor deficiency of the mutant mouse spastic: evidence for
normal glycine receptor structure and localization. J Neurosci
1986;6:1358-1364.
174. Henry B, Fox SH, Crossman AR, Brotchie JM. Mu- and delta-
opioid receptor antagonists reduce levodopa-induced dyskinesia
in the MPTP-lesioned primate model of Parkinson’s disease.
Exp Neurol 2001;171:139-146.
175. Patsopoulos NA, Ntzani EE, Zintzaras E, Ioannidis JPA. CYP2D6
polymorphisms and the risk of tardive dyskinesia in schizophre-
nia: a meta-analysis. Pharmacogenet Genomics 2005;15:151-158.
176. Liévens JC, Woodman B, Mahal A, et al. Impaired glutamate
uptake in the R6 Huntington’s disease transgenic mice.
Neurobiol Dis 2001;8:807-821.
177. Kanyó B, Argyelán M,Dibó G,et al. [Imaging of dopamine trans-
porter with Tc99m-Trodat-SPECT in movement disorders].
Ideggyogy Sz 2003;56:231-240 (in Hungarian).
178. de Lago E, Fernández-Ruiz J, Ortega-Gutiérrez S, et al. UCM707,
an inhibitor of the anandamide uptake, behaves as a symptom
control agent in models of Huntington’s disease and multiple scle-
rosis, but fails to delay/arrest the progression of different motor-
related disorders. Eur Neuropsychopharmacol 2006;16:7-18.
179. Pamplona FA, Ferreira J, Lima OM de, et al. Anti-inflammatory
lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabi-
noid receptor. Proc Natl Acad Sci 2012;109:21134-21139.
180. Daigle TL, Kearn CS, Mackie K. Rapid CB1 cannabinoid receptor
desensitization defines the time course of ERK1/2 MAP kinase
signaling. Neuropharmacology 2008;54:36-44.
181. Aso E, Sánchez-Pla A, Vegas-Lozano E, Maldonado R, Ferrer I.
Cannabis-based medicine reduces multiple pathological processes
in AβPP/PS1 mice. J Alzheimers Dis 2015;43:977-991.
182. Cheng D, Spiro AS, Jenner AM, Garner B, Karl T. Long-term
cannabidiol treatment prevents the development of social recogni-
tion memory deficits in Alzheimer’s disease transgenic mice. J
Alzheimers Dis 2014;42:1383-1396.
183. Calì T, Ottolini D, Brini M. Mitochondrial Ca2+ and neurodegen-
eration. Cell Calcium 2012;52:73-85.
184. Currais A. Ageing and inflammation –acentral role for mitochon-
dria in brain health and disease. Ageing Res Rev 2015;21:30-42.
185. Hsieh H-L, Yang C-M. Role of redox signaling in neuroinflam-
mation and neurodegenerative diseases. Biomed Res Int
2013;2013:484613.
186. Sosa-Ortiz AL, Acosta-Castillo I, Prince MJ. Epidemiology of
dementias and Alzheimer’s disease. Arch Med Res 2012;43:600-
608.
187. Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade
hypothesis. Science 1992;256:184-185.
188. Latta CH, Brothers HM, Wilcock DM. Neuroinflammation in
Alzheimer’s disease; a source of heterogeneity and target for per-
sonalized therapy. Neuroscience 2014;pii:S0306-4522(14)00820-
3.
189. Lozano D, Gonzales-Portillo GS, Acosta S, et al.
Neuroinflammatory responses to traumatic brain injury: etiology,
clinical consequences, and therapeutic opportunities.
Neuropsychiatr Dis Treat 2015;11:97-106.
190. Ruhoy IS, Saneto RP. The genetics of Leigh syndrome and its
implications for clinical practice and risk management. Appl
Clin Genet 2014;7:221-234.
191. Heneka MT, Carson MJ, Khoury J El, et al. Neuroinflammation in
Alzheimer’s disease. Lancet Neurol 2015;14:388-405.
