Compositions and methods using a combination of oleuropein and fisetin for use in cartilage degeneration

Abstract

The present invention relates to the use of a composition comprising an effective amount of oleuropein and / or its metabolites in combination with fisetin and / or derivatives for the maintenance of joint health or the prevention or treatment of joint disorders in an individual. In particular, the present invention relates to a composition comprising an effective amount of oleuropein and / or its metabolites in combination with fisetin and / or derivatives for use in the prevention or treatment of cartilage degeneration in an individual.

Description

[Technical field]

[0001] The present invention relates to joint health, and in particular to the use of a composition comprising a combination of oleuropein and / or its metabolites and fisetin and / or its metabolites for the prevention or treatment of joint disorders or the maintenance of joint health. [Background technology]

[0002] Osteoarthritis (OA) is a prevalent disease with significant socio-economic impact. Osteoarthritis is a degenerative disease of the articular cartilage of the joints and is the most common form of arthritis, affecting 10% of the adult population. OA is the main cause of disability and healthcare costs in the elderly worldwide. Progressive degeneration and loss of articular cartilage are among the main features of the pathology, accompanied by other changes in joint structures, such as synovial proliferation, sclerosis and thickness of the subchondral bone, osteophyte formation around the joint, ligament laxity, and muscle atrophy, all of which contribute to the clinical symptoms of OA. These symptoms include severe pain, stiffness, reduced joint motion, and disability. Since articular cartilage relies exclusively on its resident cells, chondrocytes, for the maintenance of the extracellular matrix, impaired chondrocyte function and survival leads to articular cartilage failure.

[0003] Recent ex vivo studies have reported mitochondrial dysfunction in human OA chondrocytes, and analysis of mitochondrial electron transport chain activity in these cells showed reduced activity of complexes I, II, and III, as well as low ATP production, compared to normal chondrocytes. This mitochondrial dysfunction may affect several pathways involved in cartilage degeneration, such as oxidative stress, impaired chondrocyte biosynthesis and proliferation response, increased cytokine-induced chondrocyte inflammation and matrix catabolism, mineralization of the cartilage matrix, and increased chondrocyte apoptosis (Blanco et al. "The role of mitochondria in osteoarthritis" Nat. Rev. Rheumatol. 7, 161-169 (2011)).

[0004] Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across the inner membrane, providing the signaling potential for the molecule. Furthermore, mitochondrial Ca2+ may play a role in regulating ATP production in mitochondria and contribute to the orchestration of cellular metabolic homeostasis. (Glancy, B. and RS Balaban (2012) "Role of mitochondrial Ca2+ in the regulation of cellular energetics." Biochemistry 51(14):2959-2973).

[0005] Although the number of individuals suffering from OA is increasing, there is still no cure, and current drug therapies are still symptomatic treatments that focus on alleviating symptoms. For example, pain and inflammation are treated using analgesics (such as acetaminophen) and nonsteroidal anti-inflammatory drugs (NSAIDs). Furthermore, the use of these drugs often comes with side effects, such as gastrointestinal or cardiovascular risks. Because current treatments for OA do not prevent or cure OA, chondrocyte apoptosis is an effective target for regulating cartilage degeneration. Summary of the Invention

[0006] The present inventors have surprisingly demonstrated that a combination of oleuropein (or oleuropein aglycone) and fisetin synergistically activates mitochondrial function at the cellular level via increasing mitochondrial calcium.

[0007] Therefore, the object of the present invention is to provide a composition for use in improving joint health. In particular, the object of the present invention is to provide a composition that improves joint health by preventing or treating cartilage degeneration, and that solves the above-mentioned problems of the prior art regarding side effects such as gastrointestinal and / or cardiovascular risks.

[0008] Thus, one aspect of the present invention relates to a composition comprising an effective amount of a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives for use in the prevention or treatment of cartilage degeneration in an individual.

[0009] Another aspect of the invention relates to a method of preparing a composition for use according to the invention.

[0010] In a final aspect, the invention relates to a kit comprising, in one or more containers, an effective amount of a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives.

[0011] Additional features and advantages are described herein, and will be apparent from the drawings and detailed description that follow. [Brief description of the drawings]

