Multiple Sclerosis Resource Centre

Welcome to the Multiple Sclerosis Resource Centre. This website is intended for international healthcare professionals with an interest in Multiple Sclerosis. By clicking the link below you are declaring and confirming that you are a healthcare professional

You are here

The disease-modifying effects of a Sativex-like combination of phytocannabinoids in mice with experimental autoimmune encephalomyelitis are preferentially due to Δ9 tetrahydrocannabinol acting through CB1 receptors

Multiple Sclerosis and Related Disorders, Volume 4, Issue 6, November 2015, Pages 505 - 511

Abstract

Sativex®, an equimolecular combination of Δ9-tetrahydrocannabinol-botanical drug substance (Δ9-THC-BDS) and cannabidiol-botanical drug substance (CBD-BDS), is a licensed medicine that may be prescribed for alleviating specific symptoms of multiple sclerosis (MS) such as spasticity and pain. However, further evidence suggest that it could be also active as disease-modifying therapy given the immunomodulatory, anti-inflammatory and cytoprotective properties of their two major components. In this study, we investigated this potential in the experimental autoimmune encephalitis (EAE) model of MS in mice. We compared the effect of a Sativex-like combination of Δ9-THC-BDS (10 mg/kg) and CBD-BDS (10 mg/kg) with Δ9-THC-BDS (20 mg/kg) or CBD-BDS (20 mg/kg) administered separately by intraperitoneal administration to EAE mice. Treatments were initiated at the time that symptoms appear and continued up to the first relapse of the disease. The results show that the treatment with a Sativex-like combination significantly improved the neurological deficits typical of EAE mice, in parallel with a reduction in the number and extent of cell aggregates present in the spinal cord which derived from cell infiltration to the CNS. These effects were completely reproduced by the treatment with Δ9-THC-BDS alone, but not by CBD-BDS alone which only delayed the onset of the disease without improving disease progression and reducing the cell infiltrates in the spinal cord. Next, we investigated the potential targets involved in the effects of Δ9-THC-BDS by selectively blocking CB1 or PPAR-γ receptors, and we found a complete reversion of neurological benefits and the reduction in cell aggregates only with rimonabant, a selective CB1 receptor antagonist. Collectively, our data support the therapeutic potential of Sativex as a phytocannabinoid formulation capable of attenuating EAE progression, and that the active compound was Δ9-THC-BDS acting through CB1 receptors.

Highlights

 

  • A Sativex-like phytocannabinoid mixture may serve as a disease modifier in EAE mice.
  • Its effects are preferentially due to Δ9-tetrahydrocannabinol.
  • The effect of Δ9-tetrahydrocannabinol is exerted through activation of CB1 receptors.

Abbreviations: AEA - anandamide, BBB - blood–brain barrier, CB1 receptor - cannabinoid type-1 receptor, CB2 receptor - cannabinoid type-2 receptor, CBD-BDS - cannabidiol botanical drug substance, CFA - complete Freund’s adjuvant, CNS - central nervous system, CSF - cerebrospinal fluid, CUPID - Cannabinoid Use in Progressive Inflammatory Brain Disease, EAE - experimental autoimmune encephalomyelitis, FAAH - fatty acid amide hydrolase, MOG - myelin oligodendrocyte glycoprotein, MS - multiple sclerosis, PBS - phosphate-buffered saline, PPAR - peroxisome proliferator-activated receptor, Δ9-THC-BDS - Δ9-tetrahydrocannabinol botanical drug substance, TMEV - Theiler's murine encephalomyelitis virus.

Keywords: Phytocannabinoid-based medicines, Sativex, CB1 receptors, Therapeutic effects, Multiple sclerosis, EAE mice.

1. Introduction

Multiple sclerosis (MS) is the most common cause of neurological decline in young adults ( Noseworthy et al., 2000 ). Clinically, MS is characterized by symptoms that range from spasticity, dystonia, tremor, ataxia and pain ( Compston and Coles, 2008 ), which are caused by the death of oligodendrocytes and axonal damage triggered by immune cell infiltration, inflammation, demyelination and degenerative events affecting mainly the spinal cord ( Friese et al., 2014 ). Effective disease-modifying treatments for MS patients remain elusive, although some promise has been generated with the development of new immunomodulatory and anti-inflammatory therapies (e.g. natalizumab, fingolimod, interferon-β, glatiramer; see Ransohoff et al., 2015 ).

