IT202000020839A1 - "NEW COMPOSITION FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES" - Google Patents

Description

Description of the patent for industrial invention entitled:

?New composition for the treatment of neurodegenerative diseases?

FIELD OF THE INVENTION

The present invention relates to the field of medicament for treating inflammatory disease. The present invention ? directed to the combination of cannabidiol (CBD) and alpha-lipoic acid, preferably at a specific dosage and ratio, and to its application with ?entourage-like effect? (?entourage effect-like?) in neurodegenerative disorders. Said pharmaceutical composition and related pharmaceutical combination are used in the treatment of the inflammatory disease.

PRIOR ART

Cannabis has a long history of use in medicine, in diseases such as: asthma; depression; epilepsy; tiredness; glaucoma; insomnia; migraine; nausea; ache; rheumatism; tetanus; (Doyle and Spence, 1995; Zuardi, 2006). In the middle In the 1960s, researchers in Israel identified ?9-Tetrahydrocannabinol (?9-THC) as the major psychoactive agent found in the cannabis plant (Mechoulam and Gaoni, 1967). This discovery led to intense cannabinoid research in the 1970s, and renewed interest in the potential therapeutic effects of cannabinoids.

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

Later they were also approved for the treatment of anorexia associated with weight loss in patients with acquired immune deficiency syndrome (AIDS). This progression has followed the model of Western medicine in which individual chemical constituents of plants historically used in medicine are isolated and then proprietary drugs are developed.

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

The warning? what cannabis ? been used for a long time as an intoxicating drug in the absence of need? medical, and most of the organizations that have lobbied to legalize it in the medical field, has recognized that such legalization is? it was also a step forward for legalization for non-medical purposes.

This question? interesting why? there are several important nuances in each approach, which inevitably complicate the answer from both a regulatory and a scientific point of view. Furthermore, in conversations about the medicinal use of cannabis/cannabinoids, there seem to be strong ideological beliefs among patients, doctors and carers, tending to favor only the use of botanical cannabis or pharmaceutical cannabinoids. There are also a number of popular misconceptions (described below) associated with the two approaches that call for better public education as cannabinoid drugs become increasingly popular. common.

CANNABIS AND CANNABINOIDS

The cannabis plant has been shown to be chemically rich, with over 565 known constituents, belonging to 23 classes of compounds (ElSohly and Slade, 2005; ElSohly and Gul 2014; Radwan et al. 2017). Perhaps the most class of compounds? wanted in cannabis ? that of cannabinoids. At the time of this writing, 120 different phytocannabinoids, molecules derived from unique cannabis plants, have been identified, many of which directly modulate the endocannabinoid system. These naturally occurring cannabinoids are distributed into ten subclasses, including ?9- and ?8-THC, cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabinodiol (CBND), cannabielsoin (CBE), cannabicyclol (CBL ), cannabitriol (CBT), and other various types (30 known).

THC is produced as an acid (?9-Tetrahydrocannabinolic, ?9-THCA) in the glandular trichomes of the leaves and inflorescent bracts of the plant and undergoes decarboxylation with age. or heating to form ?9-THC (Turner et al., 1980). The THC? typically the chemical constituent pi? abundant of the cannabis flower, and ? by far the most common cannabinoid studied and well understood. However, cannabinoids are not the only active components of cannabis. Other constituents that may contribute in some way to the effects of cannabis are Terpenes (120 known); Nitrogen 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); phenanthrene (4 known); spiroindans (2 known); xanthones (1 known) and biphenyls (1 known).

In addition to plant-derived phytocannabinoids, hundreds of synthetic exogenous 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, a synthetically derived ?9-THC oral formulation, and ZYN002, a transdermal synthetic CBD gel manufactured by Zynerba Pharmaceuticals), as well as new molecules not found in nature (for example, WIN 55,212-2, JWH-018, AM-2201, AMB-FUBINACA). There are two common misconceptions that we often hear in relation to synthetic versus natural phytocannabinoids. One ? that there are differences in the effects of a single phytocannabinoid molecule (e.g. CBD) depending on whether it is a synthetic phytocannabinoid or a plant derivative. This shouldn't be the case, as the chemistry ? an exact science as to chemical composition and structure. The circumstances in which there? could be true with respect to botanical versus synthetic cannabinoid products would be limited to cases where either substance contains impurities that contribute to the overall pharmacological or toxicological effect, or due to the inappropriate designation of synthetically derived isomers as true replicas of the naturally derived cannabinoids. In these cases, the differences would be due to the impure extraction of cannabinoids of botanical origin or to missteps in the synthesis of the cannabinoid.

The other common misconception? that synthetic cannabinoids, which do not occur naturally in cannabis, are more? harmful phytocannabinoids. This largely stems from the current problems associated with the illicit sales of synthetic CB1 agonists (Ffattore and Fratta, 2011; Vandrey et al., 2012). The potential harm associated with any newly synthesized drug? directly linked to its pharmacological effects (pharmacology, specificity and affinity of receptors, potency) in the organism. Indeed, one of the advantages of pursuing the development of a botanical cannabis drug over synthesized phytocannabinoids? that the cannabis plant has a very well established and positive safety profile.

