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Endocannabinoids Control Spasticity In A Multiple Sclerosis Model

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David Baker,* Gareth Pryce,* J. Ludovic Croxford,* Peter Brown, "  Roger G. Pertwee, "¡
Alexandros Makriyannis, § Atmaram Khanolkar, § Lorna Layward, Filomena Fezza, # Tiziana
Bisogno, # and Vincenzo Di Marzo#


*Neuroinflammation Group, Institute of Neurology, University College London, U.K.; " The
Medical Research Council Human Movement and Balance Unit, National Hospital for
Neurology and Neurosurgery, London, U.K.;"¡Biomedical Sciences, Institute of Medical
Sciences, University of Aberdeen, U.K.; §Department of Pharmaceutical Sciences and Molecular
and Cell Biology, Center for Drug Discovery, University of Connecticut, Storrs, Conn.;
Multiple Sclerosis Society of Great Britain and Northern Ireland, London, U.K.;
#Endocannabinoid Research Group, Instituto per la Chimica di Molecole di Interesse Biologico,
Consiglio Nazionale delle Ricerche, Arco Felice, Naples, Italy.
Corresponding author: Dr. David Baker Neuroinflammation Group, Department of
Neurochemistry, Institute of Neurology, University College London, 1 Wakefield Street,
London, WC1N 1PJ, U.K. E-mail: d.baker@ion.ucl.ac.uk; and Dr. Vincenzo Di Marzo,
Endocannabinoid Research Group, Instituto per la Chimica di Molecole di Interesse Biologico,
Consiglio Nazionale delle Ricerche, via Toiano 6, 80072, Arco Felice, Naples, Italy. E-mail:
vdimarzo@icmib.na.cnr.it
ABSTRACT
Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic
relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated
with nerve damage, increased levels of the endocannabinoids, anandamide
(arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA
congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these
compounds were found in normal and non-spastic CREAE mice. While exogenously
administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of
endocannabinoid re-uptake and hydrolysis–probably through the enhancement of endogenous
levels of AEA, and, possibly, 2-arachidonoyl glycerol–significantly ameliorated spasticity to an
extent comparable with that observed previously with potent cannabinoid receptor agonists.
These studies provide definitive evidence for the tonic control of spasticity by the
endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other
neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which
exhibit little psychotropic activity.

Multiple sclerosis (MS) is a chronic, demyelinating disease of the central nervous system
(CNS), where individuals accumulate neurological damage and paresis. In addition,
troublesome signs often develop, such as pain, spasticity, and tremor, which are
difficult to treat. This condition has prompted some patients to find alternative medicines and to
perceive benefit from cannabis use (1). The effects of cannabis and cannabinoids are mediated
through cannabinoid receptors (CB) of the type 1 (CB1), expressed mainly in the CNS but also in
peripheral tissues, and type 2 (CB2), expressed almost uniquely in immune cells (2). Recently,
we have demonstrated that exogenous agonists of CB receptors, in particular CB1, can inhibit
spasticity in the Biozzi ABH mouse chronic relapsing experimental allergic encephalomyelitis
(CREAE) model of MS (3). This autoimmune demyelinating disease is induced actively by
sensitization to CNS myelin (4). Systemic agonists will not discriminate between CB receptors
located in centers associated with control of pain, motor, or cognitive functions and therefore
may induce unwanted psychoactive effects that will limit medical alleviation of pain/motor
deficits. More important to note, we demonstrated that CB antagonism transiently exacerbated
spasticity (3). Whilst inverse agonism of antagonists (2) may explain the latter observation,
alternatively this finding suggested that endogenous CB ligands are limiting the spasticity that
occurs during CREAE. This finding would predict that spasticity would be associated with
changes in the equilibrium of the endocannabinoid system (5), which could be manipulated for
therapeutic benefit by using inhibitors of endocannabinoid degradation. This study demonstrates
that the endocannabinoid system exhibits tonic control of spasticity in an MS-like condition.