192. Di Iorio G, Lupi M, Sarchione F, et al. The endocannabinoid
system: a putative role in neurodegenerative diseases. Int J High
Risk Behav Addict 2013;2:100-106.
193. Maroof N, Pardon MC, Kendall DA. Endocannabinoid signalling
in Alzheimer’s disease. Biochem Soc Trans 2013;41:1583-1587.
Molecular Targets of Cannabidiol
Author's personal copy

194. Elmes MW, Kaczocha M, Berger WT, et al. Fatty acid-binding
proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocan-
nabinol (THC) and cannabidiol (CBD). J Biol Chem 2015;290:
8711-8721.
195. Xu Y, Yan J, Zhou P, et al. Neurotransmitter receptors and cogni-
tive dysfunction in Alzheimer’s disease and Parkinson’s disease.
Prog Neurobiol 2012;97:1-13.
196. Magen I, Avraham Y, Ackerman Z, Vorobiev L, Mechoulam R,
Berry EM. Cannabidiol ameliorates cognitive and motor impair-
ments in bile-duct ligated mice via 5-HT1A receptor activation. Br
J Pharmacol 2010;159:950-957.
197. Pazos MR, Mohammed N, Lafuente H, et al. Mechanisms of
cannabidiol neuroprotection in hypoxic-ischemic newborn pigs:
role of 5HT(1A) and CB2 receptors. Neuropharmacology
2013;71:282-291.
198. Esposito G, Scuderi C,Valenza M, et al. Cannabidiol reduces Aβ-
induced neuroinflammation and promotes hippocampal
neurogenesis through PPARγinvolvement. PloS One 2011;6:
e28668.
199. Cho H-Y, Gladwell W, Wang X, et al. Nrf2-regulated
PPAR{gamma} expression is critical to protection against acute
lung injury in mice. Am J Respir Crit Care Med 2010;182:170-
182.
200. Juknat A, Pietr M, Kozela E, et al. Differential transcriptional
profiles mediated by exposure to the cannabinoids cannabidiol
and Δ9-tetrahydrocannabinol in BV-2 microglial cells. Br J
Pharmacol 2012;165:2512-2528.
201. Lauckner JE, Jensen JB, Chen H-Y, Lu H-C, Hille B, Mackie K.
GPR55 is a cannabinoid receptor that increases intracellular calci-
um and inhibits M current. Proc Natl Acad Sci U S A 2008;105:
2699-2704.
202. Henstridge CM, Balenga NAB, Ford LA, Ross RA, Waldhoer M,
Irving AJ. The GPR55 ligand L-alpha-lysophosphatidylinositol
promotes RhoA-dependent Ca2+ signaling and NFAT activation.
FASEB J 2009;23:183-193.
203. Fric J, Zelante T, Wong AYW, Mertes A, Yu H-B, Ricciardi-
Castagnoli P. NFAT control of innate immunity. Blood
2012;120:1380-1389.
204. Vashishta A, Habas A, Pruunsild P, Zheng J-J, Timmusk T,
Hetman M. Nuclear factor of activated T-cells isoform c4
(NFATc4/NFAT3) as a mediator of antiapoptotic transcription in
NMDA receptor-stimulated cortical neurons. J Neurosci 2009;29:
15331-15340.
205. Wu H-Y, Hudry E, Hashimoto T, et al. Amyloid beta induces the
morphological neurodegenerative triad of spine loss, dendritic
simplification, and neuritic dystrophies through calcineurin acti-
vation. J Neurosci 2010;30:2636-2649.
206. Abdul HM, Sama MA, Furman JL, et al. Cognitive decline in
Alzheimer’s disease is associated with selective changes in
calcineurin/NFAT signaling. J Neurosci 2009;29:12957-12969.
207. Deutsch SI, Burket JA, Benson AD. Targeting the α7 nicotinic
acetylcholine receptor to prevent progressive dementia and im-
prove cognition in adults with Down’s syndrome. Prog
Neuropsychopharmacol Biol Psychiatry 2014;54:131-139.
