EP4680213A1 - Composition containing beta-caryophyllene, docosahexaenoic acid and eugenol for use as analgesic and anti-inflammatory antioxidant for the central and peripheral nervous system - Google Patents

Abstract

A composition containing p-caryophyllene, docosahexaenoic acid and eugenol for use as analgesic and anti-inflammatory antioxidant for the central and peripheral nervous system to be administered by oral route as analgesic for chronic pain conditions, chronic inflammatory pain, chronic neuralgia and as anti-inflammatory antioxidant for the central and peripheral nervous system is described.

Description

COMPOSITION CONTAINING BETA-CARYOPHYLLENE, DOCOSAHEXAENOIC ACID AND EUGENOL FOR USE AS ANALGESIC AND ANTI-INFLAMMATORY ANTIOXIDANT FOR THE CENTRAL AND PERIPHERAL NERVOUS SYSTEM

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DESCRIPTION

The present invention relates to a composition containing [3-caryophyllene, docosahexaenoic acid and eugenol for use as analgesic and antiinflammatory antioxidant for the central and peripheral nervous system. In particular, the composition containing p-caryophyllene, docosahexaenoic acid and eugenol is administered by oral route and it is effective as analgesic for chronic pain conditions, chronic inflammatory pain, chronic neuralgia and as antiinflammatory antioxidant for the central and peripheral nervous system.

Background Art

It is estimated that about 10% of the world population suffers from chronic pains [Wong et al., Examining the effects of low back pain and mental health symptoms on healthcare utilisation and costs: A protocol for a population-based cohort study. BMJ Open 2019, 9, e031749]. All pain syndromes, including arthritis, backache, cervical pain, fibromyalgia, interstitial cystitis, migraine, neuropathic pain, bursitis and vulvodynia, have a specific inflammatory profile. The “pain theory” states that the origin of all pain is inflammation and the response to inflammation. Activation of pain receptors, transmission and modulation of pain signals, neuroplasticity and central sensitization are a continuum of inflammation and inflammatory response [Omoigui S. The biochemical origin of pain-proposing a new law of pain: the origin of all pain is inflammation and the inflammatory response. Part 2 of 3-a unifying law of pain. Med Hypotheses.

2007, 69(1), 70-82],

Specialized primary afferent neurons having the function to detect noxious chemical, thermal and mechanical stimuli are called nociceptors. Their cellular bodies, which are mainly in trigeminal nerve and in dorsal root ganglion (DRG), provide sensory innervation to almost all tissues, except brain parenchyma. Specialized receptors, channels and synthetic pathways contribute to define the specificity of particular nociceptor subtypes, allowing the detection and signaling of both acute and persistent noxious stimuli. Two main receptor-channels have been identified. The first one is TRPV1 (transient receptor potential cation channel subfamily V member 1), which is considered as an integrator of multiple noxious stimuli since it is modulated by several inflammation products [Schumacher et al., TRPV1 splice variants: structure and function.

RECTIFIED SHEET (RULE 91) ISA/EP Front Biosci (Landmark Ed). 2010,15(3):872-82], The second channel bound to the transient potential expressed on nociceptors is TRPA1 (transient receptor potential cation channel subfamily A member 1), which is considered as a gatekeeper for inflammation. TRPA1 is thought to play an important and maybe complementary role to TRPV1 in the development and maintainance of inflammatory pain conditions; in fact, TRPA1 is activated by both exogenous and endogenous inflammatory mediators [e.g. reactive oxygen species (ROS)]. TRPA1 has been linked to persistent models of inflammatory pain, to mechanical and thermal ipersensitivity, to inflammatory muscle pain and to pain due to pancreatitis [Bautista eta/., TRPA1 : A gatekeeper for inflammation. Annu Rev Physiol. 2013,75,181-200; Koivisto etal., TRPA1 : a transducer and amplifier of pain and inflammation. Basic Clin Pharmacol Toxicol. 2014,114(1):50-5],

In chronic pain, immune system cells strongly affect the neuronal function not only in inflamed tissues but also in damaged peripheral nerves and in CNS.

Signs and symptoms of inflammation include cell migration, edema, fever, erythema, pain and hyperalgesia. A spontaneous pain often develops along with inflammation development and the inflamed tissue may show hyperalgesia, that is an increase of pain following stimulation, comprising an increase of pain following noxious stimuli as well as a lowering of pain threshold (allodynia).

The cells contributing to inflammatory pain are mast cells, which release inflammatory mediators, in particular cytokines [Stassen etal. Classical and alternative pathways of mast cell activation. Crit. Rev. Immunol. 2002, 22, 115-140] and activated macrophages which can release many inflammatory mediators, such as pro-inflammatory cytokines, in particular tumor necrosis factor a (TNF-a) and interleukin-1 (IL-10), nerve growth factor (NGF), nitric oxide (NO) and prostanoids. Macrophage activation seems to have a remarkable effect on the subsequent recruitment and activation of other types of cells (neutrophils) at the inflammation site [Thomazzi et al., Tumor necrosis factor, interleukin-1 and interleukin-8 mediate the nociceptive activity of supernatant of LPS- stimulated macrophages. Mediators Inflamm. 1997, 6, 195-200]. Neutrophils are the first type of inflammatory cell which leaks into the tissues from blood and control acute and early inflammatory responses. They can produce several inflammatory factors, including lipoxygenase products, NO, cytokines and chemokines, and it is known that preventing the accumulation of neutrophils in inflammation reduces the associated inflammatory pain [Chou et al., The antiinflammatory and analgesic effects of baicalin in carrageenan-evoked thermal hyperalgesia. Anesth. Analg. 2003, 97, 1724-1729]. T and B

RECTIFIED SHEET (RULE 91) ISA/EP cells can produce inflammatory cytokines and chemokines too and contribute to natural immune responses [Sibilia J., Novel concepts and treatments for autoimmune disease: ten focal points. Joint Bone Spine 2004, 71 ,511-517].