MATERIALS AND METHODS
CREAE induction
Biozzi ABH mice were from stock bred at the Institute of Ophthalmology, UCL London, or were
purchased from Harlan Olac (Bicester, U.K.). CREAE was induced following subcutaneous
injection of 1 mg of syngeneic spinal cord homogenate emulsified in Freund's complete adjuvant
(Difco, Poole, U.K.) on day 0 and day 7 as described previously (4). Animals developed a
relapsing-remitting disease progression and between 60—80 days post-inoculation developed
evidence of spasticity (incidence 50%—60%) (3). Similarly treated CREAE animals that had not yet
demonstrated tremor, hind-limb, or tail spasticity were used as non-spastic controls.
Assessment of endocannabinoid levels
The spinal cords were expelled rapidly from the cervical spinal column by using hydrostatic
pressure applied through a 19-gauge needle inserted into the lumbar column via a phosphatebuffered
saline-filled syringe. Brains were dissected from the cranium. All tissues were frozen in
liquid N2 within 60 s from death (6). AEA, PEA, and 2-AG levels in lipid extracts from mouse brain
and spinal cord were assessed by using isotope-dilution gas chromatography/mass spectroscopy
essentially as described previously (7, 8). Results were compared by one-way analysis of variance
(ANOVA), incorporating a Bonferroni t-test. Statistical analysis was performed by using
SigmaStat V2 software (SPSS Inc., Chicago, Ill.).
Assessment and Modulation of Spasticity
Quinpirole, rolipram, and R(+)WIN-55,212 were purchased from RBI/Sigma (Poole, U.K.).
Anandamide, PEA and 2-AG were purchased from Cayman chemical (Ann Harbor Mich.). AM404
(9), AM374 (10), and VDM11 (11) were synthesized as previously described. The CB receptor
antagonists SR141617A and SR144465 (2) were supplied by Sanofi Research (Montpellier,
France). Ethanolic solutions were evaporated under vacuum and dissolved in PBS:tween 80
(Sigma.) (3) to be administered as a single intraperitoneal or intravenous tail injection. The
resistance to flexion of individual hind limbs was measured against a strain gauge (5—8 readings per
time point) as described previously (3). The results were expressed as a mean ± SEM per group, and
the data were analyzed by using repeated measures, ANOVA incorporating a pair-wise Tukey posthoc
test.
RESULTS AND DISCUSSION
Endocannabinoid levels in spastic mice
At baseline in normal ABH mice (Fig. 1), whole brains and spinal cords contained similar levels
of the endocannabinoids arachidonoylethanolamide (AEA/anandamide, ~29—33 pmol/g) and 2-
arachidonoyl glycerol (2-AG, ~5—7 nmol/g) and the non-CB receptor binding, cannabimimetic
metabolite (2) palmitoylethanolamide (PEA, ~220—240 pmol/g), as found previously in rat CNS
tissue (7, 8). These levels were not changed significantly in non-spastic CREAE remission
animals (Fig. 1), despite the fact that these animals had experienced 2—3 paralytic episodes and
would contain demyelinated lesions and axonal loss in the spinal cord (4). In comparison with
normal animals, however, endocannabinoids were present in significantly (P<0.05) elevated
amounts in the brain of spastic mice (Fig. 1). Although brain levels were relatively unchanged
for AEA compared (P>0.05) with non-spastic mice, there was a modest increase of AEA
(P<0.05) in spastic brains compared with levels in normal brains. However, there was a marked
increase (~200%) of AEA (P<0.01) and PEA (P<0.05) within the spinal cord of spastic mice
(Fig. 1). This site is one in which major pathological change occurs during CREAE in ABH mice
(4, 6). The brain during CREAE is relatively unaffected, but animals do develop lesions within
the cerebellum (4), an area implicated in the control of tone. Because of the low levels of AEA
(picomoles/gram), subtle changes in the amounts of this metabolite may not be detected as
readily in whole brain, as opposed to that in isolated brain regions (8). However, 2-AG is found
at 200—800 times higher levels than AEA and here an increase (~70%) in 2-AG levels was found
readily in brain and spinal cords (P<0.05) of spastic animals compared with non-spastic controls
(Fig. 1). Although this finding may suggest that 2-AG could serve as a more-easily detectable
indicator of endocannabinoid activity during spastic disease, 2-AG has not been detected in
micro-dialysates from rat striatum (12) or in human cerebrospinal fluid (CSF) (13), despite its
relative abundance in CNS tissues (5, 7, 8). This condition is due possibly to tissue
compartmentalization of 2-AG (13), to its very limited release from neuronal cells (14) or to its
rapid esterification into phospholipid membranes and hydrolysis (15, 16). This study indicates
that the endocannabinoids are up-regulated locally in areas of CREAE-induced damage.
