Compositions and methods for cartilage degeneration using combination of oleuropein and fisetin

Abstract

The present invention relates to the use of a composition comprising an effective amount of oleuropein and / or a metabolite thereof in combination with fisetin and / or a derivative for maintaining joint health in a subject or preventing or treating joint disorders in a subject. In particular, the present invention relates to a composition comprising an effective amount of oleuropein and / or a metabolite thereof in combination with fisetin and / or a derivative for use in the prevention or treatment of cartilage degeneration in a subject.

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 preventing or treating joint disorders or maintaining joint health. Background Art

[0002] Osteoarthritis (OA) is a highly prevalent disease with important socioeconomic impact. It 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 health care costs in the elderly worldwide. The progressive degeneration and loss of articular cartilage are the main pathological features, along with changes in other joint structures, such as synovial hyperplasia, sclerosis and thickening of subchondral bone, osteophyte formation at the joint margins, ligament laxity and muscle atrophy, all of which lead to the clinical symptoms of OA. These symptoms include severe pain, stiffness, loss of joint motion and disability. Because articular cartilage relies only on its intrinsic cells (chondrocytes) to maintain the extracellular matrix, the impairment of chondrocyte function and survival will lead to the failure of articular cartilage.

[0003] Recent in vitro 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, and reduced ATP production compared to normal chondrocytes. This mitochondrial dysfunction may affect several pathways associated with cartilage degradation, including oxidative stress, defects in chondrocyte biosynthesis and growth responses, cytokine-induced chondrocyte inflammation and increased matrix catabolism, cartilage matrix calcification, and increased chondrocyte apoptosis (Blanco et al., "The role of mitochondria inosteoarthritis" Nat. Rev. Rheumatol. 7, 161-169 (2011).

[0004] Mitochondria are the main source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across their inner membrane, providing a signaling potential for this molecule. In addition, mitochondrial Ca2+ plays a role in the regulation of ATP production in mitochondria and potentially contributes to the coordination 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] Despite the increase in the number of individuals suffering from OA, there is still no cure, and current medical treatments remain merely conservative, focusing on relieving symptoms. For example, painkillers (such as acetaminophen) and nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat pain and inflammation. In addition, the use of these drugs is often associated with side effects such as gastrointestinal or cardiovascular risks. Because current treatments for OA do not prevent or cure OA, chondrocyte apoptosis would be an effective target for regulating cartilage degradation. Summary of the invention

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

[0007] Therefore, one object of the present invention relates to providing a composition for improving joint health. Specifically, one object of the present invention is to provide a composition for improving joint health by preventing or treating cartilage degradation, and solving the above-mentioned problems in 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 oleuropein and / or its metabolites in combination with fisetin and / or its derivatives for use in preventing or treating cartilage degeneration in an individual.

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

[0010] In a final aspect, the present invention relates to a kit comprising, in one or more containers, effective amounts 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 following figures and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Representing the chemical structures of fisetin (A) and oleuropein (B)

[0013] Figure 2The effect of the combination of fisetin and oleuropein is shown to be greater than the effect of fisetin or oleuropein alone in terms of mitochondrial activation by mitochondrial Ca2+ elevation in HeLa cells. 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 increase in integrated mitochondrial calcium induced by 100 μM histamine. The results are expressed as the mean + / - SEM from n = 6 experiments. * indicates a statistically significant difference in the measured value relative to the theoretical difference in mitochondrial calcium, P < 0.05 (one-way ANOVA test).

[0014] Figure 3 is a graph showing that fisetin acts synergistically with oleuropein to activate mitochondria through 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 (the sum of the fisetin effect and the oleuropein effect) was calculated from Figure 2 ) and the combination (fisetin + oleuropein, from Figure 2 The actual measured effects (extracted from the data of ) were compared. Results are presented as mean + / - SEM from n=6 experiments. * indicates a statistically significant difference in measured values ​​relative to the theoretical difference in mitochondrial calcium, P<0.05 (Student's t-test).

