Neuroprotection in Experimental Autoimmune Encephalomyelitis and Progressive Multiple Sclerosis by Cannabis-Based Cannabinoids

Abstract

Multiple sclerosis (MS) is the major immune-mediated, demyelinating, neurodegenerative disease of the central nervous system. Compounds within cannabis, notably Δ9-tetrahydrocannabinol (Δ9-THC) can limit the inappropriate neurotransmissions that cause MS-related problems and medicinal cannabis is now licenced for the treatment of MS symptoms. However, the biology indicates that the endocannabinoid system may offer the potential to control other aspects of disease. Although there is limited evidence that the cannabinoids from cannabis are having significant immunosuppressive activities that will influence relapsing autoimmunity, we and others can experimentally demonstrate that they may limit neurodegeneration that drives progressive disability. Here we show that synthetic cannabidiol can slow down the accumulation of disability from the inflammatory penumbra during relapsing experimental autoimmune encephalomyelitis (EAE) in ABH mice, possibly via blockade of voltage-gated sodium channels. In addition, whilst non-sedating doses of Δ9-THC do not inhibit relapsing autoimmunity, they dose-dependently inhibit the accumulation of disability during EAE. They also appear to slow down clinical progression during MS in humans. Although a 3 year, phase III clinical trial did not detect a beneficial effect of oral Δ9-THC in progressive MS, a planned subgroup analysis of people with less disability who progressed more rapidly, demonstrated a significant slowing of progression by oral Δ9-THC compared to placebo. Whilst this may support the experimental and biological evidence for a neuroprotective effect by the endocannabinoid system in MS, it remains to be established whether this will be formally demonstrated in further trials of Δ9-THC/cannabis in progressive MS.

Full text

INVITED REVIEW
Neuroprotection in Experimental Autoimmune
Encephalomyelitis and Progressive Multiple Sclerosis
by Cannabis-Based Cannabinoids
Gareth Pryce &Dieter R. Riddall &David L. Selwood &
Gavin Giovannoni &David Baker
Received: 26 October 2014 /Accepted: 10 December 2014
#Springer Science+Business Media New York 2014
Abstract Multiple sclerosis (MS) is the major immune-me-
diated, demyelinating, neurodegenerative disease of the cen-
tral nervous system. Compounds within cannabis, notably
Δ9-tetrahydrocannabinol (Δ9-THC) can limit the inappropri-
ate neurotransmissions that cause MS-related problems and
medicinal cannabis is now licenced for the treatment of MS
symptoms. However, the biology indicates that the
endocannabinoid system may offer the potential to control
other aspects of disease. Although there is limited evidence
that the cannabinoids from cannabis are having significant
immunosuppressive activities that will influence relapsing
autoimmunity, we and others can experimentally demonstrate
that they may limit neurodegeneration that drives progressive
disability. Here we show that synthetic cannabidiol can slow
down the accumulation of disability from the inflammatory
penumbra during relapsing experimental autoimmune
encephalomyelitis (EAE) in ABH mice, possibly via blockade
of voltage-gated sodium channels. In addition, whilst non-
sedating doses of Δ9-THC do not inhibit relapsing autoim-
munity, they dose-dependently inhibit the accumulation of
disability during EAE. They also appear to slow down clinical
progression during MS in humans. Although a 3 year, phase
III clinical trial did not detect a beneficial effect of oral Δ9-
THC in progressive MS, a planned subgroup analysis of
people with less disability who progressed more rapidly, dem-
onstrated a significant slowing of progression by oral Δ9-
THC compared to placebo. Whilst this may support the ex-
perimental and biological evidence for a neuroprotective ef-
fect by the endocannabinoid system in MS, it remains to be
established whether this will be formally demonstrated in
further trials of Δ9-THC/cannabis in progressive MS.
Keywords Cannabinoid .Cannabidiol .Experimental
autoimmune encephalomyelitis .Multiple sclerosis .
Neuroprotection .Δ9-tetrahydrocannabinol
Introduction
Multiple sclerosis (MS) is a major immune-mediated, demy-
elinating and neurodegenerative disease of the central nervous
system (CNS), which affects about 2-3 million people world-
wide (Compston and Coles 2002,2008). Disease is often
associated with relapsing-remitting neurological attacks and
the progressive, slow worsening of disability, typically over
many years. Demyelination and axonal and neuronal loss
leads to a variety of different cognitive, sensory and motor
problems that accumulate as disease progresses due to lesions
within different neural pathways of the CNS (Compston and
Coles 2002). At present there is no cure, although there are
some disease modifying therapies (DMT) that can slow down
the development of CNS lesions and neurological relapses
Electronic supplementary material The online version of this article
(doi:10.1007/s11481-014-9575-8) contains supplementary material,
which is available to authorized users.