Therefore, clinical parameters; that is, spasticity versus non-spasticity and lesion
load/topography, need to be considered when samples from MS patients are examined for human
confirmation of these animal data. As post-mortem artifactual increases of endocannabinoids
may mask changes in human CNS tissue (17), studies in humans will rely on examination of the
CSF. Further, studies are also warranted to evaluate whether the levels of functional receptors for endocannabinoids [to date, AEA has been found to bind to the two CB receptors, as well as to
vanilloid receptors] (2, 18, 19) are likewise changed from the non-spastic condition to
compensate for the effects of the disease.
Endocannabinoids inhibit spasticity
The lack of changes of enodcannabinoid levels between normal and non-spastic mice and
elevations of endocannabinoids in spastic mice may provide an explanation to previous
observations obtained by using CB receptor antagonists (3). In this previous study, it was shown
that exogenously administered SR141617A and SR144465, two antagonists selective for CB1
and CB2 receptors, failed to affect resistance to flexion of hind limbs (muscle tone) in normal
and non-spastic CREAE mice, in contrast to spastic animals, which showed a transient elevation
in resistance to flexion of the hind limbs with both antagonists (3). On the basis of the results
described above, these observations can now be explained by hypothesizing that the two
antagonists block the action of disease-limiting endocannabinoids, whose levels are elevated
during spastic disease. Although it is speculated that endocannabinoid levels may increase in an
attempt to compensate for the spastic defect, it is possible that endocannabinoids, or other
unrelated fatty acid amides, could be elevated transiently as a mere consequence of spasticity,
such as increased motor activity (12, 20) or tissue damage (5), rather than exerting a
compensatory effect on this sign. While exogenous administration of methanandamide (AM356),
an enzymatically stable AEA analog (21), can limit experimental spasticity (3), to suggest a
cause-and-effect relationship between endocannabinoids and inhibition of spasticity, the effect of
exogenous AEA, 2-AG and PEA was investigated here. All three substances had the capacity to
significantly (P<0.01) ameliorate spasticity, although they had differing efficacy profiles (Fig. 2).
Whilst AEA and PEA maximally inhibited spasticity within 10—30 min, exogenous 2-AG
induced inhibition with a relatively slower onset. (10 mg/kg i.v. Fig. 2, and 1 mg/kg i.v. n = 13
limbs, data not shown). This finding was somewhat surprising because endocannabinoids are
susceptible to rapid inactivation through enzymatic hydrolysis (5, 15). Although different
cannabinoids have different pharmacokinetics (3), this observation may also suggest that 2-AG is
not directly mediating the inhibition, and that this condition could result in part from the actions
of other bioactive metabolite(s). For example, 2-AG may also act by slowly inhibiting the
degradation of endogenous AEA, thereby increasing its levels (15, see below). Alternatively, 2-
AG activates CB2 receptors more efficaciously than AEA, and this different mechanism of action
may also explain the different profile of spasticity inhibition observed here for the two
endocannabinoids. As for PEA, this endogenous compound does not exhibit CB1 or CB2 agonist
activity (2) but may also be capable of enhancing endocannabinoid actions, as shown in suppression
of signs of hyperalgesia (22) and inhibition of breast cancer cell proliferation (23), through not fully
understood effects (23). Similar to AEA, the levels of this metabolite were found here to be raised in
CREAE mice spinal cord (Fig. 1) and to transiently ameliorate spasticity (3) (Fig. 2). Thus, PEA
appears to be linked somewhere to the endocannabinoid system, even though it is not an
endocannabinoid itself.
