Neuroprotection with the cannabigerol quinone derivative VCE-003.2 and its analogs CBGA-Q and CBGA-Q-Salt in Parkinson's disease using 6-hydroxydopamine-lesioned mice

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Highlights

  • The CBG derivative VCE-003.2 afforded neuroprotection in 6-OHDA-lesioned mice.

  • Additional derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection, but their effects were lower.

  • In vitro studies confirmed the relevance of PPAR-γ receptors for all these effects.

Abstract

The quinone derivative of the non-psychotropic cannabinoid cannabigerol (CBG), so-called VCE-003.2, has been recently investigated for its neuroprotective properties in inflammatory models of Parkinson's disease (PD) in mice. Such potential derives from its activity at the peroxisome proliferator-activated receptor-γ (PPAR-γ). In the present study, we investigated the neuroprotective properties of VCE-003.2 against the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA), in comparison with two new CBG-related derivatives, the cannabigerolic acid quinone (CBGA-Q) and its sodium salt CBGA-Q-Salt, which, similarly to VCE-003.2, were found to be active at the PPAR-γ receptor, but not at the cannabinoid CB1 and CB2 receptors. First, we investigated their cytoprotective properties in vitro by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-003.2 at a concentration of 20 μM, which was not reversed by the blockade of PPAR-γ receptors with GW9662, supporting its activity at an alternative site (non-sensitive to classic antagonists) in this receptor. We also found CBGA-Q and CBGA-Q-Salt being cytoprotective in this cell assay, but their effects were completely eliminated by GW9662, thus indicating that they are active at the canonical site in the PPAR-γ receptor. Then, we moved to in vivo testing using mice unilaterally lesioned with 6-OHDA. Our data confirmed that VCE-003.2 administered orally (20 mg/kg) preserved tyrosine hydroxylase (TH)-positive nigral neurons against 6-OHDA-induced damage, whereas it completely attenuated the astroglial (GFAP) and microglial (CD68) reactivity found in the substantia nigra of lesioned mice. Such neuroprotective effects caused an important recovery in the motor deficiencies displayed by 6-OHDA-lesioned mice in the pole test and the cylinder rearing test. We also investigated CBGA-Q, given orally (20 mg/kg) or intraperitoneally (10 mg/kg, i.p.), having similar benefits compared to VCE-003.2 against the loss of TH-positive nigral neurons, glial reactivity and motor defects caused by 6-OHDA. Lastly, the sodium salt of CBGA-Q, given orally (40 mg/kg) to 6-OHDA-lesioned mice, also showed benefits at behavioral and histopathological levels, but to a lower extent compared to the other two compounds. In contrast, when given i.p., CBGA-Q-Salt (10 mg/kg) was poorly active. We also analyzed the concentrations of dopamine and its metabolite DOPAC in the striatum of 6-OHDA-lesioned mice after the treatment with the different compounds, but recovery in the contents of both dopamine and DOPAC was only found after the treatment with VCE-003.2. In summary, our data confirmed the neuroprotective potential of VCE-003.2 in 6-OHDA-lesioned mice, which adds to its previous activity found in an inflammatory model of PD (LPS-lesioned mice). Additional phytocannabinoid derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection in 6-OHDA-lesioned mice, but their effects were lower compared to VCE-003.2, in particular in the case of CBGA-Q-Salt. In vitro studies confirmed the relevance of PPAR-γ receptors for these effects.