Both phytocannabinoids and synthetic cannabinoids can directly impact the endocannabinoid system through a variety of effects. of pharmacological mechanisms, including agonism, antagonism and allosteric modulation (for a detailed review of cannabinoid pharmacology see Pertwee, 2008; Pertwee et al., 2010). While 120 phytocannabinoids have been identified, they are limited and somewhat limited with respect to drug interaction with the endocannabinoid system. For this reason, there are clear advantages to focusing on the development of single-molecule synthetic cannabinoid drugs, simply due to the fact that pharmaceutical chemists are able to systematically modify known cannabinoid molecules to achieve very specific pharmacological effects. This type of "tuning" has the potential to produce drugs that have a very specific mechanism of action (e.g. full agonism of CB1 receptors outside the CNS), which could both improve therapeutic efficacy and reduce the adverse effects compared to phytocannabinoids such as THC, which is not? n? selective for a specific cannabinoid receptor subtype n? limited compared to crossing the blood-brain barrier.

"ENTOURAGE" EFFECT

The main rejoinder to the use of single-molecule and synthetic cannabinoid preparations? the possibility? of a unique therapeutic benefit, thanks to the dynamic interaction between the myriad of chemicals present in the cannabis plant and other combinations with phytocomplexes or phytoextracts.

Called the "entourage effect," the unique therapeutic effects of cannabis are hypothesized to be achieved through a complex synergy between phytocannabinoids and 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, together with 2-arachidonoyl glycerol (2-AG), an important endocannabinoid, they reduced the hydrolysis rate of 2-AG, which increased its activity. The term ? was then used by others (Fowler, 2003; Sanchez-Ramos, 2015) to highlight the contribution of other cannabinoids and non-cannabinoid components to the activity? of cannabis preparations. Carlini et al. (1974) provided a first example of the entourage effect by demonstrating that 2 out of 3 cannabis extracts administered more species, including humans, produced effects 2 to 4 times higher than those observed after administration of pure THC at the same doses contained in the extracts.

The limitations of the entourage effect are that, right now, it's not? unclear which compounds drive the effect, which pharmacodynamic effects of cannabis are affected, or whether this can be exploited to enhance therapeutic cannabinoid compounds. Ethan Russo has proposed very clear hypotheses about how selected terpenes contribute to the entourage effect of cannabis, but the empirical research needed to test these hypotheses with cannabis has yet to be completed (Russo, 2011). Furthermore, 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 with dronabinol (synthetic oral THC) and found that dronabinol produced cannabis-like subjective effects. herbal. Similarly, three appropriately powered randomized controlled trials failed to find differences in medicinal effects between synthetic and herbal cannabis preparations versus placebo (Haney et al., 2007; O'Neil et al., 2017 ; Strasser et al., 2006). Using these results to rule out the possibility? of an entourage effect, however, ? premature. However ? Further controlled research in this area is needed.

EXPERIMENTAL DESIGN COMBINATION

One of the fundamental principles of modern medicine? that one must be able to define drugs chemically to the greatest degree possible. A botanical drug such as cannabis requires the definition of its chemical profile and relationship with the other components. The capacity? to characterize, define and demonstrate the consistency in the chemical composition of the combination ? the biggest challenge great for drug development. Due to interactions between the constituent chemical components of the cannabis plant, it is not It is possible to generalize a positive clinical result to a product defined as cannabis botanical.

These problems make it difficult to develop a combination of botanical drugs with cannabis and other plant extracts. This ? the goal of this research.

Conversely, quality control for single molecule synthetically derived drugs? much more simple and ? the standard for the pharmaceutical industry. Are there clear standards to follow and established methods for production, testing and quality control? from start to finish. Monomolecular drugs also have great advantages in clinical trials. In single molecule studies, the drug represents only an independent variable and only a direct effect is tested. The simplicity? of the experimental design makes it more? easy to ensure that clinical trials are well powered to find effects, and interpretation of results ? relatively simple and concise. That said, the identification of target molecules requires a rigorous and lengthy preclinical screening process.

REGULATORY CONSIDERATIONS

Drug development? a complex and often misunderstood arena, and there are unique considerations with respect to cannabinoid drugs. Currently, in the United States and many other countries, cannabis and many of its constituents remain controlled substances. There? requires additional regulatory approvals, safety, and a substantial regulatory burden for any level of botanical drug development, so such as for the development of single molecule drugs, for which local regulations consider the molecule (e.g. THC, CBD) as a controlled substance. For new synthetic cannabinoids (e.g. analogues), ? It is possible that this further regulatory limitation does not apply.