G. Pryce :G. Giovannoni:D. Baker (*)
Neuroimmunology Unit, Blizard Institute, Barts and the London
School of Medicine and Dentistry, Queen Mary University of
London, 4 Newark Street, London E1 2AT, UK
e-mail: david.baker@qmul.ac.uk
G. Pryce
e-mail: g.pryce@qmul.ac.uk
G. Giovannoni
e-mail: g.giovannoni@qmul.ac.uk
D. R. Riddall :D. L. Selwood
Wolfson Institute of Biomedical Research, University College
London, London, UK
D. R. Riddall
e-mail: drr@ntlworld.com
D. L. Selwood
e-mail: d.selwood@ucl.ac.uk
J Neuroimmune Pharmacol
DOI 10.1007/s11481-014-9575-8

caused by the entry of cells of the peripheral immune system
into the CNS. These however, have relatively low efficacy, as
occurs with the beta interferons, glatiramer acetate and
teriflunomide, or higher efficacy which can be associated with
significant, sometimes life-threatening side effects, which has
been reported with fingolimod, natalizumab and alemtuzumab
(Marta and Giovannoni 2012). These can limit the nerve loss
that occurs as a consequence of these lesions (Gunnarsson
et al. 2011), however, these treatments if not started sufficient-
ly quickly following diagnosis, do not appear to control the
nerve loss associated with progressive MS. This is driven by
central inflammatory and other neurodegenerative effects that
underlie irreversible disability (Compston and Coles 2002;
Marta and Giovannoni 2012). Dysregulation of effective neu-
rotransmission leads to a number of troublesome symptoms
dependent on lesion location and include: incontinence;
spasms; spasticity and pain (Compston and Coles 2002).
These are controlled by a variety of different drugs, which
are often associated with significant sedating side effects
(Compston and Coles 2002). The failure to find adequate
treatments, leads people with MS (PwMS) to often seek
complementary or alternative medicines (CAM) to supple-
ment their prescribed medicines (Yadav et al. 2014;Masullo
et al. 2015). With the advent of the internet, use of CAM can
be widely publicised and adopted even before scientific evi-
dence can support or refute the claims of efficacy. Indeed
PwMS perceived benefit from taking cannabis for the control
of sleep disturbances, pain and spasticity (Consroeet al. 1997;
Clark et al. 2004; Chong et al. 2006). This was subsequently
supported by biology, experimental and clinical class I evi-
dence in humans to support the role of cannabinoid control of
spasticityandpaininPwMS(Bakeretal.2000,2012;
Novotna et al. 2011; Zajicek et al. 2012;Langfordetal.2013).
Symptom Control by Cannabinoids
The endocannabinoid systems regulates synaptic neurotrans-
mission and it is therefore not surprising that compounds
within cannabis can stimulate neuronal CB
1
cannabinoid re-
ceptors (CB
1
R) to control the excessive or inappropriate neu-
rotransmission that leads to symptoms of MS (Corey-Bloom
et al. 2012; Zajicek et al. 2012). Some places are now
supporting the use of medical marijuana, and cannabis ex-
tracts (Sativex/nabiximols) have become licensed medicines
for the treatment of spasticity and pain in MS (Novotna et al.
2011; Langford et al. 2013). Early reports from Europe and
the USA failed to distinguish any perceived therapeutic effi-
cacy in symptom control of MS (Consroe et al. 1997). How-
ever, in experimental models of MS-related spasticity that
occurs due to CNS autoimmunity, it could be shown that
delta9 tetrahydrocannabinol (Δ9-THC) and the CB
1
Rcon-
trolled symptoms, with no apparent effect of cannabinol
(CBD) on spasticity (Baker et al. 2000; Wilkinson et al.
2003; Pryce and Baker 2007; Pryce et al. 2014). This could
suggest that Δ9-THC is the major therapeutic chemical within
cannabis, based on the reports that cannabis in North America
may have a low CBD content (ElSohly et al. 2000; Wilkinson
et al. 2003; EMCDD 2008). However, pharmaceutical, med-
ical cannabis extracts being developed (Sativex & Cannador)
contain essentially equal proportions of Δ9-THC and CBD
(Novotna et al. 2011; Zajicek et al. 2012; Langford et al.
2013). Although it has been reported that CBD may limit
the side-effect potential of Δ9-THC within cannabis (Dalton
et al. 1976;RussoandGuy2006), little direct evidence has
been provided for such a specific ratio and contrasts with the
low CBD:Δ9-THC ratio (1:10–1:200) in many recreational
cannabis extracts (Burgdorf et al. 2011). However, it appears
that CBD is not inert and may have some medicinal value
(Mechoulam et al. 2002;RussoandGuy2006). Whilst me-
dicinal cannabis has become a licensed treatment for
symptom control, the question arises whether compounds
within cannabis have additional properties that could be
useful in the control of MS. We review the current
literature and present data to suggest that cannabis may
have utility in the control of nerve loss and disease
progression due to neuroimmunological disease.
