WO2023233185A1 - Combination of cannabidiol and alpha-lipoic acid for use in the treatment of ptsd - Google Patents

Abstract

The present invention relates to the field of medicaments. Particularly, the present invention refers to the combination of cannabidiol (CBD) and alpha-lipoic acid at a specific dosage regimen and related application with "entourage effect-like" against a neurodegenerative desease or condition. Said pharmaceutical composition being used in the treatment of inflammatory desease.

Description

COMBINATION OF CANNABIDIOL AND ALPHA-LIPOIC ACID FOR USE IN THE TREATMENT OF PTSD

TECHNICAL FIELD

The present invention relates to the field of medicaments. Particularly, the present invention refers to the combination of cannabidiol (CBD) and alpha lipoic acid at a specific dosage regimen and related application with “entourage effect-like” against a neurodegenerative disease or condition. Said pharmaceutical composition being used in the treatment of inflammatory disease.

BACKGROUND

Cannabis has a long history of use in medicine such as asthma, depression, epilepsy, fatigue, glaucoma, insomnia, migraine, nausea, pain, rheumatism and tetanus (Doyle and Spence, 1995; Zuardi, 2006). The history belongs to researchers that in Israel has identified A9- Tetrahydrocannabinol (A9-THC) as the primary psychoactive agent in the cannabis plant in the mid-1960s (Mechoulam and Gaoni, 1967). This discovery led to extensive research on cannabinoids in the 1970s, which also coincided with renewed interest in potential therapeutic effects of cannabinoids.

In 1985, a synthetic formulation of THC (dronabinol) and a synthetic analog of THC (nabilone) were approved by the U.S. Food and Drug Administration for the treatment of nausea and vomiting associated with cancer chemotherapy.

They were subsequently also approved for the treatment of anorexia associated with weight loss in patients with acquired immunodeficiency syndrome (AIDS). This progression followed the established model of western medicine in which individual chemical constituents of plants historically used in medicine are isolated and then developed into proprietary medications.

Over the past 20 years, an increasing number of countries, states, and territories have followed suit, legalizing the medicinal use of cannabis for a variety of health conditions.

The caveat here is that cannabis has long been used as an intoxicating drug in the absence of medical need, and most of the organizations that successfully lobbied to legalize medicinal use of cannabis have since acknowledged that medical cannabis legalization was a stepping stone to getting cannabis legalized for non-medicinal purposes as well.

This question is of interest because there are several important nuances to each approach that complicate the answer from both regulatory and scientific perspectives. Moreover, in conversations related to the medicinal use of cannabis/cannabinoids there seem to be strong ideological beliefs among patients, physicians, and caregivers where there is a heavy bias towards only considering use of either botanical cannabis products or pharmaceutical cannabinoids. There are also a number of popular misconceptions (detailed below) associated with the two approaches that require better public education as cannabinoid medicines becomes more commonplace.

CANNABIS E CANNABINOIDS

The cannabis plant has been shown to be chemically rich, with 565 known constituents belonging to 23 classes of compounds (ElSohly and Slade, 2005; ElSohly and Gul 2014; Radwan et al. 2017). Perhaps the most recognized class of compounds in cannabis are the namesake cannabinoids. At the time of this writing, 120 different phytocannabinoids, plant-derived molecules unique to cannabis, have been identified in the cannabis plant, many of which directly modulate the endogenous cannabinoid system. These naturally occurring cannabinoids are distributed among ten subclasses, including A9- and A8-THC, cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), Cannabinodiol (CBND), cannabielsoin (CBE), cannabicyclol (CBL), cannabitriol (CBT) and miscellaneous type (30 known).

THC is produced as an acid (A9-Tetrahydrocannabinolic acid, A9-THCA) in the glandular trichomes of the leaves and inflorescence bracts of the plant and undergoes decarboxylation with age or heating to form A9-THC (Turner et al., 1980). THC is typically the most abundant chemical constituent of the cannabis flower, and is by far the most studied and well-understood cannabinoid. However, cannabinoids are not the only active components of cannabis. Other constituents that might contribute in some way to the effects of cannabis include Terpenes (120 known); Nitrogenous compounds (33 known); Amino acids (18 known); Proteins, enzymes and glycoproteins (11); Sugars and related compounds (34); Hydrocarbons (50 known); Simple alcohols (7 known); Simple aldehydes (12 known); Simple ketones (13 known); Simple acids (20 known); Fatty acids (27 known); Simple esters and lactones (13 known); Steroids (15 known); Non-cannabinoid phenols (25 known); Flavonoids (27 known); Vitamins (1 known); Pigments (2 known); Elements (9 known); Phenanthrenes (4 known); Spiro indans (2 known); Xanthones (1 known) and Biphenyls (1 known).

In addition to plant-derived phytocannabinoids, hundreds of exogenous synthetic cannabinoids have been synthesized and characterized. These include pharmaceutical-grade synthetically derived substances that are chemically identical to the phytocannabinoids found naturally in the cannabis plant (e.g. dronabinol, an oral formulation of synthetically derived A9- THC, and ZYN002, a transdermal synthetic CBD gel produced by Zynerba Pharmaceuticals), in addition to novel molecules not found in nature (e.g., WIN 55,212-2, JWH-018, AM-2201, AMB- FUBINACA). There are two common misconceptions we often hear related to synthetic versus naturally occurring phytocannabinoids. One is that there are differences in the effects of a single molecule phytocannabinoid (e.g. CBD) based on whether it is synthetic versus plant derived. This should not be the case as chemistry is an exact science with respect to chemical composition and structure. The circumstances under which this could be true with respect to botanical cannabinoid products versus synthetic products would be limited to cases in which one of the two substances contains impurities that contribute to the overall pharmacological or toxicological effect, or due to inappropriate designation of synthetically derived isomers as being true replications of naturally derived cannabinoids. In these cases, the differences would be due to impure extraction of botanically sourced cannabinoids or missteps in the synthesis of the cannabinoid.

The other common misconception is that synthetic cannabinoids not found naturally in cannabis are more harmful than phytocannabinoids. This largely stems from the ongoing problems associated with illicit sales of synthetic CB1 full agonists (Fattore and Fratta, 2011; Vandrey et al., 2012). The potential harm associated with any newly synthesized drug is directly tied to its pharmacological effects (pharmacology, receptor specificity and affinity, potency) in the body. Indeed, one advantage of pursuing drug development of botanical cannabis or synthesized phytocannabinoids is that the cannabis plant has a very well established and positive safety profile.

Both phytocannabinoids and synthetic cannabinoids can directly impact the endocannabinoid system via a variety of pharmacological mechanisms, including agonism, antagonism, and allosteric modulation (for detailed reviews of cannabinoid pharmacology see Pertwee, 2008; Pertwee et al., 2010). Though 120 phytocannabinoids have been identified, they are finite and somewhat limited with respect to pharmacological interaction with the endocannabinoid system. Because of this, there are clear advantages of focusing on single molecule synthetic cannabinoid drug development, simply due to the fact that medicinal chemists are able to systematically modify known cannabinoid molecules in order to target very specific pharmacological effects. This type of “fine tuning” has the potential to yield medications that have a very specific mechanism of action (e.g. full agonism of CB1 receptors outside the CNS), which might both improve therapeutic efficacy and reduce adverse effects compared with phytocannabinoids such as THC, which is neither selective to a specific cannabinoid receptor subtype nor limited with respect to crossing the blood-brain barrier.