208. Lombardo S, Maskos U. Role of the nicotinic acetylcholine recep-
tor in Alzheimer’s disease pathology and treatment.
Neuropharmacology 2015;96(Pt B):255-262.
209. Vaysse PJ, Gardner EL, Zukin RS. Modulation of rat brain opioid
receptors by cannabinoids. J Pharmacol Exp Ther 1987;241:534-
539.
210. Feng Y, He X, Yang Y, Chao D, Lazarus LH, Xia Y. Current
research on opioid receptor function. Curr Drug Targets
2012;13:230-246.
211. Hroudová J, Singh N, Fišar Z. Mitochondrial dysfunctions in neu-
rodegenerative diseases: relevance to Alzheimer’sdisease.
Biomed Res Int 2014;175062.
212. Ristow M, Schmeisser K. Mitohormesis: promoting health and
lifespan by increased levels of reactive oxygen species (ROS).
Dose Response 2014;12:288-341.
213. Tapia PC. Sublethal mitochondrial stress with an attendant stoi-
chiometric augmentation of reactive oxygen species may precipi-
tate many of the beneficial alterations in cellular physiology pro-
duced by caloric restriction, intermittent fasting, exercise and die-
tary phytonutrients: BMitohormesis^for health and vitality. Med
Hypotheses 2006;66:832-843.
214. Myint A-M, Kim Y-K. Network beyond IDO in psychiatric disor-
ders: revisiting neurodegeneration hypothesis. Prog
Neuropsychopharmacol Biol Psychiatry 2014;48:304-313.
215. Figueiredo-Pereira ME, Rockwell P, Schmidt-Glenewinkel T,
Serrano P. Neuroinflammation and J2 prostaglandins: linking im-
pairment of the ubiquitin-proteasome pathway and mitochondria
to neurodegeneration. Front Mol Neurosci 2014;7:104.
216. Calder PC. Marine omega-3 fatty acids and inflammatory process-
es: Effects, mechanisms and clinical relevance. Biochim Biophys
Acta 2015;1851:469-484.
217. Pomponi MFL, Gambassi G, Pomponi M, Di Gioia A, Masullo C.
Why docosahexaenoic acid and aspirin supplementation could be
useful in women as a primary prevention therapy against
Alzheimer’s disease? Ageing Res Rev 2011;10:124-131.
218. Hamelink C, Hampson A, Wink DA, Eiden LE, Eskay RL.
Comparison of cannabidiol, antioxidants, and diuretics in revers-
ing binge ethanol-induced neurotoxicity. J Pharmacol Exp Ther
2005;314:780-788.
219. Hampson AJ, Grimaldi M, Axelrod J, Wink D. Cannabidiol and (-
)Delta9-tetrahydrocannabinol are neuroprotective antioxidants.
Proc Natl Acad Sci U S A 1998;95:8268-8273.
220. García-Arencibia M, González S, de Lago E, Ramos JA,
Mechoulam R, Fernández-Ruiz J. Evaluation of the neuroprotec-
tive effect of cannabinoids in a rat model of Parkinson’sdisease:
importance of antioxidant and cannabinoid receptor-independent
properties. Brain Res 2007;1134:162-170.
221. Itoh K, Chiba T, Takahashi S, et al. An Nrf2/small Maf heterodi-
mer mediates the induction of phase II detoxifying enzyme genes
through antioxidant response elements. Biochem Biophys Res
Commun 1997;236:313-322.
222. Mazur A, Lichti CF, Prather PL, et al. Characterization of human
hepatic and extrahepatic UDP-glucuronosyltransferase enzymes
involved in the metabolism of classic cannabinoids. Drug Metab
Dispos Biol Fate Chem 2009;37:1496-1504.
223. Fernández-Ruiz J, Sagredo O, Pazos MR, et al. Cannabidiol for
neurodegenerative disorders: important new clinical applications
for this phytocannabinoid? Br J Clin Pharmacol 2013;75:323-333.