Immune cells release many potentially algogenic substances in inflamed tissues, including TNF-a, IL-i p, NGF, prostaglandin E2 (PGE2), lipoxygenase products (leukotriene B), bradykinin, serotonin, cytokines (IL-6 and leukemia inhibitory factor), chemokines (CCL2, CXCL8 and GCSF) and NO [McMahon ef al., in Textbook of Pain (eds McMahon, S.B. & Koltzenburg, M.) Chapter 3, Elsevier, London, 2006; Omote et al., Peripheral nitric oxide in carrageenan induced inflammation. Brain Res. 2001 , 912, 171-175], The antagonism of each mentioned algogenic mediators produces a substantial antihyperalgesia, often close to 100%.

Mast cells, neutrophils, macrophages and T cells are also involved in neuropathic pain resulting from many forms of nerve damage. Several studies demonstrated a correlation between the level of TNF-a, considered as the prototype of pro- inflammatory cytokines, and the development of allodynia or hyperalgesia [George et al., Serial determination of tumor necrosis factor-a content in rat sciatic nerve after chronic constriction injury. Exp. Neurol. 1999, 160, 124-132], Other cytokines such as I L-1 p and IL-6 are involved in neurophatic pain [Schafers et al., Combined epineurial therapy with neutralizing antibodies to tumor necrosis factor-a and interleukin-1 receptor has an additive effect in reducing neurophatic pain in mice. Neurosci. Lett. 2001 , 310, 113-116; De Jongh et al., The role of interleukin-6 in nociception and pain. Anesth Analg. 2003, 96, 1096-1103], Peripheral nerve injuries leading to neuropathic pain conditions result in disease at DRG and at the damaged peripheral nerve and also in a number of changes in the central processing of the sensorial information. These changes are indirect because CNS itself is not damaged. Such central alterations include changes of the immune cell function, among which hematogen leukocytes and microglias were particularly studied. Microglias, oligodendrocytes and astrocytes form a wide group of CNS glial cells. Microglias express the same surface markers as macrophages/monocytes, they are activated by events such as CNS injuries, microbial invasion and some pain conditions, which lead to increase the production of several inflammatory cytokines, chemokynes and other substances potentially able to produce pain. Microglial activation contributes to neuropathic pain after a peripheral nerve injury. Several studies demonstrated that specific inhibitors and/or modulators of microglia can block and/or reverse neuropathic conditions [Meller et al., The possible role of glia in nociceptive

RECTIFIED SHEET (RULE 91) ISA/EP processing and hyperalgesia in the spinal cord of the rat. Neuropharmacology 1994, 33, 1471- 1478] and showed that microglia is responsible for the beginning of neuropathic pain conditions and astrocytes are involved in their maintainance. The released cytokines (TNF-a and IL-6) can be in their turn involved in microglial activation [Winkelstein etal., Nerve injury proximal or distal to DRG induces similar spinal glial activation and selective cytokine expression but differential behavioral responses to pharmacological treatment. J. Comp. Neurol. 2001 , 439, 127-139], The injuries of the peripheral nerve associated with neuropathic pain induce microglia activation in spinal cord and then astrocyte activation in the involved spinal cord segments. This process appears to be critical to the complete appearance of neuropathic pain. Under patho-physiological conditions, microglia can release several mediators, such as IL-1 p, TNF-a, PGE2 and NO, which modulate the processing of spinal pain in several ways.

Injuries and diseases directly affecting CNS can also induce a strong immune reaction. In particular, spinal cord injuries and multiple sclerosis, Parkinson’s disease and Alzheimer disease can be associated with abnormal sensitivity to pain. As in other tissues, spinal cord injury leads to local inflammatory responses and to the activation of immune cells, which are similar to those previously described, with the microglia activation, the mitochondrial disfunction and the release of pro-inflammatory cytokines and the production of reactive oxygen species [Seo et al., Exercise and neuroinflammation in health and disease. Int. Neurourol. J. 2019, 23, 82-S92]. Then, the immune cells can act at many anatomical levels: in peripheral tissues in the inflammation phase, in peripheral nerves and in spinal cord in case of peripheral neuropathy and in some forms of marrow injuries. Under these conditions a wide range of immune mediators is released, some of which can affect the pain signaling systems.

In conclusion, chronic pain, as inflammatory pain, neuropathic pain or pain associated with CNS injuries, is a debilitating disease wherein the role of immune mediators, particularly pro-inflammatory cytokines, is important.

First line pharmacological treatments for chronic pain include opioids and nonsteroidal antiinflammatory drugs (NSAIDs), both associated with severe side effects, such as tolerance/addiction for opioids and gastralgias, gastric bleeding/ulcers for NSAIDs [Alois! et al., Hormone replacement therapy in morphine-induced hypogonadic male chronic pain patients. Reproductive Biology and Endocrinology, 2011 , vol. 9, article 26, 2011;

RECTIFIED SHEET (RULE 91) ISA/EP de Maddalena et al., Opioid-induced hypogonadism: why and how to treat it. Pain Physician, 2012 vol. 15, no. 3, pp. ES111 -ES1 18],

Therefore, there exists a need for novel pharmacological approaches which limit the progression of the disease and reduce the pain without causing side effects.

Substances of plant origin and plant extracts, popularly known as natural analgesics, are of particular interest among these approaches.

The combination of two substances of natural origin, p-caryophyllene (BCP) and docosahexaenoic acid (DHA), has been studied in vitro and in vivo for its analgesic efficacy [Fiorenzani et al., In Vitro and In vivo Characterization of the New Analgesic Combination Beta-Caryophyllene and Docosahexaenoic Acid. Evid Based Complement Alternat Med. 2014, 2014, Article 596312], In the study, it has been demonstrated that BCP, alone or in combination with DHA, remarkably decreases responses to pain induced by formalin. Moreover, in vitro acute toxicity tests (on fibroblasts and astrocytes) showed that BCP is toxic at high concentrations but its toxicity is completely cancelled by the administration with DHA in a combination about 1 :1 by weigth. The Authors hypothesize that the synergical activity of the BCP+DHA combination is probably due to the fact that both substances show competitive binding/interaction for the same receptor since the modulation was observed also in vitro.

