## Beneficial effects of the phytocannabinoid Δ9-THCV in L-DOPA-induced dyskinesia in Parkinson’s disease

Isabel Espadas, Ettel Keifman, Cristina Palomo-Garo Sonia Burgaz, Concepción García, Javier Fernández-Ruiz, Rosario Moratalla

Neurobiology of Disease, 2020, 141, 104892

doi : 10.1016/j.nbd.2020.104892

A B S T R A C T

The antioxidant and CB2 receptor agonist properties of Δ9-tetrahydrocannabivarin (Δ9-THCV) afforded neuroprotection in experimental Parkinson’s disease (PD), whereas its CB1 receptor antagonist profile at doses lower than 5 mg/kg caused anti-hypokinetic effects. In the present study, we investigated the anti dyskinetic potential of Δ9-THCV (administered i.p. at 2 mg/kg for two weeks), which had not been investigated before. This objective was investigated after inducing dyskinesia by repeated administration of L-DOPA (i.p. at 10 mg/kg) in a genetic model of dopaminergic deficiency, Pitx3ak mutant mice, which serves as a useful model for testing anti-dyskinetic agents. The daily treatment of these mice with L-DOPA for two weeks progressively increased the time spent in abnormal involuntary movements (AIMs) and elevated their horizontal and vertical activities (as measured in a computer-aided actimeter), signs that reflected the dyskinetic state of these mice. Interestingly, when combined with L-DOPA from the first injection, Δ9-THCV delayed the appearance of all these signs and decreased their intensity, with a reduction in the levels of FosB protein and the histone pAcH3 (measured by immunohistochemistry), which had previously been found to be elevated in the basal ganglia in L-DOPA-induced dyskinesia. In addition to the anti-dyskinetic effects of Δ9-THCV when administered at the onset of L-DOPA treatment, Δ9-THCV was also effective in attenuating the intensity of dyskinesia when administered for three consecutive days once these signs were already present (two weeks after the onset of L-DOPA treatment). In summary, our data support the anti-dyskinetic potential of Δ9-THCV, both to delay the occurrence and to attenuate the magnitude of dyskinetic signs. Although further studies are clearly required to determine the clinical significance of these data in humans, the results nevertheless situate Δ9-THCV in a promising position for developing a cannabinoid-based therapy for patients with PD.

Keywords : Parkinson’s disease, L-DOPA, L-DOPA-induced dyskinesia, Cannabinoids, Δ9-THCV, CB1 receptors, CB2 receptors

### 1. Introduction

PD is a progressive neurodegenerative disorder whose etiology has been associated with environmental insults, genetic susceptibility, or interactions between both causes (Schapira and Jenner, 2011). The major clinical symptoms in PD are tremor, bradykinesia, postural instability and rigidity (Kim et al., 2018), symptoms that result from the severe dopaminergic denervation of the striatum caused by the progressive death of dopaminergic neurons of the substantia nigra pars compacta (Pavón et al., 2006; Sauerbier et al., 2016). Major symptoms in PD (e.g. bradykinesia) can be attenuated with dopaminergic replacement therapy using the dopamine precursor L-DOPA (Pezzoli and Zini, 2010). However, this therapy does not work in all PD patients and when used for more than 5–10 years, it loses efficacy and provokes an irreversible dyskinetic state characterized by the appearance of abnormal involuntary movements (Espay et al., 2018). Therefore, the dyskinetic side effects or able to delay/reduce these signs, in addition to delaying the progression of nigrostriatal damage in PD, remains a major challenge in PD therapy (Kulisevsky et al., 2018).