"ENTOURAGE EFFECT"

The major rejoinder to using single molecule and synthetic preparations of cannabinoids is the possibility of unique therapeutic benefit from a dynamic interaction between the myriad chemicals found in the cannabis plant and other combinations with phytocomplex or phytoextracts.

Referred to as the “entourage effect”, unique therapeutic effects of cannabis are hypothesized to be achieved through a complex synergy between phytocannabinoids and the many other secondary constituents of the plant (Ben-Shabat et al., 1998; Russo & McPartland, 2003).

The term “entourage effect” was coined by Raphael Mechoulam (Ben-Shabat et al., 1998; Mechoulam & Hanus, 2000) as he elaborated on the fact that the presence of glycerol fatty acid esters, alongside 2-arachidonoyl glycerol (2-AG), an important endocannabinoid, reduced the rate of hydrolysis of 2-AG, which enhanced its activity. The term was later used by others (Fowler, 2003; Sanchez-Ramos, 2015) to highlight the contribution of other cannabinoids and noncannabinoid constituents to the activity of cannabis preparations. Carlini et al. (1974) provided an early example of the entourage effect by demonstrating that 2 of 3 cannabis extracts, administered in multiple species, including humans, produced effects 2-4 times greater than what was observed after administration of pure THC at the same doses contained in the extracts.

The limitations of the entourage effect are that, at this time, it is not clear which compounds drive the effect, which pharmacodynamic effects of cannabis are impacted, or whether this can be harnessed for improved cannabinoid therapeutics. Ethan Russo has proposed very clear hypotheses about how select terpenes contribute to the cannabis entourage effect, but the empirical research required to test these hypotheses with cannabis has yet to be completed (Russo, 2011). Moreover, evidence for an entourage effect has not been consistently observed, and very few controlled studies have examined it systematically (Hazekamp, Ware, Muller-Vahl, Abrams, & Grotenhermen, 2013). In one study, Wachtel and colleagues (Wachtel, ElSohly, Ross, Ambre, & De Wit, 2002) directly compared oral and smoked cannabis to dronabinol (oral synthetic THC) and found that dronabinol produced similar subjective effects as herbal cannabis. Similarly, three appropriately powered randomized control trials all failed to find differences in medicinal effects between synthetic and herbal cannabis preparations when compared to placebo (Haney et al., 2007; O’Neil et al., 2017; Strasser et al., 2006). Using these findings to dismiss the potential for an entourage effect, however, is premature, and admittedly is arguing from the null hypothesis. Additional controlled research in this area is needed.

EXPERIMENTAL DESIGN COMBINATION

One of the key tenets of modern medicine is that one must be able to define medications chemically to the highest degree possible. A botanical drug such as cannabis requires definition of its chemical profile and the ratio with other components is the future. The ability to fully characterize, define, and demonstrate consistency in chemical composition of the combination is of the greatest challenges to drug development. Due to interactions between constituent chemical components of the cannabis plant, a positive clinical outcome for one defined botanical cannabis product cannot be generalized.

Though these issues make it difficult to develop a botanical drug combination with cannabis and other plant extract. This is the aim of this research.

In contrast, quality control for synthetically derived, single molecule medications is much easier, and is the norm for the pharmaceutical industry. There are clear standards to follow and established methods for manufacturing, testing, and quality control from start to finish. Single molecule medications also have major advantages in clinical testing. In single molecule studies, the study drug represents only one independent variable and one direct effect being tested. The simplicity of design makes it easier to ensure that clinical trials are well powered to find effects, and interpretation of results is relatively straightforward and concise. That being said, identification of target molecules, requires a rigorous and lengthy pre-clinical screening process.

REGULATORY CONSIDERATIONS

Pharmaceutical drug development is a complex and often misunderstood arena, and there are unique considerations with respect to cannabinoid medications. Currently, in the U.S. and many other countries, cannabis and several of its constituent components remain controlled substances. This requires extra regulatory approvals, security, and a substantial regulatory burden for any level of botanical drug development, as well as for single molecule drug development for which local regulations consider that molecule (e.g. THC, CBD) a controlled substance. For novel synthetic cannabinoids (e.g., analogues), it is possible that this additional regulatory limitation may not apply.

In some circumstances, regulations may completely sequester cannabinoid drug development. It also places treatment providers in the difficult position of trying to engage in clinical decision making related to patient use of these products in the absence of reliable information typically found in a medication package insert such as recommended dose, dose frequency, expected adverse effects, contraindications, comparative efficacy to alternative therapeutics, etc.

CANNABIDIOL (CBD)

Cannabidiol (CBD), a major nonpsychoactive component of Cannabis (Hampson, Grimaldi, Axelrod, & Wink, 1998; Mechoulam, Peters, Murillo-Rodriguez, & Hanus, 2007), has been reported to elicit interesting pharmacological effects that may have clinical applications in a number of disease states (Ligresti, De Petrocellis, & Di Marzo, 2016; Pertwee, 2009). In this regard, CBD has the potential of inhibiting cancer cell proliferation, metastasis, and tumor growth (Guzman, 2003). A number of studies also demonstrated that CBD elicits vasorelaxant responses (Stanley, Hind, & O'Sullivan, 2013), exerts anti-emetic and anti-nausea effects, decreases anxiety, and improves depressive-like behaviors (de Mello Schier et al., 2014). CBD was also reported to possess interesting anti-epileptic properties in particular for patient resistant to all conventional anti-epileptic drugs (Silvestro, Mammana, Cavalli, Bramanti, & Mazzon, 2019). In addition to that, CBD has also the potential for reducing unwanted side-effects of therapeutical treatments and to improve their tolerability.

For instance, CBD reduces the myocardial toxicity of the chemotherapeutic agent doxorubicin (Hao et al., 2015) and provides relief against LDOPA- induced dyskinesia in a Parkinson disease model (Dos-Santos-Pereira, da-Silva, Guimar~aes, & Del-Bel, 2016). CBD was also found to exert neuroprotective effects in a number of experimental settings modeling acute or chronic neurodegenerative conditions (Barata et al., 2019; Campos, Fogatja, Sonego, & Guimar~aes, 2016; Garcfa-Arencibia et al., 2007). Of interest, CBD also exhibits suppressive effects on the immune system as attested by the improvement of innate and adaptive immune responses in experimental settings that model chronic inflammatory states (Kozela et al., 2011; Lee et al., 2016). Related to that, CBD was found to reduce airway inflammation and fibrosis in experimental allergic asthma (Vuolo et al., 2019), to exert anti-inflammatory effects in nonalcoholic steatohepatitis (Huang et al., 2019) and in a viral model of multiple sclerosis (Mecha et al., 2013) and to improve clinical scores in models of experimental autoimmune encephalomyelitis (Elliott, Singh, 4 Nagarkatti, & Nagarkatti, 2018). These different effects arise from the capacity of CBD to restrain inflammatory responses mediated by immune cells, in particular pathogenic T-cells (Kozela et al., 2011), activated macrophages (Huang et al., 2019), and inflamed microglial cells (Barata et al., 2019; Martin- Moreno et al., 2011; Mecha et al., 2013). Our specific aim was, here, to further explore the mechanisms underlying the anti-inflammatory effects of CBD toward microglial cells. For that, we used a model system of microglial cells isolated from postnatal mouse brain and an activation paradigm with the bacterial inflammogen LPS. We established that CBD exerts potent anti-inflammatory effects on microglial cells by inhibiting reactive oxygen species (ROS )/NF-KB -dependent signaling events and glucosedependent synthesis of NADPH, a co-factor required for NADPH oxidase activation and ROS generation by this enzyme.

