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Sativex® is an oromucosal spray, containing equivalent amounts of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD)-botanical drug substance (BDS), which has been approved for the treatment of spasticity and pain associated to multiple sclerosis (MS). In this study, we investigated whether Sativex may also serve as a disease-modifying agent in the Theiler's murine encephalomyelitis virus-induced demyelinating disease model of MS.
A Sativex-like combination of phytocannabinoids and each phytocannabinoid alone were administered to mice once they had established MS-like symptoms. Motor activity and the putative targets of these cannabinoids were assessed to evaluate therapeutic efficacy. The accumulation of chondroitin sulfate proteoglycans (CSPGs) and astrogliosis were assessed in the spinal cord and the effect of Sativex on CSPGs production was evaluated in astrocyte cultures.
Sativex improved motor activity – reduced CNS infiltrates, microglial activity, axonal damage – and restored myelin morphology. Similarly, we found weaker vascular cell adhesion molecule-1 staining and IL-1β gene expression but an up-regulation of arginase-1. The astrogliosis and accumulation of CSPGs in the spinal cord in vehicle-infected animals were decreased by Sativex, as was the synthesis and release of CSPGs by astrocytes in culture. We found that CBD-BDS alone alleviated motor deterioration to a similar extent as Sativex, acting through PPARγ receptors whereas Δ9-THC-BDS produced weaker effects, acting through CB2 and primarily CB1 receptors.
The data support the therapeutic potential of Sativex to slow MS progression and its relevance in CNS repair.
Multiple sclerosis (MS), the most common cause of neurological disability in young adults, is a complex autoimmune disease characterized by inflammation, demyelination and axonal damage (Compston and Coles, 2008). Most patients with MS initially develop a relapsing-remitting disease course that it is followed by a secondary progressive clinical form. By contrast, 10–15% of MS patients develop a primary progressive form from the onset of the disease. Nevertheless, effective treatments for patients with either primary or secondary MS remain elusive. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a well-established model of MS (Lipton and Dal Canto, 1976) that is unique in reproducing the putative viral aetiology of MS and for studying virus-induced autoimmunity (Miller etal., 1997). TMEV is a single-stranded RNA virus, which when inoculated intracranially in susceptible mouse strains, provokes the development of a chronic-progressive demyelinating disease. Moreover, as TMEV-IDD progresses, epitope spreading produces T-cell responses against myelin peptides.
The cannabinoids are currently being studied for their potential benefits in the treatment of MS and other neuroinflammatory/neurodegenerative diseases (Pryce and Baker, 2012; Velayudhan etal., 2014). Preclinical studies have demonstrated that cannabinoids can alleviate MS-associated symptoms, such as spasticity (Baker etal., 2000), and they exhibit anti-inflammatory (Arévalo-Martín etal., 2003; Croxford and Miller, 2003), antioxidant, anti-excitotoxic and neuroprotective properties (Pryce etal., 2003; Fernández-Ruiz etal., 2010; Loría etal., 2010). Cannabinoids are also protective to oligodendrocytes in vitro and in vivo (Molina-Holgado etal., 2002; Gómez etal., 2010; Mecha etal., 2012). Sativex® is an oromucosal spray that contains an equimolecular combination of Δ9-tetrahydrocannabinol-botanical drug substance (Δ9-THC-BDS) and cannabidiol-botanical drug substance (CBD-BDS) and it has been approved for use in patients with MS-related spasticity and neuropathic pain. Indeed, safety studies with Sativex have indicated a low risk for serious adverse drug reactions (Serpell etal., 2013). Evidence for a neuroprotective effect of Sativex was recently reported in inflammatory models of Huntington disease and in a transgenic murine model of amyotrophic lateral sclerosis (Valdeolivas etal., 2012; Moreno-Martet etal., 2014). Moreover, both components of Sativex have been described to independently modify immune responses in MS animal models (Maresz etal., 2007; Kozela etal., 2011; Mecha etal., 2013b). However, there are still no clinical studies that have investigated the potential of Sativex as a disease-modifying treatment for progressive MS.