Summary of the invention

In order to further improve the analgesic efficacy of the BCP+DHA combination, the present inventor found that the addition of specific amounts of eugenol to the BCP+DHA combination significantly enhances the analgesic efficacy and the antiinflammatory antioxidant properties.

This specific combination of BCP, DHA and eugenol is particularly efficient in the treatment of chronic pain and as anti-inflammatory antioxidant for the central and peripheral nervous system since it induces the production of anti-inflammatory/pro- resolving cytokines and the decrease of pro-inflammatory cytokines.

Therefore, object of the present invention is a composition containing p-caryophyllene (BCP), docosahexaenoic acid (DHA) and eugenol for use as analgesic and antiinflammatory antioxidant for the central and peripheral nervous system. In particular, the composition contains 0.9 - 1.1 parts by weight of BCP, 0.9 - 1 .1 parts by weight of DHA and 1 .3 - 2.0 parts by weight of eugenol.

RECTIFIED SHEET (RULE 91) ISA/EP More preferably, the composition object of the present invention contains equal parts of BCP and DHA and 1.5 parts by weight of eugenol.

Brief description of the figures

Fig. 1 A - graph reporting the concentrations of IL-6 (pg/mL) dosed in the supernatant of U373-MG cells cultured in the presence of samples 1-11.

Fig. 1 B - graph reporting the %variations of IL-6 for samples 5-11 compared to positive control (IL-1 [3 - sample 4).

Fig. 2A - graph reporting the concentrations of IL-10 (pg/mL) dosed in the supernatant of U373-MG cells cultured in the presence of samples 1-11.

Fig. 2B - graph reporting the %variations of IL-10 for samples 5-11 compared to positive control (IL-1 [3 - sample 4).

Fig. 3A - graph reporting the concentrations of IL-4 (pg/mL) dosed in the supernatant of U373-MG cells cultured in the presence of samples 1-11.

Fig. 3B - graph reporting the %variations of IL-4 for samples 5-11 compared to positive control (IL-1 [3 - sample 4).

Fig. 4A - graph reporting the concentrations of TNF-a (pg/mL) dosed in the supernatant of U373-MG cells cultured in the presence of samples 1-11.

Fig. 4B - graph reporting the %variations of TNF-a for samples 5-11 compared to positive control (IL-1 [3 - sample 4).

Detailed description of the invention

Object of the present invention is a composition containing [3-caryophyllene (BCP), docosahexaenoic acid (DHA) and eugenol for use as analgesic and anti-inflammatory antioxidant for the central and peripheral nervous system. In particular, the composition contains 0.9 - 1.1 parts by weight of BCP, 0.9 - 1.1 parts by weight of DHA and 1 .3 - 2.0 parts by weight of eugenol.

More preferably, the composition object of the present invention contains equal parts of BCP and DHA and 1.5 parts by weight of eugenol.

BCP, DHA and eugenol are substances of natural origin widely known and used for years for their several beneficial properties.

B-caryophyllene

[3-caryophyllene (herein also referred to as BCP) is a bicyclic sesquiterpene widely spread in the plant kingdom, where it contributes to the unique scent of essential oils and has a primary role in the survival and evolution of higher plants. As a selective agonist of cannabinoid receptor 2 (CB2), it has several pharmacological activities,

RECTIFIED SHEET (RULE 91) ISA/EP such as antibacterial (e.g., Helicobacter pylori), antioxidant, anti-inflammatory, analgesic (e.g., neuropatic pain), anti-neurodegenerative and antitumoral properties. An increasing number of studies described multiple protective effects of BCP in several metabolic and neural disorders. In particular, these disorders are mostly characterized by chronic inflammation [Scandiffio et al., Protective Effects of (E)-p- Caryophyllene (BCP) in Chronic Inflammation. Nutrients. 2020, Oct 26, 12(11):3273]. The experimental results show the BCP ability in decreasing pro-inflammatory mediators such as TNF-a, IL-i p, IL-6, nuclear factor NF-KB, thereby ameliorating chronic pathologies characterized by inflammation and oxidative stress, in particular metabolic and neurological diseases. Through the binding with cannabinoid receptors CB2 and the interaction with the members of the family of receptors activated by peroxisome proliferator (PPAR, in particular PPAR a and y), BCP shows beneficial effects in obesity, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), diabetes, cardiovascular diseases, chronic pain and other nervous system disorders [Gertsch etal., Beta-caryophyllene is a dietary cannabinoid. Proc. Natl. Acad. Sci. USA 2008, 105, 9099-9104; Youssef et al., Beta-caryophyllene protects against diet-induced dyslipidemia and vascular inflammation in rats: Involvement of CB2 and PPAR-y receptors. Chem. Biol. Interact. 2019, 297, 16-24].

Therefore, it is already widely known that BCP acts in several molecular pathways involved in generating inflammatory conditions and that it is able to decrease several pro-inflammatory mediators, including IL-ip, IL-6, TNF-a, NF-KB. Published data suggest that BCP is able to exercise its strong anti-inflammatory effects through multiple mechanisms, mostly started by BCP binding to CB2 receptors.