[0012] [Figure 1] 1 shows the chemical structures of fisetin (A) and oleuropein (B). [Diagram 2]1 is a graph showing that the effect of the combination of fisetin and oleuropein on mitochondrial activation via mitochondrial Ca2+ rise in HeLa cells is greater than that of fisetin or oleuropein alone. The bar graph shows the effect of oleuropein (3 μM, black), fisetin (3 μM, gray), and the combination of 3 μM fisetin + 3 μM oleuropein on the integrated mitochondrial calcium rise induced by 100 μM histamine. Results are expressed as mean ± SEM of n=6 experiments. * indicates statistically significant difference at P<0.05 (one-way ANOVA test) for the difference between measured and theoretical values ​​of mitochondrial calcium. [Diagram 3] 1 is a graph showing that fisetin synergizes with oleuropein to activate mitochondria via mitochondrial Ca2+ elevation in HeLa cells. To quantify the synergistic effect of the combination of oleuropein and fisetin on mitochondrial activation, the expected theoretical effect (sum of fisetin effect and oleuropein effect, extracted from data in FIG. 2) was compared with the actual measured effect of this combination (fisetin+oleuropein, extracted from data in FIG. 2). Results are expressed as mean±SEM of n=6 experiments. * indicates statistical significance at P<0.05 (Student's t-test) for the difference between measured and theoretical values ​​in mitochondrial calcium. [Figure 4]Graph showing that oleuropein (aglycone form) synergizes with fisetin to activate mitochondria via mitochondrial Ca2+ elevation in a chondrocyte model mimicking osteoarthritis (SW1353 cells, treated with interleukin-1β). Inset shows the effect of oleuropein (aglycone form, 1 μM, gray), fisetin (10 μM, black), and the combination of 1 μM oleuropein aglycone + 10 μM fisetin on the integrated mitochondrial calcium elevation elicited by 5 mM caffeine. Data in inset are mean + / - SEM from n=11 cell replicate samples from three independent experiments. Inset, * indicates statistically significant difference between quercetin + fisetin combination and any other indicated condition at P<0.05 (one-way ANOVA test). The data in the inset were used to evaluate the expected theoretical effect (the sum of oleuropein aglycone and fisetin effects) and the actual measured effect of this combination (oleuropein aglycone + fisetin) in the main figure. AUC is the area under the curve. Results are expressed as mean + / - SEM from n=11 cell replicates from 3 independent experiments for each condition. * indicates statistical significance at P<0.05 (Student's t-test) for the difference between measured and theoretical values ​​in mitochondrial calcium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition Before discussing the present invention in further detail, the following terms and general technical knowledge will first be defined.

[0014] In the context of the present invention, unless otherwise stated, the percentages referred to are weight / weight percentages.

[0015] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y".

[0016] Numeric ranges used herein are intended to include all numbers and subsets of numbers contained within the range, whether or not expressly disclosed. Moreover, these numerical ranges should be construed to provide support for claims directed to any number or subset of numbers within the range. For example, a disclosure of 1-10 should be construed to correspond to ranges of 1-8, 3-7, 4-9, 3.6-4.6, 3.5-9.9, etc.

[0017] The terms "prevent" and "prevention" refer to administering a composition disclosed herein to a subject not exhibiting any symptoms of the condition to reduce or prevent the onset of at least one symptom associated with the condition. Additionally, "prevention" includes reducing the risk, incidence, and / or severity of a condition or disorder.

[0018] As used herein, an "effective amount" is an amount that treats or prevents a deficiency, treats or prevents a disease or medical condition in an individual, or, more generally, reduces the symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0019] "Animal" includes mammals, including, but not limited to, rodents; aquatic mammals; domestic animals, such as dogs, cats, and other pets; livestock, such as sheep, pigs, cows, and horses; and humans. When "animal", "mammal", or plurals thereof are used, these terms also apply to any animal in which, depending on the context, a demonstrated or intended effect can be demonstrated, for example, by the animal benefiting from improved mitochondrial calcium transport. Although the term "individual" or "subject" is often used herein to refer to a human, the disclosure is not so limited. Thus, the term "individual" or "subject" refers to any animal, mammal, or human that can benefit from the methods and compositions disclosed herein.

[0020] The term "pet" refers to any animal that can benefit from or enjoy the compositions provided by the present disclosure. For example, a pet may be an animal such as a bird, bovine, canine, equine, feline, caprine, wolf, murine, ovine, or porcine, although a pet may be any suitable animal. The term "companion animal" refers to a dog or cat.

[0021] A "subject" or "individual" is a mammal, preferably a human. The term "elderly" in the human context means an age of at least 60 years, preferably greater than 63 years, more preferably greater than 65 years, and most preferably greater than 70 years. The term "older adult" in the human context means a postnatal age of 45 years or more, preferably greater than 50 years, and more preferably greater than 55 years, and includes elderly people. The term "older adult" in the human context means a postnatal age of 45 years or more, preferably greater than 50 years, and more preferably greater than 55 years, and includes elderly people.

[0022] An "oral nutritional supplement" or "ONS" is a composition that contains at least one macronutrient and / or at least one micronutrient, e.g., in a sterile liquid, semisolid, or powder form, and is intended to supplement other nutritional intakes, such as those from food. Non-limiting examples of commercially available ONS products include MERITENE®, BOOST®, NUTREN®, and SUSTAGEN®. In some embodiments, the ONS can be in a liquid form that can be consumed without the addition of additional liquid, e.g., the amount of liquid is one serving of the composition.

[0023] "Kit" means that the components of the kit are physically associated in or with one or more containers and are considered a single unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, packaging of any type or design or material, overwraps, shrink wraps, affixed components (e.g., stapled or glued), or combinations thereof.

[0024] Unless otherwise specified or clearly implied to the contrary by the stated context, any reference to a single feature or limitation of the invention is intended to include the corresponding multiple features or limitations, and any reference to multiple features or limitations is intended to include the corresponding single feature or single limitation.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0026] Compositions for use Joint diseases can involve inflammation to varying degrees. In some diseases, such as rheumatoid arthritis (RA), inflammation is a central component. In other diseases, such as OA, inflammation is less prominent. However, all diseases have a catabolic component that destroys articular cartilage.

