Analgesic compositions comprising a synergistic combination of active ingredients

Abstract

Compositions with analgesic activity are described comprising as active ingredients spilanthol or an extract containing it in synergistic association with an acylethanolamide and / or vitamin K2.

Description

[0001] ANALGESIC COMPOSITIONS COMPRISING A SYNERGISTIC COMBINATION OF ACTIVE INGREDIENTS

[0002] The invention relates to a pharmaceutical, veterinary or nutraceutical composition comprising an association of spilanthol with acylethanolamides and / or vitamin K2.

[0003] BACKGROUND OF THE INVENTION

[0004] The Acmella oleracea. (formerly known as Spilanthes acmella) also known as toothache plant, paracress, and Jambu, is an annual herbaceous plant belonging to the Asteraceae family traditionally used by the indigenous populations of South America as a remedy to reduce toothache.

[0005] The Acmella oleracea is native to the tropical and subtropical regions of South America, in particular Brazil and Peru, where it is identified with the common name of Jambu. Thanks to its ease of cultivation and adaptability, the plant has spread to other parts of the world. It is often cultivated for ornamental purposes and used in local traditional medicine for its many benefits.

[0006] The Acmella oleracea extract most commonly used in phytotherapy and nutraceuticals is a hydroalcoholic extract of aerial parts, especially flowers and leaves, rich in biologically active secondary metabolites, including alkamides or N-alkylamides (spilanthol), flavonoids, polyphenols and tannins.

[0007] Spilanthol is an alkamide, responsible for the biological properties attributable to the extract, in particular anti-inflammatory properties, modulating the inflammatory response and reducing the production of pro-inflammatory cytokines (Rondanelli et al Phytotherapy 2020, Volume 140, 104419; Yien et al., Natural Product Research 2023, Volume 37, 18, 2023, 3136- 31442022; Jayashan et al. Appl. Sci. 2024, 14, 3755), as well as for its antioxidant, analgesic (Rondanelli et al Phytotherapy 2020 Volume 140, 2020, 104419) and antimicrobial effects (Spinozzi et al., Plants 2022, 11, 2721).

[0008] The hydroalcoholic extract of A. oleracea is therefore used in food supplements, herbal preparations and cosmetics. The dosage forms most commonly used as a supplement are capsules or tablets containing standardized extracts of the plant's active ingredients.

[0009] Thanks to its analgesic properties, Acmella oleracea is used to relieve pain, both of inflammatory and neuropathic origin.

[0010] Numerous scientific evidences support the powerful analgesic properties of alkamides, which compounds are capable of interacting with pain receptors in the central and peripheral nervous system, decreasing sensitivity to pain. Specifically, spillanthol inhibits the vanilloid receptor TRPV1 involved in pain signaling. Preclinical studies have shown that Acmella oleracea extract can significantly reduce inflammatory and neuropathic pain, offering natural relief without the side effects associated with traditional medications. In summary, Acmella oleracea is a valuable resource in nutraceuticals and phytotherapy. It can be a valuable support of natural origin for the treatment of various inflammatory disorders and pain, improving the quality of life.

[0011] Acylethanolamides, such as anandamide, palmitoylethanolamide, oleoyl ethanol ami de, and prostamides, are natural compounds that play physiological roles as lipid and autacoid neurotransmitters .

[0012] In particular, palmitoylethanolamide (PEA) has aroused interest for its antiinflammatory and analgesic properties.

[0013] PEA acts primarily through modulation of cannabinoid and non-cannabinoid receptors, such as the proliferator-activated peroxisome receptor alpha (PPAR-a). This mechanism helps reduce inflammation and pain without the psychotropic effects associated with traditional cannabinoids.

[0014] PEA has been proposed for the treatment of chronic pain, neuropathic pain, pain associated with chronic inflammatory diseases, rheumatoid arthritis, intestinal inflammation, neurodegenerative diseases (Gabrielsson, L. et al. Br J Clin Pharmacol. 2016, 8 2(4):932-42; Skaper, S. D. et al. Inflammopharmacology 2014; 22(2):79-94).

