Sativex-like Combination of Phytocannabinoids is Neuroprotective in Malonate-Lesioned Rats, an Inflammatory Model of Huntington's Disease: Role of CB1 and CB2 Receptors

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by motor abnormalities, cognitive dysfunction, and psychiatric symptoms.1 The primary cause of the disease is a mutation in the huntingtin gene consisting of a CAG triplet repeat expansion translated into an abnormal polyglutamine tract in the amino-terminal portion of this protein that becomes toxic for striatal and cortical neuronal subpopulations.2 At present, there is no specific pharmacotherapy to alleviate motor and cognitive symptoms and/or to arrest/delay disease progression in HD. Thus, even though a few compounds have produced encouraging effects in preclinical studies (i.e., minocycline, coenzyme Q10, unsaturated fatty acids, inhibitors of histone deacetylases) none of the findings obtained in these studies have yet led on to the development of an effective medicine.3 Importantly, therefore, following on from an extensive preclinical evaluation using different experimental models of HD, clinical tests are now being performed with cannabinoids. The preclinical studies with cannabinoids demonstrated preservation of striatal neurons by several agonists against different cytotoxic stimuli that operate in HD pathogenesis,4,5 effects that were exerted through multiple mechanisms of action, some of which involve the activation of CB1 and/or CB2 receptors and others of which do not. For example, cannabinoids with antioxidant profile, that is, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), protected striatal neurons against toxicity caused by the mitochondrial complex II inhibitor 3-nitropropionic acid (3NP) that primes oxidative injury.6,7 However, the activity of Δ9-THC at the CB1 and CB2 receptors enables this phytocannabinoid to induce neuroprotection in other experimental models, for example, the transgenic mouse model of HD, R6/2, in which the effects of Δ9-THC are likely produced through the activation of CB1 receptors8 and possibly through the activation of CB2 receptors too, as selective agonists of this receptor type preserved striatal neurons in this genetic model9 and also in malonate-lesioned rats,10 a model priming pro-inflammatory events. Selective agonists of CB18,11 and CB29 receptors also preserved striatal neurons in in vitro or in vivo excitotoxic models. By contrast, other studies showed no effects in R6/1 mice using Δ9-THC, the synthetic agonist HU-210 and the inhibitor of the endocannabinoid metabolism URB597.12 All these data suggest that the evaluation of cannabinoids as disease-modifying agents in patients should be necessarily conducted with a broad-spectrum cannabinoid or with combinations of various cannabinoids with different and complementary pharmacological profiles. Sativex, a cannabis-based medicine recently licensed for the treatment of spasticity and pain in multiple sclerosis patients,13,14 has an appropriate profile for HD as it can activate CB1 and CB2 receptors due to the presence of Δ9-THC, but it can also exert cannabinoid receptor-independent antioxidant properties due to Δ9-THC and, in particular, to CBD.15 We recently initiated some experiments with the combination of Δ9-THC and CBD botanical extracts present in Sativex in those animal models of HD, in which individual cannabinoid agonists have proved to be effective,6−11 with the objective of determining whether this mixture also works in these models. We have just published the first data obtained in rats subjected to 3NP intoxication,16 a model priming, as mentioned above, calpain activation and oxidative injury as major cytotoxic mechanisms, and in which pure Δ9-THC6 and CBD7 administered separately, have already been found to display neuroprotective properties. The Sativex-like combination of phytocannabinoids also preserved striatal neurons from death caused by 3NP intoxication, and this effect was, as expected, independent of CB1 and CB2 receptors.16 These findings prompted us to extend our research to rats lesioned with malonate, an acute model of HD in which striatal damage is produced primarily by apoptosis and glial activation/inflammatory events and in which selective CB2 receptor agonists have been shown to be effective.10 To this end, we lesioned rats with an intrastriatal injection of malonate and used these animals for two different sets of experiments. First, we examined the neuroprotective effects of a 1:1 combination of botanical extracts of Δ9-THC and CBD, a combination that approximates to the 1.0:0.93 mixture of these extracts that is present in Sativex. The level of neuroprotection was evaluated by measuring the following parameters: (i) the volume of edema measured by in vivo NMR imaging; (ii) the number of Nissl- and FluoroJade B-stained cells which correlate with the number of surviving and degenerating cells, respectively, in the striatal parenchyma; (iii) the presence of reactive microgliosis, by using Iba-1 immunohistochemistry, and astrogliosis labeled with GFAP immunostaining; and (iv) the expression of various biochemical markers that have been found previously to be altered in this and other HD models, i.e. inducible nitric oxide synthase (iNOS), the neurotrophin IGF-1 and the CB1 receptor.8,10,16,17 In the second set of experiments, we explored whether the neuroprotective effects observed in this model with the combination of Δ9-THC and CBD present in Sativex involved the activation of CB1 receptors, CB2 receptors, or both, using selective antagonists for these two receptors (SR141716 and AM630, respectively). For this second set of experiments, we monitored the number of Nissl-stained cells, since this constitutes a very selective and sensitive marker of malonate damage in the striatal parenchyma.