Lack of Marked Immunosuppressive Effects
of Cannabinoids in EAE
Some studies have suggested that Δ9-THC and CBD may
have an immunosuppressive activity that could provide some
DMT function (Lyman et al. 1989;Mareszetal.2007;Kozela
et al. 2011). This is seen by a reduction in the incidence and
severity of disease and/or a delay in the onset of disease in
experimental autoimmune encephalomyelitis (EAE) models
of MS (Baker et al. 2011). In contrast to some immunosup-
pressive action of 5 mg/kg CBD reported in myelin-peptide
induced EAE in C57BL/6 mice (Kozela et al. 2011) and the
observation that 5–10 mg/kg CBD, but not 2.5 or 20 mg/kg
CBD, can inhibit the development of collagen-induced arthri-
tis in DBA-1 mice (Malfait et al. 2000), we have consistently
failed to detect any immunosuppressive effect in tissue ho-
mogenate induced EAE in ABH mice in multiple experiments
across a range of doses from 0.5 to 25 mg/kg. The lack of
immunosuppressive effects were found in an initial EAE
attack (Maresz et al. 2007) or as found here (Fig. 1)inan
induced-relapse; in the latter, essentially all the animals devel-
oped EAE of comparable severity and day of onset as found in
vehicle treated animals. This suggests that CBD is unlikely to
prevent relapsing neuroimmune-autoimmunity in MS.
Differences in the ease of immunosuppression in C57BL/6
(relatively EAE-resistant) and ABH (EAE susceptible) mice
have been seen previously (Sisay et al. 2013). As such,

apparent immunosuppression observed in EAE induced in
C57BL/6 mice, where disease induction can be inconsistent,
is lost once tested in ABH mice where robust and consistent
disease is induced (Sisay et al. 2013). Furthermore, in contrast
to the relapsing-remitting nature of EAE in ABH mice (Al-
Izki et al. 2012), myelin oligodendrocyte glycoprotein-
induced EAE in C57BL/6 is typically monophasic with poor
recovery (Sisay et al. 2013). This is related to inflammation
induced neurodegeneration. Thus the benefit of CBD may
relate to a neuroprotective effect rather than an immunosup-
pressive effect. Nevertheless, it is possible to induce immuno-
suppression of EAE with ≥5mg/kgΔ9-THC in both SJL
(Lyman et al. 1989) and ABH mice, via a neuronal CB
1
R-
dependent mechanism (Maresz et al. 2007;Croxfordetal.
A
Drug-Induced
Neuroprotection
Time Post-Disease Induction (days).
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Mean Neurological Score ± SEM
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Vehicle
THC 2.5mg/kg i.p.
Period of Daily Treatment
B
C
Time of on Accelerating RotaRod (s)
0
50
100
150
200
250
300
Post- Relapse (Day 47)
**
Pre- Treatment (Day 27)
**
**
THC 10mg/kg i.p
CBD 10mg/kg i.p.
CBD 5mg/kg i.p.
Vehicle
Drug-Induced
Neuroprotection
Time Post-Disease Induction (days).
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Mean Neurological Score ± SEM
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Vehicle
CBD 5mg/kg i.p
CBD 10mg/kg i.p.
Period of Daily Treatment
Fig. 1 Neuroprotective potential
of cannabinoids during induce-
relapsing autoimmune
encephalomyelitis. EAE was
induced in Biozzi ABH mice
following immunization with
spinal cord homogenate
emulsified in Freunds complete
adjuvant on day 0 and 7. (Al-Izki
et al. 2012). Animals were
allowed to undergo a paralytic
inflammatory attack (all animals
scored 3–4) and a relapse was
induced by re-immunization on
day 28 during the first remission
(RM1). Animals (7–9/group)
were injected i.p. with either: (A,
C) 2.5 mg/kg Δ9-THC, (B, C)
5 mg/kg CBD, 10 mg/kg CBD or
ethanol:cremophor:phosphate
buffered saline from day 33
onwards. (a,b)Theresults
represent the mean± SEM daily
score based on a 0–5 scoring scale
(Al-Izki et al. 2012). The
differences between the minimal
disease score at the termination of
the experiment were analysed
using Mann Whitney U statistics.
(c) The mean±SEM activity on
an accelerating rotorod (4–
40 rpm) (Al-Izki et al. 2012)
measured on day 27 and day 47
during the second remission
(RM2). Differences between
vehicle and treatment groups
were analysed using Students t
tests ** P<0.01 compared to
vehicle treated animals

2008;deLagoetal.2012). However, we do not believe
that these effects are particularly relevant to the clinical
use of cannabis (Baker et al. 2012). This is because the
immunosuppression only occurs as doses of CB
1
Rago-
nists that cause marked sedative, cannabimimetic
(hypomotility; hypothermia and sometimes seizures) ef-
fects (Croxford et al. 2008). These probably cause
significant stress-responses that are known to be immu-
nosuppressive in EAE (Bolton et al. 1997). Importantly,
there is no solid data to suggest that doses of medical
cannabis cause significant immunosuppressive effects in
MS, following analysis of peripheral immune responses
(Killestein et al. 2003;Katonaetal.2005;Sextonetal.
2014).