[0015] Figure 4 It is a diagram showing that oleuropein (aglycone form) and fisetin act synergistically to activate mitochondria by increasing mitochondrial Ca2+ in a model of chondrocytes (SW1 353 cells, treated with interleukin-1β) simulating osteoarthritis. The 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 increase induced by 5 mM caffeine. The data in the inset are the mean + / - SEM of n = 11 cells from 3 independent experiments. In the inset, * indicates that the combination of quercetin + fisetin is statistically significantly different from any other indicated condition at P < 0.05 (single factor ANOVA test). The data in the inset are used to determine the expected theoretical effect (the sum of the oleuropein aglycone effect and the fisetin effect) and the actual measured effect of the combination (oleuropein aglycone + fisetin) in the main figure. AUC, area under the curve. Results are presented as mean + / - SEM of n=11 cell replicates from 3 independent experiments for each condition. *Indicates statistically significant difference in measured values ​​relative to theoretical difference in mitochondrial calcium, P<0.05 (Student's t-test). DETAILED DESCRIPTION

[0016] definition

[0017] Before discussing the present invention in further detail, the following terms and conventions are first defined.

[0018] In the context of the present invention, percentages mentioned are weight / weight percentages, unless otherwise indicated.

[0019] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y", or "X and Y".

[0020] The numerical ranges used herein are intended to include every value and subset of values ​​contained within the range, whether or not specifically disclosed. In addition, these numerical ranges should be understood to provide support for claims involving any value or subset of values ​​within the range. For example, the disclosure of 1 to 10 should be understood to support ranges of 1 to 8, 3 to 7, 4 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0021] The term "prevention" refers to administering a composition disclosed herein to a subject who does not show any symptoms of the disorder to reduce or prevent the development of at least one symptom associated with the disorder. In addition, "prevention" includes reducing the risk, incidence and / or severity of a disorder or disorder.

[0022] As used herein, an "effective amount" is an amount that treats or prevents a defect, treats or prevents a disease or medical condition in an individual, or more generally, reduces symptoms, manages disease progression, or provides a nutritional, physiological or medical benefit to the individual.

[0023] "Animal" includes, but is not limited to, mammals, including, but not limited to, rodents; aquatic mammals; livestock, such as dogs, cats, and other pets; farm animals, such as sheep, pigs, cattle, and horses; and humans. When "animal," "mammal," or their plural forms are used, these terms also apply to any animal that is capable of having the effect indicated or intended to be indicated by the context of the paragraph, such as an animal that benefits from improved mitochondrial calcium import. Although the terms "individual" or "subject" are often used herein to refer to humans, the present disclosure is not limited thereto. Therefore, the terms "individual" or "subject" refer to any animal, mammal, or human that can benefit from the methods and compositions disclosed herein.

[0024] The term "pet" means any animal that can benefit from or enjoy the compositions provided by the present disclosure. For example, the pet can be an avian, bovine, canine, equine, feline, caprine, lupine, murine, ovine, or porcine animal, but the pet can be any suitable animal. The term "companion animal" means a dog or a cat.

[0025] "Subject" or "individual" is a mammal, preferably a human. In the case of humans, the term "elderly" refers to an age of at least 60 years, preferably over 63 years, more preferably over 65 years, and most preferably over 70 years, since birth. In the context of humans, the term "middle-aged and elderly" refers to an age of at least 45 years, preferably over 50 years, more preferably over 55 years, since birth, and includes elderly individuals. In the context of humans, the term "middle-aged and elderly" refers to an age of at least 45 years, preferably over 50 years, more preferably over 55 years, since birth, and includes elderly individuals.

[0026] An "oral nutritional supplement" or "ONS" is a composition comprising at least one macronutrient and / or at least one micronutrient, e.g., in sterile liquid, semisolid, or powdered form, and intended to supplement other nutritional intake, such as that from food. Non-limiting examples of commercially available ONS products include and In some embodiments, the ONS may be a beverage in liquid form that can be consumed without further addition of liquid, such as a certain amount of liquid in a serving of the composition.