[0015] The term vitamin K refers to a group of fat-soluble molecules comprising several isoforms or subtypes: vitamin KI (phylloquinone) and vitamin K2 (menaquinone); there is also a synthetic form known as vitamin K3 (menadion) which is not biologically active in humans.

[0016] The different forms of vitamin K have a common central structure consisting of a naphthoquinone ring with an isoprenoid side chain which, in the case of vitamin K2, can be of variable length (due to the number of repeated isoprenoid units) and for this reason it is often referred to as "MK-n" or "vitK2-n" where "n" indicates the number of isoprenoid units.

[0017] Vitamin KI is mainly present in green leafy vegetables and is involved in the process of photosynthesis.

[0018] Vitamin K2, mainly present in foods of animal origin, (especially in the form of menaquinone-7, or MK-7 or vitamin K2-7) has shown potential benefits in the treatment of peripheral neuropathies, in particular associated with vitamin B12 deficiency, diabetes and iatrogenic conditions from oncological treatment. Preliminary studies suggest that vitamin K2- 7 may improve neuropathy symptoms, such as pain, tingling, and cramps, by acting through several biological mechanisms. The MK-7 form of vitamin K2 has a longer half-life in the body than other forms of K2.

[0019] The Italian patent IT 202000024217 describes combinations of spilanthol with cannabidiol and / or boswellic acid useful for the treatment of pain, in particular neuropathic pain.

[0020] EP 3 417 846 discloses lipocomplexes in the form of microparticles comprising an emulsifier, an endocannabinoid such as PEA and a cannabinoid. Acmella is only generically mentioned along with many other possible ingredients but without any experimental support that is precisely limited to the combination of endocannabinoids with cannabinoids.

[0021] DESCRIPTION OF THE INVENTION

[0022] The combination of spilanthol or an extract containing it with acylethanolamides and / or vitamin K2 has surprisingly been found to exert synergistic analgesic effects, demonstrated in experimental models considered predictive of clinical activity.

[0023] The invention therefore relates to an oral or nutraceutical pharmaceutical composition comprising as active ingredients spilanthol in association with an acyl ethanol ami de and / or with vitamin K2 in combination with compatible carriers or excipients.

[0024] According to a first embodiment of the invention, the compositions comprise an association of spilanthol and an acylethanolamide.

[0025] According to another embodiment of the invention, the compositions comprise spilanthol, an acylethanolamide and vitamin K2.

[0026] According to yet another embodiment of the invention, the compositions comprise spilanthol and vitamin K2.

[0027] The spilanthol may be used in substantially pure or isolated form. Alternatively, the spilanthol is present in an extract of Acmella oleracea. preferably an extract of aerial parts (flowers and / or leaves) or of other species of the same genus (such as A. paniculata, A. uliginosa, A. ciliata, A. radicans, A. caulirhiza, A. bellidioides or A. decumbens).

[0028] The acylethanolamides are preferably chosen from oleoyl ethanol ami de and palmitoylethanolamide. Palmitoylethanolamide is very particularly preferred.

[0029] The compositions of the invention are suitable for oral administration, for example in the form of tablets, capsules, granules or liquid formulations, prepared in a conventional manner with pharmaceutically acceptable excipients.

[0030] In the case of tablets, suitable excipients comprise binding agents, bulking agents, lubricants, disintegrants and other conventionally used excipients or vehicles. The tablets can be single- or multi-layered and can have a coating to allow the controlled release of the active ingredients.

[0031] Liquid preparations may be in the form of solutions, emulsions, syrups or suspensions or in the form of lyophilisates to be reconstituted, before use, with water or other vehicles. Such liquid preparations can be prepared by conventional methods.

[0032] The unit dosage of spilanthol in the compositions of the invention is typically from 0.01 to 40 mg, preferably from 0.05 to 10 mg.

[0033] The dosage of acylethanolamide is between 150 and 1200 mg, preferably between 500 and 800 mg in the case of palmitoylethanolamide (preferably used in non-micronized form). In the case of oleoylethanolamide, the dosage is between 100 and 250 mg.