The potential of cannabinoids to influence the progression of MS also includes the possibility that they may be used for repair processes. In MS, repair and remyelination occur in shadow plaques (Chang etal., 2012) but, as the disease progresses, remyelination becomes less efficient (Chang etal., 2002; Franklin, 2002; Goldschmidt etal., 2009). It is unlikely that the inability to remyelinate results from the absence of oligodendrocyte precursor cells (OPCs) in the adult CNS, as OPCs are present in elderly patients even decades after having been diagnosed with the disease (Chang etal., 2012). Alternatively, the failure in remyelination might reflect changes in the local extracellular environment, specifically those affecting the extracellular matrix (ECM) proteins and chondroitin sulfate proteoglycans (CSPGs). Post-injury deposition of CSPGs may be a protective CNS response to limit damage (Silver and Miller, 2004), yet CSPGs up-regulation in chronic MS provokes scar formation (Sobel and Ahmed, 2001; Mohan etal., 2010), which inhibits both axon regeneration and remyelination (Siebert and Osterhout, 2011; Lau etal., 2012). This is why CSPGs and hyaluronan are considered to be inhibitory molecules, the accumulation of which is thought to be the main reason for the failure of remyelination (Harlow and Macklin, 2014).
TMEV represents a suitable model to elucidate the role of glial scar formation in chronic inflammatory and demyelinating CNS lesions. Chronic demyelination in the TMEV model is accompanied by alterations in spinal cord ECM and astrogliosis (Haist etal., 2012) which in turn contribute to the failure in regeneration. Accordingly, limited remyelination in TMEV has been associated with insufficient oligodendroglial differentiation (Ulrich etal., 2008). While cannabinoids have been shown to improve remyelination (Arévalo-Martín etal., 2003), there is a little information about their effects on the ECM and CSPGs.
Thus, we set out to investigate the therapeutic potential of a Sativex-like combination of phytocannabinoids (Sativex) as a disease-modifying therapy in a model of primary progressive MS. TMEV-infected mice with established symptomatology were treated with Sativex or individual Δ9-THC-BDS or CBD-BDS, which improved the motor deficits associated with the disease. Indeed, Sativex reduced cellular infiltrates, decreased microglial activity and diminished axonal damage in the TMEV-infected mice. Moreover, myelin morphology was restored and the expression of proinflammatory cytokines and adhesion molecules were down-regulated by Sativex. Astrogliosis, the accumulation of CSPGs and the expression of the synthetic enzyme xylosyltransferase-I (XT-I), was also reduced by Sativex treatment. In addition, our results support the involvement of CB1 and CB2 receptors in the beneficial effects of Δ9-THC-BDS and of PPARγ receptors in the effects of CBD-BDS in TMEV-IDD.
All animal care and experimental procedures were performed in accordance with EU (2010/63/EU) and governmental guidelines (Royal Decree 53/2013 BOE n°34 and Comunidad de Madrid: ES 280790000184) and were approved by the Ethics Committee on Animal Experimentation of the Cajal Institute (CSIC) (protocol number: 2013/03 CEEA-IC). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny etal., 2010; McGrath etal., 2010). A total of 90 animals were used in the experiments described here.
TMEV-IDD-susceptible female SJL/J mice from Harlan (Barcelona, Spain) were maintained at our in-house colony (Cajal Institute, Madrid, Spain) on a 12h light/dark cycle with ad libitum access to food and water. Four-week-old mice were intracerebrally inoculated in the right hemisphere with 2 × 106 plaque forming units of the Daniel strain of TMEV diluted in 30 μL of DMEM supplemented with 10% FCS (Lledó etal., 1999). Sham-operated mice received 30 μL of DMEM + 10% FCS alone.