DHA

The beneficial properties of co-3 polyunsaturated fatty acids (PUFAs) in the diet, in particular docosahexaenoic acid (DHA), are known since decades and their metabolic dysfunction has been related to a number of diseases, including inflammatory and neurodegenerative disorders [Echeverria et al., Docosahexaenoic acid (DHA), a fundamental fatty acid for the brain: new dietary sources. Prostaglandins Leukot Essent Fatty Acids 2017, 124, 1 -10], DHA is an important component of neural membranes, since it regulates fluidity, permeability and viscosity of synaptic membranes and plays a key role in modulating neurotransmission and synaptic function. Whilst still today it is not clear the reason why cerebral phospholipids are specifically rich in DHA and poor in DPA (docosapentaenoic acid) and EPA (eicosapentaenoic acid), the fact that the

RECTIFIED SHEET (RULE 91) ISA/EP enrichment in DHA is conserved in all species prompts the existence of a highly specific requirement for this essential fatty acid in the neuronal membrane [Farkas et al., Docosahexaenoic acid-containing phospholipid molecular species in brains of vertebrates. Proc Natl Acad Sci USA 2000, 12, 6362-6366],

Once converted into bioactive metabolites, EPA and DHA are required to resolve the inflammation through natural endogenous regulators of the immune system; in fact, the deficiency of these PUFAs is related to chronic inflammatory diseases [Zhang et al., Resolvins: anti-inflammatory and proresolving mediators derived from omega-3 polyunsaturated fatty acids. Annual Review of Nutrition 2012, 32, 203-227]. Most neuroprotective effects of omega-3 oils result from the DHA component rather than from the EPA component. DHA has strong antioxidant and anti-inflammatory properties [Cole et al., DHA may prevent age-related dementia. Journal of Nutrition 2010, 140(4), 869-874], increases also lipoxines, inhibits NF-KB and produces neuroprotectin- D and resolvins, which are important for the complete resolution of the inflammatory process so minimizing the risk of chronicization and exacerbation of pain [Kumar etal., Robbins and Cotran Pathologic Basis of Disease, W.B. Saunders, Philadelphia, Pa, USA, 2005].

DHA is essential for the CNS functions, including the neuronal growth, neuronal survival, synaptic integrity and neurotransmission as well as the protection from neuroinflammatory processes and cognitive impairment [Wu et al., The salutary effect of DHA dietary supplementation on cognition, neuroplasticity, and membrane homeostasis after brain trauma. J Neurotrauma 2011 , 10, 2113-212],

The role of DHA in inflammation is well known. In fact, DHA acts as metabolic precursor for resolvins, neuroprotectins and maresins, which are anti-inflammatory molecules involved in the spontaneous but active process of inflammation resolution mainly involving myeloid cells such as macrophages and glial cells [Serhan C.N., Proresolving lipid mediators are leads for resolution physiology. Nature 2014, 7503, 92-101], The impairment of the systemic synthesis of DHA in mice outlines dysfunctions of neuronal plasticity and cerebral inflammation. In mouse models Elovl2-/- (key enzyme for DHA synthesis), a significant underregulation of several neural plasticity factors (Arc-1 , Egr-1 and BDNF) and a concurrent overregulation of the primary inflammatory markers (TNF-a, IL-1 p, iNOS e Caspl) are found. In this model, the reintroduction of DHA in KO mice significantly increased the expression levels of Arc-1 mRNA and decreased those of IL-1 and Caspl compared to KO mice, with IL-1 p and Caspl

RECTIFIED SHEET (RULE 91) ISA/EP which have shown levels comparable to those of wildtype mice [Talamonti et al., Impairment of DHA synthesis alters the expression of neuronal plasticity markers and the brain inflammatory status in mice. FASEB J., 2020, 34(2), 2024-2040],

As already underlined, the inventive feature of the present disclosure is represented by the presence of eugenol in combination with BCP and DHA.

Eugenol is a phenolic aromatic substance, belonging to the allylbenzene class, naturally occurring in the essential oils of several plants of the Lamiaceae, Lauraceae, Myrtaceae and Myristicaceae families. In particular, it is one of the main components of clove essential oil (Syzygium aromaticum L). Eugenol is classified as GRAS (Generally Recognised as Safe) and it is widely used at industrial level as flavouring agent (both in foods and cosmetics) as well as in the pharmaceutical and dental sectors (as antimicrobial and local anesthetic). Eugenol is known to have remarkable pharmacological properties because it proved to be efficient in the treatment of several diseases of the reproductive, nervous, digestive and respiratory apparatus, is capable of regulating glucose and cholesterol levels in blood and has antihypertensive, antioxidant, anti-inflammatory, antimicrobial and antitumoral properties [Petrocelli et al., Molecules present in plant essential oils for prevention and treatment of colorectal cancer (CRC). Molecules. 2021 , 26, 885], Eugenol may interfere with a number of intracellular signaling pathways by counteracting oxidative stress and chronic inflammation. At chemical level, it has an extraordinary reducing activity because it donates hydroxy groups which react with free radicals. In view of these features, eugenol is considered as a “booster” for the immune system functionality. After administration of eugenol, a decrease in the expression of several mediators of the inflammatory process (such as TNF-a, NF-KB, COX2, I L-1 p, IL-5, IL-6, iNOS and NO) associated with an increase of antioxidant enzymes (such as superoxide dismutase, glutathione-peroxidase, catalase and glutathione-reductase) has been observed. Eugenol acts on the inflammation mediators derived from the arachidonic acid (AA) cascade, by inhibiting the production of prostaglandins and leukotrienes. In mouse model of inflammation induced by LPS, eugenol reduces the pulmonary infiltration by neutrophils/macrophages and inhibits the release of inflammatory cytokines (TNF-a, IL-1 |3 and IL-6) by inhibiting the activation of the NF-KB signaling pathway [Nisar et al., Pharmacological Properties and Health Benefits of Eugenol: A Comprehensive Review. Oxidative medicine and cellular longevity. 2021 , 2497354]. Eugenol

RECTIFIED SHEET (RULE 91) ISA/EP also shows antipyretic activity [Feng et al., Eugenol: antipyretic activity in rabbits. Neuropharmacology. 1987, 26(12), 1775-1778],