[0027] The inventors have shown that providing a combination of oleuropein and / or its metabolites with fisetin and / or derivatives synergistically improves altered mitochondrial function, for example in osteoarthritis.

[0028] That is, in a first aspect, the present invention relates to a composition comprising an effective amount of a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives for use in improving joint health, e.g., for preventing or treating cartilage degeneration in an individual.

[0029] In another manner, this aspect of the invention can be described as the use of an effective amount of a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives in the manufacture of a medicament for the prevention or treatment of cartilage degeneration in an individual.

[0030] Use for preventing or treating cartilage degeneration is synonymous with use for inhibiting or reducing cartilage degeneration.

[0031] Thus, embodiments of the present invention include compositions comprising an effective amount of a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives for use in the prevention or treatment of cartilage degeneration.

[0032] A further embodiment of the invention comprises a composition for use according to the invention, wherein the composition further comprises calcium.

[0033] Raw Materials Key Bioactive Compounds Oleuropein and fisetin are the primary bioactive molecules according to the present invention.

[0034] Oleuropein is a polyphenol present in the fruits, roots, stems and more specifically the leaves of plants belonging to the Oleaceae family, in particular the olive (Olea europaea).

[0035] In one embodiment, at least a portion of the oleuropein is obtained by extraction, for example from one or more of the stems, leaves, fruits, or kernels of a plant belonging to the family Oleaceae, preferably from a plant belonging to the family Oleaceae, such as olive (olive tree), plants of the genus Ligustrum, plants of the genus Syringa, plants of the genus Fraximus, plants of the genus Jasminum, plants of the genus Osmanthus, etc. Additionally or alternatively, at least a portion of the oleuropein and / or metabolites can be obtained by chemical synthesis.

[0036] Non-limiting examples of suitable metabolic products of oleuropein include oleuropein aglycone, hydroxytyrosol, elenolic acid, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

[0037] Fisetin (7,3',4'-flavone-3-ol) (see Figure 1) is a polyphenol found in many plants and is used as a yellow / ochre colorant. Fisetin is also found in many fruits and vegetables, such as strawberries, apples, persimmons, grapes, onions, and cucumbers.

[0038] In one embodiment, at least a portion of the fisetin is obtained by known means, such as, for example, extraction of fisetin from plant / vegetable / fruit sources. Additionally or alternatively, at least a portion of the fisetin and / or its metabolites may be obtained by chemical synthesis.

[0039] Non-limiting examples of suitable metabolites of fisetin include its glucuronidated form, its sulfated form, its derivatives, and mixtures thereof. In a preferred embodiment, the derivative is gerardol.

[0040] Fisetin may be derived from any suitable source and may be isolated and / or chemically synthesized.

[0041] In a preferred embodiment, oleuropein and fisetin and derivatives are obtained from plant sources. For example, oleuropein can be obtained from olive plants. For example, fisetin can be obtained from strawberries, apples, persimmons, grapes, onions, cucumbers, etc.

[0042] The effective amount of each of oleuropein and / or its metabolites and fisetin and / or its derivatives varies depending on the specific composition, the age and condition of the recipient, and the specific disorder or disease being treated.However, in a general embodiment, an individual can be administered 0.001 mg to 1.0 g / day, preferably 0.01 mg to 0.9 g / day, more preferably 0.1 mg to 750 mg / day, more preferably 0.5 mg to 500 mg / day, and most preferably 1.0 mg to 200 mg / day.Furthermore, the inventors have found that the active dose of oleuropein or derivatives in the combination can be reduced for equivalent efficacy.

[0043] In some embodiments, the combination of oleuropein or metabolites and fisetin or derivatives is administered in a composition further comprising calcium. At least a portion of the calcium may be one or more calcium salts, such as calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluconate, calcium lactate, or mixtures thereof. In general embodiments, the individual is administered between 0.1 g and 1.0 g of calcium per day, preferably between 125 mg and 950 g of calcium per day, more preferably between 150 mg and 900 mg of calcium per day, more preferably between 175 mg and 850 mg of calcium per day, and most preferably between 200 mg and 800 mg of calcium per day.

[0044] In an alternative embodiment, the combination of oleuropein and fisetin can be administered sequentially with calcium in a separate composition.The term "sequentially" means that calcium and at least one oleuropein or metabolite thereof are administered sequentially, such that at a first time, at least one oleuropein or metabolite thereof is administered without calcium, and at a second time (before or after the first time), calcium is administered without the combination of oleuropein and fisetin.The time between consecutive administrations can be, for example, a second or a few seconds, minutes, or hours on the same day; a day or a few days, or a few weeks on the same month; or a month or a few months on the same year.

[0045] At least one oleuropein or metabolite thereof and fisetin or a derivative may be formulated in a specific ratio. In some embodiments, the formulation may include these components in the following exemplary ratios: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, each of which may be fisetin:OLE in some embodiments and OLE:fisetin in other embodiments. Preferably, the ratio of OLE:fisetin is between 1:1 and 1:10.

[0046] In some embodiments, oleuropein or a metabolite thereof and fisetin or a derivative thereof are the only polyphenols in a composition and / or the only polyphenols administered to an individual.