Some specific targets within the so-called endocannabinoid system are being also investigated for the possible development of cannabinoid-based disease-modifying therapies for MS (Baker and Pryce, 2008 and Sánchez and García-Merino, 2012). In fact, the relation of cannabinoids with MS is not new and has its origin in the late 1980s and early 1990s when it was evident that MS patients frequently self-medicated with illegal cannabis to alleviate pain, spasticity, sleep disturbance and other specific MS symptoms (Consroe et al, 1997 and Pertwee, 2002). This anecdotal evidence was extensively investigated, with the generation of solid preclinical ( Baker et al., 2000 ) and clinical (Collin et al, 2007 and Wade et al, 2010) data that supported the potential use of cannabinoid compounds targeting the endocannabinoid signalling system as a therapy for specific MS symptoms (e.g. spasticity, pain; Rog, 2010 ; Baker et al., 2012 ). In fact, a cannabis-based medicine, Sativex® (GW Pharmaceuticals, Cambridge, UK), an oromucosal spray formed by an equimolecular combination of Δ9-tetrahydrocannabinol-botanical drug substance (Δ9-THC-BDS) and cannabidiol-botanical drug substance (CBD-BDS), showed a clinically significant improvement in spasticity (30% or higher reduction from baseline) in clinical trials ( Collin et al., 2007 ). Associated MS symptoms such as sleep disturbance, bladder problems and loss of motility also showed clear improvements ( Russo et al., 2007 ), whereas safety studies indicated a low risk for serious adverse drug reactions ( Serpell et al., 2013 ). At present, Sativex is already a licensed medicine in numerous countries for use in patients with MS-related spasticity. However, it appears now evident that the usefulness of Sativex in the MS clinic might be extended in the future to the control of disease progression ( de Lago et al., 2009 ), given the immunomodulatory, anti-inflammatory and cytoprotective properties showed by the two components of Sativex in isolation (Lyman et al, 1989, Arévalo-Martín et al, 2003, Pryce et al, 2003, Pryce et al, 2015, Maresz et al, 2007, Kozela et al, 2011, and Mecha et al, 2013), as well as by other phyocannabinoids or derivatives (e.g. Δ8-THC, Wirguin et al., 1994 ; cannabigerol quinone, Granja et al., 2012 ; Carrillo-Salinas et al., 2014 ), synthetic agonists (e.g. WIN55,212-2, Pryce et al., 2003 ; Mestre et al., 2009 ; de Lago et al., 2012 ; HU-210, Aarabi et al., 2011 ), and inhibitors of the endocannabinoid inactivation ( Loría et al., 2010 ), in preclinical models of MS. It is possible that the disease-modifying effects of cannabinoids may be due to an enhancement of those endogenous protective responses that specific components of the endocannabinoid system experience in MS. However, the opposite interpretation is also possible, with an effect that would correct those deficits in the endocannabinoid system that may have an influence in MS pathogenesis. In this sense, most of studies have concentrated on compounds targeting the cannabinoid type-1 (CB1) and/or type-2 (CB2) receptors, as well as fatty acid amide hydrolase (FAAH), the enzyme that degrades the endocannabinoid anandamide (AEA), which has been found to be specific markers of plaque cell subtypes in human MS ( Benito et al., 2007 ). Elevated levels of AEA have been found in active MS lesions ( Eljaschewitsch et al., 2006 ), lymphocytes and cerebrospinal fluid (CSF) samples ( Centonze et al., 2007 ) or in plasma from MS patients ( Jean-Gilles et al., 2009 ), in concordance with the increased levels of AEA found in the spinal cord and brain of CREAE mouse model of MS ( Baker et al., 2001 ). In contrast, other authors found lowered endocannabinoid levels in the CSF of MS patients during relapsing periods of the disease ( Di Filippo et al., 2008 ).

Given that MS appears to produce a broad-spectrum of alterations in endocannabinoid elements, a cannabinoid-based therapy designed to correct or enhance these alterations should be based on a multitarget compound or a combination of compounds with complementary pharmacological profiles. In this context, Sativex fulfils this criteria and the fact that it is already approved for symptom-alleviating effects is a clear advantage. This possibility has been scarcely investigated in experimental models of MS, although the anti-inflammatory and neuroprotective potential of Sativex has been confirmed in models of Huntington’s disease ( Valdeolivas et al., 2012 ). A recent clinical trial investigated cannabinoids as disease modifiers in a cohort of patients with progressive MS (CUPID: Cannabinoid Use in Progressive Inflammatory Brain Disease) with negative results ( Zajicek et al., 2013 ). However, this study was conducted only with dronabinol (synthetic Δ9-THC), and not combined with other cannabinoids like CBD. Although this study showed that dronabinol has no overall effect on the progression of MS in its progressive phase ( Zajicek et al., 2013 ), it encouraged the use of experimental models that better represent progressive MS to continue investigating whether cannabinoids might change the disease course of progressive MS.

We recently initiated some studies with the Sativex-like combination of phytocannabinoids, compared to its two components alone, in experimental murine models of MS. We first used Theiler’s murine encephalomyelitis virus (TMEV) model of MS, in which we found a relevant efficacy of Sativex in delaying disease progression and promoting repair processes, an effect that was completely reproduced by CBD-BDS alone acting through the activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) and, to a lesser extent, by Δ9-THC-BDS acting primarily through the activation of CB1 receptors but also partially through CB2 receptors ( Feliú et al., in press ). The present study is a continuation of this previous one using an alternative murine model of MS, the experimental autoimmune encephalomyelitis (EAE), which also provides a useful model to reproduce MS progression and the underlying pathological mechanisms ( Croxford et al., 2011 ). After immunization with a portion of the myelin oligodendrocyte glycoprotein (MOG), the blood–brain barrier (BBB) becomes permeable to auto-reactive lymphocytes and several days later mice exhibit typical MS relapses with symptoms such as weight loss and progressive tail and hindlimb weakness and paralysis. In these mice, we investigated first the disease-modifying effects of a Sativex-like combination of phytocannabinoids (a 1:1 combination of Δ9-THC-BDS (10 mg/kg) and CBD-BDS (10 mg/kg)), in comparison with the effects of each component alone at 20 mg/kg. In a second experiment, we investigated the involvement of CB1 or PPAR-γ receptors, using selective antagonists of these receptors, in the effects of the most active phytocannabinoid component.