Sham or TMEV-IDD mice were treated daily with vehicle alone or a 1:1 combination of botanical extracts enriched with either Δ9-THC botanical extract (Δ9-THC-BDS) containing 67.1% Δ9-THC, 0.3% CBD, 0.9% cannabigerol, 0.9% cannabichromene and 1.9% other phytocannabinoids or CBD: CBD botanical extract (CBD-BDS) containing 64.8% CBD, 2.3% Δ9-THC, 1.1% cannabigerol, 3.0% cannabichromene and 1.5% other phytocannabinoids (kindly provided by GW Pharmaceuticals Ltd., Cambridgeshire, UK). The dose of Sativex administered was 10mg·kg−1 which corresponds to 5mg·kg−1 of Δ9-THC-BDS plus 5mg·kg−1 of CBD-BDS. Cannabinoids were prepared in a Tween-80 saline solution (1:16) gassing it with N2 to avoid oxidation and they were administered i.p. once daily from days 70 to 80 post-infection when the signs of disease were evident. The duration of the treatment was based on previous studies using the TMEV-IDD model (Docagne etal., 2007). Additional groups of mice received Δ9-THC-BDS (5mg·kg−1) or CBD-BDS (5mg·kg−1) administered separately. In an additional experiment, mice treated with Δ9-THC-BDS also received the CB1 receptor antagonist AM251 (2mg·kg−1) or the CB2 receptor antagonist AM630 (2mg·kg−1) (Tocris Bioscience, Bristol, UK) 30min previously, whereas mice administered with CBD-BDS were treated 30min beforehand with the PPARγ inhibitor T0070907 (5mg·kg−1) (Cayman Chem, Ann Arbor, MI, USA). In all experiments and after 10 days of treatment, the motor activity was evaluated and the animals were killed with an overdose of anaesthetic for tissue collection.
Locomotor activity was evaluated in mice using an activity cage (Activity Monitor System, Omnitech Electronics Inc., Columbus, OH, USA) coupled to a digiscan analyser. The number of times that the animals broke the horizontal or vertical sensor beams was measured in two 5min sessions.
Mice were anaesthetized with pentobarbital (Dolethal, 50mg·kg−1 body weight, i.p.) and perfused transcardially. The spinal cord was fixed overnight in 4% paraformaldehyde (PFA) prepared in 0.1M phosphate buffer (PB) and cryoprotected in sucrose solution in 0.1M PB (15% followed by a 30%). Coronal and longitudinal spinal cord cryostat sections (15 and 30μm thick; cryostat Leica Microsystems CM1900, Barcelona, Spain) were then processed for immunohistochemistry.
Free-floating spinal cord cryostat sections were washed with 0.1M phosphate buffer (PB), PB + 0.2% Triton X-100 (PBT) and then blocked for 1h at room temperature in blocking buffer (PBT and 5% normal goat serum: Vector Laboratories, Inc., Burlingame, CA, USA) after inhibiting the endogenous peroxidase for 3,3′ diaminobenzidine tetrahydrochloride (DAB) staining. The sections were then incubated overnight at 4°C with the primary antibody (Table1). For colourimetric immunohistochemistry, microglial cells in spinal cord sections (30μm thick) were stained with Iba-1 in blocking buffer. T lymphocytes were stained for CD4 and CD8 expression. We also assessed vascular cell adhesion molecule-1 (VCAM-1) staining. For immunofluorescence, SMI32 antibody was used to detect axonal damage (sections of 15μm thick). In longitudinal and transversal 30μm sections, axons were stained with Neurofilament-H and RIP antibodies. Astrocytes were stained with GFAP antibody, proliferative astrocyte CSPGs were stained with vimentin antibody and CSPGs with an anti-chondroitin sulfate antibody (CS56). After incubation with the primary antibody, the sections were rinsed with PBT and were incubated for 1h with biotinylated antibodies (Vector Laboratories, Inc.). For immunofluorescence, an Alexa 488 Fluor-conjugated goat anti-rabbit antibody (for Neurofilament-H), goat anti-mouse antibody (for SMI-32 and GFAP; Molecular Probes Inc., Eugene, OR, USA) and Texas Red-conjugated donkey anti-mouse IgM (for CS56; Jackson Immunoresearch Europe, Suffolk, UK) were used. For DAB immunostaining, the sections were incubated for 1h with a biotin–peroxidase complex (Vector Laboratories, Inc.) and with the chromogen DAB (Sigma-Aldrich, St. Louis, MO, USA). In all cases, the specificity of staining was confirmed by omitting the primary antibody.
For immunocytochemistry, astrocytes maintained in culture for 48h were fixed with PFA 4% for 20min, incubated with blocking buffer and stained overnight at 4°C for neurocan, using an antibody that recognizes the N-terminal and full-length protein. The following day, the cells were rinsed and incubated for 1h with Alexa 594 Fluor-conjugated goat anti-mouse antibody (Molecular Probes Inc.).