Eugenol has also a neuroprotective effect. Thanks to its hydrophobic properties, orally administered eugenol crosses the blood-brain barrier and acts in situ to inhibit lipid peroxidase and enhance endogenous antioxidant mechanisms [Singh et al., In vivo antioxidative and neuroprotective effect of 4-allyl-2-methoxyphenol against chlorpyrifos induced neurotoxicity in rat brain. Mol Cell Biochem 2014, 388, 61-74], For instance, in a mouse model of cerebral damage induced by aluminum, the eugenol neuroprotective role occured thanks to its antioxidant and antiapoptotic potential, as well as to its neurotrophic property [Said ef al., Neuroprotective effects of eugenol against aluminium induced toxicity in the rat brain. Arh Hig Rada Toksikol. 2017 Mar 1 , 68(1), 27-37],

The eugenol ability to inhibit apoptosis and pro-inflammatory cytokine secretion and the resulting protection of neuronal cells is linked to its ability to counterbalance oxidative stress and to regulate inflammatory cytokine expression. In fact, eugenol inhibits the pro-inflammatory mediators in activated macrophages and the activation of NF-KB induced by TNF-a in cell lines of human acute myeloblastic leukemia [Li et al., Inhibitory action of eugenol compounds on the production of nitric oxide in RA W264.7 macrophages. Biomed Res 2006, 27, 69-74; Chainy et al., Anethole blocks both early and late cellular responses transduced by tumor necrosis factor: effect on NF-kappaB, AP-1 , JNK, MAPKK and apoptosis. Oncogene 2000, 19, 2943-50], Overall, eugenol exerts multiple, direct and indirect, actions to restore a normal neurotrophic and serotoninergic balance. It also normalizes the acetylcholinesterase level and shows a beneficial effect on glycogen basal synthesis in mouse primary astrocytes [Sartorius et al., Cinnamon extract improves insulin sensitivity in the brain al lowers liver fat in mouse models of obesity. PLoS One 2014, 9:e92358], Moreover, eugenol has likely a direct or indirect effect in preserving cerebral astrocytes from induced neurotoxicity.

In a mouse model of neuronal degeneration similar to Parkinson’s disease due to the treatment with a neurotoxin (6-OHDA), eugenol was able to improve motor condition and to counterbalance weight loss [Moreira Vasconcelos ef al. , Eugenol and its association with levodopa in 6-hydroxydopamine-induced hemiparkinsonian rats: Behavioural and neurochemical alterations. Basic Clin Pharmacol Toxicol. 2020 127(4): 287-302], It is known that damage caused by 6-OHDA is associated with a significant increase of lipid peroxidation rates and nitrites/nitrates content [Souza et al., Neuroprotective effects of sulphated agaran from marine alga Gracilaria cornea in rat 6-hydroxydopamine Parkinson’s disease model: behavioural, neurochemical and transcriptional alteration. Basic Clin

RECTIFIED SHEET (RULE 91) ISA/EP Pharmacol Toxicol. 2017, 120, 159-170], The treatment with eugenol was able to counteract these effects, confirming other results related to the decrease in the production levels of nitric oxide and derivatives thereof as well as of malonaldheyde rates, a byproduct of lipid oxidation [Prasad et al., Neurorestorative effects of eugenol, a spice bioactive: evidence in cell model and its efficacy as an intervention molecule to abrogate brain oxidative dysfunctions in the streptozoticin diabetic rat. Neurochem Int. 2016, 95, 24-36], As known, eugenol may have direct or indirect antioxidant effects. Its direct action is linked to the capture of free hydroxy radicals, which may interfere with the formation of intermediate fatty acids, a possible mechanism for preventing lipid peroxidation [Ito et al., Antioxidant action of eugenol compounds: role of metal ion in the inhibition of lipid peroxidation. Food Chem Toxic. 2005, 43, 461-466], Moreover, a possible action of eugenol in preventing the degradation of IKB-O, a protein inhibiting NF-KB, has been hypothesized, which indirectly culminates with NF-KB inhibition and decrease in iNOS expression, so decreasing the levels of nitric oxide derivatives [Irie Y„ Effects of eugenol on the central nervous system: its possible application to treatment of Alzheimer’s disease, depression, and Parkinson’s disease. Cur Bioact Com pd. 2006, 2, 57-66], On the contrary, the indirect action of eugenol is linked to its possible action in inducing glutathione-S- transferase, leading to an increase of glutathione available levels and to the formation of reduced glutathione (GSH), an important endogenous antioxidant [Vidhya et al., Antioxidant effect of eugenol in rat intestine. Indian J Exp Biol. 1999, 37, 1192-1195], In Parkinson mouse model, a decrease of GSH content in brain has been observed. The treatment of Parkinson’s disease with levodopa is often associated to side effects such as dyskinesias [Smith et al., Striatal mRNA expression patterns underlying peak dose L-Dopa-induced dyskinesia in the 6-OHDA hemiparkinsonian rat. Neuroscience 2016, 324, 238-251], The treatment with eugenol in combination with levodopa at a low dose reduced these side effects, suggesting a possible neuroprotective action.

Combination

The combination of BCP, DHA and eugenol underlying the present invention has as its peculiar feature the presence of eugenol in specific amounts which result in a synergical effect compared to BCP and DHA alone.

In particular, said amount is within the range of 1.3 - 2.0 parts by weight.

It is worth noting that by significantly increasing the eugenol amount (e.g., by tripling it) a substantial improvement compared to the combination BCP+DHA alone is no longer observed, so loosing the synergical effect of the eugenol addition.

RECTIFIED SHEET (RULE 91) ISA/EP Said synergical effect appears with a significant decrease in pro-inflammatory cytokines (IL-6 and TNF-a) and a concurrent increase in anti-inflammatory cytokines (IL-10 and IL-4) as proved in vitro on a cell line of human astrocytes after inflammatory stimulus with I L-1 p (Figures 1-4).

Formulation

In the composition object of the present invention, the three substances BCP, DHA and eugenol can be used in isolated and/or purified form, optionally also of synthetic origin, or in the form of extracts or essential oils of natural origin containing them.