In some circumstances, regulations can completely limit the development of cannabinoids. Additionally, drug providers find themselves in the difficult position of engaging in clinical decision-making regarding patients' use of these products, in the absence of reliable information typically found in a drug's package insert, such as recommended dose, frequency, doses, expected adverse effects, contraindications, comparative efficacy versus alternative therapies, etc.

CANNABIDIOL (CBD)

Cannabidiol (CBD), an important non-psychoactive component of cannabis (Hampson, Grimaldi, Axelrod, & Wink, 1998; Mechoulam, Peters, Murillo-Rodriguez, & Hanus, 2007), has been attributed interesting pharmacological effects that may have clinics in different pathological states (Ligresti, De Petrocellis, & Di marzo, 2016; Pertwee, 2009). In this regard, CBD has the potential to inhibit cancer cell proliferation, metastasis, and tumor growth (Guzm?n, 2003). A number of studies have also shown that CBD elicits vaso-relaxant responses (Stanley, Hind, & O'Sullivan, 2013), exerts anti-emetic and anti-nausea effects, decreases anxiety, and improves depressive behaviors (de Mello Schier et al., 2014). The CBD ? also in possession of interesting properties? antiepileptic drugs, in particular for patients resistant to all conventional antiepileptic drugs (Silvestro, Mammana, Cavalli, Bramanti, & Mazzon, 2019). In addition to this, CBD also has the potential to reduce unwanted side effects of therapeutic treatments and improve their tolerability.

For example, does CBD reduce the toxicity myocardial infarction of the chemotherapeutic agent doxorubicin (Hao et al., 2015) and provides relief against L-DOPA-induced dyskinesia in a model of Parkinson's disease (Dos-Santos-Pereira, da-Silva, Guimar~aes, & Del- Bel, 2016). The CBD ? also capable of exerting neuroprotective effects in a range of experimental settings that model acute or chronic neurodegenerative conditions (Barata et al., 2019; Campos, Foga?a, Sonego, & Guimar~aes, 2016; Garc?a-Arencibia et al ., 2007). CBD also exhibits suppressive effects on the immune system, as evidenced by the enhancement of innate and adaptive immune responses in experimental settings that model chronic inflammatory states (Kozela et al., 2011; Lee et al., 2016). In relation to this, the CBD ? able to reduce airway inflammation and fibrosis in allergic asthma (Vuolo et al., 2019), exert anti-inflammatory effects in nonalcoholic steatohepatitis (Huang et al., 2019), and in a viral model of multiple sclerosis ( Mecha et al., 2013) and improve clinical outcomes in experimental autoimmune encephalomyelitis models (Elliott, Singh, 4 Nagarkatti, & Nagarkatti, 2018). These different effects derive from the ability? of CBD to curb inflammatory responses mediated by immune cells, especially pathogenic T cells (Kozela et al., 2011), activated macrophages (Huang et al., 2019), and inflamed microglial cells (Barata et al., 2019 ; Martín-Moreno et al., 2011; Mecha et al., 2013). Our specific goal in this case was to further explore the mechanisms underlying the anti-inflammatory effects of CBD on microglial cells. For this, we used a microglial cell model system isolated from postnatal mouse brain and an activation paradigm with the bacterial inflammatory agent LPS. We established that CBD exerts potent anti-inflammatory effects on microglial cells by inhibiting NF-?B-dependent reactive oxygen species (ROS) signaling events and glucose-dependent synthesis of NADPH, a co-factor required for the activation of NADPH oxidase and the generation of ROS by this enzyme.

The medical use of Cannabis sativa (Mechoulam R. 1986), ? always been known for its medical purposes and benefits. Based on the evidence collected, the pharmacological effects of Cannabis include anti-nociception, anti-inflammatory, anticonvulsant, antiemetic, as well as such as recreational use, which has largely limited its medical application (Iversen L.). About 120 phytocannabinoids have been identified in the plant, including ?9-tetrahydrocannabinol (THC) ? the main psychoactive component. Chemically, phytocannabinoids occur both as a terpene fused to an alkyl-substituted resorcinol and as a benzopyran ring system. Since the discovery of THC, a large number of synthetic cannabinoids, similar or distinct in structure to phytocannabinoids, have contributed to the identification and cloning of cannabinoid receptor 1 (CB1R), and more? late discovery of the receptor 2 (CB2R). They are located differently throughout the body: CB1 ? strongly concentrated in neurons of the brain and least concentrated in the amygdala, hypothalamus, nucleus accumbens, thalamus, periaqueductal gray matter (PAG), and spinal cord. CB2 ? strongly concentrated in immune cells, including microglia and astrocytes (De Leo et al., 2005; Luongo et al., 2010).

Many functions are regulated and controlled by the CB1 receptor such as: mobility? of the gastrointestinal tract, the secretion of gastric acid fluids, the secretion of neurotransmitters and hormones, the control of appetite from the hypothalamus in the CNS, the regulation of energy balance and food intake. While CB1 activation reduces neurotransmitter release, CB2 activation inhibits microglial activation and reduces neuroinflammation.