Cannabinoid Dose
THC 0.0mg/kg 0.25mg/kg 2.5mg/kg 0.0mg/kg 0.0mg/kg 2.5mg/kg 2.5mg/kg
CBD 0mg/kg 0mg/kg 0mg/kg 5mg/kg 10mg/kg 5mg/kg 10mg/kg
Mean Clinical Score During Remission
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RM1 Pre-Relapse
RM2 Post-Relapse
*
**
A
*
*
Cannabinoid Dose
THC 0.0mg/kg 0.25mg/kg 2.5mg/kg 0.0mg/kg 0.0mg/kg 2.5mg/kg 2.5mg/kg
CBD 0mg/kg 0mg/kg 0mg/kg 5mg/kg 10mg/kg 5mg/kg 10mg/kg
Mean Rotorod Score During Remission
0
50
100
150
200
250
300
RM1 Pre-Relapse
RM2 Post-Relapse
*
*
B
Fig. 2 Neuroprotective potential
of cannabinoids during induce-
relapsing autoimmune
encephalomyelitis. EAE was
induced in Biozzi ABH mice
following immunization with
spinal cord homogenate
emulsified in Freunds complete
adjuvant on day 0 and 7. (Al-Izki
et al. 2012) Animals were allowed
to undergo a paralytic
inflammatory attack (all score 3–
4) and a relapse was induced by
re-immunization on day 28
during the first remission (RM1).
Animals (5-9/group) were
injected i.p. with either
0.25 mg/kg Δ9-THC, 2.5 mg/kg
Δ9-THC, 5 mg/kg CBD,
10 mg/kg CBD a combination of
Δ9-THC and CBD or
ethanol:cremophor:phosphate
buffered saline from day 33
onwards. (a) The results represent
the mean minimum score during
remission±SEM daily score
based on a 0–5 scoring scale (Al-
Izki et al. 2012). These were
analysed using Mann Whitney U
statistics. (b)Themean±SEM
activity on an accelerating rotorod
(Al-Izki et al. 2012) measured on
day 27 and day 47 during the
second remission (RM2).
*P<0.05, ** P<0.01 compared
to vehicle treated animals, using
Students ttest

Neuroprotective Effect of Cannabis-Plant Based
Cannabinoids in EAE
In contrast to the limited immunosuppressive action of
cannabis-based cannabinoids that inhibit the development of
paralytic EAE (Maresz et al. 2007; Baker et al. 2011), we and
others have shown that CB
1
R agonists, including Δ9-THC and
endocannabinoids, can induce a neuroprotective effect (Pryce
et al.2003; Croxford et al. 2008;Webbetal.2008;Hasseldam
and Johansen 2010; Hernández-Torres et al. 2014;Bernal-
Chico et al. 2015). In EAE, this is seen by a better functional
recovery and a reduced accumulation of disability following
paralytic attack (Croxford et al. 2008; Baker et al. 2011;Al-Izki
et al. 2014). As such, Δ9-THC facilitated a dose-dependent
enhanced recovery from the effects of the inflammatory pen-
umbra (Al-Izki et al.2014) developing during paralytic, relaps-
ing EAE (Figs. 1and 2) at a stage that allows us to dissociate
neuroprotective and immunosuppressive effects (Baker et al.
2011; Al-Izki et al. 2012). This was seen by significantly
(P<0.01) less accumulation of neurological disability, as
assessed using neurological score (Figs. 1a and 2a)andless
loss of motor co-ordination as assessed using rotorod activity
(Figs. 1c and 2b), which is consistent with neuroprotection
observed in previous studies (Pryce et al. 2003). This is prob-
ably mediated by multiple mechanisms including; alterations in
neural excitotoxicity, oxidative stress and changes in the glial
neuroimmune responses (Pryce et al. 2003; Docagne et al.
2007;Rossietal.2011a; Ribeiro et al. 2013; Musella et al.
2014). Whilst we have not found any symptomatic benefit or
immunosuppressive action of CBD in acute (Baker et al.2000;
Maresz et al. 2007) and relapsing EAE (Fig. 1b), interestingly
there was a significantly (P<0.05) better clinical recovery,
indicative of a neuroprotective effect (Figs. 1b and 2a) follow-
ing administration of 5 mg/kg and particularly 10 mg/kg CBD
i.p. (Fig. 2a) when administered shortly before relapse. This
clinical effect was also reflective of better rotorod activity
(Figs. 1c and 2b). For technical reasons, it was not possible to
measure spinal nerve content in these experiments to defini-
tively demonstrate an effect on nerve survival. However, based
on the clinical score and notably the rotorod score, which has a
very strong positive correlation with spinal nerve content (Al-
Izki et al. 2012,2014), it appeared evident that both Δ9-THC
and CBD alone had neuroprotective potential.
A neuroprotective effect of Δ9-THC was not surprising as
CB
1
R agonists, including Δ9-THC, have previously been
shown to have neuroprotective effects as shown histologically,
in other stages of EAE (Pryce et al.2003; Croxford et al. 2008).
Likewise, this is consistent with the observation that CB
1
R-
deficient mice have been shown to accumulate more neurode-
generation and disability as a consequence of neuroimmune
attack than wild type animals (Pryce et al. 2003; Rossi et al.