Cannabinoid-based compounds have been recently proposed as promising therapies in PD given their potential as symptom-alleviating and disease-modifying agents (Fernández-Ruiz, 2009; González- Aparicio and Moratalla, 2014; Fernández-Ruiz et al., 2015; Aymerich et al., 2018; Antonazzo et al., 2019; Cristino et al., 2020; Junior et al., 2020). As regards to the first of these two options, the blockade of the CB1 receptor, which is highly abundant in basal ganglia structures, may be effective in reducing the motor inhibition typical of PD patients, which is concordant with the overactivity of the cannabinoid system observed in PD patients and animal models of this disease (reviewed in Fernández-Ruiz, 2009). However, the preclinical studies conducted so far have demonstrated that the efficacy of CB1 receptor blockade was restricted to specific circumstances, e.g. the use of low doses, strong nigral damage (Fernández-Espejo et al., 2005; González et al., 2006; Kelsey et al., 2009), conditions that were not reproduced in the only clinical trial conducted so far with a CB1 receptor blocker, which included a population of patients that were all good-responders to LDOPA (Mesnage et al., 2004). Therefore, this potential therapeutic strategy merits further clinical investigation, this time with PD patients that respond poorly to L-DOPA (approximately 15–20% of patients are poor responders to L-DOPA and it appears that, in general, they may correspond to those having tremor as the key symptom rather than rigidity and bradykinesia (Mohl et al., 2017)).

Some cannabinoids have been reported to protect nigral neurons from death caused by different insults in various experimental models of PD (reviewed in Fernández-Ruiz, 2009; Fernández-Ruiz et al., 2015; Aymerich et al., 2018; Antonazzo et al., 2019; Cristino et al., 2020; Junior et al., 2020). These include the phytocannabinoids, Δ9-THC and CBD, the synthetic cannabinoid receptor agonist CP55,940 and the
anandamide analog AM404 (reviewed in Fernández-Ruiz, 2009; Fernández-Ruiz et al., 2015; Aymerich et al., 2018). A priori these compounds acted through antioxidant mechanisms that seem to be independent of CB1 or CB2, although compounds also targeting the CB2 receptor afforded neuroprotection in MPTP- (Price et al., 2009; Chung et al., 2016), LPS- (García et al., 2011; Gómez Gálvez et al., 2016) or rotenone-lesioned (Javed et al., 2016) mice, with controversial results in 6-OHDA-lesioned rodents (García-Arencibia et al., 2007; Ternianov et al., 2012). The benefits obtained with CB2 agonists appeared to depend predominantly on the activation of receptors located in activated astrocytes and/or reactive microglial cells, which would result to be upregulated in the pathology in an attempt to limit the generation of proinflammatory factors (García et al., 2011; Concannon et al., 2015, 2016; Gómez-Gálvez et al., 2016; Navarrete et al., 2018). However, a contribution of CB2 receptors located in a few neuronal subpopulations, e.g. nigrostriatal neurons (García et al., 2015), pallidothalamic neurons (Lanciego et al., 2011), in the basal ganglia cannot be ruled out. In addition, cannabinoids that activate the PPAR nuclear receptors, in particular at the PPAR-γ type, were also neuroprotective in LPS-lesioned mice (García et al., 2018), and those targeting GPR55 may also be beneficial (Celorrio et al., 2017). By contrast, selectively activating the CB1 receptor, which may elicit ataxia as an adverse effect and/or aggravate major parkinsonian symptoms (e.g. bradykinesia), given the hypokinetic effects associated with the activation of this receptor (Fernández-Ruiz, 2009), has been found not to protect against 6-OHDAinduced damage in pharmacological studies (García-Arencibia et al., 2007). However, CB1 receptor-deficient mice display an increased vulnerability to 6-OHDA lesions (Pérez-Rial et al., 2011), which indicates certain neuroprotective potential exerted by this receptor type.

Therefore, these previous data provide good evidence that a cannabinoid having antioxidant properties and the ability to activate CB2 and PPAR-γ receptors and/or to target GPR55, but to also block CB1 receptors, might serve to alleviate parkinsonian symptoms and to arrest/ delay neurodegeneration in this disease (see Fernández-Ruiz, 2009; Fernández-Ruiz et al., 2015; Aymerich et al., 2018; Antonazzo et al., 2019; Cristino et al., 2020; Junior et al., 2020, for review).