2. Materials and methods

2.1. Animals, treatments and sampling

The experiments were performed according to European regulations for experimental work with animals (directive 2010/63/EU), were approved by the Ethical Committee for Animal Research of the Complutense University, and followed the principles of the ARRIVE guidelines to which this journal is adhered ( Amor and Baker, 2012 ). Female C57BL/6 mice (6–8 week-old) were purchased from Harlan Laboratories (Barcelona, Spain) and housed in our animal facilities with controlled photoperiod (12 h light/dark cycle), temperature (22±1 °C) and relative humidity (40–60%). They had free access to standard food and water. EAE was induced using a previously-published method ( Mendel et al., 1995 ). Briefly, mice were injected in each flank with an emulsion containing 200 mg of the 35–55 portion of MOG (Advanced Biotechnology Centre, Imperial College, London, UK) and 4 mg/ml of Mycobacterium tuberculosis (H37RA DIFCO Lab, Detroit, MI, USA) in a 1:1 mixture with incomplete Freund’s adjuvant (Sigma/Aldrich, Madrid, Spain) prepared in phosphate-buffered saline (PBS). This injection was repeated after 7 days. To enhance the inflammatory response, mice received 1.5 mg/ml of Pertussis toxin (Sigma/Aldrich, Madrid, Spain) prepared in saline and administered i.p. on days 0 and 2. Control animals were obtained by inoculation with the same emulsion (complete Freund's adjuvant, CFA) without MOG. After inoculation, mice were scored daily using the following scale: 0, no clinical signs; 1, limp tail; 2, hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; and 5, moribundity or death. Neurological signs first appeared around day 11–12 after inoculation showing a worsening pattern compared to control animals inoculated with CFA that progress up to day 19–20 post-inoculation (first peak of neurological clinical) followed by a remission phase and subsequent relapse (see Fig. 1 ). EAE mice were distributed in the different treatment groups with the criteria to have the same proportion of animals as regards to the post-inoculation time at which symptoms first appear. At that time point, EAE mice were injected daily with a Sativex-like combination of Δ9-THC-BDS and CBD-BDS (1:1; 10 mg/kg weight for each phytocannabinoid), Δ9-THC-BDS (20 mg/kg), CBD-BDS (20 mg/kg) or vehicle (Tween 80-saline). Δ9-THC-BDS was provided by GW Pharmaceuticals Ltd. (Cambridge, UK) and it contained 67.1% Δ9-THC, 0.3% CBD, 0.9% cannabigerol, 0.9% cannabichromene, and 1.9% other phytocannabinoids, whereas CBD-BDS was also provided by GW Pharmaceuticals Ltd. (Cambridge, UK) and it contained 64.8% CBD, 2.3% Δ9-THC, 1.1% cannabigerol, 3.0% cannabichromene, and 1.5% other phytocannabinoids. The total dose of cannabinoid administered in the Sativex-like combination was always 30.8 mg/kg (equivalent to 10 mg/kg of pure CBD+10 mg/kg of pure Δ9-THC). These doses were selected according to previous studies showing the most active ones for each cannabinoid administered individually in mice and, in particular, our recent experience in a viral model of MS ( Feliú et al., in press ). We assume that tetrad effects are acutely present in our mice at these doses of Δ9-THC, as already found in our previous study ( Feliú et al., in press ), but these effects do not interfere with the analysis of clinical score that was carried out immediately before of cannabinoid injections. A number of 7 subjects in controls and 8 individuals in EAE groups were used. A second set of experiments aimed to further explore the target for the most active phytocannabinoid in the first experiment. To this end, animals were treated with Δ9-THC-BDS (10 mg/kg weight) alone or combined with the selective CB1 receptor antagonist SR141716 (5 mg/kg weight; kindly provided by Sanofi-Aventis, Montpellier, France) or the PPAR-γ inhibitor T0070907 (5 mg/kg weight; purchased from Tocris Cookson Ltd., Bristol, UK). SR141716 and T0070907 were injected 30 min before Δ9-THC-BDS, or vehicle, following the same schedule as the previous experiment. Again, a number of 7 controls and 8 subjects in EAE groups were used. Researchers blinded to the treatment evaluated the neurological status of all animals. Immediately after neurological evaluation, at day 31 in the first experiment and at day 18 in the second one (the second experiment only prolonged up to have the necessary statistical significance in neurological scores, following ARRIVE principles; Amor and Baker, 2012 ), animals were perfused with cold PBS and their spinal cords were removed and fixed in 4% paraformaldehyde overnight followed by a cryoprotective treatment with 20% sucrose. They were used for histological analyses (n=4–5 subjects per experimental group selected from those having a clinical score closer to the mean of each experimental group).

Fig. 1

Fig. 1 Effects of a Sativex-like combination of Δ9-THC-BDS (10 mg/kg) and CBD-BDS (10 mg/kg) (panel A) or its two components administered separately at a dose of 20 mg/kg (panel B for Δ9-THC-BDS and panel C for CBD-BDS) on the neurological score of EAE mice and controls at days 10−31 post-inoculation (CFA-treated; values of neurological score always = 0). Values are means±SEM of 7–8 subjects per experimental group. Data were assessed by Kruskall–Wallis test followed by Dunn’s Multiple Comparison test (p<0.05, ⁎⁎p<0.01, ⁎⁎⁎p<0.005 versus EAE mice treated with vehicle). Panel D contains representative microphotographs showing the presence of cell aggregates, identified by Nissl staining and indicated by arrows, in the spinal cord of EAE mice and controls (CFA-treated) after the above treatments.

2.2. Histological analyses

Spinal cords fixed in 4% paraformaldehyde and cryoprotected in 20% sucrose were sliced (20 μm thick) with a cryostat and collected on TESPA-coated slides. Slides were used to determine the presence of cell aggregates using Nissl staining ( Alvarez et al., 2008 ), as well as the characteristics of these cells using immunohistochemical analysis with Iba-1 and CD11b, which serve as markers of resident microglia and reactive macrophages. In this case, sections were incubated overnight at 4 °C with: (i) monoclonal anti-rabbit Iba-1 (Wako Pure Chemical Industries Ltd., Osaka, Japan) used at 1:1000, or (ii) monoclonal anti-mouse CD11b antibody (AbD Serotec, Oxford, UK) used at 1:200. After incubation with the corresponding primary antibody, sections were washed in 0.1 M PBS and incubated for 2 h at room temperature with the appropriate biotin-conjugated anti-rat (1:200; Millipore, Temecula, CA, USA) or biotin-conjugated anti-rabbit (1:200; Sigma/Aldrich, Madrid, Spain) secondary antibodies. The reaction was revealed with the Vectastain® Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Negative control sections were obtained using the same protocol with omission of the primary antibody. All sections for each immunohistochemical procedure were processed at the same time and under the same conditions. A Nikon Eclipse 90i microscope and a Nikon DXM 1200F camera were used for slide observation and photography, and all image processing was done using ImageJ, the software developed and freely distributed by the US National Institutes of Health (Bethesda, MD, USA).