2011a).Hereitmaybeassociatedwiththeinhibitionofexces-
sive glutamatergic signals that can lead to downstream,
excitotoxic nerve damage, via neural CB
1
R-dependent mecha-
nisms (Pryce et al. 2003; Docagne et al. 2007; Musella et al.
2014). In addition, Δ9-THC also exhibits anti-oxidant proper-
ties (Hampson et al. 1998) and inhibits calcium and sodium ion
channels (Howlett et al. 2002) that can also limit nerve cell
death due to toxic ion concentrations within the disease CNS.
In addition, there may be actions through the inhibition of
microglial cell activity, which could be via CB
2
R(Howlett
et al. 2002; Docagne et al. 2007;Correaetal.2009). Likewise,
CBD has been found to offer neuroprotective potential in a
variety of different experimental paradigms (Mecha et al. 2012,
2013). Although a number of studies indicate that CBD has
neuroprotective anti-oxidant effects (El-Remessy et al. 2003;
Hayakawa et al. 2007), we show here that CBD also appears to
inhibit Na
+
ion channel activity (Fig. 3).
We used veratrine-evoked uptake of [
14
C]-guanidine flux
assay and inhibition of scorpion venom facilitated [
12
H]-
batrachotoxin-B (a sodium channel ligand; BTX-B) binding
using rat cerebral cortex synaptosomes to measure sodium
channel activity (Fig. 3.Garthwaite et al. 2002). In this assay,
veratrine holds the Na
+
channels in an open state and the influx
of [
14
C] guanidine through the channels and into the synapto-
somes is measured (Garthwaite et al. 2002). YC-1, a soluble
guanylylcyclase activator and Na
+
channel inhibitor (Garthwaite
et al. 2002) served as a positive control. This had an IC
50
of
16.2 μM in veratrine-evoked uptake of [
14
C]-guanidine flux
assay (Fig. 3a). This is comparable to the response reported
previously for YC-1 and showed a similar activity to Sipatrigine
(BW619C89. IC
50
=14.8 μM). Lamotrigine (IC
50
=186.2 μM),
which is a clinical sodium channel inhibitor used as an anti-
convulsive agent to treat epilepsy, had a lower inhibitory capac-
ity (Garthwaite et al. 2002). Cannabidiol, surprisingly, was over
ten times more active than YC-1, in this veratrine-evoked uptake
of [
14
C]-guanidine flux assay with an IC
50
of 0.9 μM(Fig.3a).
In contrast, nabilone, a potent CB
1
R/CB
2
R receptor agonist,
used to treat emesis in humans was inactive (>100 μM) in the
assay (Fig. 3a). Likewise nabilone was relatively inactive in
blockage of titrated BTX-B binding, whereas cannabidiol po-
tently (IC
50
of 3–4μM) inhibited binding of BTX-B to synap-
tosomes (Fig. 3b) and was again about ten times more potent
than YC-1 (IC
50
of 3–45.7 μM) and compared well with
Sipatrigine (IC
50
of 14.8 μM) and Lamotrigine (IC
50
of
159.6 μM Garthwaite et al. 2002). Our studies on sodium
channel blockage are supported and extended by other recent
studies that have also shown activity of CBD on sodium chan-
nels including Na
v
1.1, Na
v
1.2 and others. (Hill et al. 2014).
Sodium channel inhibitors can exhibit marked neuroprotective
effects in induced-relapsing EAE through effects on nerves and
microglia (Waxman 2002; Al-Izki et al. 2014; Morsali et al.
2013). Sodium channel blockers used to treat epilepsy may also
have some neuroprotective potential in MS (Gnanapavan et al.
2013). This is perhaps consistent with the mechanism of action
of CBD, although clinically CBD does not appear to have the

side-effect potential of some clinical sodium channel blockers,
which limited drug compliance and the perceived success of
clinical trials of this compound class in MS (Kapoor et al. 2010;
Gnanapavan et al. 2013). However, CBD is reported to have
additional ionic effects within mitochondria, and inhibits pro-
inflammatory activities of microglia and other modes of action
(Ryan et al. 2009; Mecha et al. 2012; Espejo-Porras et al. 2013;
Iannotti et al. 2014), which could also add to a neuroprotective
potential and in addition CBD has been reported to limit oligo-
dendrocyte damage (Mecha et al. 2012).
It was interesting however that there appeared to be no
additive effect following co-administration of both CBD with
Δ9-THC in 2:1–4:1 CBD:Δ9-THC ratios (Fig. 2). Further-
more, the neuroprotective effect of Δ9-THC appeared to be
abrogated by the presence of CBD (Fig. 2). It has been reported
that the presence of CBD limits the sedative and psychoactive
effect of Δ9-THC (Dalton et al. 1976; Russo and Guy 2006).