The medical use of Cannabis sativa (Mechoulam R. 1986), has always been known for its medical and beneficial purposes. Based on the records found the pharmacological effects of Cannabis include anti-nociception, anti-inflammation, anticonvulsant, anti-emetic, as well as recreational use, which has largely limited its medical application (Iversen E.). Approximately 120 phytocannabinoids have been identified in the plant, and among those, the A9- tetrahydrocannabinol (THC) is the main psychoactive component. Chemically, phytocannabinoids occur either as a terpene fused to an alkyl- substituted resorcinol, or as a benzopyranic ring system. After the discovery of THC, a large number of synthetic cannabinoids, similar or distinct in the structures to phytocannabinoids, helped the identification and the cloning of the cannabinoid receptor 1 (CB 1R), and later on the cannabinoid receptor 2 (CB2R) discovery. They are differently localized throughout the body: CB 1 is highly expressed in brain neurons and are less expressed in the amygdala, hypothalamus, nucleus accumbens, thalamus, periacqueductal grey matter (PAG) and the spinal cord. CB2 is highly expressed on immune cells including microglia and astrocytes (De Leo et al., 2005; Luongo et al., 2010). Many functions are regulated and controls by CB 1 receptor such as: mobility of GI tract, secretion of gastric acids fluids, secretion of neurotransmitter and hormones, control appetite from the hypothalamus in the CNS, regulation of the energy balance and food intake. While, CB1 activation reduces neurotransmitter release, CB2 activation inhibits microglial activation and reduces neuroinflammation.

Evidence from preclinical studies have already shown the beneficial properties of cannabidiol CBD, including neuroprotective effects in Central Nervous System (CNS) disorders (Fernandez-Ruiz et al., 2013; De Gregorio et al., 2018; Schonhofen et al., 2018). CBD interacts with the endocannabinoid system, our biological system composed of endocannabinoids, which are endogenous lipid-based retrograde neurotransmitters that bind to cannabinoid receptors (CB 1 and CB2). Surprisingly, CBD binds with very low affinity CB1 and CB2, instead it inhibits the anandamide uptake and enzymatic hydrolysis (Lastres-Becker et al., 2005), and decreases adenosine reuptake (Carrier et al., 2006). These are all important steps of the endocannabinoid pathways, through which CBD is believed to exert neuroprotective effects. Recent preclinical research has suggested that cannabis and in particular CBD may have a beneficial effect in rodent models ofpost-traumatic stress disorder (PTSD). Generally, Post-traumatic stress disorder (PTSD) is described as a chronic psychopathology characterized by debilitating alterations in mood and cognition following the experience of an intense traumatic life event. According to the DSM-V, twenty PTSD symptoms are classified into four symptom clusters: (i) intrusion (including flashbacks and nightmares), (ii) experience of negative alterations in mood and cognition, (iii) avoidance of memories of the trauma, and (iv) hyperarousal and hyperreactivity (including lack of concentration or irritability) (Steenkamp et al., 2016; Pai et al., 2017). Although the pathophysiology of PTSD has not yet been definitively described, a number of factors are suspected to contribute to the development of this disorder. One hypothesis relates PTSD to dysregulated memory retrieval through the process of reconsolidation and impaired extinction of aversive memories (Parsons RG, Ressler KJ. 2013). Psychotherapy is recommended as treatment for PTSD accompanied by drug treatment such as selective serotonin reuptake inhibitors, serotonin/norepinephrine reuptake inhibitors, antiadrenergic agents, and second-generation antipsychotics. However, the investigation of new pharmacological tools are needed to enhance the pharmacological efficacy and minimize the side effects. Stress and environmental factors play a fundamental role in developing maladaptation and behavioral abnormalities. Indeed, stressful events negatively affect several neuroendocrine systems, which can cause deep repercussions on both cognitive and emotional processing (McEwen et al., 2015; Pagliaccio et al., 2015; Herman et al., 2016). PTSD is complicated by major depressive disorders (Shalev,2001). This impressive comorbidity rate can be partially explained by the presence of overlapping symptoms between the two disorders. Other disorders observed in PTSD patients are enhanced vulnerability to substance and/or alcohol abuse, generalized anxiety, or even attempted suicide (Spinhovenet al., 2014; Gradus et al., 2017; Lento et al., 2018). In fact, another significant consequence of worsening PTSD symptoms is the increased risk for suicide and suicidal ideations (Pompili et al., 2013). Suicide is rarely caused by any single factor, but rather, is determined by multiple factors. In addition to comorbidity with psychiatric conditions (e.g. PTSD, depression) and prior suicide attempts, other contributing factors consist in personality disorder/traits, life stressors and social and economic problems (Stone et al., 2018). Thus, new effective therapies for PTSD complicated by suicidal ideations, as well as, unveiling novel PTSD biomarkers are in high demand.

The lockdown due to the pandemic COVID-19 outbreak could be one of the cause for the appearing of the post- traumatic stress disorders.

Animal models are useful tools to investigate the aetiology of diseases, their course and, ultimately, to develop new pharmacological treatments (Lanzas et al., 2010). Even though animals do not develop PTSD many fundamental physiological and basic behavioral responses can reproduce neurobio logical components associated with these psychopathologies processes, which are involved in the onset and manifestation of this psychopathology. For instance, post-weaning prolonged social isolation induces abnormal forms of behavior in mice; an effect directly related to increased glucocorticoid responses (Toth et al., 2011). Socially isolated mice show a peculiar phenotype, including: an exacerbation of aggressive behavior and an increase in anxiety- and depressive-like behaviors (Locci and Pinna, 2017). Behavioral deficits following protracted social isolation are associated with a number of physical and neuronal dysfunctions, including impairment of the Hypothalamic-pituitary-adrenal axis HPA axis, neurotransmitter systems, neuropeptides, neurohormones, and neurotropic factors (Nin et al., 2011a).