2.3. Statistics

All data were subjected to Kruskall–Wallis test, followed by Dunn's Multiple Comparison test.

3. Results

3.1. Effects of a Sativex-like combination of phytocannabinoids in EAE mice

Mice treated with MOG exhibited a progressive pattern of EAE induction with neurological disabilities, typical of the neurological phase of this disease, that initiate 11 days post-inoculation and progress during the following days ( Fig. 1 A–C). Control animals (CFA-treated) exhibited no neurological decline at all days examined (data not shown). The pharmacological treatments with the Sativex-like combination, or with Δ9-THC-BDS or CBD-BDS alone, were initiated in an early stage of the disease, administering the first dose at 11 days post-inoculation just when the first neurological symptoms appeared ( Fig. 1 A–C). As expected, the administration of the three treatments, Sativex-like combination, Δ9-THC-BDS alone or CBD-BDS alone, had a positive effect in delaying symptoms onset ( Fig. 1 A–C). However, only the Sativex-like combination ( Fig. 1 A) and the Δ9-THC-BDS ( Fig. 1 B) succeeded in maintaining a reduction in the neurological disability, then improving disease progression of EAE mice. This was not produced by CBD-BDS, as its beneficial effects in delaying the appearance of neurological disability disappeared from day 21 post-inoculation ( Fig. 1 C).

The analysis by Nissl staining of the spinal cord of MOG-induced EAE showed the presence of cell aggregates presumably formed by infiltrated lymphocytes and recruited macrophages in the white matter ( Fig. 1 D). In previous studies ( de Lago et al., 2012 ), we demonstrated that these aggregates are mainly formed by activated microglial cells and recruited peripheral macrophages. In agreement with the neurological improvement, these aggregates were reduced in EAE mice treated with either Δ9-THC-BDS or the Sativex-like combination ( Fig. 1 D), thus indicating that these treatments limited the infiltration of activated lymphocytes through the BBB. Also in agreement with the poor response found with CBD-BDS, there was no reduction in the presence of these aggregates in EAE mice treated with CBD-BDS, at least not at the time at which the samples were collected (day 31 post-inoculation), a stage at which the differences in the neurological score between EAE mice treated with vehicle or CBD-BDS were minimal ( Fig. 1 C).

3.2. Pharmacological targets involved in the effect of Δ9-THC-BDS in EAE mice

The above results support that the beneficial effects of the Sativex-like combination of Δ9-THC-BDS and CBD-BDS appear to be likely due to Δ9-THC-BDS. Next, we wanted to identify the pharmacological targets that could mediate these beneficial effects. In our previous study in TMEV-inoculated mice, the effects of CBD-BDS, which was the most active phytocannabinoid, were mediated by the activation of PPAR-γ whereas those of Δ9-THC-BDS were primarily mediated by the activation of CB1 receptors ( Feliú et al., in press ). Therefore, we investigated both targets in the present study and again using a strategy with selective antagonists for both receptors. We found that the blockade of the CB1 receptor with rimonabant completely reversed the beneficial effects of Δ9-THC-BDS on neurological decline in EAE mice ( Fig. 2 A), whereas the inhibition of PPAR-γ signalling with T0070907 was not effective ( Fig. 2 B). These different responses were highly evident when comparing the neurological score of all experimental groups at the day 18 post-inoculation, proving the complete reversion of Δ9-THC-BDS effects by rimonabant and the absence of change with T0070907. We did not include any experiment with a CB2 receptor antagonist, as we have previously described that this receptor is not involved in disease-modifying effects of cannabinoids in EAE mice ( de Lago et al., 2012 ).

Fig. 2

Fig. 2 Effects of Δ9-THC-BDS (20 mg/kg) administered alone or in combination with rimonabant (SR141716; 5 mg/kg; panel A) or T0070907 (5 mg/kg; panel B) on the neurological score of EAE mice and controls (CFA-treated; values of neurological score always=0). Values are means±SEM of 7–8 subjects per experimental group. Data were assessed by Kruskall–Wallis test followed by the Dunn’s Multiple Comparison test (p<0.05, ⁎⁎p<0.01 versus EAE mice treated with Δ9-THC-BDS; p<0.05, p<0.01 versus EAE mice treated with T0070907 and Δ9-THC-BDS).

Next, we analysed using Nissl staining the presence of cell aggregates in the spinal cord after the different treatments. As expected, treatment with Δ9-THC-BDS resulted in a complete disappearance of these aggregates ( Fig. 3 A). The co-administration of Δ9-THC-BDS with rimonabant proved again the presence of cell aggregates ( Fig. 3 A), thus confirming that the effects of Δ9-THC-BDS were mediated by the activation of CB1 receptors. We also treated animals with Δ9-THC-BDS combined with T0070907 and cell aggregates were absent ( Fig. 3 A), thus indicating that PPAR-γ receptors do not appear to be implicated in the effects of Δ9-THC-BDS. In this experiment, we also conducted immunohistochemistry for Iba-1, a marker of microglia, and CD11b, a marker of infiltrated macrophages, and the results confirmed the data obtained with Nissl staining, with: (i) high presence of Iba-1- and CD11b-immunostained cells in the spinal cord of EAE mice treated with vehicle; (ii) a complete disappearance of these immunostained cells in EAE mice treated with Δ9-THC-BDS alone or combined with T0070907; and (iii) the presence again of Iba-1- and CD11b-immunostained cells in EAE mice treated with Δ9-THC-BDS combined with rimonabant ( Fig. 3 B and C).

Fig. 3

Fig. 3 Representative microphotographs representing the effects of Δ9-THC-BDS (20 mg/kg) administered alone or in combination with rimonabant (SR141716; 5 mg/kg) or T0070907 (5 mg/kg) on the presence of cell aggregates, identified by Nissl staining (panel A), and Iba-1 (panel B) or CD11b (panel C) immunostaining, in the spinal cord of EAE mice and controls (CFA-treated).