Evidence from preclinical studies has already demonstrated the properties benefits of cannabidiol CBD, including neuroprotective effects in central nervous system (CNS) disorders (Fern?ndez-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 (CB1 and CB2). Surprisingly, CBD binds with very low affinity CB1 and CB2, on the other hand, inhibit anandamide uptake and enzymatic hydrolysis (Lastres-Becker et al., 2005), and decrease adenosine reuptake (Carrier et al., 2006). These are all important steps in the endocannabinoid pathways, through which CBD is thought to exert neuroprotective effects. Recent preclinical research has suggested that cannabis and especially CBD may have a beneficial effect in rodent models of post-traumatic stress disorder (PTSD). In general, post-traumatic stress disorder (PTSD) ? 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 symptoms of PTSD are classified into four groups of symptoms: (i) intrusion (including flashbacks and nightmares), (ii) experience of negative alterations in mood and cognition, (iii) escape from memories of the trauma, and (iv) hyperreactivity? and hyperreactivity (including lack of concentration or irritability) (Steenkamp et al., 2016; Pai et al., 2017). Even if the pathophysiology of PTSD is not been definitively described, a number of factors are suspected to contribute to the development of this disorder. One hypothesis relates PTSD to disordered memory retrieval through the process of reconsolidation and altered extinction of adverse memories (Parsons RG, Ressler KJ. 2013). Psychotherapy? recommended as a treatment for PTSD accompanied by pharmacological treatment such as selective serotonin reuptake inhibitors, serotonin/norepinephrine reuptake inhibitors, antiadrenergic agents and second generation antipsychotics. However, ? It is necessary to study new pharmacological tools to improve pharmacological efficacy and minimize side effects. Stress and environmental factors play a fundamental role in the development of maladaptation and behavioral abnormalities. In fact, stressful events negatively affect several neuroendocrine systems, which can cause profound repercussions on both cognitive and emotional processes (McEwen et al., 2015; Pagliaccio et al., 2015; Herman et al., 2016). PTSD? complicated by severe depressive disorders (Shalev, 2001). This staggering rate of comorbidity? can? be partially explained by the presence of overlapping symptoms between the two disorders. Other disorders seen in PTSD patients are the major 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? the increased risk of suicide and suicidal ideation (Pompili et al., 2013). Suicide ? rarely caused by a single factor, but ? rather determined by multiple factors. In addition to the comorbidity with psychiatric conditions (eg, PTSD, depression) and previous suicide attempts, other contributing factors are disorder or personality traits, life stressors, and social and economic problems (Stone et al., 2018) . Therefore, new effective therapies for PTSD complicated by suicidal ideation, such as as, the presentation of new PTSD biomarkers? much in demand.

The isolation due to the COVID-19 epidemic could be one of the causes of the emergence of post-traumatic stress disorder.

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

The Social Isolation (SI) model, which exposes rodents to a sustained and possibly intense or mild stressor, offers a putative animal model for investigating the development of social vulnerability. to PTSD. In rodents, SI can be considered a distressing event that induces behavioral deficits, even if the duration of isolation can vary from one laboratory to another. A pattern of social isolation leading to PTSD could be: isolation in individual cages for 3-4 weeks starting on the 21st? postnatal day; mice that are socially isolated for 3-4 weeks after 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 anxious behavior and aggression and the expression of aggressive behavior, reaching a plateau between the 4th and 6th week of isolation (Pibiri et al., 2008; Pinna et al., 2008; Guidotti et al., 2001; Pinna et al., 2003; Rau et al., 2005; Locci and Pinna, 2019b). Individual detention? is also a powerful stressful condition that can increase susceptibility? to develop behavioral dysfunction when rodents are exposed to an additional acute traumatic stressor, to a series of stressful stimuli such as periodic water and/or food deprivation, exposure to cold temperatures, physical restraint and inversion of light/dark cycle. Other stresses can be induced such as prolonged single stress consisting of three stressors that are administered in succession: restraint stress (2 h), forced swimming (20 min), and exposure to diethyl ether (Liberzon et al. al., 1997, 1999) or the social defeat stress model? mostly performed in male rodents by a resident-intruder test, resulting in aggressive behavior and social stress for the intruder (Bj?rkqvist, 2001; Hammels et al., 2015).

MICROGLIAL CELL

Our specific goal in this case was to further explore the mechanisms underlying the anti-inflammatory effects of CBD towards microglial cells. For this, we used a model system of microglial cells isolated from postnatal mouse brain and activation with the bacterial inflammatory agent LPS (lipopolysaccharide).