Immunofluorescence images were acquired on a Leica TCS SP5 confocal microscope and for immunohistochemistry, with a Zeiss Axiocam high resolution digital colour camera. Individual optical sections were examined analysing five to six sections from at least five to six animals per group. Staining was quantified using Image J software (NIH, Bethesda, MD, USA), maintaining the threshold intensity constant to compare the experimental and control images obtained within the experiments. The data are presented as the percentage of the total area stained with respect to the sham-operated animals.
The spinal cord sections were stained with haematoxylin and eosin (H&E) to analyse the infiltrates in the parenchyma and with LFB to define myelin integrity. Inflammatory infiltrates were evaluated by scoring the infiltrate in the spinal cord on a scale of 0 to 4: a score of 0 reflects the absence of infiltrates; 4 reflects the maximal infiltrate; while the intermediate scores of 1, 2 and 3 define the increasing infiltrate density in the tissue.
For LFB staining, free-floating spinal cord sections (15μm thick) were washed with 0.1M PB. Tissue samples were dehydrated successively in an ethanol series from 70 to 95% and the sections were then incubated in LFB solution at 56oC in an oven overnight. The following day, the excess stain was rinsed off from the sections with 95% ethanol and the slides were contrasted in lithium carbonate solution for 30 s. Finally, the sections were dehydrated, cleared with xylene and cover slipped.
Astrocyte cultures were prepared from postnatal Wistar pups (0–2 days of age) as described previously (Mecha etal., 2011). After isolation, the astrocytes were plated in poly-D-lysine-coated 6-well plates at a density of 6 × 105cells·mL−1 for Western blotting and PCR analysis or in 24-well plates with coverslips at a density of 5 × 104cells·mL−1 for immunocytochemistry and grown in DMEM medium supplemented with 5% horse serum, 5% FBS, 100U·mL−1 penicillin and 100mg·mL−1 streptomycin. The medium was replaced 3h later, adding cytosine-d-arabinofuranoside (Sigma-Aldrich). After 4 days in vitro, the cells were maintained for 1h in DMEM before they were then exposed for 24, 48 or 72h to TGFβ1 and bFGF (10ng·mL−1 each; Peprotech, Rocky Hill, NJ, USA) and with Sativex (100nM: 50 CBD-BDS + 50 Δ9-THC-BDS; 0.50μM: 0.25 CBD-BDS + 0.25 Δ9-THC-BDS; 1μM: 0.5 CBD-BDS + 0.5 Δ9-THC-BDS) or the vehicle alone. The cells harvested at 24h were processed for PCR analysis to analyse the mRNA expression of brevican and XT-I. Immunocytochemical studies were performed on astrocytes harvested after 48h, whereas the supernatants were taken from cells incubated with the stimuli for 72h. These supernatants were analysed by Western blots to evaluate the amount of ECM secreted into the medium.
Total RNA was extracted from spinal cords or astrocyte cultures using RNeasy mini columns (Qiagen, Manchester, UK), avoiding genomic DNA contamination by DNase I degradation (DNase I, Sigma-Aldrich). The RNA yield was determined using a Nanodrop spectrophotometer (Thermo Scientific; Wilmington, DE, USA) and the total RNA (1μg in 20μL) was reverse transcribed into cDNA using poly-dT primers and a reverse transcription kit (Promega Biotech Ibérica, S.L., Madrid, Spain). Real-time PCR was carried out with SYBR® and the oligonucleotide primer sequences (Applied Biosystems, Warrington, UK) used are given in Table2. After an initial incubation at 50°C for 2min and 95°C for 10min, PCR amplification was performed over 40 cycles of 95°C for 15s and 60°C for 1min. The samples were assayed in triplicate on an Applied Biosystems PRISM 7500 Sequence detection system. To rule out genomic DNA contamination, a control sample using RNA that had not been reversed transcribed was used as the template for each set of extractions. Gene expression was calculated using the 2−ΔΔCt method and the relative expression was quantified by calculating the ratio between the values obtained for each gene of interest and those of the 18S gene. The results are expressed as a percentage with respect to the sham operated mice for each time point.