4. Discussion

The results of this study conducted in EAE mice support the potential of the phytocannabinoid combination present in Sativex®, a cannabis-based medicine already licenced for the treatment of MS spasticity, as a potential disease modifier in this demyelinating disorder. This efficacy is based on the capability of Sativex to attenuate the clinical decline experienced by EAE mice, as well as to reduce the presence of cell aggregates (typically microglial cells and macrophages) derived from cell infiltration in the spinal cord of these animals. These results add to our previous positive data obtained in an alternative murine model of MS, the TMEV-inoculated mice, also conducted with the Sativex-like combination of Δ9-THC-BDS and CBD-BDS ( Feliú et al., in press ), as well as to other studies conducted with these phytocannabinoids administered in pure form and individually, e.g. Δ9-THC (Lyman et al, 1989, Arévalo-Martín et al, 2003, Pryce et al, 2003, Pryce et al, 2015, and Maresz et al, 2007), CBD (Kozela et al, 2011, Mecha et al, 2013, and Pryce et al, 2015), or with additional cannabinoids, e.g. Δ8-THC ( Wirguin et al., 1994 ), cannabigerol quinone (Granja et al, 2012 and Carrillo-Salinas et al, 2014), WIN55,212-2 (Pryce et al, 2003, Mestre et al, 2009, and de Lago et al, 2012), HU-210 ( Aarabi et al., 2011 ), and inhibitors of the endocannabinoid inactivation ( Loría et al., 2010 ). It remains, however, to be established whether lower doses of these botanical drug substances are immunosuppressive as previous studies have related Δ9-THC-induced immunosuppression at doses above 5 mg/kg to cannabimimetic effects ( Croxford et al., 2008 ). Collectively, all this evidence supports that the current indication of Sativex for the treatment of MS spasticity might be extended with potential efficacy in relation with the disease progression, which would imply a much more relevant benefit for MS patients that would need to be investigated in further clinical trials. It is true that the issue has been already investigated in MS patients in a clinical trial named CUPID with negative results ( Zajicek et al., 2013 ), but this clinical study used dronabinol (synthetic Δ9-THC) instead the phytocannabinoid combination present in Sativex, and an important finding derived from the present work, our previous study conducted also with Sativex in TMEV-inoculated mice ( Feliú et al., in press ), and those studies of other authors using different cannabinoids and experimental models, is the variability of effects which support the need to go to the clinical testing with multi-target compounds or with combinations of complementary profiles in the selected cannabinoids. This is a critical point, even in the present study, in which we found that the most active compound (in fact, much more active, as CBD was almost inactive in our study in agreement with previous studies also conducted in EAE mice; see Maresz et al., 2007 ) was Δ9-THC, which is a non-selective cannabinoid agonist with activity also at receptors of the PPAR family and also a cannabinoid receptor-independent antioxidant profile. By contrast, in our previous study conducted in TMEV-inoculated mice ( Feliú et al., 2015 ), the most active compound was CBD, which does not activate CB1/CB2 receptors but it is active at PPAR-γ and possesses a high antioxidant activity, with Δ9-THC being also active, but to a lesser extent and acting through CB2 and, in particular, CB1 receptors ( Feliú et al., in press ). This is in agreement with the efficacy showed by CBD in the study conducted by Kozela et al. (2011) , although this study was carried out in EAE mice in which we found poor effects of CBD in the present study, although these authors used a unique administration of CBD in contrast with our chronic treatment. Irrespective of these possible differences, it appears likely that the data collected in both models with the two components of Sativex, together with the previous studies of other authors (see Sánchez and García-Merino, 2012 , for review), support the advantage of Sativex for clinical studies, because of its broad-spectrum action, against the use of individual cannabinoids. In addition, our two studies, conducted in different but complementary models of MS from clinical and neuropathological points of view, have also demonstrated that the effects of the two phytocannabinoids that are present in Sativex are exerted through different targets and mechanisms. Thus, the activation of CB1 receptors appears to be a key point in EAE mice, as previously demonstrated (Pryce et al, 2003, Maresz et al, 2007, and de Lago et al, 2012), which explains the efficacy of Δ9-THC and the lack of effects of CBD, which is not active at this receptor, in these mice (present study). Our observation that the effects of Δ9-THC-BDS were reversed when CB1 receptors were blocked with rimonabant, but not when PPAR-γ receptors were blocked with T0070907, strongly supports the importance of CB1 receptors. By contrast, the activation of CB1 receptors, although capable to attenuate disease progression in TMEV-inoculated mice, was not the key mechanism is this MS model, in which the maximal effects were obtained after the administration of CBD-BDS and these effects were reversed by the inhibition of PPAR-γ signalling ( Feliú et al., in press ). It is important to remark that PPAR-γ receptors had been already identified as a promising disease modifying target in MS in studies conducted with classic activators of these receptors (e.g. pioglitazone), not with cannabinoids (Feinstein et al, 2002 and Klotz et al, 2009). The recent demonstration that certain cannabinoids, including mainly CBD but Δ9-THC too, may also activate these receptors ( O’Sullivan and Kendall, 2010 ) provides an interesting support to the idea of using a broad-spectrum cannabinoid-based treatment in MS patients, with activity at the classic targets within the endocannabinoid system, e.g. CB1/CB2 receptors, but also at the PPAR-γ signalling, and the combination of Sativex appears to be an excellent option for such purpose. Lastly, it is important to remark that, despite the Δ9-THC-induced activation of CB1 receptors may be considered a pharmacological problem for prolonged clinical uses, because of its associated psychoactive effects, the safety in prolonged treatments of Sativex, which generate low levels of Δ9-THC in plasma having none to mild side effects, has been documented (Collin et al, 2007, Hilliard et al, 2012, Serpell et al, 2013, and Flachenecker et al, 2014).