[0034] The dosage of vitamin K2 is between 10 and 300 mcg.

[0035] The compositions of the invention are useful for the treatment of pain in general, in particular pain related to inflammatory states or neuropathic pain, in human or veterinary medicine.

[0036] For the intended uses, the frequency of administration may vary from 1 to 3-4 times per day. The dose administered may vary depending on the age and weight of the patient and also the severity of the clinical condition being treated, at the discretion of the physician or veterinarian.

[0037] The invention is illustrated in more detail in the following examples.

[0038] Example 1: Coated Tablets

[0039] Composition for tablet

[0040] • Palmitoylethanolamide (PEA): 600 mg

[0041] • of Acmella titrated at 10% in spilanthol: 14 mg (equivalent to 1.4 mg spilanthol)

[0042] • Excipients: o Microcrystalline cellulose: 200 mg o Com starch: 80 mg o Povidone (K30): 20 mg o Magnesium stearate: 10 mg o Colloidal Anhydrous Silica: 5 mg

[0043] Composition for coating

[0044] • Hydroxypropylmethylcellulose (HPMC): 10 mg

[0045] • Macrogol 400: 5 mg • Dye (optional): as needed

[0046] Example 2: HPMC capsules

[0047] Composition for capsule (0 size)

[0048] • Palmitoylethanolamide (PEA): 150 mg

[0049] • Acmella extract titrated at 30% in spilanthol: 6.67 mg (equivalent to 2 mg spilanthol)

[0050] • Microcrystalline cellulose: 143.33 mg

[0051] • Com starch: 50 mg

[0052] • Colloidal Anhydrous Silica: 10 mg

[0053] • Magnesium stearate: 5 mg

[0054] Example 3: Soft Gelatin Capsules

[0055] Composition for capsule

[0056] Palmitoylethanolamide (PEA): 300 mg

[0057] Acmella extract titrated at 1% in spilanthol: 5 mg (equivalent to 0.05 mg spilanthol)

[0058] Sunflower oil : 150 mg

[0059] Sunflower lecithin: 50 mg

[0060] Glycerine: 30 mg

[0061] Soft gelatine capsule composition

[0062] Gelatine: 200 mg

[0063] Glycerine: 80 mg

[0064] Purified water: 50 mg

[0065] Dye (optional): as needed

[0066] Example 4: Sachets

[0067] Composition for sachet

[0068] • Palmitoylethanolamide (PEA): 1200 mg

[0069] • Acmella extract titrated at 20% in spilanthol: 5 mg (equivalent to 1 mg spilanthol)

[0070] • Sorbitol: 1370 mg

[0071] • Microcrystalline cellulose: 500 mg

[0072] • Citric acid: 300 mg

[0073] • Natural flavour (lemon): 75 mg

[0074] • Xanthan gum: 30 mg • Colloidal Anhydrous Silica: 15 mg

[0075] • Stevia or Erythritol (non-caloric sweetener): 10 mg.

[0076] Example 5: Coated tablets

[0077] Composition for tablet

[0078] • Palmitoylethanolamide (PEA): 600 mg

[0079] • Acmella extract titrated at 1% in spilanthol: 14 mg (equivalent to 0.14 mg of spilanthol)

[0080] • Vit. K2-7 Tit. 0.2% 10 mg (Eq. 20mcg)

[0081] • Excipients: o Microcrystalline cellulose: 200 mg o Corn starch: 70 mg o Povidone (K30): 20 mg o Magnesium stearate: 10 mg o Colloidal Anhydrous Silica: 5 mg

[0082] Composition for coating

[0083] • Hydroxypropylmethylcellulose (HPMC): 10 mg

[0084] • Macrogol 400: 5 mg

[0085] • Dye (optional): as needed

[0086] Example 6: Sugar-free sachets

[0087] Composition for sachet

[0088] Palmitoylethanolamide (PEA): 1200 mg

[0089] Acmella extract titrated at 2% in spilanthol: 5 mg (equivalent to 1 mg of spilanthol)

[0090] Vit. K2-7 (Powder Tit. 0.2%) 100 mg (Eq. 200 mcg Vit. K2-7)