As already underlined, the weight ratios among BCP, DHA and eugenol are specific and represent an essential feature of the composition object of the present invention. In fact, the amount of eugenol is present in excess with respect to BCP and DHA but this excess must be within the range 1.3 - 2.0 for an optimal result in terms of synergical efficacy. By increasing the amount of eugenol, the efficacy of the combination decreases, as clearly shown by the comparison between the combinations BCP:DHA:eugenol=1 :1 :1.5 and BCP:DHA:eugenol=1 :1 :5.0 (Figures 1- 4).

As already reported, eugenol is naturally occurring in essential oils from several plants, in particular, it is one of the main components of clove essential oil (Syzygium aromaticum L.). Clove essential oil is widely used in perfume, cosmetic, health, medical, flavor and food industries. Eugenol represents at least 50% of clove essential oil. The remaining 10-40% consists of eugenyl acetate, BCP and a-humulene. Therefore, the use of clove essential oil, containing both eugenol and BCP, would be particularly advantageous from a practical point of view for the preparation of the composition object of the present invention.

However, the commonly used clove essential oil does not allow to achieve the desired weight ratio between eugenol and BCP.

The present inventor has found that the specific weight ratio between BCP and eugenol can be achieved by using essential oils with high titer (e.g., 80%) in BCP and eugenol, which are obtained by steam distillation.

Such oils are then mixed in suitable amounts to achieve the desired ratio and the mixture is preferably microencapsulated according to conventional techniques before the final formulation.

RECTIFIED SHEET (RULE 91) ISA/EP A particularly preferred example of the mixture of oils enriched in BCP and eugenol is represented by an oil comprised of: 60% w/w clove oil with high titer in eugenol (80%) and 40% w/w clove oil with high titer in BCP (80%).

Then, 100 g of this “EUG/BCP” oil contain 48 g of eugenol and 32 g of BCP (eugenol: BCP ratio = 1.5:1).

DHA is preferably used in the form of powder with >75% purity grade.

A particularly preferred example is microencapsulated DHA in powder with 75.8% titer, commercialized by Nutraceutica srl.

Then, the final formulation in its preferred form having a ratio BCP:DHA:eugenol = 1 :1 :1.5 can be obtained by mixing 100g of microencapsulated “EUG/BCP” oil with 42,7 g of microencapsulated DHA powder.

Then, one or more carriers and/or excipients, depending on the selected final formulation form, may be optionally added to the resultant mixture, containing the combination BCP, DHA and eugenol according to the invention.

The composition object of the present invention is orally administered and therefore the carriers/excipients will be suitably selected among those commonly used for oral administration forms, such as, e.g., tablets, capsules, softgels and solutions/suspensions.

Preferably, the composition object of the present invention is administered in a microencapsulated form or as an emulsion filled in soft gelatine capsules (softgels) containing a unit dosage of combination between 350 and 1000 mg, e.g., 350 mg (100 mg BCP, 100 mg DHA and 150 mg eugenol), 437.5 mg (125 mg BCP, 125 mg DHA and 187.5 mg eugenol), 525 mg (150 mg BCP, 150 mg DHA and 225 mg eugenol), etc.

The composition object of the present invention is preferably administered 1-3 times a day, still more preferably twice a day.

EXPERIMENTAL PART

Study goal

Testing inflammatory/pro-analgesic and anti-inflammatory/pro-resolution response after inflammatory stimulus (I L-1 p) on human astrocyte cell line.

The production of pro- and anti-inflammatory cytokines was tested in the presence of the inflammatory stimulus alone and under conditions of co-culture with eugenol (2.5 pM and 0.75 pM), [3-caryophyllene (BCP - 0.4 pM), docosahexaenoic acid (DHA - 0.25 pM) and their combinations.

RECTIFIED SHEET (RULE 91) ISA/EP Materials and methods

The cell line U373-MG (derived from human astrocytoma) was cultured in DM EM (Dulbecco’s Modified Eagle’s Medium), supplemented with 10% fetal bovine serum, 1% L-glutaminostreptomycin and 1% non-essential amino acids, and incubated at 37°C under an atmosphere containing 5% CO2. At confluence, the cells were washed with 0.1 M PBS, removed from the support by means of a trypsin solution, centrifuged at 1500 rpm for 5 minutes at room temperature and re-suspended in complete medium (1 :15 dilution). In each well of a 24-well plate, 15000 suspended cells were seeded in complete medium. The medium volume of each well was brought to 1 mL and the cells were kept in an incubator at 37°C and 5% CO2 for 24 hours.

At the end of the incubation, each sample was added with, respectively:

Sample 1 : negative control

Sample 2: 0.1 % ethanol (vehicle control)

Sample 3: 0.1% DMSO (vehicle control)

Sample 4: I L-1 (3 1 ng/mL (positive control, inflammatory stimulus only)

Sample 5: Eugenol [2.5 pM] + I L-1 fB [1 ng/mL]

Sample 6: Eugenol [0.75 pM] + I L-113 [1 ng/mL]

Sample 7: BCPI [0.4 pM] + IL-113 [1 ng/mL]

Sample

Sample

Sample 10: BCP [0.4 pM] + Eugenol [2.5 pM] + DHA [0.25 pM] + IL-1 p [1 ng/mL] Sample 11 : BCP [0.4 pM] + Eugenol [0.75 pM] + DHA [0.25 pM] + IL-ip [1 ng/mL] The correspondences between the micromolar concentrations and the w/V concentrations are the following:

P-caryophyllene 0.4 pM = 81.744 pg/L

- DHA 0.25 pM = 82.122 pg/L

Eugenol 2.5 pM = 410 pg/L

Eugenol 0.75 pM = 123.15 pg/L

Therefore, Sample 10 contains BCP, DHA and eugenol in the ratio 1 :1:5 by weight while Sample 11 contains BCP, DHA and eugenol in the ratio 1 :1 :1.5 by weight.

The plate was then incubated for further 24 hours at 37°C, 5% CO2. After collection of the supernatant of each sample, it was analyzed using Bio-Plex Multiplex Immunoassay System (Bio-Rad Laboratories srl, Segrate, Ml, Italy).