In summary, our data support the disease-modifying potential of Sativex® as a phytocannabinoid formulation capable to attenuate EAE progression, and they also indicate that the active compound in this experimental model of MS was Δ9-THC-BDS acting by targeting CB1 receptors. These data add to previous studies conducted also with the Sativex-like combination of phytocannabinoids, but also with other cannabinoids, in the sense to strength the potential of this cannabis-based medicine for the treatment of disease progression in MS and presumably in other demyelinating disorders, extending its already approved uses for the alleviation of spasticity.

Conflict of interest

Authors have formal links with GW Pharmaceuticals that funds some of their research.

Acknowledgements

This work has been supported by grants from CIBERNED (CB06/05/0089), MINECO (SAF2009/11847 to J.F.R. and SAF2010-17501 to C.G.), Red Española de Esclerosis Múltiple (REEM; RD12/0032/0008 to C.G.), CAM (S2011/BMD-2308 to J.F.R. and C.G.) and GW Pharmaceuticals. These agencies had no further role in study design, the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication. Miguel Moreno-Martet was a predoctoral student supported by the FPU Programme of the Ministry of Education, Culture and Sports (MECD). Authors are indebted to Yolanda García-Movellán for administrative assistance.

References

  • Aarabi et al., 2011 M.H. Aarabi, M.E. Shahaboddin, K. Parastouei, M. Motallebi, A. Jafarnejad, M. Mirhashemi, G.A. Hamidi. Evaluation of 11-hydroxy-Δ8-THC-dimethylheptyl effects on cytokines profile and locomotor tests in experimental autoinmune encephalomyelitis. J. Med. Plant Res.. 2011;5:4244-4250
  • Alvarez et al., 2008 F.J. Alvarez, H. Lafuente, M.C. Rey-Santano, V.E. Mielgo, E. Gastiasoro, M. Rueda, R.G. Pertwee, A.I. Castillo, J. Romero, J. Martínez-Orgado. Neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol in hypoxic-ischemic newborn piglets. Pediatr. Res.. 2008;64:653-658
  • Amor and Baker, 2012 S. Amor, D. Baker. Checklist for reporting and reviewing studies of experimental animal models of multiple sclerosis and related disorders. Mult. Scler. Rel. Disord.. 2012;1:111-115
  • Arévalo-Martín et al., 2003 A. Arévalo-Martín, J.M. Vela, E. Molina-Holgado, J. Borrell, C. Guaza. Therapeutic action of cannabinoids in a murine model of multiple sclerosis. J. Neurosci.. 2003;23:2511-2516
  • Baker et al., 2000 D. Baker, G. Pryce, J.L. Croxford, P. Brown, R.G. Pertwee, J.W. Huffman, L. Layward. Cannabinoids control spasticity and tremor in a multiple sclerosis model. Nature. 2000;404:84-87
  • Baker et al., 2001 D. Baker, G. Pryce, J.L. Croxford, P. Brown, R.G. Pertwee, A. Makriyannis, A. Khanolkar, L. Layward, F. Fezza, T. Bisogno, V. Di Marzo. Endocannabinoids control spasticity in a multiple sclerosis model. FASEB J. 2001;15:300-302
  • Baker and Pryce, 2008 D. Baker, G. Pryce. The endocannabinoid system and multiple sclerosis. Curr. Pharm. Des.. 2008;14:2326-2336
  • Baker et al., 2012 D. Baker, G. Pryce, S.J. Jackson, C. Bolton, G. Giovannoni. The biology that underpins the therapeutic potential of cannabis-based medicines for the control of spasticity in multiple sclerosis. Mult. Scler. Relat. Disord.. 2012;1:64-75
  • Benito et al., 2007 C. Benito, J.P. Romero, R.M. Tolón, D. Clemente, F. Docagne, C.J. Hillard, C. Guaza, Romero J. Cannabinoid CB1. and CB2 receptors and fatty acid amide hydrolase are specific markers of plaque cell subtypes in human multiple sclerosis. J. Neurosci.. 2007;27:2396-2402
  • Carrillo-Salinas et al., 2014 F.J. Carrillo-Salinas, C. Navarrete, M. Mecha, A. Feliú, J.A. Collado, I. Cantarero, M.L. Bellido, E. Muñoz, C. Guaza. A cannabigerol derivative suppresses immune responses and protects mice from experimental autoimmune encephalomyelitis. PLoS One. 2014;9:e94733
  • Centonze et al., 2007 D. Centonze, M. Bari, S. Rossi, C. Prosperetti, R. Furlan, F. Fezza, V. De Chiara, L. Battistini, G. Bernardi, S. Bernardini, G. Martino, M. Maccarrone. The endocannabinoid system is dysregulated in multiple sclerosis and in experimental autoimmune encephalomyelitis. Brain. 2007;130:2543-2553
  • Collin et al., 2007 C. Collin, P. Davies, I.K. Mutiboko, S. Ratcliffe. for the Sativex Spasticity in MS Study Group. Randomized controlled trial of cannabis-based medicine in spasticity caused by multiple sclerosis. Eur. J. Neurol.. 2007;14:290-296
  • Compston and Coles, 2008 A. Compston, A. Coles. Multiple sclerosis. Lancet. 2008;372:1502-1517
  • Consroe et al., 1997 P. Consroe, R. Musty, J. Rein, W. Tillery, R. Pertwee. The perceived effects of smoked cannabis on patients with multiple sclerosis. Eur. Neurol.. 1997;38:44-48
  • Croxford et al., 2008 J.L. Croxford, G. Pryce, S.J. Jackson, C. Ledent, G. Giovannoni, R.G. Pertwee, T. Yamamura, D. Baker. Cannabinoid-mediated neuroprotection, not immunosuppression, may be more relevant to multiple sclerosis. J. Neuroimmunol.. 2008;193:120-129
  • Croxford et al., 2011 A.L. Croxford, F.C. Kurschus, A. Waisman. Mouse models for multiple sclerosis: historical facts and future implications. Biochim. Biophys. Acta. 2011;1812:177-183
  • Di Filippo et al., 2008 M. Di Filippo, L.A. Pini, G.P. Pelliccioli, P. Calabresi, P. Sarchielli. Abnormalities in the cerebrospinal fluid levels of endocannabinoids in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry. 2008;79:1224-1229
  • Eljaschewitsch et al., 2006 E. Eljaschewitsch, A. Witting, C. Mawrin, T. Lee, P.M. Schmidt, S. Wolf, H. Hoertnagl, C.S. Raine, R. Schneider-Stock, R. Nitsch, O. Ullrich. The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells. Neuron. 2006;49:67-79
  • Feinstein et al., 2002 D.L. Feinstein, E. Galea, V. Gavrilyuk, C.F. Brosnan, C.C. Whitacre, L. Dumitrescu-Ozimek, G.E. Landreth, H.A. Pershadsingh, G. Weinberg, M.T. Heneka. Peroxisome proliferator-activated receptor-gamma agonists prevent experimental autoimmune encephalomyelitis. Ann. Neurol. 2002;51:694-702
  • Feliú et al., 2015 A. Feliú, M. Moreno-Martet, M. Mecha, F.J. Carrillo-Salinas, E. de Lago, J. Fernández-Ruiz, C. Guaza. A Sativex-like combination of phytocannabinoids as a disease-modifying therapy in a viral model of multiple sclerosis. Br. J. Pharmacol.. 2015;172:3579-3595
  • Flachenecker et al., 2014 P. Flachenecker, T. Henze, U.K. Zettl. Nabiximols (THC/CBD oromucosal spray, Sativex®) in clinical practice--results of a multicenter, non-interventional study (MOVE 2) in patients with multiple sclerosis spasticity. Eur. Neurol.. 2014;71:271-279