This may be compatible with the report that CBD has some
CB
1
R antagonist potential, which could alter some behavioural
effects of Δ9-THC (Thomas et al. 2007;Vannetal.2008). As
Log Concentration (M)
-9 -8 -7 -6 -5 -4
[
12
H] Batrachotoxin-B Binding(CPM)
0
500
1000
1500
2000
2500
3000
3500
YC-1
Cannabidiol
Nabilone
B
Log Concentration (M)
-9 -8 -7 -6 -5 -4
[
14
C]-Guanidine Flux (CPM)
0
1000
2000
3000
4000
5000
6000
7000
YC-1
Cannabidiol
Nabilone
A
Fig. 3 Sodium ion channel
Inhibitory activity of cannabidiol.
Various concentrations of CBD,
nabilone or YC-1 were incubated
with rat cerebral cortex
synaptosomes in the presence of
(a)veratrineand[
14
C]-guanidine
or (b) Scorpion venon and [
12
H]-
Batrachotoxin-B (Garthwaite
et al. 2002). Uptake of guanidine
or inhibition of binding of
batrachotoxin-B was assessed
using liquid scintillation
spectroscopy (Garthwaite et al.
2002). The results of CBD
affinities were repeated with
comparable results

such this could counteract the CB
1
R-mediated beneficial ef-
fects of Δ9-THC. Whether this impacts dosing of medical
cannabis is difficult to properly address in the absence of
human data. Although it been suggested that the influence of
CBD on CB
1
R-mediated effects of Δ9-THC are of marginal
significance at the concentrations of CBD in typical US of
smoked marijuana (Varvel et al. 2006), it may be relevant that
long-term follow-up in symptom-control trials suggested that
oral Δ9-THC but not oral 1:1 CBD:Δ9-THC cannabis extracts
(Cannador) containing comparable levels of Δ9-THC, could
limit the accumulation of disabilities in MS (Zajicek et al.
2005). This would be consistent with a neuroprotective effect
of Δ9-THC (Carroll et al. 2012), which was formally investi-
gated in a recent trial in MS (Zajicek et al. 2013).
Cannabinoids for the Control of Progression in Multiple
Sclerosis
Genetic depletion of CB
1
R in mice is associated with the
development of neurodegeneration (Pryce et al. 2003;Rossi
et al. 2011a). Furthermore, antagonism of CB
1
Rinhumans
Time (Weeks)
0 12 24 36 48 60 72 84 96 108 120 132 144 156
Proportion of Subjects with Stable EDSS (%)
0
10
20
30
40
50
60
70
80
90
100
Dronabinol (THC)
Placebo
P<0.01
Time (Weeks)
0 12 24 36 48 60 72 84 96 108 120 132 144 156
Proportion of Subjects with Stable EDSS (%)
0
10
20
30
40
50
60
70
80
90
100
A
Dronabinol (THC)
Placebo
Cohort of 5.5 EDSS or Less
Total Trial Cohort (EDSS 4.0-6.5)
B
Fig. 4 Inhibition of progressive
multiple sclerosis by oral
tetrahydrocannabinol. People
with progressive MS were
enrolled into a placebo-controlled
double blind clinical trial to assess
efficacy of twice daily oral
tetrahydrocannabinol (Grey)toa
maximum of 28 mg/day verses
vegetable oil placebo (Black)
capsules (Zajicek et al. 2013). The
probability of progression of
EDSS was performed for the (a)
total population (n=320Δ9-THC
and n=162 placebo) or (b)
participants with a baseline EDSS
score of 5·5 or lower (n=76 Δ9-
THC and n=34 placebo). The
results represent Kaplan-Meier
estimates of the proportion of
people with stable EDSS over
time weekly. The plot shows
timings of first events of
progression. Figures have been
presented and analysed in Zajicek
J. et al. Lancet Neurol
2013:12:857–865+online
supplement. Reproduced with
permission from Elsevier

augments glutamatergic excitability (Oliviero et al. 2012),
which is known in excess to cause excitotoxicity in nerves.
It is of interest therefore that a non-coding; genetic variant of
the CB
1
R gene has been be associated with more rapid pro-
gression and neurodegeneration during inflammatory attack in
MS (Rossi et al. 2011b,2013). Endocannabinoid stimulation
of cannabinoid receptors has also been reported to be neuro-
protective (Eljaschewitsch et al. 2006). This suggests that
agonism of the CB
1
R by exogenous delivered cannabinoids
should have neuroprotective potential. However, unfortunate-
ly, it was reported that daily treatment with oral Δ9-THC had
no overall effect on the progression of MS in the progressive
phase of MS (Zajicek et al. 2013.Fig.4a). This may question
previous suggestions of a neuroprotective effect in MS
(Zajicek et al. 2005) and the accumulated experimental biol-
ogy (Baker et al. 2012). However, similar to the failure of Δ9-
THC in this progression trial, likewise, it was originally re-
ported that oral Δ9-THC and cannabis extract (Cannador) had
no effect on spasticity in a symptom-control trial (Zajicek et al.