[0047] The composition may comprise an effective amount of at least one oleuropein or metabolite thereof. For example, a serving or dose of the composition may comprise an effective amount, and a package may comprise one or more servings or doses. Optionally, the composition may further comprise calcium.

[0048] In another embodiment, oleuropein and / or derivatives may be provided by any of the compositions and methods disclosed in WO 2019 / 092068 and WO 2019 / 092066, entitled "Bioconversion of oleuropein" and "Method of selecting a probiotic," respectively, and WO 2019 / 092069, entitled "Homovanilyl alcohol (HVA), HVA isomer, methods of making compositions comprising such compounds, and methods of using such compounds," each of which is incorporated herein by reference in its entirety.

[0049] Raw Materials Further Bioactive Compounds The composition for use according to the invention may also comprise at least one further biologically active compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibres, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

[0050] The term "biologically active" in the context of this application means that the compound contributes to the health of an individual or provides an effect to the human body beyond satisfying basic nutritional needs.

[0051] At least one further bioactive compound may be of natural origin, i.e. the compound may be obtained from an extract of a plant, animal, fish, fungus, algae, or microbial fermentation. Minerals are also considered to be of natural origin within this definition.

[0052] In a preferred embodiment, the enzyme may be, for example, a protease, such as trypsin, or an enzyme extract, such as bromelain.

[0053] Nutritional composition The composition for use according to the invention may be a nutritional or pharmaceutical composition and may be for human or veterinary use.

[0054] Thus, in a preferred embodiment, the composition for use according to the invention is a nutritional composition.

[0055] By "nutritional composition" is meant in the context of this application a composition that is a source of nutrition for an individual.

[0056] The nutritional formulations or compositions of the present invention may be a complete or incomplete source of nutrition.

[0057] As used herein, "complete nutrition" includes nutritional formulations and compositions that contain sufficient variety and levels of macronutrients (protein, fat, and carbohydrates) and sufficient micronutrients to provide the sole nutritional resource for the animal to which it is administered, such that the patient can obtain 100% of their nutritional needs from such a complete nutritional composition.

[0058] As used herein, "incomplete nutrition" includes nutritional formulations or compositions that do not contain sufficient levels of macronutrients (protein, fat, and carbohydrates) or sufficient micronutrients to be the sole source of nutrition for the animal to which it is administered. Partially complete or incomplete nutritional compositions can be used as dietary supplements.

[0059] The combination of oleuropein and fisetin can be administered in any composition suitable for human and / or animal consumption. In a preferred embodiment, such combination is administered orally or enterally (e.g., tube feeding) to an individual. For example, such combination can be administered to an individual as a drink, food product, capsule, tablet, powder, or suspension.

[0060] Non-limiting examples of suitable compositions include food compositions, dietary supplements, dietary supplements (e.g., liquid ONS), complete nutritional compositions, beverages, pharmaceuticals, oral dietary supplements, medical foods, nutraceuticals, Foods for Special Medical Purposes (FSMP), powdered nutritional formulations to be reconstituted with water or milk prior to ingestion, food additives, medicaments, drinks, pet foods, and combinations thereof.

[0061] Ingredients for nutritional composition Protein Source In one embodiment, the composition for use according to the invention comprises a protein source. The protein source may be a food protein, including but not limited to animal protein (such as milk protein, meat protein, or egg protein), plant protein (such as soy protein, wheat protein, rice protein, and pea protein), or combinations thereof. In one embodiment, the protein is selected from the group consisting of whey, chicken, corn, caseinate, wheat, flax, soy, locust bean, pea, or combinations thereof.

[0062] Carbohydrate Source In one embodiment, the composition comprises a carbohydrate source. Any suitable carbohydrate may be used in the composition of the present invention, including, but not limited to, starch, sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, modified starch, amylose starch, tapioca starch, maize starch, xylitol, sorbitol, or combinations thereof.

[0063] fat source In one embodiment, the composition comprises a fat source. The fat source can comprise any suitable fat or fat mixture. For example, the fat source can include, but is not limited to, vegetable fat (e.g., olive oil, corn oil, sunflower oil, high oleic sunflower oil, rapeseed oil, canola oil, hazelnut oil, soybean oil, palm oil, coconut oil, blackcurrant seed oil, borage oil, and lecithin), animal fat (e.g., dairy fat), or combinations thereof. The fat source can also be a crude product of the above fat (e.g., olive oil containing polyphenols).

[0064] Flavorings, etc. In addition, compositions for use according to the invention may also include natural or artificial flavors, for example fruit flavors such as banana, orange, peach, pineapple or raspberry, or other botanical flavors such as vanilla, cocoa, coffee, etc.

[0065] Nutritional Composition Format In addition to the primary bioactive ingredient, and any further bioactive ingredients, and optionally one or more of a protein source, a carbohydrate source, and a fat source, the nutritional compositions of the present invention may optionally contain any number of additional optional food ingredients, including conventional food additives (artificial or natural), such as, for example, one or more acidulants, additional thickeners, buffers or pH adjusters, chelating agents, colorants, emulsifiers, additives, flavorings, minerals, osmotic agents, pharma-ceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizing agents, and / or vitamins. The optional ingredients may be added in any suitable amount.

[0066] The nutritional compositions of the present invention may be provided in any suitable format.