224. Reddy PH. Is the mitochondrial outermembrane protein VDAC1
therapeutic target for Alzheimer’s disease? Biochim Biophys Acta
2013;1832:67-75.
225. Manczak M, Sheiko T, Craigen WJ, Reddy PH. Reduced VDAC1
protects against Alzheimer’s disease, mitochondria, and synaptic
deficiencies. J Alzheimers Dis 2013;37:679-690.
226. Keinan N, Pahima H, Ben-Hail D, Shoshan-Barmatz V. The role
of calcium in VDAC1 oligomerization and mitochondria-
mediated apoptosis. Biochim Biophys Acta 2013;1833:1745-
1754.
227. De Stefani D, Bononi A, Romagnoli A, et al. VDAC1 selectively
transfers apoptotic Ca2+ signals to mitochondria. Cell Death
Differ 2012;19:267-273.
228. Mato S, Victoria Sánchez-Gómez M, Matute C. Cannabidiol in-
duces intracellular calcium elevation and cytotoxicity in oligoden-
drocytes. Glia 2010;58:1739-1747.
229. Ryan D, Drysdale AJ, Lafourcade C, Pertwee RG, Platt B.
Cannabidiol targets mitochondria to regulate intracellular Ca2+
levels. J Neurosci 2009;29:2053-2063.
Bih et al.
Author's personal copy

230. Ho KW, Ward NJ, Calkins DJ. TRPV1: a stress response protein
in the central nervous system. Am J Neurodegener Dis 2012;1:1-
14.
231. Luo Z, Ma L, Zhao Z, et al. TRPV1 activation improves exercise
endurance and energy metabolism through PGC-1αupregulation
in mice. Cell Res 2012;22:551-564.
232. Medvedeva YV, Kim M-S, Usachev YM. Mechanisms of
prolonged presynaptic Ca2+ signaling and glutamate release in-
duced by TRPV1 activation in rat sensory neurons. J Neurosci
2008;28:5295-5311.
233. Gellerich FN, Gizatullina Z, Gainutdinov T, et al. The control of
brain mitochondrial energization by cytosolic calcium: the mito-
chondrial gas pedal. IUBMB Life 2013;65:180-190.
234. Gupta S, Sharma B. Pharmacological benefits of agomelatine and
vanillin in experimental model of Huntington’sdisease.
Pharmacol Biochem Behav 2014;122:122-135.
235. Gomes CV, Kaster MP, Tomé AR, Agostinho PM, Cunha RA.
Adenosine receptors and brain diseases: neuroprotection and neu-
rodegeneration. Biochim Biophys Acta 2011;1808:1380-1399.
236. Smathers RL, Petersen DR. The human fatty acid-binding protein
family: evolutionary divergences and functions. Hum Genomics
2011;5:170-191.
237. Ohrfelt A, Andreasson U, Simon A, et al. Screening for new bio-
markers for subcortical vascular dementia and Alzheimer’sdis-
ease. Dement Geriatr Cogn Disord Extra 2011;1:31-42.
238. Shioda N, Yabuki Y, Kobayashi Y, Onozato M, Owada Y,
Fukunaga K. FABP3 protein promotes α-synuclein oligomeriza-
tion associated with 1-methyl-1,2,3,6-tetrahydropiridine-induced
neurotoxicity. J Biol Chem 2014;289:18957-18965.
239. Pahnke J, Fröhlich C, Krohn M, Schumacher T, Paarmann K.
Impaired mitochondrial energy production and ABC transporter
function-A crucial interconnection in dementing proteopathies of
the brain. Mech Ageing Dev 2013;134:506-515.
240. Krohn M, Lange C, Hofrichter J, et al. Cerebral amyloid-β
proteostasis is regulated by the membrane transport protein
ABCC1 in mice. J Clin Invest 2011;121:3924-3931.
241. Xiong H, Callaghan D, Jones A, et al. ABCG2 is upregulated in
Alzheimer’s brain with cerebral amyloid angiopathy and may act
as a gatekeeper at the blood-brain barrier for Abeta(1-40) peptides.
