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

Abstract

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

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Acknowledgments

The authors thank the support of the National MS Society (USA) and the MS Society (UK). We thank Prof. John Zajicek and Susan Ball, Plymouth, UK for providing access to data from the CUPID trial.

Conflicts of Interest

None.

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Correspondence to David Baker.

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Materials and Methods

Induction of autoimmune experimental encephalomyelitis protocols consistent with the ARRIVE guidelines have been published previously (Al-Izki et al. 2012; Baker and Amor 2012). Animal studies were approved following local ethical and United Kingdom Government, Home Office review in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Full working protocols of the methods and doses and use of cannabinoids in animals have been reported previously (Pryce et al. 2003; Croxford et al. 2008; Al-Izki et al. 2012). Briefly, 6–8 week adult Biozzi ABH mice (Al-Izki et al. 2012) were injected with spinal cord homogenate in Freunds adjuvant on day 0 and 7 to induce experimental autoimmune encephalomyelitis with onset around day 15–19 post-inoculation (p.i.) and again during remission from paralytic attack on day 28 p.i. to induce a relapse 7–8 days later (Al-Izki et al. 2012). Animals were scored daily: 0 = normal; 1 = limp tail; 2 = impaired righting reflex; 3 = hindlimb paresis; 4 = hindlimb paralysis; 5 = moribund. Scores were assessed using Mann Whitney U statistics (Al-Izki et al. 2012). The motor co-ordination was assessed on an accelerating (0–40 rpm/5 min) rotorod and analysed using Students t test following normality and equal variance tests (Al-Izki et al.2012). Synthetic Δ9-THC and CBD were purchased from Δ9-THC Pharm GmbH. Frankfurt, Germany and were diluted in alcohol:cremophor:phosphate buffered saline (1:1:18). Various doses injected in 0.1 ml intraperitioneally (i.p). as described previously (Pryce et al. 2003; Croxford et al. 2008). These were administered shortly before anticipated relapse (Al-Izki et al. 2012, 2014).

Veratrine induced flux of [ 14 C]guanidine in synaptosomes has been reported previously (Pauwels et al. 1986; Garthwaite et al. 2002). Briefly veratrine (100 μg/ml final concentration), and rat cerebral cortex synaptosomes (4 mg/ml, wet weight) were incubated in the absence or presence of compound at 37 °C for 5 min in polypropylene test tubes. Uptake was initiated by the addition of pre-warmed [14C]-guanidine (final concentration 1 μCi/ml) and stopped 2 min later by the addition of 10 ml of ice-cold wash medium as described previously (Pauwels et al. 1986). Incubates were immediately filtered under vacuum through GF/C filters by using a Brandel harvester. The incubation tubes were rinsed with 5 ml of ice-cold wash buffer, which was then used to wash the filter. Filters were transferred to minivials (Beckman Coulter, Fullerton, CA) with the use of a Brandel deposit/dispense system and subsequently counted by liquid scintillation spectroscopy with Picofluor40 liquid scintillator (Garthwaite et al. 2002). Cannabidiol; YC-1 (5-[1-phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (Cayman, Chem Ann Arbor, Michigan, USA); Nabilone (Cambridge Labs; Newcastle, UK); Lamotrigine (6-(2,3-Dichlorophenyl)-1,2,4-triazine-3,5-diamine. Tocris, Bristol, UK) and Sipatrigine (BW619C89. 2-(4-Methyl-1-piperazinyl)-5-(2,3,5-trichlorophenyl)-4-pyrimidinamine. Tocris Ltd) were diluted with medium from 10 mM stock solutions.

Batrachotoxin-B (BTX-B) Binding. This was performed as described previously (Garthwaite et al. 2002). Binding was initiated by the addition of synaptosomes (final concentration 10 mg/ml, wet weight) to a mixture of test compound and 10 nM [3H]Batrachotoxin-B in the absence or presence of scorpion venom (25 μg/ml final concentration). Samples were mixed and incubated for 90 min at 25 °C. Ice-cold wash medium (5 ml) was added and then the samples subjected to vacuum filtration through GF/C filters by using a Brandel harvester. Incubation tubes were rinsed with 5 ml of ice-cold wash buffer, which was then used to wash the filter. Radioactivity in the filter was counted as described above.

Randomised, double-blind, placebo-controlled trial of Δ9-THC in people with progressive MS has been reported previously (Zajicek et al. 2013), with an International Standard Randomised Controlled Trial number 62942668. Human studies were approved by the South and West Devon Research Ethics Committee and done in accordance with Good Clinical Practice guidelines. Eligible patients provided written informed consent before participation as International Standard Randomised Controlled Trial (ISRCTN 62942668). Briefly, 18–65 year old humans with primary or secondary progressive MS (Expanded disability status scale (EDSS) Score4.0–6.5), not on current disease modifying therapy (DMT), were enrolled into the study. These were randomised to oral dronabinol (Δ9-THC) starting at 3.5 mg twice a day escalated to a maximum of 28 mg/day depending on tolerability (n = 329) or vegetable oil placebo in gelatin capsules (n = 164). These were supplied by Insys Therapeutics (Phoenix, AZ, USA). Analysis of the total population (n = 493) or subgroup analysis on time to progression in those participants with a baseline EDSS score of 5 · 5 or lower (n = 110) was performed using a log-rank test to compare probability of progression between treatment groups (Zajicek et al. 2013).

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Pryce, G., Riddall, D.R., Selwood, D.L. et al. Neuroprotection in Experimental Autoimmune Encephalomyelitis and Progressive Multiple Sclerosis by Cannabis-Based Cannabinoids. J Neuroimmune Pharmacol 10, 281–292 (2015). https://doi.org/10.1007/s11481-014-9575-8

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Keywords

  • Cannabinoid
  • Cannabidiol
  • Experimental autoimmune encephalomyelitis
  • Multiple sclerosis
  • Neuroprotection
  • Δ9-tetrahydrocannabinol