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

CANNABIDIOL (CBD) NEUROPROTECTIVE EFFECT

CBD has also been shown to exert neuroprotective effects in a range of experimental settings that model acute or chronic neurodegenerative conditions (Barata et al., 2019; Campos, Foga?a, Sonego, & Guimaraes, 2016; Garc?a-Arencibia et al. , 2007). Of interest, CBD also exhibits suppressive effects on the immune system, as evidenced by the enhancement 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

Alpha-lipoic acid (also referred to as ?-Lipoic acid or LA), a naturally occurring enzyme cofactor with beneficial properties antioxidants and iron chelators (Persson HL, 2003), ? been used as a therapeutic agent for many chronic diseases such as diabetes mellitus (DM) and associated peripheral neuropathy. Importantly, ? LA has also been found to provide neuroprotection against Alzheimer's disease (AD), as it can easily penetrate the blood-brain barrier (BBB). The therapeutic effect of LA for AD ? was found incidentally in clinical trials which 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 SAMP8 mice and aged rats (Farr SA, 2003); reduced hippocampal-dependent memory deficits in the Tg2576 model of AD without affecting ?-amyloid (A?) levels or plaque deposition (Quin JF, 2007), and restored glucose metabolism and plasticity? synaptic in the triple transgenic mouse model of AD (Sancheti H, 2014). In vitro studies related to the mechanisms of AD have revealed that LA can inhibit the formation of amyloid fibrils (fA?) and the stabilization of fA? preformed, as well as protecting cultured hippocampal neurons from neurotoxicity? induced by fA? and iron/hydrogen peroxide (Lowell MA, 2003).

Based on the above facts, ? It has been proposed that these actions are mediated by potent antioxidants (Rochette L, 2013), anti-inflammatories (Maczurek A, 2008) and properties anti-amyloidogenic. Also, isn't LA itself? only an effective free radical scavenger, but the disulfide bond and ring structure with five LA atoms lead to powerful scavenging capabilities. antioxidants and a good activity of iron-chelation.

These multiple effects suggest that LA ? a promising therapeutic agent for AD, but the exact cellular and molecular mechanisms remain unknown, especially with regards to the ability of LA to control Tau pathology and neuronal damage.

In this context, we have previously shown that the hyperphosphorylation state of Tau can be reversed by iron chelation in a mouse model of AD (Guo C, 2013). Indeed, LA plays many different roles in the pathogenic pathways of dementia, acting as a neuroprotective agent. LA could mitigate the damage from free radicals and reduce the activities? inflammatory and, therefore, could have a positive effect on neuronal ferroptosis, which ? a recently discovered form of iron and ROS dependent cell death (Yie Y, 2016), why? ferroptosis can? also be prevented by ferrostain-1, lipophilic antioxidants, and iron binders, such as deferoxamine. In a recent study, P301S mice encoding the human P301S mutation were injected with LA for 10 weeks to investigate whether LA could actually alleviate the AD-related tauopathy state and whether ferroptosis inhibition is a potential mechanism for restore impaired cognition.

DESCRIPTION OF THE INVENTION

COMBINATION OF CANNABIDIOL AND ALPHA-LIPOIC ACID

The combination of alpha lipoic acid with CBD? a valid support against the progression of neurodegenerative pathologies (Alzheimer's, cognitive decline, etc.). The therapeutic combination is developed with CBD from 20 to 1800 mg / day alpha-lipoic acid from 50 mg to 6000 mg / day.

The dosages were tested based on published animal model studies and considering their lipophilicity suitable for blood-brain barrier passage and possible related pharmaceutical forms: capsules, tablets, softgels, oral oleolites and parenteral nutrition to support nutrition.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention ? a pharmaceutical composition comprising cannabidiol and alpha-lipoic acid in a ratio of between 1:0.03 and 1:300 w/w, respectively, and wherein the total weight of cannabidiol and alpha-lipoic acid is ? ranging from 70 to 7800 mg.

According to a preferred embodiment, in the pharmaceutical composition the ratio of cannabidiol and alpha-lipoic acid ? ranging from 1:0.125 to 1:75 wt/wt.

According to a more embodiment preferred, in the pharmaceutical composition the ratio of cannabidiol and alpha-lipoic acid ? ranging from 1:1 to 1:15 weight/weight.

According to one embodiment, the pharmaceutical composition comprises from 20 mg to 1800 mg of cannabidiol and from 50 mg to 6000 mg of alpha-lipoic acid.

According to a more embodiment preferred, the pharmaceutical composition comprises from 40 mg to 800 mg of cannabidiol and from 100 mg to 3,000 mg of alpha-lipoic acid.

According to an embodiment even more? preferred, the pharmaceutical composition comprises from 40 mg to 100 mg of cannabidiol and from 100 mg to 600 mg of alpha-lipoic acid.

According to a preferred embodiment, the pharmaceutical composition further comprises one or more? pharmaceutically acceptable excipients.

According to a preferred embodiment, the pharmaceutical composition ? in the form of capsules, tablets, oral oils and parenteral nutrition to support nutrition.

According to a preferred embodiment, the pharmaceutical composition ? suitable for administration to humans or animals.

According to a preferred aspect cannabidiol and alpha-lipoic acid can be administered simultaneously or sequentially. Therefore ? It is possible to foresee a separate administration of cannabidiol and alpha-lipoic acid.