Identical amounts (1mL) of astrocyte supernatant were precipitated overnight at −20°C in 4vol acetone. The proteins were pelleted by centrifugation at 11,180× g, dried and resuspended in 50μL chondroitinase ABC buffer (50mM Tris pH 8.0, 60mM sodium acetate, 0.02% BSA) and treated for 3h at 37°C with 0.1U·mL−1 chondroitinase ABC from Proteus vulgaris (Sigma-Aldrich). This reaction was stopped by adding Laemmli sample buffer and boiling for 10min. The proteins (15μL of the protein supernatant) were then resolved on a 6% SDS-polyacrylamide gel and transferred at 4°C to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were incubated for 20min in citrate buffer at 95°C for antigen retrieval and then washed with Tris-buffered saline (TBS) followed by further washes with TBS with 0.1% Tween® 20 (TBST). The membranes were blocked for 1h in 5% BSA (Gibco-Invitrogen S.A., Barcelona, Spain) in TBST and they were probed overnight for neurocan (Table1). The membranes were then washed in blocking solution and incubated with a secondary HRP conjugated goat anti-mouse IgG antibody for 1h (1:8000; Bio-Rad, Hercules, CA, USA). The membranes were washed with TBST and TBS. Protein bands were visualized by enhanced chemiluminescence detection and the amount of protein was estimated by densitometry (GS800 calibrated densitometer; Bio-Rad).
Data are expressed as the mean ± SEM. One-way anova followed by the Bonferroni and Tukey's post hoc test or non-parametric Kruskal–Wallis test or unpaired two-tailed Student's t-test were used to determine the statistical significance. The level of significance was set at P ≤ 0.05.
We studied the effect of Sativex and of the two components, Δ9-THC-BDS and CBD-BDS, individually in the TMEV-IDD model of murine primary progressive MS. To this end, 70 days after TMEV infection, mice were treated for 10 consecutive days with Sativex (10mg·kg−1: 5mg·kg−1 Δ9-THC-BDS and 5mg·kg−1 CBD-BDS), with CBD-BDS (5mg·kg−1) or with Δ9-THC-BDS (5mg·kg−1) or an equivalent amount of the vehicle alone and their motor activity was then assessed in an activity cage. As expected, TMEV infection dramatically reduced both horizontal and vertical activity in the chronic phase of the model, whereas Sativex treatment abolished these motor deficits (Figure1A). The same degree of motor improvement was seen after the treatment with CBD-BDS alone, whereas Δ9-THC-BDS provoked a weaker improvement (Figure1B). These data indicate that Δ9-THC-BDS and CBD-BDS, used independently or in combination (in a Sativex-like mixture), restored motor function in TMEV-infected mice, although a greater influence in the effect of Sativex can be attributed to CBD-BDS. These treatments had no effect on sham animals (data not shown).
The different contribution of each phytocannabinoid to the activity of Sativex may be related to differences in their potential targets. Thus, to determine how the CB1 and CB2 receptors influenced the effects of Δ9-THC-BDS, this phytocannabinoid was co-administered with a selective antagonist for the CB1 (AM251) or CB2 (AM630) receptor. In addition, as CBD appears to act through PPARγ (Esposito etal., 2011), and only negligibly through the CB1 and CB2 receptors (Izzo etal., 2009; Mecha etal., 2013b), CBD-BDS was also administered along with the PPARγ antagonist, T0070907. Our data indicated that the positive effect of Δ9-THC-BDS on motor deterioration was significantly blocked by the CB1 receptor antagonist AM251 and only partially by the CB2 receptor antagonist AM630 (Figure2A). Similarly, the beneficial effect of CBD-BDS was significantly attenuated by the PPARγ antagonist (Figure2B). These data suggest the involvement of CB1 receptors and, to a lesser extent, of CB2 receptors in ameliorating the effect of motor disturbances in TMEV-IDD mice provoked by Δ9-THC-BDS and an equivalent role of PPARγ in the effects of CBD-BDS. Moreover, we saw that Sativex treatment tended to up-regulate the gene expression of CB1 and CB2 receptors and of PPARγ in TMEV animals (data not shown). Accordingly, these three targets might be involved in the action of Sativex on TMEV-IDD consistent with the idea of a broad-spectrum effect of this combination of phytocannabinoids.