[0067] Examples of nutritional composition formats in which the compositions for use according to the present invention may be supplied include solutions, ready-for-consumption compositions (e.g. ready-to-drink compositions or instant beverages), liquid meals, soft drinks, juices, sports drinks, dairy drinks, milkshakes, yogurt drinks, soups, etc.

[0068] In other embodiments, the nutritional compositions may be provided in the form of a concentrate, powder, or granules (e.g., effervescent granules) that can be diluted with water or other liquids, such as milk or fruit juice, to produce a ready-to-use composition.

[0069] Further formats of the nutritional compositions include baked products, dairy products, desserts, confectionery products, cereal bars, and breakfast cereals. Examples of dairy products include milk and milk products, yogurt and other cultured dairy products, ice cream, and cheese. Examples of baked products include bread, biscuits, and cakes.

[0070] In one embodiment, the composition for use according to the invention may also be available in a wide variety of formats designed as animal food, in particular for dogs or cats, whether in wet, semi-moist or dry form, in particular in the form of a biscuit.

[0071] Route of administration The nutritional composition of the present disclosure may be administered by any method suitable for administration to humans, particularly to any part of the digestive tract. Enteral administration, oral administration, and administration through a tube or catheter are all included in the present disclosure. The nutritional composition of the present invention may also be administered by a method selected from oral, enteral, sublingual, sublabial, buccal, topical, etc.

[0072] The nutritional composition of the present invention may be administered in any convenient dosage form, including, for example, tablets, capsules, liquids, chewables, softgels, sachets, powders, syrups, suspensions, emulsions, and solutions. In soft capsules, the active ingredients are preferably dissolved or suspended in a suitable liquid, such as fatty oils, paraffin oil, or liquid polyethylene glycol. If desired, stabilizers may be added.

[0073] When the nutritional composition is administered by tube feeding, the nutritional composition may be used for short-term tube feeding or long-term tube feeding.

[0074] Inhibition or reduction of cartilage degeneration Cartilage degeneration can be the result of pathology (either chronic or acute), trauma, or a combination thereof.

[0075] Cartilage degeneration occurs in both disease states dominated by inflammation (eg, rheumatoid arthritis) and in those in which inflammation is less prominent (eg, osteoarthritis).

[0076] Trauma can also result in the initiation of the cartilage degeneration process, for example, rupture of a ligament in the knee leads to instability of the knee joint and the degeneration process is initiated.

[0077] Trauma in the context of this application refers to physiological damage caused by an external cause, such as a fall or impact with a car, etc. Trauma may also be the accumulation of small injuries over time, the so-called "wear and tear".

[0078] Treating trauma with surgery is often preferred, and in one embodiment, the invention relates to a method of treatment for treating trauma by surgery and by administering a composition of the invention.

[0079] Thus, embodiments of the use according to the present invention include use for inhibiting or reducing cartilage degeneration, the cartilage degeneration being the result of pathology or trauma.

[0080] Examples of conditions which involve cartilage degeneration and for which the compositions of the invention may therefore be useful include osteoarthritis, rheumatoid arthritis, gout and pseudogout, septic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, Still's disease, psoriasis (psoriatic arthritis), reactive arthritis, Ehlers-Danlos syndrome, hemochromatosis, hepatitis, Lyme disease, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), Henoch-Schonlein purpura, Hyperimmunoglobulinemia D with recurrent fever, sarcoidosis, TNF receptor-associated periodic syndrome, Wegener's granulomatosis (and many other vasculitic syndromes), familial Mediterranean fever, and systemic lupus erythematosus.

[0081] In a preferred embodiment, the compositions of the invention are for use in inhibiting or reducing cartilage degeneration in RA and / or OA.

[0082] In a further preferred embodiment, the compositions of the invention are for use in inhibiting or reducing cartilage degeneration in OA.

[0083] Furthermore, without wishing to be bound by theory, it has been observed that while inflammation often leads to cartilage degeneration in joints, cartilage degeneration also occurs in situations where the inflammatory component is much less severe, perhaps even negligible.

[0084] For example, trauma to a joint may be sufficient to initiate cartilage degeneration without the significant inflammatory component present in, for example, RA. Trauma may include, for example, a torn ligament, or an impact injury to a joint (e.g., knee, finger).

[0085] In another example, OA is primarily a joint degenerative disease, with a lesser inflammatory component.

[0086] Thus, in one embodiment the invention relates to a composition for use according to the invention for inhibiting or reducing cartilage degeneration, wherein the cartilage degeneration occurs in the context of trauma or a pathology with little or no inflammatory component, such as, for example, OA.

[0087] Use to combat early degenerative events The hypertrophy suggests catabolic activity of chondrocytes that are not of normal phenotype.

[0088] Thus, in one embodiment, the present invention relates to a composition according to the invention comprising a combination of oleuropein and / or its metabolites with fisetin and / or derivatives for use in inhibiting or reducing chondrocyte hypertrophy, which is one early event indicative of cartilage degeneration.

[0089] Treatment or prevention of age-related loss of mobility Compositions for use according to the invention have been shown to inhibit or reduce proteolytic activity.

[0090] Aging leads to cartilage degeneration.