Another item? a pharmaceutical composition comprising cannabidiol and alpha-lipoic acid for use in the reduction or treatment of at least one of:

- post-traumatic stress disorder (PTSD),

- inflammatory processes,

- depression related to the reduction of inflammatory processes,

- sociability related to the reduction of inflammatory processes,

- anxiety related to the reduction of inflammatory processes,

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

- aggression related to the reduction of inflammatory processes,

- apathy related to the reduction of inflammatory processes, or

- ?new object recognition test? related to the reduction of inflammatory processes.

Preferably, ? envisaged is the pharmaceutical composition comprising cannabidiol and alpha-lipoic acid for use in the alleviation or treatment of post-traumatic stress disorder (PTSD).

According to a preferred embodiment, the pharmaceutical composition comprising cannabidiol and alpha-lipoic acid for the above use, is as defined above.

In particular, the composition of the invention allows to reduce the activity? cerebral microglial, thus reducing the inflammatory processes.

According to a preferred embodiment, ? the use of a pharmaceutical composition as defined above is preferred, said composition comprising: - from 20 mg to 1800 mg of cannabidiol and from 50 mg to 6000 mg of alpha-lipoic acid, or - comprising from 40 mg to 800 mg of cannabidiol and 100 mg to 3000 mg of alpha lipoic acid, or

- comprising 40 mg to 100 mg of cannabidiol and 100 mg to 600 mg of alpha-lipoic acid, or

- including one or more pharmaceutically acceptable excipients;

to be administered daily to the patient.

Another item? a dosage regimen of administering a pharmaceutical composition comprising 20 mg to 1800 mg of cannabidiol and 50 mg to 6000 mg of alpha-lipoic acid per day.

According to a preferred embodiment, ? a dosage regimen comprising 40 mg to 800 mg of cannabidiol and 100 mg to 3000 mg of alpha-lipoic acid per day is preferred.

According to a further preferred embodiment, ? a dosing regimen comprising 40 mg to 100 mg of cannabidiol and 100 mg to 600 mg of alpha-lipoic acid per day is preferred.

According to a more embodiment favorite, ? preferred a dosage regimen comprising about 50 mg of cannabidiol and 600 mg of alpha-lipoic acid per day, based on a 60 kg human being.

According to a preferred aspect, with reference to the above use, said cannabidiol and alpha-lipoic acid can be administered simultaneously or sequentially. Therefore ? It is possible to foresee a separate administration of cannabidiol and alpha-lipoic acid.

EXPERIMENTAL PART ACTIVITY? MICROGLIAL

To evaluate the anti-inflammatory potential of CBD, microglial cells activated by the bacterial cell wall component LPS (10 ng/ml), a prototypical agonist for Toll-like receptor 4 (TLR4; D?ring et al. , 2017). After 24 hours of treatment, LPS strongly stimulated the production/release of the pro-inflammatory cytokine TNF-? is it an entity? lower than that of IL-1?, a cytokine produced in response to inflammasome activation (Mouton-Liger et al., 2018; Weigt, Palchevskiy, Belperio, 2017). When cultures were exposed to 1 and 10 ?M CBD, the production of TNF-? and IL-1? LPS-induced? been efficiently reduced. CBD ? appeared only slightly less effective at 1 ?M than at 10 ?M. At 0.1 ?M, however, CBD did not significantly impact cytokine production, suggesting that a threshold concentration ? been required for anti-inflammatory effects in the present configuration. Of note, the CBD vehicle alone had no effect on LPS response. A reference anti-inflammatory drug, the glucocorticoid dexamethasone (DEX; 2.5 µM) mimicked the inhibitory effects of CBD on cytokine release. Upon LPS treatment, we also measured the release of glutamate, a neurotransmitter that operates as a non-cytokine mediator of microglial cell inflammation. LPS-induced glutamate release? useful for checking the values in the presence of CBD 10 ?M. CBD ? was least effective at 1 ?M, but still reduced extracellular glutamate levels by half. Note that DEX ? been ineffective for reducing LPS-induced glutamate release.

In agreement with the data obtained in a previous study (Dos-Santos-Pereira et al., 2018), a 24-hour treatment with 10 ng/ml LPS resulted in an approximately 20% increase in the number of DAPI nuclei (4 ',6-diamidin-2-phenylindole) that ? indicative of a small proliferative effect of the inflammatory agent. Despite its potential to block cell proliferation reported elsewhere (McAllister et al., 2011), CBD has failed to reduce the number of DAPI nuclei under LPS treatment. Finally, CBD (10 µM) also led to reduced expression of the phenotypic activation marker Iba-1 in LPS-exposed microglial cells. This inhibitory effect was detectable through the quantification of Iba-1 expression by western immunoblotting. ? was confirmed by visual inspection of cultures of LPS-treated microglial cells exposed or unexposed to CBD and then sequentially processed for fluorescence immunodetection of CD11b and Iba-1.