2003). Likewise, other cannabis extracts (Sativex) failed to
alter the Ashworth scale as a measure of spasticity in symptom
control trials in MS (Wade et al. 2003). However, through
adapting clinical trial design, duration and the outcome mea-
sures, it has been found that cannabis can indeed control
symptoms of MS (Zajicek et al. 2005,2012; Novotna et al.
2011; Corey-Bloom et al. 2012). This indicates that trial
design is critical in the detection of therapeutic effects and
the translation of animal studies into human benefit (Baker
and Amor 2014). In the trial of Δ9-THC in progressive MS,
lower than expected progression rates occurred and so may
have affected the ability to detect clinical change, which
would require longer trials (Zajicek et al. 2013). Clinical
progression in MS is assessed using the Expanded Disability
Status Score (EDSS). This is a motor score ranging from
health (EDSS Score 0); walking without aid or rest for
500 m (EDSS Score 4), walking with an aid/cane (EDSS
score 6); essentially restricted to a wheel chair (EDSS 7); to
death (EDSS 10). However, this neurological rating system is
not linear and PwMS can progress at variable rates (Leray
et al. 2010). However, progression between from EDSS 3 to
EDSS 6 is more consistent and potentially more rapid (Leray
et al. 2010). Therefore, trials enriched for this subset of PwMS
may have a greater chance of detecting differences. Whilst it
was clear that daily Δ9-THC, at the doses tested, did not slow
progression (Fig. 4a), it is of immense interest that analysis of
a subset of people with MS with an EDSS ≤5.5 demonstrated
that Δ9-THC significantly (P<0.01) slowed disease progres-
sion (Fig. 4b; Zajicek et al. 2013). This would be consistent
with the accumulating biological knowledge and supportive
experimental evidence in animal models (Baker et al. 2000,
2012) and would strongly suggest that cannabinoids indeed
have the potential to control neuroimmune processes that lead
to neurodegeneration. Furthermore, the presence of Δ9-THC
in the blood, presumably due to recreational cannabis use, was
associated with a better prognosis following traumatic brain
injury (Nguyen et al. 2014). This possibly suggests a neuro-
protective effect from cannabinoid use in humans.
Conclusions
Despite the potential promise of cannabis to control progres-
sion in MS, it remains to be established whether similar trials
of Δ9-THC in progressive MS, using a revised trial design,
will be repeated to deliver licensed treatments. This may be
difficult for investigator-led academic studies due to the per-
ceived failure of the original trial (Zajicek et al. 2013), making
it difficult to raise the significant funds required to undertake
similar studies. This failure in humans probably also stifles
support for further basic science research in this area. Follow-
ing the initial failure of academic led-trials in spasticity
(Zajicek et al. 2003), interest was maintained because of
commercial development of alternative products. However,
in contrast to the short symptom control trials of a few weeks
duration (Novotna et al. 2011; Corey-Bloom et al. 2012),
phase III trials in progressive MS will probably require a
further 5–6 years to do a 3 year trial that recruits sufficiently
large numbers of people for the trials (estimated to be n=375
with 90 % power to detect an 18 % treatment effect. Zajicek
et al. 2013). Such a study would suffer from competitive
recruitment to other pharmaceutical company and academic-
investigator led trials in progressive MS, where a perceived
failure has not yet occurred. Importantly, unless pharmaceuti-
cal companies are involved, it will be difficult to perform
further studies, typically two phase III trials, to a level actually
required for regulatory approval and licensing (Giovannoni
et al. 2015). However, because of poor patent protection of
oral Δ9-THC as a potential medicine for MS, coupled with
MS drug-pricing structures, where symptom control drugs are
significantly cheaper than current DMT, it means that there
will probably belittle major pharmaceutical interest in funding
and undertaking these studies using a symptom control drug.
The proliferation of outlets supplying relatively cheap,
legalised, medical marijuana that is occurring, notably in the
USA, which is the major commercial market in MS, under-
mines both the chance of commercial development in this area
and the chances of recruiting to placebo-controlled trials, in
regions where medical cannabis is readily available. Whether
retrospective analysis of large numbers of long-term cannabis
users and non-users for symptom control can detect effects on
progression remains to be determined. However, development
of patent-protected formulations of cannabinoids or non-
cannabis pharmaceuticals such as endocannabinoid modula-
tors may be one way to develop commercial interest that could
exploit the cannabinoid biology to help deliver a treatment of
progression in MS.

Acknowledgments The authors thank the support of the National MS
Society (USA) and the MS Society (UK). We thank Prof. John Zajicek
and Susan Ball, Plymouth, UK for providing access to data from the
CUPID trial.
Conflicts of Interest None.