[0091] Thus, the present invention relates to a composition of the invention for use in inhibiting or reducing cartilage degeneration associated with aging.

[0092] In another embodiment, the invention relates to a composition of the invention for use in inhibiting or reducing collagen degeneration in age-related cartilage degeneration, e.g. for use in inhibiting or preventing collagen II degeneration in age-related cartilage.

[0093] Cartilage degeneration can contribute to joint stiffness and pain, resulting in decreased mobility in the patient.

[0094] The compositions for use according to the invention may in other embodiments be used for i) maintaining or improving joint function, including cartilage function, during ageing; ii) reducing joint pain, including inflammatory pain and / or nociceptive pain.

[0095] In a further embodiment, the present invention relates to a composition for use according to the present invention for improving mobility in a subject, such as an adult or elderly mammal.

[0096] Thus, in a preferred embodiment, the compositions according to the invention may be for use to improve the activity and / or mobility of an individual, for example by preventing or treating osteoarthritis and / or by inhibiting or reducing cartilage degeneration.

[0097] Other preferred embodiments relate to compositions for use according to the invention, which use is for preventing cartilage degeneration and thus maintaining healthy joints, or for maintaining or improving mobility, for preventing or reducing joint pain (inflammatory and / or nociceptive pain). In a further embodiment, the compositions of the invention may be used to maintain the condition of cartilage.

[0098] Subject group The subject group of the composition for use according to the present invention may be any mammalian animal, for example, suffering from one or more of the pathologies associated with cartilage degeneration mentioned herein, and thus exhibiting cartilage degeneration.Cartilage degeneration can be detected by visual means, such as X-ray photography.Alternatively, the product of cartilage degeneration can be detected in body fluids.For example, one or more collagen II epitopes, such as (Coll2-1, Coll2-1 NO2, CTX-II), can be detected in a sample, such as, for example, a plasma sample or a urine sample.

[0099] Another subject group may be any mammalian animal that has not yet exhibited cartilage degeneration, but is at risk for cartilage degeneration, e.g., at risk for OA, RA, or any of the conditions involving cartilage degeneration mentioned herein. In a preferred embodiment, the present invention relates to a composition according to the present invention comprising a combination of oleuropein or a metabolite thereof and fisetin or a derivative, for use in inhibiting or reducing premature cartilage degeneration, administered to this subject group.

[0100] Certain embodiments of the invention relate to compositions for use to improve activity and / or mobility of an individual, for example by preventing or treating osteoarthritis and / or by inhibiting or reducing cartilage degeneration in elderly or aging individuals.

[0101] In a further embodiment, the composition for use according to the invention may be for use in a mammal, such as a human, or a pet. Examples of pets include cats, dogs, and horses.

[0102] Although the present invention may be useful in many different age groups, in a preferred embodiment, the compositions for use to improve mobility according to the present invention are targeted to the geriatric population, particularly healthy aging and / or senior mammals.

[0103] Method for producing the nutritional composition of the present invention The present invention relates in a further aspect to a method for producing a nutritional composition for use according to the invention, comprising the steps of: The present invention relates to a method comprising the steps of: preparing raw materials for a nutritional composition comprising a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives; and mixing the raw materials so that the nutritional composition comprises a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives.

[0104] Pharmaceutical Compositions for Use In a further embodiment, the present invention relates to a composition for use in inhibiting or preventing cartilage degeneration according to the present invention, which is a pharmaceutical composition.

[0105] By pharmaceutical is meant a composition, as distinct from a nutritional composition, in which the substance is used on or in the body as a medicine to prevent, diagnose, mitigate, treat or cure a disease in a human or animal. In accordance with the present invention, the pharmaceutical can be used to inhibit or reduce cartilage degeneration.

[0106] The medicament may be for human use or may be a veterinary composition suitable for example for dogs, cats or horses, particularly thoroughbred horses.

[0107] In one preferred embodiment, the pharmaceutical composition of the present invention comprises a combination of oleuropein or a metabolite thereof and fisetin or a derivative.

[0108] In another preferred embodiment, the pharmaceutical composition of the present invention comprises oleuropein or a metabolite thereof and fisetin or a derivative.

[0109] The present invention further relates to the use of a medicament according to the invention, as described herein as the use of a composition of the invention.

[0110] A pharmaceutical composition for use according to the invention, comprising a combination of oleuropein or a metabolite thereof and fisetin or a derivative in combination with at least one additive selected from the group consisting of pharma- ceutically acceptable additives. Procedures for the preparation of pharmaceutical compositions according to the invention can be easily found by those skilled in the art, for example in the handbook Remington's Pharmaceutical Sciences, Mid. Publishing Co, Easton, Pa., USA. Physiologically acceptable additives, vehicles and adjuvants are also described in the handbook entitled "Handbook of Pharmaceutical Excipients, Second edition, American Pharmaceutical Association, 1994". To formulate pharmaceutical compositions according to the invention, those skilled in the art will have the advantage of referring to the latest edition of the European Pharmacopoeia or the United States Pharmacopoeia (USP). Those skilled in the art will have the advantage of referring in particular to the fourth edition of the European Pharmacopoeia "2002" or the US Pharmacopoeia USP 25-NF 20 edition.