The social isolation model by exposing rodents to a protracted and, probably, severe or mild stressor, social isolation (SI) offers a putative animal model to investigate the development of vulnerability to PTSD. In rodents, SI can be considered a distressing event that induces behavioral deficits, even though the length of isolation varies among several laboratories. A social isolation model that leads to PTSD could be: isolation in individual cages for 3-4 weeks from 21th postnatal day; Mice that are socially isolated for 3-4 weeks post-weaning (PN21) express a number of behavioral deficits relevant to model aspects of human mood disorders (reviewed in Pinna and Rasmusson, 2012; Zelikowsky et al., 2018; Aspesi and Pinna, 2019; Locci and Pinna, 2019b). including increased anxiety-like behavior and aggression and the expression of aggressive behavior, reaches a plateau between week 4 and 6 of isolation (Pibiri et al., 2008; Pinna et al., 2008 ; Guido tti et al. , 2001 ; Pinna et al. , 2003 ; Rau et al. , 2005 ; Locci and Pinna, 2019b) . Individual housing is likewise a powerful stressful condition that may increase the susceptibility to develop behavioral dysfunctions when rodents are additionally exposed to an acute traumatic stressor, to a series of stressful stimuli such as periodic deprivation of water and/or food, exposure to low temperatures, physical restraint and reversal of the light/dark cycle. Other stresses can be induced such as the single prolonged stress that consists in three stressors that are administered in succession: restraint stress (2 h), forced swimming (20 min) and exposure to diethyl ether (Liberzon et al., 1997, 1999) or the social defeat stress model is mostly performed in male rodents by a resident-intruder test, which results in aggressive behavior and social stress for the intruder (Bjbrkqvist, 2001; Hammels et al., 2015). MICROGLIAL CELL

Our specific aim was, here, to further explore the mechanisms underlying the antiinflammatory effects of CBD toward microglial cells. For that, we used a model system of microglial cells isolated from postnatal mouse brain and an activation paradigm with the bacterial inflammogen LPS.

We established that CBD exerts potent anti-inflammatory effects on microglial cells by inhibiting reactive oxygen species (ROS )/NF-KB -dependent signaling events and glucose dependent synthesis of NADPH, a co-factor required for NADPH oxidase activation and ROS generation by this enzyme.

CANNABIDIOL (CBD) NEUROPROTECTIVE EFFECT

CBD was also found to exert neuroprotective effects in a number of experimental settings modeling acute or chronic neurodegenerative conditions (Barata et al., 2019; Campos, Fogaca, Sonego, & Guimaraes, 2016; Garcfa-Arencibia et al., 2007). Of interest, CBD also exhibits suppressive effects on the immune system as attested by the improvement of innate and adaptive immune responses in experimental settings that model chronic inflammatory states (Kozela et al., 2011; Lee et al., 2016).

ALPHA LIPOIC ACID a-Lipoic acid (LA), a naturally occurring enzyme cofactor with antioxidant and iron chelator (Persson HL, 2003) properties, has been used as a therapeutic agent for many chronic diseases such as diabetes mellitus (DM) and the associated peripheral neuropathy. Importantly, LA was also found to provide neuroprotection against Alzheimer Disease (AD), as it can easily penetrate the blood-brain barrier (BBB). The therapeutic effect of LA for AD was found by chance in clinical trials that demonstrated that LA supplementation could moderate the cognitive functions of patients with AD and related dementias (Hager K, 2007). In previous animal studies, LA supplementation improved cognition and memory in aged SAMP8 mice and aged rats (Farr S.A., 2003), reduced hippocampal-dependent memory deficits in the Tg2576 model of AD without affecting P-amyloid (AP) levels or plaque deposition (Quin J.F., 2007), and restored glucose metabolism and synaptic plasticity in the triple transgenic mouse model of AD (Sancheti H, 2014). In vitro studies relevant to AD mechanisms have revealed that LA can inhibit the formation of AP fibrils (fAP) and the stabilization of preformed fAp, as well as protect cultured hippocampal neurons against neurotoxicity induced by AP and iron/hydrogen peroxide (Lowell MA, 2003).

Based on the above facts, it has been proposed that these actions are mediated through potent antioxidants (Rochette L, 2013), anti-inflammatory (Maczurek A, 2008), and anti- amyloidogenic properties. Moreover, LA itself is not only an efficient free radical scavenger, but the disulfide bond and five-membered cyclic structure of LA lead to powerful antioxidant capacity and good iron-chelation activity. These multifaceted effects suggest that LA is a promising therapeutic agent for AD, but the exact cellular and molecular mechanisms remain unknown, especially with respect to the ability of LA to control Tau pathology and neuronal damage. In this context, we previously showed that the hyperphosphorylation status of Tau can be reversed by iron chelation in an AD mouse model (Guo C, 2013). In fact, LA plays many different roles in the pathogenic pathways of dementia, acting as a neuroprotective agent. LA might attenuate free radical damage and reduce inflammatory activities and, hence, might have a positive effect on neuronal ferroptosis, which is a recently discovered form of cell death dependent on iron and ROS (Yie Y, 2016), because ferroptosis can also be prevented by ferrostain-1, lipophilic antioxidants and iron chelators, such as deferoxamine. In the present study, P301S mice encoding the human P301S mutation were injected with LA for 10 weeks to study whether LA could effectively alleviate the state of AD-related tauopathy and whether the inhibition of ferroptosis is a potential mechanism of restoring impaired cognition.

BRIEF DESCRIPTION OF THE INVENTION

COMBINATION OF CANNABIDIOL AND ALPHA LIP QIC ACID

The combination between alpha lipoic acid and CBD is a plastic membrane and a conduction support for neural stimuli. The Applicant has now found an specific pharmaceutical composition and specific dosage through which advantageous therapeutic effect can be obtained, as experimentally tested.

In a first aspect the invention relates to a pharmaceutical composition as reported in the attached claims.

In a second aspect the invention relats to a pharmaceutical composition for use as reported in the attached claims.

Dosages were tested considering the suitable lipophily to to cross the blood-brain barrier and the possible related pharmaceutical forms: capsules, tablets, softgels, oral oleolites and parenteral nutrition to support nutrition.

Advantageously, the pharmaceutical composition of the invention provides a allows the restoration and provide support to the neuronal stumulus conduction and therefore it is effective to the treatment of neurodegenerative diseases and conditions. Particularly, the specific selected pharmaceutical composition allows to achieve a synergistic effect, which is higher and better than the effect obtainable with CBD and alpha lipoic acid considered individually. Such results being experimentally obtained through immunohistochemical analysis and biochemical analysis on cerebral cortex. The same positive effect being detected as for the hippocalmpus. With reference to amygalda, glia functionality is clearly recvovered, and accordingly the impact in terms of aggression and inflammation are considerably reduced.

Further features and advantages of the pharmaceutical composition of the invention will be evident from the description of the embodiment of the invention, which is to be intended as illustrative, but not limiting, purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Immunofluorescence reaction for MAP2 (green fluorescence, neurons), GFAP (cyano fluorescence, astrocytes), Ibal (red fluorescence, microglia) in social (a), asocial (b), CBD 2.5mg/Kg (c), CBD 5mg/Kg, (d) CBD lOmg/Kg (e), ALPHA LIPOIC ACID (ALA) (g), CBD 2.5mg/Kg + ALA 10 mg/Kg (i) cortex of mice. Nuclei were counterstained with DAPI (blue fluorescence). Objective magnification: 40x.

Figure 2. JNK signalling in the cortical TIF. Data are represented as mean ± SEM and were analysed with one-way ANOVA test, followed by post hoc test Tukey. Significance relative to social: *p<0.05; **p<0.01.