H&E staining revealed that infection with TMEV provoked the infiltration of immune cells into the spinal cord (Figure3A). Treatment with Sativex partly diminished this infiltration (Figure3B) and specifically decreased the accumulation of CD4 and CD8 lymphocytes into the parenchyma (Figure3C, ,D).D). Sativex also reduced the expression of VCAM-1 (Figure3E) and ICAM-1 (Figure3F), both involved in peripheral immune cell transmigration. These results suggest that Sativex may restrict the permissiveness of immune cell infiltration into the CNS parenchyma. Indeed, no infiltrates were detected in sham-operated mice treated with Sativex (data not shown).
Microglial/macrophage activation plays a critical role in TMEV-IDD and thus, we analysed the effect of Sativex on Iba-1 immunoreactivity in the spinal cord of TMEV-infected mice. As expected, Iba-1 staining revealed increased microglial reactivity in the spinal cord at chronic phases of the disease following TMEV infection. Sativex administration significantly reduced the intensity of this microglial labelling (Figure4A, ,B).B). Moreover, no microglia reactivity was detected in Sham mice administered Sativex (data not shown).
The reduction of microglia reactivity was accompanied by a significant reduction of IL-1β mRNA levels (Figure4C) and a tendency to reduce TNF-α and IFN-γ (Figure4D, ,E).E). Sativex treatment also up-regulated the expression of Arg-1 and IL-10 mRNA showed a tendency to increase (Figure4F, ,G).G). These data suggest that Sativex could act as an immunomodulator, not only limiting the inflammation in TMEV-IDD mice but also promoting an anti-inflammatory environment. No effect on these genes was observed in Sham animals treated with the Sativex (data not shown).
Demyelinated areas that are evident upon LFB staining can be observed in TMEV-IDD infected mice at chronic phases of the disease. However, demyelination was diminished in TMEV-infected mice treated with Sativex as it preserved the morphology of the myelin sheaths in these infected mice (Figure5A). To further assess the neuroprotective potential of Sativex, we evaluated axonal damage in TMEV-infected mice, as reflected by Neurofilament-H and SMI32 staining. Axonal damage in the white matter tracts was clearly evident in the TMEV-infected animals that received the vehicle alone, yet it was absent in the Sham mice. Treatment with Sativex restored Neurofilament-H staining (Figure5B), reflecting the preservation of the axonal package (Figure5C). Similarly, the axonal swelling evident when longitudinal spinal cord sections of TMEV-infected animals that received the vehicle alone were stained with Neurofilament-H (Figure6A) was less frequent and milder following Sativex administration, as confirmed in longitudinal spinal cord sections labelled with SMI32 (Figure6B).
To address whether the treatment with Sativex affected astrogliosis and the expression of CSPGs in TMEV-IDD at the chronic phases of the disease, we analysed its effects on astroglial reactivity using GFAP and vimentin staining as an indicator of reactive astrocytes (Nash etal., 2011). Likewise, the accumulation of CSPGs was studied by staining with CS56 and analysing the brevican gene expression in the spinal cord. There was prominent astrogliosis [Figure7: GFAP (A,B) and vimentin (C) staining] associated with the accumulation of CSPGs (CS56) in the spinal cord of vehicle-treated TMEV-infected mice (Figure7D). However, treatment with Sativex reduced both astrogliosis (Figure7B) CS56 staining (Figure7D) and brevican gene expression (Figure7E) in TMEV-infected mice, suggesting that it may modulate glial scar formation.
As astrocytes are the main source of CSPGs (Susarla etal., 2011), we assessed the capability of Sativex to alter the production of CSPGs by astrocytes in culture. Exposure to the Sativex not only reduced the mRNA expression of brevican and XT-I in activated astrocytes (TGFβ1 and bFGF) after 24h (Figure8A), but it also reduced the neurocan protein staining at an intracellular and extracellular level in astrocytes by immunocytochemistry (Figure8B). Most importantly, Sativex diminished the release of neurocan by activated astrocytes when assessed at 72h (Figure8C, ,D).D). An overall inhibitory effect of Sativex on CSPG production by astrocytes occurred at a dose of 0.50μM. However, a dose of 100nM (50nM for each phytocannabinoid) also achieved a significant reduction in the expression of brevican mRNA and the dose of 1.0μM (0.5μM for each phytocannabinoid) diminished the expression of XT-I mRNA within 24h. Together, these data suggest that astrocytes could represent a new target for Sativex activity, modulating the production of CSPG.