Introduction

Phytocannabinoids, the active constituents of the cannabis plant, and also endocannabinoids and synthetic cannabinoids have been proposed as promising neuroprotective agents, a property derived from their pleiotropism and ability to activate numerous pharmacological targets within the so-called endocannabinoid system, but also outside this signaling system (reviewed in Fernández-Ruiz, 2019). Such neuroprotective potential is exerted by the combination of different cannabinoid capabilities, in particular the inhibition of several insults that damage neurons, for example excitotoxicity, oxidative stress and glial reactivity/inflammatory events (reviewed in Aymerich et al., 2018), but also the promotion of prosurvival events such as the elimination of protein aggregates (reviewed in Costa et al., 2016) and the replacement of neurons and other neural cells (reviewed in Oddi et al., 2020). All those events are critical in the pathogenesis of those CNS disorders involving neuronal deterioration and death (reviewed in Fernández-Ruiz et al., 2015; Paloczi et al., 2018; Aymerich et al., 2018). These activities enable cannabinoids to serve as neuroprotective agents in accidental brain damage, e.g., stroke (Kolb et al., 2019), brain trauma (Shohami et al., 2011), spinal injury (Arevalo-Martin et al., 2016), but also in chronic progressive disorders such as Alzheimer's disease (Maroof et al., 2013), amyotrophic lateral sclerosis (Giacoppo and Mazzon, 2016), Huntington's disease (Sagredo et al., 2012), multiple sclerosis (Mecha et al., 2020), cerebellar ataxias (Gómez-Ruiz et al., 2019), prion diseases (Iuvone et al., 2009), and Parkinson's disease (PD) (Junior et al., 2020).

PD is one of the disorders that has attracted most research with cannabinoids to date, with the objective to generate new therapies based on these compounds aimed at alleviating specific parkinsonian symptoms and/or at delaying disease progression (reviewed in Fernández-Ruiz et al., 2015; Aymerich et al., 2018; Antonazzo et al., 2019; Baul et al., 2019). For example, cannabinoids targeting the cannabinoid receptor type-1 (CB1) have been found to reduce, mainly in preclinical studies, parkinsonian signs such as bradykinesia and immobility (Fernández-Espejo et al., 2005; González et al., 2006; Kelsey et al., 2009), tremor (Sañudo-Peña et al., 1999) and/or L-DOPA-induced dyskinesia (Junior et al., 2020). Targeting the CB1 receptor has been also proposed as neuroprotective therapy (Iuvone et al., 2007; Chung et al., 2011; Pérez-Rial et al., 2011; Nguyen et al., 2016), but most of cannabinoids proposed as disease modifiers work: (i) against inflammatory events by targeting the cannabinoid receptor type-2 (CB2) (Price et al., 2009; García et al., 2011; Javed et al., 2016; Gómez-Gálvez et al., 2016; Chung et al., 2016; Aymerich et al., 2016; Wang et al., 2018), but also the peroxisome proliferator-activated receptor-γ (PPAR-γ) (Carroll et al., 2012; García et al., 2018; Burgaz et al., 2019) and even an orphan receptor, GPR55, which has been recently associated with the endocannabinoid system (Celorrio et al., 2017); and (ii) against oxidative injury based on cannabinoid receptor-independent effects (Lastres-Becker et al., 2005; Jimenez-Del-Rio et al., 2008) and possibly on their proposed ability to modulate the signaling of the antioxidant transcription factor Nrf-2 (Gugliandolo et al., 2018).

