VCE-003.2, a novel cannabigerol derivative, enhances neuronal progenitor cell survival and alleviates symptomatology in murine models of Huntington’s disease

Abstract

Cannabinoids have shown to exert neuroprotective actions in animal models by acting at different targets including canonical cannabinoid receptors and PPARγ. We previously showed that VCE-003, a cannabigerol (CBG) quinone derivative, is a novel neuroprotective and anti-inflammatory cannabinoid acting through PPARγ. We have now generated a non-thiophilic VCE-003 derivative named VCE-003.2 that preserves the ability to activate PPARγ and analyzed its neuroprotective activity. This compound exerted a prosurvival action in progenitor cells during neuronal differentiation, which was prevented by a PPARγ antagonist, without affecting neural progenitor cell proliferation. In addition, VCE-003.2 attenuated quinolinic acid (QA)-induced cell death and caspase-3 activation and also reduced mutant huntingtin aggregates in striatal cells. The neuroprotective profile of VCE-003.2 was analyzed using in vivo models of striatal neurodegeneration induced by QA and 3-nitropropionic acid (3NP) administration. VCE-003.2 prevented medium spiny DARPP32+ neuronal loss in these Huntington’s-like disease mice models improving motor deficits, reactive astrogliosis and microglial activation. In the 3NP model VCE-003.2 inhibited the upregulation of proinflammatory markers and improved antioxidant defenses in the brain. These data lead us to consider VCE-003.2 to have high potential for the treatment of Huntington’s disease (HD) and other neurodegenerative diseases with neuroinflammatory traits.

Introduction

Cannabinoids the main active compounds of marijuana (Cannabis sativa) and its endogenous counterparts anandamide and 2-arachidonoyl glycerol have attracted the interest of the scientific community in the last decade owing to their prominent effects on neurodegenerative and neuroinflammatory conditions1. Cannabinoids exert neuroprotective actions in various experimental models of neurodegenerative diseases and most of their effects are mediated via the presynaptic CB1 receptor. CB1 receptor levels notably diminish at early stages of Huntington’s disease (HD) prior to the characteristic atrophy and neurodegeneration of medium spiny neurons (MSNs)2,3. In addition, the activation of CB1 receptor by Δ9-tetrahydrocannabinol (THC), the most abundant psychoactive compound of Cannabis sativa, exerts a disease attenuating action diminishing MSN neuronal loss4. Moreover, CB1 receptor signaling promotes neural progenitor cell proliferation and regulates neural cell differentiation5. Unfortunately the clinical implications for the neuroprotective cannabinoid actions are hampered by the psychoactive consequences of CB1 receptor signaling, which is the most abundantly expressed G-protein coupled receptor in the CNS. Therefore CB1 receptor targeting compounds exert undesired psychoactive actions by altering neurotransmitter release.

In addition to THC, more than 100 plant-derived compounds are known to exist and for most of them their pharmacological profile is still far to be understood6. Among them, cannabidiol (CBD) and cannabigerol (CBG) constitute non-psychoactive cannabinoid compounds that do not bind to CB1 receptors and have also been tested as potential candidates for pharmacological therapies in HD patients and experimental models7,8. CBD alone or combined with THC (1:1), as formulated in sublingual spray Sativex (GW Pharmaceuticals) prevents neurodegeneration in animal models of toxin-induced striatal neurodegeneration8. These results prompted the development of a recent clinical trial to assess the safety of Sativex administration in HD patients (ClinicalTrials.gov, NCT01502046) that evidenced the lack of negative consequences of chronic Sativex administration9. More recently, we have found that CBG activated PPARγ in striatal cells thereby alleviating symptomatology and neuroinflammation in murine models of HD7.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor implicated in the regulation of lipid metabolism and glucose homeostasis and it is the target for glitazones, a class of oral antidiabetic drugs10. However, PPARγ is broadly expressed and has been recognized to play a key role in inflammatory processes and neurodegenerative diseases. In this sense, it has been shown that thiazolidinediones (TZDs) are neuroprotective in mutant huntingtin (mHtt) expressing cells and reduce mHtt aggregates in the brain11,12,13, thus supporting the concept that PPARγ may be a valid target for the management of HD. In addition, targeting PPARγ owing to its regulatory role of neural progenitor cell proliferation and differentiation14 constitutes a promising candidate to promote neural repair in neurodegenerative conditions.

TZDs, including the widely used drug rosiglitazone (RZG), are strong activators of PPARγ and have come under scrutiny because of their clinical side effects such as weigh gain, fluid retention, osteoporosis and other disorders15. However, more subtle modulation of PPARγ by small molecules such as cannabinoids may provide anti-inflammatory and neuroprotective activities resulting in more favourable outcomes.

In a step forward to improve the pharmacological profile and efficacy of natural cannabinoids different chemical modifications have been introduced and evaluated. For instance, quinone derivatives such as HU-331 and VCE-003 have been developed from CBD and CBG respectively16,17. HU-331 exerts a potent antitumoral activity by targeting DNA polymerase II, while VCE-003 has been identified as an immunosuppressant that exerts a protective action in models of multiple sclerosis (the Theiler’s murine encephalomyelitis virus and experimental autoimmune encephalomyelitis) through a PPARγ dependent pathway16,18. However, VCE-003 is a potential electrophilic compound that may complicate its development for chronic treatments. In this report we have developed a second generation of cannabigerol quinone derivatives such as VCE-003.2, which retain the ability to bind and activate PPARγ. VCE-003.2 is a non thiol-reactive compound with low adipogenic activity that shows neuroprotective and antiinflammatory activities in HD models in vivo and in vitro.

Results

VCE-003.2 is a non-thiol trapping derivative of CBG

We have previously shown that CBG oxidation to quinol (VCE-003) increases the PPARγ binding activity of this natural cannabinoid16. VCE-003 is also a potent immunosuppressor, which may be due to the electrophilic (thiol-trapping) nature of this quinone derivative18. In order to dissect the potential thiol-trapping activity of VCE-003 from the rest of its biological profile, the compound was subjected to C-H functionalization by the introduction of a nitrogen function as in VCE-003.2 (Fig. 1a). The Michael reactivity of VCE-003 and its analogue VCE-003.2 was investigated using a cysteamine recovery assay inspired by the cysteamine-trapping assay, an NMR method based on the treatment of a thiophylic compound with cysteamine in DMSO19. We found that VCE-003.2 can be recovered unscathed while in contrast VCE-003 was undetectable in the residue, indicating that it had irreversibly formed polar and not extractable adducts with cysteamine (Supplementary information). In addition, we found that VCE-003 induced reactive oxygen species (ROS) and activated the Nrf2 pathway, whereas none of these bioactivities were induced by VCE-003.2 (see Supplementary Fig. S1)

https://www.nature.com/articles/srep29789

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