J Neurosci 2009;29:5463-5475.
242. Martorana A, Koch G. Is dopamine involved in Alzheimer’sdis-
ease? Front Aging Neurosci 2014;6:252.
243. Caraci F, Battaglia G, Sortino MA, et al. Metabotropic glutamate
receptors in neurodegeneration/neuroprotection: still a hot topic?
Neurochem Int 2012;61:559-565.
244. Lund-Katz S, Phillips MC. High density lipoprotein structure-
function and role in reverse cholesterol transport. Subcell
Biochem 2010;51:183-227.
245. Poirier J, Miron J, Picard C, et al. Apolipoprotein E and lipid
homeostasis in the etiology and treatment of sporadic
Alzheimer’s disease. Neurobiol Aging 2014;35(Suppl. 2):S3-S10.
246. Benyamin R, Trescot AM, Datta S, et al. Opioid complications
and side effects. Pain Physician 2008;11(2 Suppl.):S105–S120.
247. Sindrup SH, Jensen TS. Efficacy of pharmacological treatments of
neuropathic pain: an update and effect related to mechanism of
drug action. Pain 1999;83:389-400.
248. Costa B, Giagnoni G, Franke C, Trovato AE, Colleoni M.
Vanilloid TRPV1 receptor mediates the antihyperalgesic effect
of the nonpsychoactive cannabinoid, cannabidiol, in a rat model
of acute inflammation. Br J Pharmacol 2004;143:247-250.
249. Costa B, Trovato AE, Comelli F, Giagnoni G, Colleoni M. The
non-psychoactive cannabis constituent cannabidiol is an orally
effective therapeutic agent in rat chronic inflammatory and neuro-
pathic pain. Eur J Pharmacol 2007;556:75-83.
250. Maione S, Piscitelli F, Gatta L, et al. Non-psychoactive cannabi-
noids modulate the descending pathway of antinociception in
anaesthetized rats through several mechanisms of action. Br J
Pharmacol 2011;162:584-596.
251. Ward SJ, Ramirez MD, Neelakantan H, Walker EA. Cannabidiol
prevents the development of cold and mechanical allodynia in
paclitaxel-treated female C57Bl6 mice. Anesth Analg 2011;113:
947-950.
252. Ward SJ, McAllister SD, Kawamura R, Murase R, Neelakantan H,
Walker EA. Cannabidiol inhibits paclitaxel-induced neuropathic
pain through 5-HT(1A) receptors without diminishing nervous
system function or chemotherapy efficacy. Br J Pharmacol
2014;171:636-645.
253. Sałat K, Filipek B. Antinociceptive activity of transient receptor
potential channel TRPV1, TRPA1, and TRPM8 antagonists in
neurogenic and neuropathic pain models in mice. J Zhejiang
Univ Sci B 2015;16:167-178.
254. Costa B, Siniscalco D, Trovato AE, et al. AM404, an inhibitor of
anandamide uptake, prevents pain behaviour and modulates cyto-
kine and apoptotic pathways in a rat model of neuropathic pain. Br
J Pharmacol 2006;148:1022-1032.
255. Lehto SG, Weyer AD, Zhang M, et al. AMG2850, a potent and
selective TRPM8 antagonist, is not effective in rat models of in-
flammatory mechanical hypersensitivity and neuropathic tactile
allodynia. Naunyn Schmiedebergs Arch Pharmacol 2015;388:
465-476.
256. Baddack U, Frahm S, Antolin-Fontes B, et al. Suppression of
peripheral pain by blockade of Cav2.2 channels in nociceptors
induces RANKL and impairs recovery from inflammatory arthri-
tis. Arthritis Rheumatol 2015;67:1657-1667.
257. Jayamanne A, Jeong HJ, Schroeder CI, Lewis RJ, Christie MJ,
Vaughan CW. Spinal actions of ω-conotoxins, CVID, MVIIA and
related peptides in a rat neuropathic pain model. Br J Pharmacol
2013;170:245-254.