Inhibition of endocannabinoid degradation in the control of spasticity
Having established that the endocannabinoids are up-regulated during spastic CREAE, a causal
relationship to the anti-spastic effect was sought through manipulation of endocannabinoid levels and action in vivo (Fig. 2). AEA was the first endocannabinoid described, and its
biosynthetic/degradation pathways have been best described (5, 24). Because exogenous applied
naturally occurring cannabimimetic metabolites–in particular AEA–can limit spasticity (Fig.
2), then augmenting the levels of endogenous AEA might have a therapeutic effect. One route
would be to induce its synthesis. AEA has been reported to regulate dopamine-mediated
locomotor activity, whereas D2 receptor agonism with quinpirole induced the release of AEA and
augmented the dopamine-inhibiting effect of exogenous AEA in mice (12). However, no
amelioration of spasticity was evident following administration of quinpirole alone (1 mg/kg i.v.)
(Fig. 3) and 10 mg/kg i.p. (data not shown, n = 8 limbs). This finding suggests that such
stimulation either fails to induce sufficient AEA to control spasticity or that AEA production was
altered in an anatomical site away from the spastic lesion (20).
Blockade of degradation with specific inhibitors may serve as an alternative means of increasing
the bio-availability of the endocannabinoids. Furthermore, this strategy would provide some
selectivity, as these inhibitors would particularly target areas where elevated levels of the
endocannabinoids are being produced and utilized. AEA, and possibly 2-AG, are removed
actively from the extracellular space into the cytosol through specific mechanisms (9, 15) and
undergo breakdown by fatty acid amide hydrolase (FAAH) (5, 15, 24, 25). We found that
spasticity could be ameliorated by injection (10 mg/kg i.v.) of either the competitive re-uptake
inhibitor AM404 (9) or the selective FAAH inhibitor, AM374 (10), both of which have been
shown to enhance AEA neuromodulatory actions (9, 26) (Fig. 3A). No additive effect was
evident by using a combination of both reagents (n = 18, data not shown). These compounds
have very low affinity for cannabinoid receptors (9, 10) and have never been shown to behave as
CB agonists (27). In fact, there was no evidence for cannabimimetic effects (hypothermia) of
AM404 or AM374 at the doses used here in vivo (data not shown). Furthermore, significant
(P<0.001) anti-spasticity effects were also evident by using doses of AM404 (2.5 mg/kg) and
AM374 (1 mg/kg) likely to be sub-threshold for CB1 agonist control of spasticity (Fig. 3B) (3).
These inhibitory effects had a rapid onset before a slow return of CREAE signs over the next few
hours (Fig. 2) and were comparable with those obtained following effective CB receptor agonism
(3). Both AEA and AM404 may also behave as vanilloid receptor (VR1) agonists (18, 19), and
other VR1 agonists have been found to reduce bladder hyper-reactivity in MS (28). Whilst the
role of VR1, if any, in control of spasticity has yet to be demonstrated, a similar inhibition
(P<0.001) of spasticity (Fig. 3B) by the extremely selective anandamide transporter inhibitor
VDM11 (10 mg/kg i.v.), which has essentially no CB or VR-1 agonist activity (11), further
supports the hypothesis that endocannabinoids mediate control of spasticity via CB receptors.