Figure 3. Immunofluorescence reaction for MAP2 (green fluorescence, neurons), GFAP (cyano fluorescence, astrocytes), Ibal (red fluorescence, microglia) in social (a), asocial (b), CBD 2.5mg/Kg (c), CBD 5mg/Kg, (d) CBD lOmg/Kg (e), alpha lipoic acid (ALA) (g), CBD 2.5mg/Kg + ALA (i) amygdala of mice. Nuclei were counterstained with DAPI (blue fluorescence). Objective magnification: 40x.

Figure 4. Immunofluorescence reaction for MAP2 (green fluorescence, neurons), GFAP (cyano fluorescence, astrocytes), Ibal (red fluorescence, microglia) in social (a), asocial (b), CBD 2.5mg/Kg (c), CBD 5mg/Kg, (d) CBD lOmg/Kg (e), alpha lipooic acid (ALA) (g), CBD 2.5mg/Kg + ALA 10 mg/Kg (i) amygdala of mice. Nuclei were counterstained with DAPI (blue fluorescence). Objective magnification: 40x.

DETAILED DESCRIPTION OF THE INVENTION

For the scopes of the invention, the following definitions used in the present description and attached claims are provided.

As intended herein, the term patient and subject can be used interchangeably.

As intended herein, alpha lipoic acid can be insidated also as “ALA”.

Therefore, according to a first aspect the present invention relates to a pharmaceutical composition comprising cannabidiol and alpha lipoic acid, wherein cannabidiol is in an amount from 60 to 900 mg and wherein alpha lipoic acid is in an amount from 300 to 900 mg.

According to another preferred aspect, cannabidiol is in an amount from 120 to 720 mg and wherein alpha lipoic acid is in an amount from 450 a 750 mg. According to a further preferred aspects, cannabidiol is in an amount from 150 to 600 mg and wherein alpha lipoic acid is in an amount from 550 a 650 mg. According to a further preferred aspects, cannabidiol is in an amount from 150 to 600 mg and wherein alpha lipoic acid is in an amount of 600 mg. Preferably, cannabidiol is in an amount of 150 mg and wherein alpha lipoic acid is in an amount of 600 mg. Such values being referred to an average weight of 60 kg of a patient to be treated.

According to anoher preferred aspect, the pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients.

According to another preferred aspect, the pharmaceutical composition is in the form of capsules, tablets, softgels, oral oleolites and parenteral nutrition to support nutrition.

As above mentioned, the pharmaceutical composition of th invention is particularly effective in the treatment of neurodegenerative diseases and conditions. The pharmaceutical composition allowing the reduction of brain microglia activity, thereby reducing inflammatory processes.

In a further aspect, the invention relates to a pharmaceutical composition as defined above for use in reducing or treating:

- post-traumatic stress disorder (PTSD), or

- inflammatory processes, or

- depression related to the reduction of inflammatory processes, or

- sociability related to the reduction of inflammatory processes, or

- anxiety related to the reduction of inflammatory processes, or

- anxiety and obsessive behaviour related to the reduction of inflammatory processes, or

- aggressiveness related to the reduction of inflammatory processes, or

- apathy related to the reduction of inflammatory processes, or

- “novel object recognition test” related to the reduction of inflammatory processes.

Preferably, the pharmaceutical composition of the invetion is suitable to be administered to human beings or animals. According to a preferred aspect, the pharmaceutical composition is administered daily to the patient. Preferably, the patient is a human being. According to a preferred aspect, cannabidiol and alpha lipoic acid are administered by simultaneous, alternate or separate administration.

According to another preferred aspect, the composition is administered orally.

Accoring to another preferred aspect, the dosage regimen of administration involves the administration amount of cannabidiol from 1 to 15 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid from 5 to 20 mg per kg with respect to the weight of the subject to be treated. According to a further preferred aspect, cannabidiol is in an amount from 2 to 12 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid from 7.5 to 17.5 mg per kg with respect to the weight of the subject to be treated. According to a further preferred aspect, cannabidiol is in an amount from 2.5 a 10 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid from 9 to 15 mg per kg with respect to the weight of the subject to be treated. According to a further preferred aspect, cannabidiol is in an amount from 2.5 a 10 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid 10 mg per kg with respect to the weight of the subject to be treated. Preferably, cannabidiol is in an amount from 2.5 or 5 or 10 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid 10 mg per kg with respect to the weight of the subject to be treated. Preferably, the subject to be treated is an animal or a human being, ore preferalby a human being.

Other features of the pharmaceutical composition “for use” are as described above, when referred to the pharmaceutical composition according to the first asppect of the invention.

All the features, aspects and embodiments reported above and related to the present invention are to be intended for illustrative, but not limiting, purpose only. Therefore, the skilled person will promptly understand that variations and modifications can be made withouth departing from the scope of the present invention.

EXAMPLES

EXPERIMENTAL PART - 1

MICROGLIA ACTIVITY To evaluate the anti-inflammatory potential of CBD, it has been used microglial cells activated by the bacterial cell wall component LPS (10 ng/ml), a prototypical agonist for Toll- like receptor-4 (TLR4; Dbring et al., 2017). After 24 hr of treatment, LPS strongly stimulated the production/release of the pro-inflammatory cytokine TNF-a and to a lesser extent that of IL-ip, a cytokine produced in response to inflammasome activation (Mouton- Liger et al., 2018; Weigt, Palchevskiy, Belperio, 2017). When the cultures were exposed to 1 and 10 pM of CBD, the production of TNF-a and IL-ip induced by LPS was curtailed efficiently (Figure la,b). CBD appeared only slightly less efficacious at 1 pM than at 10 pM. At 0.1 pM, however, CBD had no significant impact on cytokine production, suggesting that a threshold concentration was required for anti-inflammatory effects in the present setting. Note that CBD vehicle had no effect on its own on the LPS response. A reference anti-inflammatory drug, the glucocorticoid dexamethasone (DEX; 2.5 pM) mimicked the inhibitory effects of CBD on cytokine release. Upon LPS treatment, we also measured the release of glutamate, a neurotransmitter operating as a non-cytokine mediator of microglial cell inflammation (Figure 1c). Glutamate release induced by LPS returned to control values in the presence of 10 pM CBD. CBD was less effective at 1 pM but it still reduced extracellular glutamate levels by half. Note that DEX was ineffective to reduce glutamate release induced by LPS.

Consistent with data obtained in a previous study (Dos-Santos-Pereira et al., 2018), a 24 hr treatment with 10 ng/ml LPS led to about a 20% increase in the number of DAPI+ nuclei, which is indicative of a small proliferative effect of the inflammogen. Despite its potential to restrain cell proliferation reported elsewhere (McAllister et al., 2011), CBD failed to reduce the number of DAPI+ nuclei under LPS treatment (Figure Id). Finally, CBD (10 pM) also led to reduced expression of the phenotypic activation marker Iba-1 in microglial cells exposed to LPS (Figure le). This inhibitory effect was detectable by quantification of Iba-1 expression by western immunoblotting. It was confirmed through a visual inspection of LPS-treated microglial cell cultures, exposed or not to CBD and then processed sequentially for CD 11b and Iba- 1 fluorescence immunodetection

MICROGLIA STUDY

Thanks to the possibility of studying some cellular microglial models from mouse brain explants, it has been evaluated the cytotoxicity and on human models of Human monocytic leukemia cell line (THP-1) we evaluated the MTT reduction through a photometric method to determine the release of cytokines (LPS stimulation) by ELISA methods the following endpoints: IL-1 beta, TNF-alpha, IL-6 release with the combinations listed below.