MICROGLIAL STUDY

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

COMBINATION OF INGREDIENTS

The combination involves the use of rat microglial cells in which different combinations of the substances are administered and tested against the control: unstimulated, untreated microglial cells and a positive control: LPS-stimulated microglial cells. The reference ? status: LPS-stimulated microglial cells treated with dexamethasone or corticosteroids (FAS).

The combination involves evaluating:

? LPS-stimulated microglial cells treated with 1 natural compound alone (3 concentrations)

? LPS-stimulated microglial cells with 1 natural compound (concentrations) alpha-lipoic acid (3 concentrations)

STUDY OF ANIMAL MODELS

The interest of the study will be that of investigating whether treatment with the compound provided could exert beneficial effects on cognitive and behavioral dysfunctions associated with a preclinical model of PTSD (Post Traumatic Stress Disorders).

? a behavioral situation such as:

? Tail suspension to evaluate depression,

? Three-chamber test to evaluate social interaction,

? Field test or marble buyring test for anxiety and OCD,

? Resident-intruder test for aggression

? Novelty suppressed feeding test for apathy and anhedonia? New object cognition test to measure cognition

Depression: tail suspension test

This test will come used to assess depressive-like behavior. Mice will be suspended individually by their tails on a horizontal bar (55 cm from the floor) using an adhesive tape placed approximately 4 cm from the tail end. of the tail. The duration of immobility, recorded in seconds, will be? monitored during the last 4 minutes of the 6-minute test through a time logger. The time of immobility? will be defined as the absence of escape-oriented behavior. Mice will be considered immobile when they show no body movement, passively suspended, and completely immobile. (Belardo et al. 2019)

Sociability: sociability test three chambers

This test? used for the assessment of social interaction using a three-chamber social interaction apparatus. Sar? built a custom plexiglass three-chamber box as follows: the entrances in the two dividing walls have a sliding cover to control access to the chambers from the outside. The test consists of two consecutive stages each of 5 and 10 min. During the first 5-min stage of adaptation, the mouse will be? allowed to freely explore the three chambers of the apparatus, noting in this stage any preference of innate sides. After that, the mouse will be gently encouraged into the central chamber and here briefly confined by closing the doors of the side chambers. During subsequent 10-min stadium sessions, a custom stainless steel barred cup (6.5cm ? 15cm) will be used. placed upside down in one of the side chambers. A stranger never met before, previously accustomed, will be? placed in an identical cup upside down in the other room. Time spent sniffing each cup upside down, time spent in each chamber, and number of entries into each chamber will be recorded. (Belardo et al. 2019)

Anxiety: open field test

This test? been commonly used to evaluate the? activity? movement and anxiety. The mice will be placed in an OFT arena (80 ? 80 ? 15 cm3), and the activity? outpatient (total walking distance in centimeters), frequency, and total duration of core zone visits will be recorded for 20 minutes and analyzed. (De Gregorio et al. 2019) Anxiety and obsessive-compulsive behavior: Marble-burying

In this test the mice will be placed singly in a cage (21 ? 38 ? 14 cm long x wide x high) containing a 5 cm layer of sawdust and glass beads (1.5 cm in diameter) arranged in three rows. Mice will be left undisturbed for 15 min in penumbra conditions. An experimenter, blinded to treatment, will count? the time spent in the search behavior and the number of ?bits of marbles? (at least two or three covered in sand). At the end of the test the animal will be? removed in its cage. (Guide et al. 2015)

Aggressiveness: intruder-resident test

Mice will be placed singly for one week in the Plexiglas cage to establish a familiar territory and increase the aggression of the resident experimental mouse. To begin, the food containers will be removed and an intruding mouse, of the same sex but different species, will appear. placed in a resident family cage and resident intruder interactions will be analyzed for 10 min. Aggressive behavior of socially resident isolated mice will be characterized by an initial pattern of activity? exploration around the intruder, who will be? followed by body lifting and tail flapping, accompanied within seconds by grappling and/or vicious biting attacks. The number of these attacks and latency to first attack during the 10 min observation will be recorded. (Belardo et al. 2019)

Apathy: test of power repressed by novelty?