Based on this previous experimental evidence, a family of cannabinoid analogs that have been recently investigated for their therapeutic profile for PD are the quinone derivatives of cannabigerol (CBG) (García et al., 2018; Burgaz et al., 2019). The lead compound in this series is VCE-003.2, which behaves as a PPAR-γ activator (Díaz-Alonso et al., 2016), while retaining the lack of CB1/CB2 activity of the CBG template (Granja et al., 2012; Carrillo-Salinas et al., 2014). We have been particularly interested in exploring the potential of this non-thiophilic CBG quinone derivative against inflammation-driven neuronal deterioration in LPS-lesioned mice (García et al., 2018; Burgaz et al., 2019), the experimental model of PD that better reproduces inflammation as a pathogenic event in this disease. VCE-003.2 worked as an anti-inflammatory and neuroprotective agent in this in vivo PD model after both i.p. (García et al., 2018) and oral (Burgaz et al., 2019) administration, and its effects were confirmed to be mediated by the activation of the PPAR-γ receptor (García et al., 2018). We also demonstrated its anti-inflammatory profile in cultured BV2 cells stimulated with LPS, as well as its neuroprotective effects in cultured M-213 neuronal cells incubated with conditioned media generated from cultured BV2 cells stimulated with LPS (García et al., 2018). In these in vitro studies (García et al., 2018), we observed that PPAR-γ-mediated effects of VCE-003.2 implied its binding to a functional alternative binding site different from the canonical binding site used by glitazones (Hughes et al., 2014). In the same study (García et al., 2018), VCE-003.2 was also found to apparently serve as neuroprotectant after i.p. administration in 6-OHDA-lesioned mice, a model of PD characterized by mitochondrial dysfunction and oxidative stress, but having a more modest inflammatory response. However, the results of this experiment were preliminary and needed further replication. This is one of the objectives of our present study, focusing on the neuroprotective properties of VCE-003.2, but using 6-OHDA-lesioned mice instead of LPS-lesioned animals, as well as using an oral administration instead of the i.p. route used in the previous study (García et al., 2018). We also investigated new compounds, CBGA-Q and a water-soluble CBGA-Q-Salt, which should be active at the PPAR-γ too. The inclusion of these additional compounds had the objective to determine whether, compared to VCE-003.2, they could have advantages, for example being more potent than VCE-003.2 at the PPAR-γ receptor, or having additional activities at the CB1/CB2 receptors which are also useful in PD. Even, in the case of CBGA-Q-Salt, which is a more water soluble derivative, whether this may facilitate its administration and pharmacokinetics resulting in a better bioavailability. Another potential advantage for the two new derivatives is the synthesis method, as VCE-003.2 requires two-step synthesis and CBGA-Q only one step.

Section snippets

Chemical synthesis of the different compounds investigated

The aminoquinone derivative of CBG, 6-(3,7-dimethyl-octa-2,6-dienyl)-5-hydroxy-3-pentyl-2-ethylamino-[1,4]benzoquinone, so-called VCE-003.2, was synthesized as described previously (Díaz-Alonso et al., 2016). In this study, we also included two new compounds, CBGA-Q and CBGA-Q-Salt (see chemical structures in Fig. 1).

For the synthesis of CBGA-Q, cannabigerol acid ((Z)-3-(3,7-dimethylocta-2,6-dienyl)-2,4-dihydroxy-6-pentylbenzoic acid) (0.995 g; 2.76 mmol) was dissolved in THF (10 mL) at 20 °C

Determination of PPAR-γ/CB1/CB2 receptor binding for CBGA-Q and CBGA-Q-Salt

One important objective of this study was to generate novel analogs of VCE-003.2 with possibly a better pharmacological profile. Thus, we synthesized the compounds CBGA-Q and CBGA-Q-Salt. CBGA-Q is derived from CBGA in a one-step synthesis and CBGA-Q-Salt is a water-soluble sodium salt CBGA-Q derivative. Both derivatives were first analyzed by their activity at the PPAR-γ receptor showing similar binding and transcriptional activities compared to VCE-003.2 (Table 1). We also conducted

Discussion

Our current study is an extension of previous reports aimed at exploring the neuroprotective potential of the non-thiophilic CBG quinone derivative VCE-003.2 in experimental models of PD (García et al., 2018; Burgaz et al., 2019). The novelties of this follow-up study are: (i) the use of the 6-OHDA model, which was explored before only in a pilot study (García et al., 2018, Supplementary data); (ii) the use of an oral route for administration, which may facilitate the clinical development of

Declaration of competing interest

The authors declare that they have not any conflict of interest in relation with this study.

Acknowledgements

This work has been supported by grants from CIBERNED (CB06/05/0089), MINECO-Biomedicina (SAF2015-68580-C2-1-R), MINECO Retos-Colaboración (RTC-2014-1877-1) and MICIU-Biomedicina (RTI2018-098885-B-100). Part of the study was also funded by Emerald Health Pharmaceuticals (San Diego, USA). 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. Sonia Burgaz and

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