258. Gao X, Lu Q, Chou G, et al. Norisoboldine attenuates inflamma-
tory pain via the adenosine A1 receptor. Eur J Pain 2014;18:939-
948.
259. Li L, Hao JX, Fredholm BB, Schulte G, Wiesenfeld-Hallin Z, Xu
XJ. Peripheral adenosine A2A receptors are involved in
carrageenan-induced mechanical hyperalgesia in mice.
Neuroscience 2010;170:923-928.
260. Fornal CA, Metzler CW, Gallegos RA, VeaseySC, McCreary AC,
Jacobs BL. WAY-100635, a potent and selective 5-
hydroxytryptamine1A antagonist, increases serotonergic neuronal
activity in behaving cats: comparison with (S)-WAY-100135. J
Pharmacol Exp Ther 1996;278:752-762.
261. Staton PC, Hatcher JP, Walker DJ, et al. The putative cannabinoid
receptor GPR55 plays a role in mechanical hyperalgesia associat-
ed with inflammatory and neuropathic pain. Pain 2008;139:225-
236.
262. Terrando N, Yang T, Ryu JK, et al. Stimulation of the alpha 7
nicotinic acetylcholine receptor protects against neuroinflamma-
tion after tibia fracture and endotoxemia in mice. Mol Med
2015;20:667-675.
263. AlSharari SD, Freitas K, Damaj MI. Functional role of alpha7
nicotinic receptor in chronic neuropathic and inflammatory pain:
Studies in transgenic mice. Biochem Pharmacol 2013;86:1201-
1207.
264. Gong S, Liang Q, Zhu Q, et al. Nicotinic acetylcholine receptor α7
subunit is involved in the cobratoxin-induced antinociception in
an animal model of neuropathic pain. Toxicon 2015;93:31-36.
265. Papke RL, Bagdas D, Kulkarni AR, et al. The analgesic-like prop-
erties of the alpha7 nAChR silent agonist NS6740 is associated
with non-conducting conformations of the receptor.
Neuropharmacology 2015;91:34-42.
266. Hasani R Al-, Bruchas MR. Molecular mechanisms of opioid
receptor-dependent signaling and behavior. Anesthesiology
2011;115:1363-1381.
Molecular Targets of Cannabidiol
Author's personal copy

267. Vowles KE, McEntee ML, Julnes PS, Frohe T, Ney JP, van der
Goes DN. Rates of opioid misuse, abuse, and addiction in chronic
pain: a systematic review and data synthesis. Pain 2015;156:569-
576.
268. Zahari Z, Ismail R. Influence of Cytochrome P450, family 2, sub-
family D, polypeptide 6 (CYP2D6) polymorphisms on pain sen-
sitivity and clinical response to weak opioid analgesics. Drug
Metab Pharmacokinet 2014;29:29-43.
269. Campos AC, Guimarães FS. Evidence for a potential role for
TRPV1 receptors in the dorsolateral periaqueductal gray in the
attenuation of the anxiolytic effects of cannabinoids. Prog
Neuropsychopharmacol Biol Psychiatry 2009;33:1517-1521.
270. Moussaieff A, Rimmerman N, Bregman T, et al. Incensole acetate,
an incense component, elicits psychoactivity by activating TRPV3
channels in the brain. FASEB J 2008;22:3024-3034.
271. Uslaner JM, Smith SM, Huszar SL, et al. T-type calcium channel
antagonism produces antipsychotic-like effects and reduces
stimulant-induced glutamate release in the nucleus accumbens of
rats. Neuropharmacology 2012;62:1413-1421.
272. Gangarossa G, Laffray S, Bourinet E, Valjent E. T-type calcium
channel Cav3.2 deficient mice show elevated anxiety, impaired
memory and reduced sensitivity to psychostimulants. Front
Behav Neurosci 2014;8:92.
273. Price DL, Bonhaus DW, McFarland K. Pimavanserin, a 5-HT2A
receptor inverse agonist, reverses psychosis-like behaviors in a
rodent model of Alzheimer’s disease. Behav Pharmacol 2012;23:
426-433.