Despite intense research, there are still no treatments available to achieve clinical modification of progressive MS. The approval of Sativex for treatment of spasticity and neuropathic pain in MS opens the door to study the therapeutic potential of Sativex as a disease-modifying agent. Accordingly, we have assessed the efficacy of Sativex in controlling disease progression in a model of primary progressive MS, the TMEV-IDD model (Mecha etal., 2013a).
There is a wealth of data from clinical trials that support the efficacy and safety of Sativex in patients with MS-related spasticity (Collin etal., 2007; Wade etal., 2010; Sastre-Garriga etal., 2011; García-Merino, 2013). However, to date, there are no clinical studies evaluating Sativex as a disease-modifying agent of progressive MS. The closest clinical study is the CUPID trial, the results of which were recently published (Zajicek etal., 2013). This study showed that dronabinol (Δ9-THC) administration to patients with progressive MS did not slow the course of the disease. However, the failure to detect such an effect may reflect the high attrition rate and the lower rate of disease progression than initially expected.
The study performed here is the first to address the effect of Sativex on primary progressive MS in a recognized experimental model, providing highly promising results in terms of reduced axonal damage. Indeed, this result is consistent with the proposed neuroprotective profile of Sativex and its components (Valdeolivas etal., 2012; Moreno-Martet etal., 2014). When administered alone, Δ9-THC or CBD has positive effects on disease progression in EAE, CREAE and TMEV-IDD models. A clinical and histological improvement provoked by Δ9-THC, predominantly reducing inflammation, was first described in EAE rats (Lyman etal., 1989). Here, we also found that Δ9-THC-BDS ameliorated the progression of disease in TMEV-IDD mice, with a major participation of CB1 receptors, even though blocking CB2 receptors partly prevented the benefits elicited by this phytocannabinoid. The activation of CB1 receptors probably mediates the neuroprotective effects of Δ9-THC as this receptor fulfils an important role in the defence against excitotoxicity (Marsicano etal., 2003) and excitotoxic mechanisms are prominent in TMEV-IDD (Docagne etal., 2007). Nevertheless, the participation of CB2 receptors is not unexpected given their key involvement in the control of glial activation and given that inflammatory events are also important in TMEV-IDD mice (Arévalo-Martín etal., 2003; 2008). Indeed, it was previously demonstrated that Δ9-THC suppressed CNS autoimmune inflammation with the participation of both CB1 and CB2 receptors in CREAE mice (Maresz etal., 2007).
With regard to CBD, the other component of Sativex, this phytocannabinoid provokes a reduction in disease severity in both autoimmune (EAE mice) and viral (TMEV-IDD mice) models of MS, as well as in a murine collagen-induced arthritis model (Malfait etal., 2000; Kozela etal., 2011; Mecha etal., 2013b). The attenuation of disease progression in MS models involved the reduction of immune cell infiltrates and the decrease in microglial activation following systemic CBD treatment (5mg·kg−1), the same dose of CBD-BDS used in this study. These findings contrast with the earlier failure to find an effect of CBD in the CREAE model of MS (Maresz etal., 2007), which might reflect differences in the strain of mice and/or in the antigen used. The influence of genetic background on EAE in mice deficient in some key endocannabinoid genes has recently been shown (Sisay etal., 2013). It should be noted that CBD is not particularly active at classic CB1 and CB2 receptors and, while it appears to be able to inhibit the inactivation of endocannabinoids (Bisogno etal., 2001), its mechanisms of action are quite diverse and extend to targets beyond the cannabinoid system (see Fernández-Ruiz etal., 2013). One of these targets includes the nuclear receptors of the PPAR family, in particular PPARγ (O'Sullivan and Kendall, 2010; Esposito etal., 2011). Here, we found that the inhibition of PPARγ completely blocked the motor benefits of CDB-BDS, clearly indicating the participation of these nuclear receptors in the effects of CBD-BDS on TMEV-IDD and presumably when it is combined with Δ9-THC-BDS in Sativex. This is consistent with studies showing that activation of PPARγ by selective agonists ameliorated MS symptomatology (Feinstein etal., 2002; Klotz etal., 2009; Mestre etal., 2009).