This test is used to evaluate anxiety. Will the latency be measured as the time it takes for mice to consume 3 pieces of food pellets spread across the central area of an unfamiliar arena (80 ? 80 ? 30 cm3) after 48-hour food deprivation, as described previously. Before the test, the power supply latency will be? observed in the familiar domestic cage. A time of cut-off time sar? of 10 minutes. (De Gregorio et al. 2019)

New object recognition test

Novel Object Recognition (NOR) task sar? used to measure learning and long-term memory. Two identical objects will be placed in the arena during a 6 min sample phase. Subsequently, one of the objects will be? exchanged with a new object and the memory sar? assessed by comparing the time spent exploring the new object versus the time spent exploring the familiar object during a 5 min test phase. One week prior to the NOR experiments, the animals will experience handling through the experimenter and arena habituation for 2 and 3 consecutive days, respectively. In the handling procedure sar? including exposure to the transparent plexiglass tunnel with a length of 12 cm and a diameter of 6 cm. Using this tunnel, an animal will be able to transferred to a new cage and after a few minutes back to the familiar cage again. For adaptation, mice will be placed in an empty arena (38 ? 38 ? 30 cm, PVC) for 5 min. During all experiments the arena will be? illuminated with 60?90 Lux (lm/m2). For the NOR experiments custom-made pieces of plastic (Polyoxymethylene, POM) will be used, with different shapes (cones: diameter 4 cm, height 6 cm, pyramids: 4 ? 4 ? 4?6 cm) and different colors (white, black , red). Objects will be thoroughly cleaned with 70% ethanol followed by distilled water between experiments to remove olfactory cues. During the sample phase, on the first day of the NOR test, the mice will be allowed to explore two identical black and white objects (or two cones or two pyramids) for 6 min. For the short-delay test phase (1.5 h) a sample of the objects will be? replaced by a new one (cone-pyramid and vice versa) and exploration (L. Bolz et al.). The pattern separation during the recognition of the new object will be? measured for 5 min. For the long-delayed test phase (24 h) the new object will be? replaced by yet another new item. The positioning of the new object in 24 hours will be? always different from the one in 1,5 h, or first left then right, or vice versa. Consequently, the placement of the familiar object changes between the two phases of the test. Objects with the same color but different shapes will be considered to be similar to the reference object. Objects with different shape and with different colors (red, black, white) will be considered to be distinct from the original sample objects. To analyze the strictly active exploration the time will be? measured manually using a digital stopwatch. Active exploration will be defined as direct sniffing or whisking at objects or direct nose contact. Climbing on objects will not be possible. counted as exploration. The relative exploration will be quantified by normalizing the difference between the exploration time of the new (Tn) and familiar (Tf) object by the total exploration time (Ttot) to calculate the NOR discrimination index: NOR index = (Tn?Tf)/Ttot .

Below is the summary table 1 which correlates the dosages on animal models with those on humans intended as a standard at 60 kg.

TABLE 1

Conversion formula:

HEDmg/kg= Animal-dose mg/kg*Animal Km/human Km

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

*Km ? a standard value (see FDA guideline).

Claims (13)

1. A pharmaceutical composition comprising cannabidiol and alpha-lipoic acid in a ratio of 1:0.03 to 1:300 w/w, respectively, and wherein the total weight of cannabidiol and alpha-lipoic acid is ? ranging from 70 to 7800 mg. 2. The pharmaceutical composition according to claim 1, wherein the ratio of cannabidiol to alpha lipoic acid is ? ranging from 1:0.125 to 1:75 wt/wt. 3. The pharmaceutical composition according to claim 2, wherein the ratio of cannabidiol to alpha lipoic acid is ? ranging from 1:1 to 1:15 weight/weight. The pharmaceutical composition according to claim 1, comprising from 20 mg to 1800 mg of cannabidiol and from 50 mg to 6000 mg of alpha-lipoic acid. The pharmaceutical composition according to claim 4, comprising from 40 mg to 800 mg of cannabidiol and from 100 mg to 3,000 mg of alpha-lipoic acid. The pharmaceutical composition according to claim 5, comprising from 40 mg to 100 mg of cannabidiol and from 100 mg to 600 mg of alpha-lipoic acid. 7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising one or more? pharmaceutically acceptable excipients. 8. The pharmaceutical composition according to any one of claims 1 to 7, wherein said pharmaceutical composition is in the form of capsules, tablets, softgels, oral oleolites and parenteral nutrition to support nutrition. 9. A pharmaceutical composition comprising cannabidiol and alpha-lipoic acid for use in the reduction or treatment of at least one of: - post-traumatic stress disorder (PTSD), - inflammatory processes, - depression related to the reduction of inflammatory processes, - sociability related to the reduction of inflammatory processes, - anxiety related to the reduction of inflammatory processes, - anxiety and obsessive behavior related to the reduction of inflammatory processes, - aggression? related to the reduction of inflammatory processes, - apathy related to the reduction of inflammatory processes, or - ?new object recognition test? related to the reduction of inflammatory processes. 10. The pharmaceutical composition for use according to claim 9, wherein said pharmaceutical composition is defined according to any one of claims 1 to 8. The pharmaceutical composition for use according to claim 9, wherein a pharmaceutical composition as defined according to any one of claims 4 to 7 is administered daily to the patient. 12. A dosage regimen of administration of a pharmaceutical composition comprising 20 mg to 1800 mg of cannabidiol or 50 mg to 6000 mg of alpha lipoic acid per day. The administration dosage regimen according to claim 12, comprising 40 mg to 800 mg of cannabidiol and 100 mg to 3000 mg of alpha-lipoic acid per day.