COMBINATION OF INGREDIENTS

The combination involves the use of rat microglia cells in which different combinations of substances are administered and verified with respect to the control: unstimulated and untreated microglia cells and a positive control: LPS -stimulated microglia cells. The reference was: LPS- stimulated microglia cells treated with Dexamethasone or corticosteroids (FAS).

The combination involved evaluating:

• LPS -stimulated microglia cells treated with 1 natural compound alone (3 concentrations)

• LPS -stimulated microglia cells treated with 1 natural compound (concentrations) + transresveratrol (3 concentrations)

ANIMAL STUDY

The focus of the study is to investigate the pharmaceutical composition of the invention in term of beneficial effects on the behavioral and cognitive dysfunctions associated with a preclinical model of PTSD (Post Traumatic Stress Disorders).

It has been tested behavioral situation like:

• tail suspension to evaluate depression,

• three chambers test to evaluate social interaction,

• open field test or murble buyring test for anxiety and obsessive compulsive behaviour,

• resident-intruder test for aggressiveness • novelty suppressed feeding test for apathy and anedonia

• novel object cognition test for measuring cognition

Depression: Tail Suspension Test

This test will be used to evaluate the depressive-like behavior. Mice will be individually suspended by the tail on a horizontal bar (55 cm from floor) using adhesive tape placed approximately 4 cm from the tip of the tail. The duration of immobility, recorded in seconds, will be monitored during the last 4 min of the 6-minute test by a time recorder. Immobility time will be defined as the absence of escape-oriented behavior. Mice will be considered immobile when they will not show any body movement, hung passively and completely motionless. (Belardo et al. 2019) Sociability: Three Chambers Sociability Test

This test is used for the evaluation of social interaction using a three-chambered social interaction apparatus. A plexi-glass three-chambered box will be custom-built as follows: doorways in the two dividing walls has sliding covers to control access to the outer-side chambers. The test consists of two consecutive stages of 5 and 10 min each. During the 5-min first stage of habituation the mouse will be allowed to freely explore the three chambers of the apparatus, detecting at this stage any innate side preference. After that, the mouse will be gently encouraged into the central chamber and confined there briefly by closing the side chamber doors. During the following 10- min stage sessions, a custom-made stainless-steel barred cup (6.5 cm x 15 cm) will be placed upside down in one of the side chambers. A never before-met intruder, previously habituated, will be placed into an upside down identical cup in the other chamber. The time spent sniffing each upside-down cup, the time spent in each chamber and the number of entries into each chamber will be recorded. (Belardo et al. 2019)

Anxiety: Open Field Test

This test is commonly used to evaluate motor activity and anxiety. Mice will be placed in an OFT arena (80 x 80 x 15 cm³), and ambulatory activity (total distance travelled in centimeter), frequency, and total duration of central zone visits will be recorded for 20 minutes and analyzed. (De Gregorio et al. 2019)

Anxiety and Obsessive and compulsive behavior: Marble-burying

In this test mice will be individually placed in a cage (21 x 38 x 14 cm length x width x height) containing 5 cm layer of sawdust bedding and fifteen glass marbles (1.5 cm in diameter) arranged in three rows. Mice will be left undisturbed for 15 min under dim light. An observer blind to the treatment will count the time spent in digging behavior and the number of marbles buried (at least two or third buried in the sawdust). At the end of the test the animal will be removed to its own cage. (Guida et al. 2015)

Aggressiveness: Resident intruder test

Mice will be individually housed for 1 week in Plexiglas cages to establish a home territory and to increase the aggression of the resident experimental mice. To begin, food containers will be removed and an intruder mouse of the same gender but different species will be placed in a resident home cage and resident-intruder interactions will be analyzed for 10 min. The aggressive behavior of resident socially isolated mice will be characterized by an initial pattern of exploratory activity around the intruder, which will be followed by rearing and tail rattle, accompanied in few seconds by wrestling and/or a violent biting attack. The number of these attacks and latency to the first attack during 10 min observation period will be recorded. (Belardo et al. 2019)

Apathy: Novelty- suppressed feeding test

This test is used to evaluate the anxiety. Latency will be measured as the time it takes a mouse to consume 3 chow pellets spread across the central area of an unfamiliar arena (80 x 80 x 30 cm³) after 48-hour food deprivation, as previously described. Before the test, feeding latency will be observed in the familiar home cage. Cutoff time will be 10 minutes. (De Gregorio et al. 2019) Novel object recognition test

Novel Object Recognition (NOR) task will be used for measuring learning and long-term memory. Two identical objects will be placed into the arena during a 6 min sample phase. Subsequently, one of the objects will be exchanged by a new object and memory will be assessed by comparing the time spent exploring the novel object as compared with the time spent exploring the familiar object during a 5 min test phase. One week before the NOR experiments, the animals will experience handling by the experimenter and habituation to the arena for 2 and 3 consecutive days, respectively. In the handling procedure will be included exposure to a transparent plexiglas tunnel with a length of 12 cm and a diameter of 6 cm. Using this tunnel, an animal will be transferred into a new cage and after a few minutes back again to home cage. For habituation, mice will be placed into the empty arena (38 x 38 x 30 cm, PVC) for 5 min. During all experiments the arena will be illuminated with 60-90 Lux. For NOR experiments custom-built plastic pieces (Polyoxymethylen, POM), will be used with different shapes (cones: 4 cm diameter, 6 cm height, pyramids: 4 x 4 x 4-6 cm) and different color (white, black, red). The objects will be cleaned thoroughly with 70% ethanol followed by distilled water between trials to remove olfactory cues. During the sample phase on the first day of the NOR test, the mice will be allowed to explore the two identical white or black objects (either two cones or two pyramids) for 6 min. For the shortdelay test phase (1.5 h) one of the sample objects will be replaced by a new one (cone by pyramid or vice versa) and exploration (L. Bolz et al.). Pattern separation during novel object recognition will be measured for 5 min. For the long-delay test phase (24 h) the new object will be again replaced by another new object. The location of the novel object at 24 h will be always different from that at 1.5 h, either first left then right, or vice versa. Consequently, the location of the familiar object also switches between the two test phases. Objects with the same color, but different shapes will be considered to be similar to sample object. Objects with both, different shape and different colours (red, black, white) will be considered to be distinct from the original sample objects. To analyze strictly active exploration the time will be measured manually using a digital stopwatch. Active exploration will be defined as direct sniffing or whisking towards the objects or direct nose contact. Climbing over the objects will be not counted as exploration. The relative exploration will be quantified by normalizing the difference between the exploration time of the novel (Tn) and familiar object (Tf) by the total time of exploration (Ttot) to calculate the NOR discrimination index: NOR index = (Tn-Tf)/Ttot.

Formula for the conversion: HEDmg/kg= Animal-dose mg/kg*AnimalKm/HumanKm

*human equivalent dose (HED) based on body surface area

*Km is a standard value (See FDA Guideline).