One phytocannabinoid with such a pharmacological profile is Δ9- THCV. It is antioxidant and has been found to produce signs of CB1 receptor antagonism (when used at doses lower than 3 mg/kg (Pertwee, 2008)), but also CB2 receptor activation with significant potency (García et al., 2011). It also appears to have certain agonist activity at the GPR55 (Morales et al., 2017). Using different experimental models of PD, we have demonstrated that Δ9-THCV alleviates motor inhibition in 6-OHDA-lesioned rodents by blocking CB1 receptors at low doses, with this effect being similar to that of the classic CB1 receptor antagonist/ inverse agonist, rimonabant (García et al., 2011). It was also able to preserve nigral neurons against different degenerative stimuli in 6-OHDA- and LPS-lesioned mice due to its antioxidant properties and CB2 agonist activity (García et al., 2011). Its effects in 6-OHDA-lesioned mice were equivalent to those observed with the antioxidant phytocannabinoid CBD (Lastres-Becker et al., 2005; García-Arencibia et al., 2007), whereas those found in LPS-lesioned mice were similar to those found with HU-308, a selective CB2 receptor agonist (Gómez-Gálvez et al., 2016).

In the present study, we have investigated for the first time whether Δ9-THCV, used at a low dose, is also anti-dyskinetic, a relevant property for any antiparkinsonian agent. Previous studies implicated modulation of endocannabinoid signaling, in particular targeting the CB1 receptor, in delaying/reducing L-DOPA-induced dyskinesia (reviewed in Fernández-Ruiz, 2009; Fernández-Ruiz et al., 2015; Aymerich et al., 2018). For example, CB1 receptor-deficient mice showed less severe dyskinetic signs when lesioned with 6-OHDA and treated with L-DOPA in comparison with wild-type animals (Pérez-Rial et al., 2011). This supports the hypothesis that pharmacological blockade of this receptor may be beneficial for L-DOPA-induced dyskinesia, which has already been investigated in some studies using experimental models (Segovia et al., 2003; Cao et al., 2007; Gutiérrez-Valdez et al., 2013). Benefits were also found after activation of the CB1 receptor (Segovia et al., 2003; Morgese et al., 2007; Martinez et al., 2012), demonstrating the extreme complexity of the role exerted by this modulatory system in the basal ganglia. In any case, this potential has not been corroborated yet in clinical studies (Carroll et al., 2004). A recent study has added the CB2 receptor to those endocannabinoid targets susceptible to serve for treating L-DOPA-induced dyskinesia (Rentsch et al., 2020). The current study was designed to explore the benefits of Δ9-THCV against L-DOPAinduced dyskinesia elicited in a genetic model of dopaminergic deficiency, Pitx3ak mutant mice, which has been widely used to test antidyskinetic agents (Hwang et al., 2005; Solís et al., 2015; Suárez et al., 2018). These mice are characterized by hypomorphic expression of the transcription factor gene Pitx3 due to a spontaneous mutation in the aphakia locus affecting the lens-brain-specific promoter, and leaving intact the muscle-specific promoter (Luk et al., 2013; Del Río-Martín et al., 2019). The hypomorphic Pitx3 expression severely affects differentiation of dopamine neurons with a dramatic reduction in number of neurons in the substantia nigra pars compacta and strong lack of dopamine in the striatum (Alberquilla et al., 2020). Our study investigated first whether the treatment with Δ9-THCV administered daily in parallel to L-DOPA was associated with a delay in the appearance of dyskinetic movements, and also whether Δ9-THCV was able to reduce dyskinetic signs when administered once the L-DOPA-induced dyskinesia was already established. In addition, given that the experimental model used is the first time that was investigated in relation with cannabinoids, our
study also included an analysis of the status of major endocannabinoid elements in wildtype and Pitx3ak mutant mice at the age that animals were used for pharmacological treatments.

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