[0111] Advantageously, the pharmaceutical composition as defined above is suitable for oral, parenteral or intravenous administration. When the pharmaceutical composition for use according to the invention comprises at least one pharma- ceutically acceptable or physiologically acceptable excipient, in particular, the excipient is suitable for administering the composition by the oral route or the excipient is suitable for administering the composition by the parenteral route.

[0112] The pharmaceutical compositions for use according to the invention may be available indifferently in solid or liquid form. For oral administration, solid pharmaceutical compositions in the form of tablets, capsules or gelatin capsules will be preferred.

[0113] If in liquid form, the pharmaceutical composition may suitably be in the form of an aqueous or non-aqueous suspension, or a water-in-oil or oil-in-water emulsion.

[0114] The solid pharmaceutical forms may contain, as vehicles, adjuvants or additives, at least one diluent, one flavoring, one solubilizer, one lubricant, one suspending agent, one binder, one disintegrating agent and one encapsulating agent. Such compounds are, for example, magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, gelatin, cellulosic materials, cocoa butter, etc. The liquid forms of the compositions may contain water, optionally in admixture with propylene glycol or polyethylene glycol, and may also contain colorants, flavors, stabilizers and thickeners.

[0115] Combination with known treatments In the absence of disease-modifying OA drugs (DMOADs), alternative treatments and prevention of OA may be achieved through nutrition.

[0116] From histological data, it can be seen that oleuropein has a greater effect on OA score than compounds that mainly affect degeneration.Therefore, the effectiveness of oleuropein may be due to its combined effect on inflammation and degeneration.Therefore, in a preferred embodiment, the composition of the present invention that shows to inhibit or reduce degeneration can be combined with the treatment to inhibit or reduce inflammation.

[0117] Treatment method The present invention also relates to a method for preventing or treating cartilage degeneration, such as a pathology in which cartilage degeneration occurs or a trauma associated with cartilage degeneration, comprising administering an effective amount of a composition according to the present invention to an individual in need thereof. For example, the method comprises administering an effective amount of a composition comprising a combination of oleuropein or a metabolite thereof and fisetin or a derivative.

[0118] As used herein, an "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0119] The effective amount of a composition of the invention required to achieve a therapeutic effect may, of course, vary depending on the particular composition, the route of administration, the age and condition of the subject, and the particular disorder or disease being treated.

[0120] The invention further provides a method for preventing or treating a condition involving cartilage degeneration, such as OA or RA, inhibiting or reducing cartilage degeneration, inhibiting or reducing collagen degeneration in cartilage, inhibiting or reducing collagen II degeneration in cartilage, which method comprises administering to an individual an effective amount of a composition for use according to the invention.

[0121] In one embodiment, the method of treatment according to the invention relates to the prevention or treatment of osteoarthritis.

[0122] The method of treatment according to the invention may be in a mammal, such as a human or a pet, such as a dog, cat and / or horse.

[0123] In certain embodiments, the composition of the invention administered in the treatment method may be one or more nutritional compositions of the invention and / or a pharmaceutical composition of the invention.

[0124] kit The disclosure also provides kits comprising a combination of oleuropein and / or its metabolites in combination with fisetin and / or derivatives in one or more containers. In embodiments of the kit, the one or more containers include at least one first container storing oleuropein and / or metabolites separately from fisetin and / or derivatives stored in at least one second container, and the kit further includes instructions for combining the oleuropein with fisetin to form a unit dosage form.

[0125] In one embodiment of the kit, the combination may be provided together as one or more prepackaged unit dosage forms, e.g., as separate containers each containing a dry powder, such that each container contains one prepackaged unit dosage form.

[0126] In another embodiment, the kit can include multiple compositions to be mixed together to form one or more of the compositions disclosed herein.For example, the kit can contain two or more dry powders in separate containers that are related to each other, each of which contains a portion of the final unit dosage form.As a non-limiting example of such an embodiment, the kit can include one or more first containers that contain oleuropein, and also one or more second containers that contain fisetin.The contents of one of the first containers can be mixed with one of the second containers to form at least a portion of the unit dosage form of the composition.

[0127] The above dosing examples do not require uninterrupted daily administration. Rather, there may be several short interruptions in administration, such as a 2-4 day break during the administration period. The ideal duration of administration of the compositions of the present invention can be determined by one of skill in the art.

[0128] Disclosure Combinations It should be noted that embodiments and features described in the context of one aspect of the invention also apply to other aspects of the invention.

[0129] Compositions for use according to the invention are described herein under different parameters, such as ingredients, form of nutritional composition, use, target population, etc. It should be noted that embodiments and features described in the context of one of the parameters of a composition for use according to the invention may also be combined with other embodiments and features described in the context of another parameter, unless expressly stated otherwise.

[0130] All patent and non-patent literature cited in this application is hereby incorporated by reference in its entirety.

[0131] The invention will now be described in further detail by the following non-limiting examples. EXAMPLES

[0132] The following non-limiting examples provide experimental data supporting the compositions and methods disclosed herein.