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M, McManus D, Mallik S, Hobart J, CUPID investigator group
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Materials and Methods
Induction of autoimmune experimental encephalomyelitis protocols con-
sistent with the ARRIVE guidelines have been published previously (Al-
Izki et al. 2012; Baker and Amor 2012). Animal studies were approved
following local ethical and United Kingdom Government, Home Office
review in accordance with the United Kingdom Animals (Scientific
Procedures) Act 1986. Full working protocols of the methods and doses
and use of cannabinoids in animals have been reported previously (Pryce
et al. 2003; Croxford et al. 2008; Al-Izki et al. 2012). Briefly, 6–8week
adult Biozzi ABH mice (Al-Izki et al. 2012) were injected with spinal
cord homogenate in Freunds adjuvant on day 0 and 7 to induce experi-
mental autoimmune encephalomyelitis with onset around day 15–19
post-inoculation (p.i.) and again during remission from paralytic attack
on day 28 p.i. to induce a relapse 7–8 days later (Al-Izki et al. 2012).
Animals were scored daily: 0= normal; 1=limp tail; 2=impaired righting
reflex; 3=hindlimb paresis; 4=hindlimb paralysis; 5=moribund. Scores
were assessed using Mann Whitney U statistics (Al-Izki et al. 2012). The
motor co-ordination was assessed on an accelerating (0–40 rpm/
5 min) rotorod and analysed using Students ttest following normality
and equal variance tests (Al-Izki et al.2012). Synthetic Δ9-THC and
CBD were purchased from Δ9-THC Pharm GmbH. Frankfurt, Ger-
many and were diluted in alcohol:cremophor:phosphate buffered
saline (1:1:18). Various doses injected in 0.1 ml intraperitioneally
(i.p). as described previously (Pryce et al. 2003;Croxfordetal.
2008). These were administered shortly before anticipated relapse
(Al-Izki et al. 2012,2014).
Veratrine induced flux of [
14
C]guanidine in synaptosomes has been
reported previously (Pauwels et al. 1986; Garthwaite et al. 2002). Briefly
veratrine (100 μg/ml final concentration), and rat cerebral cortex synap-
tosomes (4 mg/ml, wet weight) were incubated in the absence or presence
of compound at 37 °C for 5 min in polypropylene test tubes. Uptake was
initiated by the addition of pre-warmed [
14
C]-guanidine (final concentra-
tion 1 μCi/ml) and stopped 2 min later by the addition of 10 ml of ice-cold
wash medium as described previously (Pauwels et al. 1986). Incubates
were immediately filtered under vacuum through GF/C filters by using a
Brandel harvester. The incubation tubes were rinsed with 5 ml of ice-cold
wash buffer, which was then used to wash the filter. Filters were trans-
ferred to minivials (Beckman Coulter, Fullerton, CA) with the use of a
Brandel deposit/dispense system and subsequently counted by liquid
scintillation spectroscopy with Picofluor40 liquid scintillator (Garthwaite
et al. 2002). Cannabidiol; YC-1 (5-[1-phenylmethyl)-1H-indazol-3-yl]-2-
furanmethanol (Cayman, Chem Ann Arbor, Michigan, USA); Nabilone
(Cambridge Labs; Newcastle, UK); Lamotrigine (6-(2,3-
Dichlorophenyl)-1,2,4-triazine-3,5-diamine. Tocris, Bristol, UK) and
Sipatrigine (BW619C89. 2-(4-Methyl-1-piperazinyl)-5-(2,3,5-
trichlorophenyl)-4-pyrimidinamine. Tocris Ltd) were diluted with medi-
um from 10 mM stock solutions.
Batrachotoxin-B (BTX-B) Binding. This was performed as described
previously (Garthwaite et al. 2002). Binding was initiated by the addition
of synaptosomes (final concentration 10 mg/ml, wet weight) to a mixture
of test compound and 10 nM [
3
H]Batrachotoxin-B in the absence or
presence of scorpion venom (25 μg/ml final concentration). Samples

were mixed and incubated for 90 min at 25 °C. Ice-cold wash medium
(5 ml) was added and then the samples subjected to vacuum filtration
through GF/C filters by using a Brandel harvester. Incubation tubes were
rinsed with 5 ml of ice-cold wash buffer, which was then used to washthe
filter. Radioactivity in the filter was counted as described above.
Randomised, double-blind, placebo-controlled trial of Δ9-THC in
people with progressive MS has been reported previously (Zajicek et al.
2013), with an International Standard Randomised Controlled Trial num-
ber 62942668. Human studies were approved by the South and West
Devon Research Ethics Committee and done in accordance with Good
Clinical Practice guidelines. Eligible patients provided written informed
consent before participation as International Standard Randomised
Controlled Trial (ISRCTN 62942668). Briefly, 18–65 year old humans
with primary or secondary progressive MS (Expanded disability status
scale (EDSS) Score4.0–6.5), not on current disease modifying therapy
(DMT), were enrolled into the study. These were randomised to oral
dronabinol (Δ9-THC) starting at 3.5 mg twice a day escalated to a
maximum of 28 mg/day depending on tolerability (n=329)orvegetable
oil placebo in gelatin capsules (n=164). These were supplied by Insys
Therapeutics (Phoenix, AZ, USA). Analysis of the total population (n=
493) or subgroup analysis on time to progression in those participants
with a baseline EDSS score of 5· 5 or lower (n=110) was performed using
a log-rank test to compare probability of progression between treatment
groups (Zajicek et al. 2013).