[0091] Sorbitol: 1370 mg

[0092] Microcrystalline cellulose: 500 mg

[0093] Citric acid: 300 mg

[0094] Natural flavour (e.g. lemon): 75 mg

[0095] Xanthan gum: 30 mg

[0096] Colloidal Anhydrous Silica: 15 mg

[0097] Stevia or Erythritol (non-caloric sweetener): 10 mg

[0098] Example 7: HPMC capsules

[0099] Composition for capsule (0 size) • Palmitoylethanolamide (PEA): 150 mg

[0100] • Acmella extract titrated at 30% in spilanthol: 6.67 mg (equivalent to 2 mg spilanthol)

[0101] • Microcrystalline cellulose: 120.33 mg

[0102] • Vitamin K2-7 (titer.1%) 5 mg (Eq. 50 mcg Vit.K2-7)

[0103] • Corn starch: 45 mg

[0104] • Colloidal Anhydrous Silica: 10 mg

[0105] • Magnesium stearate: 5 mg

[0106] Example 8: Soft Gelatin Capsules

[0107] Composition for capsule

[0108] Palmitoylethanolamide (PEA): 300 mg

[0109] Acmella extract titrated at 10% in spilanthol: 7 mg (equivalent to 0.7 mg spilanthol)

[0110] Sunflower oil: 110 mg

[0111] Sunflower lecithin: 50 mg

[0112] Glycerine: 30 mg

[0113] Vitamin K2-7 in sunflower oil (Tit. 0.15%) 67 mg (Eq. 100 mcg Vit. K2-7)

[0114] Capsule composition (12 oval size)

[0115] Gelatine: 200 mg

[0116] Glycerine: 80 mg

[0117] Purified water: 50 mg

[0118] Dye (optional): as needed

[0119] Example 9: HPMC capsules

[0120] Composition for HPMC Capsule (0 size)

[0121] • Acmella extract titrated at 10% in spilanthol: 50 mg (equivalent to 5 mg of spilanthol)

[0122] • Vitamin K2-7 0.2% ,50mg (Equivalent to 100 mcg of Vit. K2-7)

[0123] • Microcrystalline cellulose: 50 mg

[0124] • Com starch: 10 mg

[0125] • Colloidal Anhydrous Silica: 5 mg

[0126] • Magnesium stearate: 5 mg

[0127] Example 10: Synergy of associations

[0128] In vitro experiments were performed on an experimental microglia model consisting of BV-2 cells, a cell line widely used for neuroinflammation studies. Indeed, BV-2s are immortalized cells that share morphological and functional characteristics with macrophages from other body districts with regard to their antigenic profile, their phagocytic capacity and antimicrobial activity. BV-2 cells are also able to release pro-inflammatory cytokines when appropriately stimulated with pro-inflammatory cytokines and / or oxidative stress. Such stimuli are among the main triggers capable of triggering neurodegenerative disorders such as Alzheimer's and Parkinson's disease, so BV-2 is widely used as an alternative in vitro experimental model to primary microglia cells.

[0129] Assessment of TNF-a

[0130] BV-2 cells were cultured in vitro, under standard experimental conditions (37°C, 5% CO2 and controlled humidity) using RPMI 1640 culture medium supplemented with specific growth factors and 10% FCS (foetal calf serum), according to known protocols (Borgonetti, V., et al., Phytomedicine 2020; 68, 153143.).

[0131] For the evaluation of the proinflammatory cytokine TNF-a, BV-2 cells were cultured in 24-well plates at a density of 105cells per well and allowed to adhere for 24 hours.

[0132] Subsequently, the cells were treated with the substances of interest: PEA, Acmella oleracea (AC) dry extract Vitamin K2 and their AC -PEA combinations, AC -PEA+ Vitamin K2 and AC+ Vitamin K2 while cells treated only with the culture medium were used as a control. All samples were stimulated with LPS (250 ng / ml) for two hours in order to induce inflammatory stimulation and express TNF-a whose expression was evaluated by ELISA kit according to the instructions provided by the manufacturer (Invitrogen).