Although further studies are required to demonstrate that exogenous inhibitors of
endocannabinoid re-uptake (VDM11 and AM404) and hydrolysis (AM374) actually cause
amelioration of spasticity by enhancing the tissue levels of endocannabinoids, here we gained
indirect evidence that at least AM374 is acting via this mechanism. In fact, the anti-spastic effect
of AM374 (1 mg/kg i.v.) was blocked by cannabinoid receptor antagonists (SR141716A and
SR144465, both 5 mg/kg i.v.) administered 20 min prior to AM374 (Fig. 3B). Under the same
conditions, the two antagonists were shown to counteract the anti-spasticity effects of the CB
receptor agonist R(+)-WIN-55, 212 [5 mg/kg i.p., change in resistance to flexion at 30 min +7.0
± 18.1% (P>0.05, n = 11 limbs) compared with baseline in antagonist-pretreated animals, as
opposed to —35.5 ± 19.7% (P<0.05, n = 16 limbs) in vehicle-pretreated mice receiving R(+)-WIN-55, 212 alone]. These findings suggest that the inhibitory effect on spasticity by AM374,
which does not directly activate CB receptors (10), is due to enhancement of endocannabinoid
levels and subsequent stimulation of CB receptors.
Inhibition of endocannabinoid signaling exacerbates spasticity
As CB receptor antagonism very transiently exacerbates spasticity (3), we studied the effect on
spasticity of inhibiting CB receptor signaling (Fig. 3A). CB receptors are negatively coupled
through Gi/o proteins to adenylate cyclase, and the phosphodiesterase inhibitor 3-isobutyl-1-
methylxanthine inhibits receptor agonism (29). Rolipram, a selective inhibitor of cAMP-selective
phosphodiesterase IV, has anti-inflammatory effects and inhibits the immunological processes
that drive the development of EAE (30). However, in agreement with its possible counteraction
of endocannabinoid-induced inhibition of cAMP levels, rolipram (10 mg/kg i.v.) induced a
transient increase (P<0.001) in limb (Fig. 3A) and tail spasticity (n = 15/15, P<0.001 compared
with normal animals n = 0/5 (Fig. 3C—D). Furthermore, limb tremor became evident in some
mice (n = 6/15). The exacerbation was not evident in normal animals, where rolipram appeared
to have a sedative effect and was very transient (Fig. 3A), consistent with that observed
previously with CB antagonists (3). This finding again suggests that compensatory mechanisms
are activated rapidly after exacerbation of spasticity in CREAE mice and substantiates further the
involvement of the endocannabinoid system in the tonic down-regulation of this sign.
CONCLUSIONS
Whilst this study appears to provide strong evidence for a role of endocannabinoids in the
control of spasticity, the specific function of each endocannabinoid requires further elucidation
and may be assessed through the use of specific inhibitors once these are generated. The
equilibrium of the endocannabinoid system appears to be altered significantly during spastic
events in CREAE, possibly in response to abnormal neuronal signaling and/or neurodegenerative
effects in damaged nerves. However, this phenomenon does not control spasticity as adequately
as it may be possible by administering exogenous CB agonists (3)–including drugs based on
endocannabinoids that have been reported to have very low potential for physical dependence
(31)–or by manipulating endocannabinoid endogenous levels. This manipulation may minimize
some of the undesirable psychoactive effects associated with CB1 agonism and may have
implications for symptom control in MS and other neuromuscular disease conditions. Parallels
can already be seen in the treatment of depression where serotonin re-uptake inhibitors are
clinically preferable to receptor agonists. The finding of increased amounts of endocannabinoids in
these damaged tissues may open even wider horizons for therapeutic intervention in MS with little
psychotropic side effects.
ACKNOWLEDGMENTS
The authors acknowledge the support of the Multiple Sclerosis Society of Great Britain and
Northern Ireland, the Medical Research Council, the Wellcome Trust, the National Institute on
Drug Abuse (grant DA09789), the Ministero per l'Universita' e la Ricerca e Scientifica e
Tecnologica (grant 3933), and the Associazione Italiana per la Sclerosi Multipla.
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