EXPERIMENTAL PART - 2

Methods Mice were firstly divided into two groups: group-housed (social) and single-housed (asocial); wherein asocial mice being mice in which the endocannabinoids block was induced. Then, asocial mice were divided into others groups and treated with:

1) Cannabidiol (CBD) 2.5mg/Kg,

2) CBD 5mg/Kg, 3) CBD 10 mg/Kg,

4) alpha lipoic acid (ALA),

6) CBD 2.5 mg/Kg + ALA. In total, 7 experimental groups were therefore considered:

Social mice

A- social mice

A-social CBD 2.5 mg/Kg treated mice

A-social CBD: 5 mg/Kg treated mice

A-social CBD: 10 mg/Kg treated mice

A-social ALPHA LIPOIC ACID: 10 mg/kg treated mice

A-social CBD-ALA: 2.5-10 mg/kg treated mice

At the end of the bechvioural tests, mice were sacrificed for immunoistochemical and biochemical analysis.

Immunohistochemical analysis

For Immunohistochemical analysis, n=2 mice/group were deeply anesthetized and perfused intracardially with ice-cold phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PAF) in PBS. Brains were removed from the skull and post- fixed for 90 min in 4% PAF in PBS at 4 °C, transferred to 20% sucrose in PBS for 24 h at 4 °C, then frozen in n- pentane for 3 min at - 50 °C, and stored at - 80 °C until assay.

Subsequently, brain were immersed in an OCT embedding matrix for cryostat sectioning. From these brains, serially coronal sections of 20 pm, ranging from frontal cortex to posterior lobes, were obtained for immunofluorescence analysis and confocal microscope acquisition. The slices were permeabilized with a phosphate buffered saline (PBS), pH 7.4, solution containing 0.5% of Triton X-100 for 3 minutes. Then, aspecific antigen were blocked for 1 hour in PBS containing 1% BSA, 0.2% Triton X-100 and subsequently incubated overnight at 4 ⁰ C with commercial primary antibodies (chicken anti-MAP2, 1:500; mouse anti-GFAP, 1:500; rabbit anti-Ibal, 1:500) in PBS containing

1% BSA and 0.2% of Triton X-100. After PBS washes, slices were incubated with secondary antibodies (AlexFluor 488 anti-chicken antibody, AlexFluor 674 anti-mouse antibody, AlexFluor 674 anti-rat antibody, AlexFluor 568 anti-rabbit antibody) for 1 hour at room temperature. Nuclei were counterstained with DAPI. Finally, the slices were covered with the ProLong Glass Antifade Mountant for the confocal imaging.

Biochemical Analysis

For biochemical analysis, mice were euthanized by cervical dislocation; the brains were removed and specific brain areas were dissected and stored at -80°C until sample processing. Subcellular fractionation was as reported in the literature, with minor modifications. Briefly, the tissue was homogenized with a glass-glass Potter apparatus in 0.32 M ice-cold sucrose buffer containing the following concentrations (in mM): 1 HEPES, 1 MgC12, 1 EDTA, 1 NaHCO3, and 0.1 PMSF at pH 7.4, with a complete set of protease inhibitors and phosphatase inhibitors. Samples were centrifuged at lOOOxg for 10 min. The supernatant (SI) was then centrifuged at 3000xg for 15 min to obtain a crude membrane fraction (P2 fraction). The pellet was dissolved in a buffer containing 75 mM KC1 and 1% Triton X-100 plus protease and phosphatase inhibitors and centrifuged at 100,000xg for 1 h. The supernatant was stored and referred to as TSF (S4). The final pellet (P4), referred to as TIF, was homogenized in a glass Potter apparatus in 20 mM HEPES with a complete set of protease and phosphatase inhibitors and stored at -80°C until processing. Protein concentrations were quantified using the Bradford Assay: 20 pg of total homogenate and 10 pg of TIF extracted proteins were separated by 10% SDS polyacrylamide gel electrophoresis. PVDF membranes were blocked in Tris-buffered saline 5% no-fat milk powder and 0.1% Tween 20 (1 h, RT). Primary antibodies were diluted in the same buffer (incubation overnight, 4°C) using anti-PJNKs (1:1000, BK9251S, Cell Signaling, Danvers, MA, USA), anti-JNKs (1:1000 BK9252S, Cell Signaling, Danvers, MA, USA), anti-p-c-Jun (1:1000, 06-659, Millipore, Bedford, MA, USA), antic- Jun (1:1000, BK9165S, Cell Signaling, Danvers, MA, USA), anti-NMDA Receptor 2A GluN2A (1:2000, BK4205S, Cell Signaling, Danvers, MA, USA), anti-NMDA Receptor 2B GluN2B (1:2000, BK14544S, Cell Signaling, Danvers, MA, USA), anti- Glutamate Receptor 1 (AMPA subtype) GluAl (1:1000, BK13185S Millipore, Bedford, MA, USA), anti- Glutamate Receptor 2 (AMPA subtype) GluA2 (1:1000, MAB397 Millipore, Bedford, MA, USA), anti-postsynaptic density protein 95 (1:2000, CAY-10011435-100, Cayman Chemical Company, Ann Arbor, Michigan, USA), anti- Drebrin (1:1000, BSR-M05530, Boster, Pleasanton, CA, USA), anti- Shank3 (1:1000, 64555, Cell Signaling, Danvers, MA, USA), and anti-Actin (1:5000, MAB1501 Millipore, Bedford, MA, USA). Blots were developed using horseradish peroxidase-conjugated secondary antibodies (SC-2357, SC- 516102, Santa Cruz Biotechnology, CA, USA) and the ECL chemiluminescence system (Biorad). Western blots were quantified by densitometry using Quantity One software (Bio-Rad, Hercules, California, USA).

Results

With the purpose to investigate the effects of the aforementioned treatments on the PTSD and anxiety disorders we decided to focus the attention on three crucial brain areas involved in these disorders i.e. cerebral cortex, amygdala and hippocampus. In particular, we investigate with immunoistochemical analysis the following markers: Microtubule association protein 2 (MAP2) for neurons detection; Glial Fibrillar Acid Protein (GFAP) for astrocytes imaging and Ionized calcium-binding adapter molecule 1 (Ibal) for microglia investigations. Concerning the biochemical evalutaion, we analysed the neuronal part of cortex and hippocampus, analysing the post-synaptic proteins enriched fractions (TIF), considering the main scaffold proteins of the excitatory synapses and glutamate rceptors. In addition, we focused on the activation of one of the most invovled signaling in the response to stress stimuli in the brain: the c-jun N-terminal kinase pathway.

Cerebral cortex

Immunohistochemical Analysis

Concerning cerebral cortex, the GFAP positivity was evident in social animals while a markedly decrease labelling can be observed in the asocial group (Fig. la, b). These results support the idea that PTSD and anxiety disorders could affect astrocytes ratio in the brain parenchyma causing brain circuits dysfunctions. No relevant ameliorations were observed with the CBD and ALA treatments (Fig. Ic-g). A recovery of astrocytic immunopositive fibers can be observed mainly in animals treated with CBD+ALA suggesting that the combination of these two compound could restore the homeostasis of brain circuits. However, in these conditions, the immunopositivity appeared less evident than the social conditions suggesting an overall reaction of astrogliosis an thus an anti-inflammatory effect of the treatments. Considering microglia, Ibal positive cells can be observed in social animals. Microglia was located and visible along all the cortical layers and displayed the typical branched phenotype of the resting state (Fig. la). In the asocial animal, microglia immunopositive cells appeared less evident and the positivity was principally located in the cell soma, suggesting a phenotypic change in the activated (less branched) form (Fig. lb). The CBD alone administration seems to recover the resting phenotype morphology of microglia in a progressive fashion following increasing concentrations of treatments (Fig. Ic-e). No relevant results were obtained with ALA treatments alone (Fig. 1g). The resting phenotype recovery seems to reach the maximum effects in the experimental group treated with a combination of CBD and ALA (Fig. i). These results strongly support the neuroprotective effects of the treatments against neuroinflammation due to PTSD and anxiety disorders.