274. Zhang CG, Kim S-J. Taurine induces anti-anxiety by activating
strychnine-sensitive glycine receptor in vivo. Ann Nutr Metab
2007;51:379-386.
275. Randall-Thompson JF, Pescatore KA, Unterwald EM. A role for
delta opioid receptors in the central nucleus of the amygdala in
anxiety-like behaviors. Psychopharmacology 2010;212:585-595.
276. Rahimi A, Hajizadeh Moghaddam A, Roohbakhsh A. Central
administration of GPR55 receptor agonist and antagonist modu-
lates anxiety-related behaviors in rats. Fundam Clin Pharmacol
2015;29:185-190.
277. Maximino C, Lima MG, Olivera KRM, Picanço-Diniz DLW,
Herculano AM. Adenosine A1, but not A2, receptor blockade
increases anxiety and arousal in Zebrafish. Basic Clin Pharmacol
Toxicol 2011;109:203-207.
278. Shen H-Y, Singer P, Lytle N, et al. Adenosine augmentation ame-
liorates psychotic and cognitive endophenotypes of schizophrenia.
J Clin Invest 2012;122:2567-2577.
279. Müller N. COX-2 inhibitors as antidepressants and antipsychotics:
clinical evidence. Curr Opin Investig Drugs 2010;11:31-42.
280. Hill MN, Kumar SA, Filipski SB, et al. Disruption of fatty acid amide
hydrolase activity prevents the effects of chronic stress on anxiety and
amygdalar microstructure. Mol Psychiatry 2013;18:1125-1135.
281. Leweke FM, Piomelli D, Pahlisch F, et al. Cannabidiol enhances
anandamide signaling and alleviates psychotic symptoms of
schizophrenia. Transl Psychiatry 2012;2:e94.
282. Stangherlin EC, Nogueira CW. Diphenyl ditelluride induces
anxiogenic-like behavior in rats by reducing glutamate uptake.
Biol Trace Elem Res 2014;158:392-398.
283. Morgan CJA, Das RK, Joye A, Curran HV, Kamboj SK.
Cannabidiol reduces cigarette consumption in tobacco smokers:
preliminary findings. Addict Behav 2013;38:2433-2436.
284. Ren Y, Whittard J, Higuera-Matas A, Morris CV, Hurd YL.
Cannabidiol, a nonpsychotropic component of cannabis, inhibits
cue-induced heroin seeking and normalizes discrete mesolimbic
neuronal disturbances. J Neurosci 2009;29:14764-14769.
285. Adamczyk P, Miszkiel J, McCreary AC, Filip M, Papp M,
Przegaliński E. The effects of cannabinoid CB1, CB2 and
vanilloid TRPV1 receptor antagonists on cocaine addictive be-
havior in rats. Brain Res 2012;1444:45-54.
286. Heng L-J, Huang B, Guo H, et al. Blocking TRPV1 in nucleus
accumbens inhibits persistent morphine conditioned place prefer-
ence expression in rats. PloS One 2014;9:e104546.
287. Shapovalov G, Gkika D, Devilliers M, et al. Opiates modulate
thermosensation by internalizing cold receptor TRPM8. Cell
Rep 2013;4:504-515.
288. Tyndale RF, Droll KP, Sellers EM. Genetically deficient CYP2D6
metabolism provides protection against oral opiate dependence.
Pharmacogenetics 1997;7:375-379.
289. Miksys S, Rao Y, Hoffmann E, Mash DC, Tyndale RF. Regional
and cellular expression of CYP2D6 in human brain: higher levels
in alcoholics. J Neurochem 2002;82:1376-1387.
290. Tournier N, Chevillard L, Megarbane B, Pirnay S, Scherrmann J-
M, Declèves X. Interaction of drugs of abuse and maintenance
treatments with human P-glycoprotein (ABCB1) and breast cancer
resistance protein (ABCG2). Int J Neuropsychopharmacol
2010;13:905-915.
Bih et al.
Author's personal copy