[0133] TNF-a expression was quantified by spectrophotometer reading at the primary wavelength of 450 nm.

[0134] Statistical analyses were performed using one-way ANOVA followed by Tukey post- hoc tests (with significance level p < 0.05). The results, expressed as mean values of three independent experiments ± standard deviations, are shown in Figure 1 (**p<0.01 vs control; ***p<0.001 vs control; #p<0.05 vs PEA, Acmella and Vitamin K2 (single treatment); ###p<0.001 vs PEA, Acmella and Vitamin K2 (single treatment).

[0135] The high statistical significance of the reduction in expression levels of the cytokine TNF-a caused by the combination AC -PEA, AC -PEA- Vitamin K2 (p<0.05) and AC -Vitamin K2 (p<0.001) compared to that caused by individual treatments, shows a synergistic effect between the substances tested (PEA, vitamin K2 and Acmella oleracea).

[0136] Assessment of oxidative stress To evaluate the possible antioxidant effects of PEA, Acmella, Vitamin K2 and AC -PEA, AC -PEA + Vitamin K2 and AC + Vitamin K2 combinations, reactive oxygen species (ROS) levels were evaluated using a fluorimetric method according to the instructions provided by the manufacturer (Merck, Germany) and already described in the literature (Borgonetti, V. et al., Phytotherapy Research 2022; 36:2246-2263). In short, BV-2 cells were cultured in 96-well plates at a density of 5xl03cells per well for 24 hours. Cells were treated with the substances of interest or their combination for 4 hours and stimulated with LPS (250 ng / ml) for a further 2 hours in order to induce a condition of oxidative stress. Then the cells were incubated with a H2DCF-DA solution for 45 minutes at 37°C. In the presence of ROS, the reagent H2DCF-DA is converted into a fluorescent product, dichlorofluorescein (DCF) whose fluorescence is measured with a plate reader at a wavelength of 485 / 535 nm. Fluorescence is directly proportional to ROS levels.

[0137] Results are expressed as mean values of three independent experiments ± standard deviations and are shown in Figure 2 (**p<0.01 vs control; * **p<0.001 vs control; #p<0.05 vs PEA, Acmella and Vitamin K2 (single treatment).

[0138] The high statistical significance of the reduction in ROS levels caused by the combinations AC -PEA (p<0.05), AC -PEA +VIT K2 and AC+Vit K2 (p<0.01 vs control, #p<0.05 vs PEA, Acmella or Vitamin K2; a:p<0.01 vs vitamin K2) compared to that caused by the individual treatments, shows a synergistic effect between the tested substances (PEA, Acmella oleracea and vitamin K2).

Claims

CLAIMS1. Oral compositions comprising as active ingredients spilanthol in combination with an acylethanolamide and / or with vitamin K2 in combination with compatible carriers or excipients.

2. Compositions according to Claim 1, comprising spilanthol and an acylethanolamide.

3. Compositions according to Claim 1, comprising spilanthol, an acyl ethanol ami de and vitamin K2.

4. Compositions according to Claim 1 comprising spilanthol and vitamin K2.

5. Compositions according to Claim 1, 2 or 3 wherein the acyl ethanol ami de is palmitoylethanolamide or oleylethanolamide, preferably palmitoylethanolamide.

6. Compositions according to any one of Claims 1 to 5 wherein said spilantol is contained in an extract of Acmella oleracea.

7. Compositions according to any one of Claims 1 to 6 in the form of tablets, capsules, powders, sachets, granules or syrups.

8. Compositions according to one or more of Claims 1 to 7 wherein the unit dosage of spilanthol is between 0.01 and 40 mg, the dosage of acylethanolamide is between 150 and 1200 mg for palmitoylethanolamide and between 100 and 250 mg for oleylethanolamide and the dosage of vitamin K2 is between 10 and 300 mcg.

9. Compositions according to Claims 1-8 for use in the treatment of pain, particularly for the treatment of pain related to inflammatory states or neuropathic pain.

10. Compositions for use according to Claim 9 for treating humans or animals.

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