RECTIFIED SHEET (RULE 91) ISA/EP Each sample was tested in 4 replicates and the production of the following cytokines was evaluated: IL-6, IL-10, IL-4, IL-8 and TNF-a.

IL-6 (Figures 1A and 1B)

IL-6 is secreted by macrophages in response to specific signals, for example during an infection. IL-6 is responsible for the stimulation of protein synthesis of the acute phase and of the neutrophil production in bone marrow. It supports the growth of B cells and is antagonist to regulatory T cells. IL-6 stimulates inflammatory and autoimmune processes in several diseases such as multiple sclerosis, diabetes, atherosclerosis, depression, Alzheimer disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, reumathoid arthritis and in cerebral hemorrhages. In CNS, astrocytes are one of the primary inducible sources of IL-6. Whilst IL-6 at low concentrations has beneficial effects in CNS thanks to its neurotrophic properties, its overexpression is generally damaging, adding itself to the physiophatology associated with CNS disorders. It is known that many factors induce IL-6 overexpression by astrocytes, in particular the pro-inflammatory cytokines TNF-a and I L-113, and that their regulation is then of primary importance [Van Wagoner et a!., Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor. J Neurosci. 1999, 19(13), 5236-44], In fact, the overexpression of IL-6 mediates demyelinization, axonal damage and supports CNS inflammation and impairment during progressive autoimmune encephalomyelite. The therapeutic potential of IL-6 blockade in limiting both impairment and tissue damage in this pathology as well as in multiple sclerosis has been demostrated [Constantinescu et al., Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol.

2011 ,164(4): 1079-106],

Figures 1A and 1 B show IL-6 values (Fig. 1A) and IL-6 percent change (Fig. 1 B) in human astrocyte cell line after incubation with samples 1 -11.

Sample 1 (negative control) and samples 2-3 (vehicle controls) do not show significant differences.

Sample 4 (IL-1 only) represents the inflammatory stimulus and induces a significant IL-6 increase (P<0.05).

Samples 5-6 (eugenol), 7 (BCP) and 8 (DHA) do not result in a significant IL-6 decrease.

On the contrary, sample 9 (BCP+DHA) and samples 10-11 (BCP+DHA+Eugenol) show a significant IL-6 decrease compared to sample 4. In particular, sample 1 1

RECTIFIED SHEET (RULE 91) ISA/EP (combination of the invention) shows the highest IL-6 decrease, which is significant also compared to sample 9 (BCP+DHA) and to sample 10.

IL-10 (Figures 2A and 2B)

IL- 10 is a neuroprotective anti-inflammatory cytokine. It is produced by monocytes, mast cells and a certain subset of activated T and B cells, and plays a fundamental role in regulating inflammatory response and immune reactions. IL-10 mainly inhibits the induction mediated by LPS and by the bacterial products of the pro-inflammatory cytokines TNF-a, I L-1 (3, IL-12 and IFN-y secreted by the myeloid cells activated by TLRs (Toll-Like Receptors). In CNS, IL-10 is mainly produced by astrocytes and microglia and it is overregulated after various insults, such as experimental autoimmune encephalomyelitis, median cerebral artery occlusion, excitotoxicity and traumatic cerebral injuries. The astrocyte production of IL-10 has an impact on microglial response and lymphocyte recruitment and culminates in a beneficial effect on neuronal survival [Villacampa et al., Astrocyte-targeted production of IL-10 induces changes in microglial reactivity and reduces motor neuron death after facial nerve axotomy. Glia 2015, 63(7), 1166-84], In vitro studies demonstrated that BCP, being a CB2 receptor agonist, is capable to underregulate I L-1 p and TNF-a and to reduce iNOS expression and ROS production in primary mouse microglia, protecting it from LPS inflammatory effects. On the contrary, IL-10 and Arg1 (arginase) are overregulated. Following Arg1 increase and iNOS decrease, the generation of urea increases, while that of NO decreases and these changes clearly show that the microglial phenotype shifted towards M2 (pro-resolution of inflammation) [Askari et al., The protective effects of beta-caryophyllene on LPS-induced primary microglia M1/M2 imbalance: a mechanistic evaluation. Life Sci. 2019, 219, 40-73].

Figures 2A and 2B show IL-10 values (Fig. 2A) and IL-10 percent change (Fig. 2B) in human astrocyte cell line after incubation with samples 1 -11.

Sample 1 (negative control) and samples 2-3 (vehicle controls) do not show significant differences.

Sample 4 (IL-1 only) represents the inflammatory stimulus and does not produce significant effects compared to the control.

Sample 7 (BCP), sample 9 (BCP+DHA) and samples 10-1 1 (BCP+DHA+Eugenol) significantly increase IL-10.

In particular, sample 1 1 (combination of the invention) produces the highest IL-10 increase, which is significant also compared to sample 7, sample 9 and sample 10.

RECTIFIED SHEET (RULE 91) ISA/EP IL-4 (Figures 3A and 3B)