[0133] Example 1 To test the effect of oleuropein (or oleuropein aglycone), fisetin, and their combination in living cells, we measured mitochondrial calcium elevation in HeLa cells. HeLa cells were purchased from ATCC. HeLa cells were seeded in 96-well plates at a density of 50,000 cells / well in Minimum Essential Medium (DMEM, Gibco), high glucose, +10% fetal bovine serum. Mitochondrial calcium measurements were performed using HeLa cells (Sirion biotech) infected with an adenovirus expressing mitochondrial modified aequorin, a calcium sensor targeted in mitochondria (Montero et al., 2004). For aequorin reconstitution, 24 hours after infection, standard medium (145 mM NaCl, 5 mM KCl, 1 mM MgCl) supplemented with 1 μM wild-type coelenterazine was used. 2 , 1 mM CaCl 2 Cells were incubated for 2 hours at room temperature (22±°C) in 10 mM NaCl, 10 mM glucose, and 10 mM Hepes, pH 7.4. For treatment, compounds were added directly to cell or myotube cultures 2 hours before measurement. Luminescence was measured with a FLIPR Tetra Aequorin (Molecular Devices). Mitochondrial calcium elevation was obtained by stimulating cells with 100 μM histamine. Conversion of luminescence data to calcium concentration (Calibration) was performed using an algorithm as previously described (Alvarez & Montero, 2002). Quantification was performed using a custom module analysis based on Excel (Microsoft) and GhaphPad Prism 7.02 (GhaphPad) software.

[0134] As shown in Figure 2, the effect of the combination of oleuropein and fisetin on mitochondrial activation via mitochondrial Ca2+ elevation in HeLa cells is greater than the effect of fisetin or oleuropein alone. Also, Figure 3 shows that fisetin synergizes with oleuropein to activate mitochondria via mitochondrial Ca2+ elevation in HeLa cells.

[0135] Example 2 To verify the synergistic effect of the combination of oleuropein (aglycone form) + fisetin on a cellular model of osteoarthritis chondrocytes (chondrocytes), we measured mitochondrial calcium in SW1353 cells treated with the pro-inflammatory cytokine interleukin-1β. SW1353 cells were purchased from ATCC. SW1353 cells were seeded in 96-well plates at a density of 10,000 cells / well in 100 mm dishes. Cells were cultured in high glucose minimal essential medium (DMEM, Gibco) containing 10% fetal bovine serum and 1% penicillin-streptomycin. SW1353 cells were treated with 10 ng / mL of the pro-inflammatory cytokine interleukin-1β for 48 hours as a mimic of osteoarthritis.

[0136] Measurement of mitochondrial calcium in SW1353 cells was performed using the same procedure as used for HeLa cells. The synergistic effect of oleuropein aglycone and fisetin in an osteoarthritic chondrocyte model was quantified as described for HeLa cells.

[0137] result As shown in Figure 4, in the described cellular model of osteoarthritic chondrocytes (SW1353 cells treated with IL-1β), oleuropein aglycone synergized with fisetin to increase mitochondrial Ca 2+ Activates mitochondria through elevation.

Claims

1. A composition comprising an effective amount of oleuropein and / or its metabolites and fisetin and / or derivatives for use in the prevention or treatment of cartilage degeneration in an individual.

2. The composition according to claim 1, wherein the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, elenolic acid, homovanillyl alcohol, isohomovannillyl alcohol, glucuronide conjugates thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

3. The composition according to claim 1, wherein the derivative of fisetin is selected from the group consisting of its glucuronidated form, its sulfated form, its derivatives, and mixtures thereof.

4. The composition according to claim 1, further comprising calcium.

5. The composition according to claim 1, further comprising at least one compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fiber, probiotics, fatty acids, enzymes, minerals, trace elements, and / or vitamins.

6. The composition according to claim 1, selected from the group consisting of food compositions, dietary supplements, nutritional compositions, oral nutritional supplements, medical foods, nutraceuticals, beverages, powdered nutritional preparations that are reconstituted with water or milk before ingestion, food additives, foods for special medical purposes (FSMPs), pharmaceuticals, drinks, pet food, and combinations thereof.

7. The composition according to claim 1, which is in the form of a solid powder, powder stick, capsule, or solution.

8. The composition according to claim 1, wherein the use is i) to maintain or improve joint function, including cartilage function, in aging, and ii) to reduce joint pain, including inflammatory and / or nociceptive pain.

9. The composition according to claim 1, wherein the use is for improving the activity and / or mobility of the individual.

10. The composition according to claim 1, wherein the use is for inhibiting or reducing cartilage degeneration in osteoarthritis.

11. The composition according to claim 1, wherein the individual is a middle-aged or elderly person.

12. A method for producing a nutritional composition according to any one of claims 1 to 11, comprising the steps of: preparing one or more raw materials for the nutritional composition; oleuropein or its metabolites; fisetin and / or derivatives; and optionally further calcium; and mixing them.

13. A kit comprising one or more containers containing a combination of oleuropein and / or its metabolites and fisetin and / or derivatives.

14. The kit according to claim 13, wherein the one or more containers include at least one first container for storing the oleuropein and / or metabolites separately from the fisetin and / or derivatives stored in at least one second container, and the kit further includes instructions for mixing the oleuropein with fisetin to form a unit dosage form.

15. The kit according to claim 13 or 14, wherein each of the one or more containers contains a unit dosage form of a combination of oleuropein and / or its metabolite and fisetin and / or a derivative.

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