Biochemical Analysis

We then analysed the cortical post- synaptic enriched protein fractions to study Synaptic Dysfunction, the first neurodegenetarive event. The activation of JNK was measured as the ratio between the phosphorylated and the total form of the kinase. In this subcellular compartment, the JNK signalling was activated in the asocial compared to the control/social group (p<0.05, Fig. 2). The CBD 2.5 mg/kg treatment did not reduced this activation, but, on the contrary, increasing doses (5 and 10 mg/kg) were able to restore normal JNK activation. ALA alone and in combination with the CBD present a tendency to reduced JNK activation even if in a non statical manner. We also studied the action of JNK on one of its most important target in the post- synaptic compartment: the PSD95 scaffold protein. We found that a-social mice displayed, in line with JNK activation, a strong increased in PSD95 phosphorylation (p<0.001, Fig. 2). All treatments were able to rescue normal P-PSD95/PSD95 ratio (CBD 2.5 mg/kg p<0,05; CBD 5 mg/kg p<0.001; CBD 10 mg/kg p<0.05; ALA p<0.05; CBD+ALA p<0.05, Fig. 2).

Hippocampus

Immunohistochemical Analysis

In the hippocampus of social condition, we observed a markedly immunopositivity for GFAP along the polymorphic layer of the dentate gyrus. Astrocytes appeared regularly branched and developed (Fig. 3a). In the asocial condition astrocytes were no longer evident and the same pattern could be observed in CBD 2.5 mg/Kg, ALA singular treatments (Fig. 3c-g). Interestingly, the treatment with CBD 10 mg/Kg seemed to revert the astrocytes presence bringing the immunopositivity quite to the social levels (Fig. 3e). Also in this area, the combination of CBD + ALA exerted a positive effect on astrocytes (Fig. 3i). Regarding microglia, we observed a resting morphology in the social condition characterized by the presence of many branches around the cell. In the asocial the morphology appeared more amoeboid and less branched, a signal of an activated microglia typical of a neuro inflammatory context (Fig. 3b). No relevant ameliorations were observed with the CBD 2.5mg/Kg, ALA singular treatments (Fig. 3c, e). The resting morphology seemed to be recovered with CBD lOmg/Kg and the combination of CBD/ALA treatments (Fig. 3i).

Biochemical Analysis

Since no differences were observed between social and a-social groups, we did not show these data and we added it as supplementary information at the end of the report.

Amygdala

Immunohistochemical Analysis

As observed in cerebral cortex images, in the amygdala, astrocytes and microglia appeared more immunopositive in social animals compared to single-housed mice (Fig. 4a, b), suggesting a negative effect exert by isolation on the overall neuroglia ratio. The immunopositivity seemed to increase parallely with the increased concentration of CBD treatments (Fig. 4c-e). No relevant ameliorations were observed with the ALA alone treatments (Fig. 4g). The GFAPimmunoreactive astrocytes became evident with the combination of CBD and ALA, indicating a functional rescue of glia cells. Regarding the microglia, the cells subjected to the combined treatments showed a partial recovery of the resting-branched morphology already observed in the social condition (Fig. 4i). These results suggest a positive effect on neuroglia of both the combined treatments on asocial animals that seems to reduce the neuroinflammation that characterize PTSD and anxiety disorders. Biochemical Analysis

Since the Amygdala is a quite minute brain area, it is impossible to perform biochemical evalutaion with Western Blot.

Claims

1. A pharmaceutical composition comprising Cannabidiol and alpha lipoic acid, wherein cannabidiol is in an amount from 60 to 900 mg and wherein alpha lipoic acid is in an amount from 300 to 900 mg.

2. Composition according to claim 1, wherein cannabidiol is in an amount from 120 to 720 mg and wherein alpha lipoic acid is in an amount from 450 a 750 mg.

3. Composition according to claim 2, wherein cannabidiol is in an amount from 150 to 600 mg and wherein alpha lipoic acid is in an amount from 550 a 650 mg.

4. Composition according to claim 3, wherein cannabidiol is in an amount from 150 to 600 mg and wherein alpha lipoic acid is in an amount of 600 mg.

5. Composition according to claim 1, wherein cannabidiol is in an amount of 150 mg and wherein alpha lipoic acid is in an amount of 600 mg.

6. The pharmaceutical composition according to any one of the preceding claims, further comprising one or more pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to any one of the preceding claims, wherein said pharmaceutical composition is in the form of capsules, tablets, softgels, oral oleolites and parenteral nutrition to support nutrition.

8. A pharmaceutical composition as defined according to anyone of the claims from 1 to 7 for use in reducing or treating:

- post-traumatic stress disorder (PTSD), or

- inflammatory processes, or

- depression related to the reduction of inflammatory processes, or

- sociability related to the reduction of inflammatory processes, or

- anxiety related to the reduction of inflammatory processes, or

- anxiety and obsessive behaviour related to the reduction of inflammatory processes, or

- aggressiveness related to the reduction of inflammatory processes, or - apathy related to the reduction of inflammatory processes, or

- “novel object recognition test” related to the reduction of inflammatory processes.

9. The pharmaceutical composition for use according to the claim 8, wherein a pharmaceutical composition as defined according to anyone of the claims from 1 to 7 is administered daily to the patient.

10. The pharmaceutical composition for use according to claims 8-9, wherein a pharmaceutical composition as defined according to anyone of the claims from 1 to 7 is administered orally.

11. The pharmaceutical composition for use according to claims 8-10, in any one of the treatment as defined in claim 8, wherein the dosage regimen of administration involves the administration amount of cannabidiol from 1 to 15 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid from 5 to 20 mg per kg with respect to the weight of the subject to be treated.

12. The pharmaceutical composition for use according to claim 11, wherein the cannabidiol is in an amount from 2 to 12 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid from 7.5 to 17.5 mg per kg with respect to the weight of the subject to be treated.

13. The pharmaceutical composition for use according to claim 12, wherein the cannabidiol is in an amount from 2.5 a 10 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid from 9 to 15 mg per kg with respect to the weight of the subject to be treated.

14. The pharmaceutical composition for use according to claim 13, wherein the cannabidiol is in an amount from 2.5 a 10 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid 10 mg per kg with respect to the weight of the subject to be treated.

15. The pharmaceutical composition for use according to claim 14, wherein the cannabidiol is in an amount of 2.5 or 5 or 10 mg per kg with respect to the weight of the subject to be treated and alpha lipoic acid 10 mg per kg with respect to the weight of the subject to be treated.

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