IL-4 is a cytokine inducing the differentiation of naive helper T cells (ThO) into Th2 cells. After activation by IL-4, Th2 cells subsequently produce further IL-4 in a positive feedback cycle. IL-4 is mainly produced by mast cells, Th2 cells, eosinophils and basophils. IL-4 is a key regulator of humoral and adaptive immunity, has several biological roles, including the stimulation of activated B and T cell proliferation and the differentiation of B cells into plasmacells. IL-4 induces the class passage of B cells to IgE production, regulates class II MHC expression and inhibits the production of pro- inflammatory cytokines such as TNF-a, IFN-y and IL- 17. IL-4 may also exert astrocyte expression of CNS growth factors, such as nerve growth factor. The astrocytes clearly respond to IL-4, but the expression of their receptor and the factors which regulate the receptor expression are not well understood. IL-4 may exert pro-inflammatory as well as anti-inflammatory effects on astrocytes, depending on treatment and timing. In primary mouse astrocytes, pre-treatment with IL-4 decreases the subsequent production of NO and iNOS, as well as the secretion of TNF-a after LPS stimulus. Similarly, the concomitant treatment of human fetal primary astrocytes with IL-4 lessens NO production by those cells stimulated with IL-1|3, TNF-a or IFN-y. In another study, however, primary mouse astrocytes treated with I L-113 showed higher production of IL-6 when they were subsequently treated with IL-4, while the treatment with IL-10 and dexamethasone produced immunosuppressive effects. However, the conditioned medium of astrocytes treated with IL-4 is neuroprotective in a dosedependent manner. Then, literature data show a complex interaction between IL-1 p and IL-4 in the context of neuroprotection [Chen et al., Interleukin 4 Affects Epilepsy by Regulating Glial Cells: Potential and Possible Mechanism. Front Mol Neurosci. 2020, 4, 13:554547; Gadani et al., IL-4 in the brain: a cytokine to remember. J Immunol. 2012, 189(9), 4213-9],

Figures 3A and 3B show IL-4 values (Fig. 3A) and IL-4 percent change (Fig. 3B) in human astrocyte cell line after incubation with samples 1-11.

Sample 1 (negative control) and samples 2-3 (vehicle controls) do not show significant differences.

Sample 4 (IL-1 p only) represents the inflammatory stimulus and induces a significant IL-4 increase (P<0.05).

Samples 5-6 (eugenol), 7 (BCP), 8 (DHA), 9 (BCP+DHA) and 10 (BCP:DHA:Eugenol=1 :1 :5) induce a non-significant increase of IL-4 levels.

RECTIFIED SHEET (RULE 91) ISA/EP On the contrary, sample 11 (combination of the invention) induces a significant IL-4 increase compared both to inflammatory stimulus and sample 9.

TNF-a (Figures 4A and 4B)

TNF-a is produced by macrophages and by a wide variety of cells, including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, adipose tissue, fibroblasts and neurons. It is released in response to liposaccharide, other bacterial products and IL- ip. It has a number of actions on several organs, generally together with I L-1 p and IL-6. In hypothalamus, it activates the hypothalamus - pituitary gland - adrenal gland axis, by stimulating the release of the corticotropin-releasing hormone CRH, suppresses appetite and induces pyrosis. In liver, it stimulates the acute phase response, also inducing insulin-resistance. It is a strong chemoattractor for neutrophils and promotes the expression of adhesion molecules on endothelial cells, by favoring neutrophil migration. In macrophages, it stimulates phagocytosis and production of IL-ip and PGE2. A local increase in TNF-a concentration results in classical inflammation signs: heat, swelling, redness, pain and functionality loss. TNF- a promotes the inflammatory response which, in its turn, causes many of the clinical problems associated with autoimmune diseases such as reumathoid arthritis, ankylosing spondylitis, intestinal inflammatory diseases, psoriasis, hidradenitis suppurativa and refractory asthma.

In CNS, TNF-a may be neuroprotective, but it may lead to neurotoxic effects by microglia activation. In fact, the appearance of cognitive disorders following CNS inflammations or infections was correlated to an increase of TNF-a levels. The local TNF-a increase in hippocampal dentate gyrus activates astrocyte TNF-a type 1 receptor (TNFR1), which, in its turn, triggers an astrocyte-neuron signaling cascade resulting in a persistent functional change of hippocampal excitatory synapsis. The astrocyte signaling of TNFR1 is required for the hippocampal synapsis alteration and the concomitant memory impairment observed in experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disorders in MS, as well as in other CNS pathologies with inflammatory conditions and infections [Habbas et al., Neuroinflammatory TNFa Impairs Memory via Astrocyte Signaling. Cell, 2015, 163(7), 1730- 41],

Figures 4A and 4B show TNF-a values (Fig. 4A) and TNF-a percent change (Fig. 4B) in human astrocyte cell line after incubation with samples 1-11.

RECTIFIED SHEET (RULE 91) ISA/EP Sample 1 (negative control) and samples 2-3 (vehicle controls) do not show significant differences.

Sample 4 (IL-1 p only) represents the inflammatory stimulus and induces a significant TNF-a increase (P<0.05). Samples 5-6 (eugenol), 7 (BCP) and 8 (DHA) do not result in a significant TNF-a decrease.

On the contrary, sample 9 (BCP+DHA) and samples 10-11 (BCP+DHA+Eugenol) show a significant TNF-a decrease compared to sample 4.

The best result is achieved with sample 11 (combination of the invention) showing a significant decrease also compared to sample 9.

The results obtained in vitro on the human astrocyte cell line show that the combination of BCP, DHA and eugenol according to the present invention exerts a protective effect on the cell line treated with I L- 1 p, by inducing the production of anti- inflammatory/pro-resolving cytokines and the decrease of proinflammatory cytokines.

RECTIFIED SHEET (RULE 91) ISA/EP

Claims

1) A composition containing p-caryophyllene (BCP), docosahexaenoic acid (DHA) and eugenol for use as analgesic and anti-inflammatory antioxidant for the central and peripheral nervous system.

2) The composition according to claim 1 containing 0.9 - 1.1 parts by weight of BCP, 0.9 - 1.1 parts by weight of DHA and 1.3 - 2.0 parts by weight of eugenol.

3) The composition according to claim 1 or 2 containing equal parts of BCP and DHA and 1 .5 parts by weight of eugenol.

4) The composition according to anyone of the previous claims wherein BCP and eugenol are used in the form of clove essential oil.

5) The composition according to claim 4 wherein the clove essential oil is a mixture of clove essential oil with high titer in BCP and of clove essential oil with high titer in eugenol.

6) The composition according to claim 5 wherein the mixture of clove essential oils with high titer is in microincapsulated form.

7) The composition according to anyone of the previous claims wherein DHA is used in the form of microencapsulated powder.

8) The composition according to anyone of the previous claims in the form of oral dosage, particularly in the form of capsules.