In accordance with the diminished axonal damage, we found reduced astrogliosis and CSPGs accumulation during the late chronic phase of TMEV-IDD. It is well known that CPGSs are up-regulated in demyelinated MS lesions (Haylock-Jacobs etal., 2011; Haist etal., 2012). In particular, astrocytes at the edge of active MS lesions up-regulate CSPGs such as versican, aggrecan and neurocan (Haylock-Jacobs etal., 2011; Haist etal., 2012; Lau etal., 2012). Significantly, there are several in vitro studies indicating that neurocan and aggrecan impair the outgrowth of oligodendrocyte process, OPC differentiation and myelination (Siebert and Osterhout, 2011; Pendleton etal., 2013). Here, the enhanced expression of CSPGs in astrocytes following exposure to TGFβ (Gris etal., 2007; Susarla etal., 2011) was down-regulated by Sativex administration. We focused on neurocan and brevican, core proteins that carry inhibitory glycosaminoglycan (GAG) chains. CSPG side chain synthesis is initiated by the addition of xylose to a serine moiety of the core protein, a step carried out by XT-I, among other enzymes (Gris etal., 2007). Significantly, the up-regulation of XT-I expression in stimulated astrocytes was dampened by Sativex, which could impair CSPG synthesis and thereby provide a permissive environment for regeneration in accordance with previous studies in which XT-I targeting promotes neurite outgrowth (Hurtado etal., 2008).
The inhibitory effect of Sativex in the production of brevican and neurocan as well as in the synthesis enzyme XT-I by activated astrocytes confirm our in vivo results and support the interest of Sativex as a putative therapy in promoting repair myelin processes by reducing astroglial scar. Further work is necessary to delineate the effects of Sativex-like combination of phytocannabinoids on remyelination in experimental models of MS.
In conclusion, Sativex improved the neurological deficits evident at the chronic phases of the TMEV-IDD model of MS. These effects were fully reproduced by CBD-BDS alone, acting through PPARγ and, to a lesser extent, by Δ9-THC-BDS, acting through CB2 and primarily CB1 receptors. One potential explanation for the beneficial effects of Sativex could be the reduced transmigration of cell infiltrates into the parenchyma. Sativex also acted as an immunomodulator, decreasing microglial reactivity, the expression of proinflammatory cytokine genes and increasing levels of Arg-1 and IL-10. Moreover, Sativex diminished axonal damage and restored myelin morphology, again supporting its cytoprotective potential. Finally, we postulate that Sativex could modulate glial scar formation, reducing astrogliosis and the accumulation of CSPGs in vivo, as well as in astrocyte cultures. As CSPGs associated with inflammation have been implicated in the failure of remyelination in human MS and in murine models of demyelination, our results highlight the potential benefits of Sativex in CNS repair.
This work was supported by grants from the Ministry of the Economy and Competition SAF2010-17501 (C. G.) and SAF2009-11847 (J. F.-R.), the Comunidad de Madrid, S2011/BMD-2308, the Red Española de Esclerosis Múltiple RD12/0032/0008 (C. G.) sponsored by the Fondo de Investigación Sanitaria. GW Pharmaceuticals Ltd provided the combination of phytocannabinoids. None of the funding bodies played any role in the study design, data collection and analysis, the decision to publish or the preparation of the manuscript.
A. F. carried out the TMEV-IDD model, prepared the astrocytes cultures, determined the ECM proteins and analysed the data. M. M. performed the qRT-PCR assays. M. M.-M. was involved in motor tests. F. J. C.-S. was involved in perfusion and sections staining. M. M.-M., E. L., J. F.-R. and C. G. participated in the experimental design. A. F. and C. G. wrote the manuscript.
The authors have formal links with GW Pharmaceuticals that fund some of their research.