  • Friese et al., 2014 M.A. Friese, B. Schattling, L. Fugger. Mechanisms of neurodegeneration and axonal dysfunction in multiple sclerosis. Nat. Rev. Neurol.. 2014;10:225-238
  • Granja et al., 2012 A.G. Granja, F. Carrillo-Salinas, A. Pagani, M. Gómez-Cañas, R. Negri, C. Navarrete, M. Mecha, L. Mestre, B.L. Fiebich, I. Cantarero, M.A. Calzado, M.L. Bellido, J. Fernández-Ruiz, G. Appendino, C. Guaza, E. Muñoz. A cannabigerol quinone alleviates neuroinflammation in a chronic model of multiple sclerosis. J. Neuroimmune Pharmacol.. 2012;7:1002-1016
  • Hilliard et al., 2012 A. Hilliard, C. Stott, S. Wright, G. Guy, G. Pryce, S. Al-Izki, C. Bolton, G. Giovannoni. Evaluation of the effects of Sativex (THC BDS: CBD BDS) on inhibition of spasticity in a chronic relapsing experimental allergic autoimmune encephalomyelitis: a model of multiple sclerosis. ISRN Neurol. 2012;2012:802649
  • Jean-Gilles et al., 2009 L. Jean-Gilles, S. Feng, C.R. Tench, V. Chapman, D.A. Kendall, D.A. Barrett, C.S. Constantinescu. Plasma endocannabinoid levels in multiple sclerosis. J. Neurol. Sci.. 2009;287:212-215
  • Klotz et al., 2009 L. Klotz, S. Burgdorf, I. Dani, K. Saijo, J. Flossdorf, S. Hucke, J. Alferink, N. Nowak, M. Beyer, G. Mayer, B. Langhans, T. Klockgether, A. Waisman, G. Eberl, J. Schultze, M. Famulok, W. Kolanus, C. Glass, C. Kurts, P.A. Knolle. The nuclear receptor PPAR-γ selectively inhibits Th17 differentiation in a T cell-intrinsic fashion and suppresses CNS autoimmunity. J. Exp. Med.. 2009;206:2079-2089
  • Kozela et al., 2011 E. Kozela, N. Lev, N. Kaushansky, R. Eilam, N. Rimmerman, R. Levy, A. Ben-Nun, A. Juknat, Z. Vogel. Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice. Br. J. Pharmacol.. 2011;163:1507-1519
  • Loría et al., 2010 F. Loría, S. Petrosino, M. Hernangómez, L. Mestre, A. Spagnolo, F. Correa, V. Di Marzo, F. Docagne, C. Guaza. An endocannabinoid tone limits excitotoxicity in vitro and in a model of multiple sclerosis. Neurobiol. Dis.. 2010;37:166-176
  • Lyman et al., 1989 W.D. Lyman, J.R. Sonett, C.F. Brosnan, R. Elkin, M.B. Bornstein. Δ9- tetrahydrocannabinol: a novel treatment for experimental autoimmune encephalomyelitis. J. Neuroimmunol. 1989;23:73-81
  • Maresz et al., 2007 K. Maresz, G. Pryce, E.D. Ponomarev, G. Marsicano, J.L. Croxford, L.P. Shriver, C. Ledent, X. Cheng, E.J. Carrier, M.K. Mann, G. Giovannoni, R.G. Pertwee, T. Yamamura, N.E. Buckley, C.J. Hillard, B. Lutz, D. Baker, B.N. Dittel. Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells. Nat. Med.. 2007;13:492-497
  • Mecha et al., 2013 M. Mecha, A. Feliú, P.M. Iñigo, L. Mestre, F.J. Carrillo-Salinas, C. Guaza. 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
  • Mendel et al., 1995 I. Mendel, N. Kerlero de Rosbo, A. Ben-Nun. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells. Eur. J. Immunol. 1995;25:1951-1959
  • Mestre et al., 2009 L. Mestre, F. Docagne, F. Correa, F. Loría, M. Hernangómez, J. Borrell, C. Guaza. A cannabinoid agonist interferes with the progression of a chronic model of multiple sclerosis by downregulating adhesion molecules. Mol. Cell. Neurosci.. 2009;40:258-266
  • Noseworthy et al., 2000 J.H. Noseworthy, C. Lucchinetti, M. Rodriguez, B.G. Weinshenker. Multiple sclerosis. N. Engl. J. Med.. 2000;343:938-952
  • O’Sullivan and Kendall, 2010 S.E. O’Sullivan, D.A. Kendall. Cannabinoid activation of peroxisome proliferator-activated receptors: potential for modulation of inflammatory disease. Immunobiology. 2010;215:611-616
  • Pertwee, 2002 R.G. Pertwee. Cannabinoids and multiple sclerosis. Pharmacol. Ther.. 2002;95:165-174
  • Pryce et al., 2003 G. Pryce, Z. Ahmed, D.J. Hankey, S.J. Jackson, J.L. Croxford, J.M. Pocock, C. Ledent, A. Petzold, A.J. Thompson, G. Giovannoni, M.L. Cuzner, D. Baker. Cannabinoids inhibit neurodegeneration in models of multiple sclerosis. Brain. 2003;126:2191-2202
  • Pryce et al., 2015 G. Pryce, D.R. Riddall, D.L. Selwood, G. Giovannoni, D. Baker. Neuroprotection in experimental autoimmune encephalomyelitis and progressive multiple sclerosis by cannabis-based cannabinoids. J. Neuroimmune Pharmacol.. 2015; (in press)
  • Ransohoff et al., 2015 R.M. Ransohoff, D.A. Hafler, C.F. Lucchinetti. Multiple sclerosis−a quiet revolution. Nat. Rev. Neurol. 2015;11:134-142
  • Rog, 2010 D.J. Rog. Cannabis-based medicines in multiple sclerosis--a review of clinical studies. Immunobiology. 2010;215:658-672
  • Russo et al., 2007 E.B. Russo, G.W. Guy, P.J. Robson. Cannabis, pain, and sleep: lessons from therapeutic clinical trials of Sativex, a cannabis-based medicine. Chem. Biodivers.. 2007;4:1729-1743
  • Sánchez and García-Merino, 2012 A.J. Sánchez, A. García-Merino. Neuroprotective agents: cannabinoids. Clin. Immunol.. 2012;142:57-67
  • Serpell et al., 2013 M.G. Serpell, W. Notcutt, C. Collin. Sativex long-term use: an open-label trial study in patients with spasticity due to multiple sclerosis. J. Neurol.. 2013;260:285-295
  • Valdeolivas et al., 2012 S. Valdeolivas, V. Satta, R.G. Pertwee, J. Fernández-Ruiz, O. Sagredo. Sativex-like combination of phytocannabinoids is neuroprotective in malonate-lesioned rats, an inflammatory model of Hungtinton’s disease: role of CB1 and CB2 receptors.. ACS Chem. Neurosci.. 2012;3:400-406
  • Wade et al., 2010 D.T. Wade, C. Collin, C. Stott, P. Duncombe. Meta-analysis of the efficacy of Sativex (Nabiximols), on spasticity on people with multiple sclerosis. Mult. Scler.. 2010;16:707-714
  • Wirguin et al., 1994 I. Wirguin, R. Mechoulam, A. Breuer, E. Schezen, J. Weidenfeld, T. Brenner. Suppression of experimental autoimmune encephalomyelitis by cannabinoids. Immunopharmacology. 1994;28:209-214
  • Zajicek et al., 2013 J. Zajicek, S. Ball, D. Wright, J. Vickery, A. Nunn, D. Miller, M. Gomez Cano, D. McManus, S. Mallik, J. Hobart. CUPID investigator group. Effect of dronabinol on progression in progressive multiple sclerosis (CUPID): a randomised, placebo-controlled trial. Lancet Neurol.. 2013;12:857-865
  • de Lago et al., 2009 E. de Lago, M. Gómez-Ruiz, M. Moreno-Martet, Fernández-Ruiz J. Cannabinoids. multiple sclerosis and neuroprotection. Expert Rev. Clin. Pharmacol.. 2009;2:645-660
  • de Lago et al., 2012 E. de Lago, M. Moreno-Martet, A. Cabranes, J.A. Ramos, J. Fernández-Ruiz. Cannabinoids ameliorate disease progression in a model of multiple sclerosis in mice, acting preferentially through CB1 receptor-mediated anti-inflammatory effects. Neuropharmacology. 2012;62:2299-2308

Footnotes

a Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain

b Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain

c Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain

d Neuroimmunology Group, Functional and Systems Neurobiology Department, Instituto Cajal, CSIC, Madrid, Spain

Corresponding author at: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense, Ciudad Universitaria s/n, 28040 Madrid, Spain.


Search this site

Stay up-to-date with our monthly e-alert

If you want to regularly receive information on what is happening in MS research sign up to our e-alert.

Subscribe »

About the Editors

  • Prof Timothy Vartanian

    Timothy Vartanian, Professor at the Brain and Mind Research Institute and the Department of Neurology, Weill Cornell Medical College, Cornell...
  • Dr Claire S. Riley

    Claire S. Riley, MD is an assistant attending neurologist and assistant professor of neurology in the Neurological Institute, Columbia University,...
  • Dr Rebecca Farber

    Rebecca Farber, MD is an attending neurologist and assistant professor of neurology at the Neurological Institute, Columbia University, in New...

This online Resource Centre has been made possible by a donation from EMD Serono, Inc., a business of Merck KGaA, Darmstadt, Germany.

Note that EMD Serono, Inc., has no editorial control or influence over the content of this Resource Centre. The Resource Centre and all content therein are subject to an independent editorial review.

The Grant for Multiple Sclerosis Innovation
supports promising translational research projects by academic researchers to improve understanding of multiple sclerosis (MS) for the ultimate benefit of patients.  For full information and application details, please click here

Journal Editor's choice

Recommended by Prof. Brenda Banwell

Causes of death among persons with multiple sclerosis

Gary R. Cutter, Jeffrey Zimmerman, Amber R. Salter, et al.

Multiple Sclerosis and Related Disorders, September 2015, Vol 4 Issue 5