[0027] "Kit" means that the components of the kit are physically associated in or with one or more containers and are considered a unit for manufacture, distribution, sale or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, outer packaging, shrink wrap, attached components (e.g., binding components, adhesive components, etc.), packaging of any type, design or material, or any combination thereof.

[0028] All singular features or limitations mentioned in the present invention shall include the corresponding plural features or limitations, and vice versa, unless otherwise indicated or clearly implied to the contrary in the context of the reference to these contents.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0030] Composition used

[0031] Joint diseases may be accompanied by inflammation to a greater or lesser degree. In some diseases, inflammation is the most important component, such as in rheumatoid arthritis (RA). In other diseases, such as OA, inflammation appears less prominent. However, both diseases have a catabolic component, in which the articular cartilage is broken down.

[0032] The present inventors have shown that providing oleuropein and / or its metabolites in combination with fisetin and / or derivatives synergistically improves mitochondrial function that is altered, for example, in osteoarthritis.

[0033] Thus, the present invention in a first aspect 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, such as preventing or treating cartilage degeneration in an individual.

[0034] 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 preventing or treating cartilage degeneration in an individual.

[0035] The use for preventing or treating cartilage degeneration is synonymous with the use for inhibiting or reducing cartilage degeneration.

[0036] Thus, embodiments of the present invention include a composition comprising an effective amount of oleuropein and / or its metabolites in combination with fisetin and / or its derivatives for use in preventing or treating cartilage degradation.

[0037] A further embodiment of the present invention comprises a composition for use according to the present invention, wherein the composition further comprises calcium.

[0038] Ingredients - Main Bioactive Compounds

[0039] Oleuropein and fisetin are the main bioactive molecules according to the present invention.

[0040] Oleuropein is a polyphenol present in plants belonging to the family Oleaceae, and in particular in the fruit, roots, trunk and more particularly in the leaves of olives.

[0041] In one embodiment, at least a portion of oleuropein is obtained by extraction, for example by extraction from plants such as plants belonging to the family Oleaceae, preferably from plants belonging to the family Oleaceae such as olive (olive tree), plants of the genus Ligustrum, plants of the genus Syringa, plants of the genus Fraxinus, plants of the genus Jasminum and plants of the genus Oleaceae One or more of the stems, leaves, fruits or pits. Additionally or alternatively, at least a portion of oleuropein and / or its metabolites may be obtained by chemical synthesis.

[0042] Non-limiting examples of suitable metabolites of oleuropein include oleuropein aglycone, hydroxytyrosol, olive acid, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

[0043] Fisetin (7,3',4'-flavon-3-ol) (see Figure 1 ) is a polyphenol found in many plants where it is used as a yellow / ochre colorant. It is also present in many fruits and vegetables, such as strawberries, apples, persimmons, grapes, onions and cucumbers.

[0044] In one embodiment, at least a portion of fisetin is obtained by known means, such as by extraction from a plant / vegetable / fruit source of fisetin. Additionally or alternatively, at least a portion of fisetin and / or its metabolites may be obtained by chemical synthesis.

[0045] 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.

[0046] Quercetin may be derived from any suitable source, and may be isolated and / or chemically synthesized.

[0047] In a preferred embodiment, oleuropein and fisetin and derivatives are obtained from plant sources. For example, oleuropein can be obtained from olive plants, and fisetin can be obtained from strawberries, apples, persimmons, grapes, onions, cucumbers, etc.

[0048] The effective amount of each of oleuropein and / or its metabolites and fisetin and / or its derivatives varies with the specific composition, the age and condition of the recipient, and the specific disorder or disease being treated. However, in general embodiments, 0.001 mg to 1.0 g per day, preferably 0.01 mg to 0.9 g per day, more preferably 0.1 mg to 750 mg per day, more preferably 0.5 mg to 500 mg per day, and most preferably 1.0 mg to 200 mg per day can be administered to an individual. In addition, the inventors have found that the active dose of oleuropein or a derivative in the combination can be reduced to obtain an equivalent efficacy.

[0049] In some embodiments, the combination of oleuropein or a metabolite and fisetin or a derivative is administered in a composition that also comprises 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 gluconate, calcium gluconate, calcium lactate, or a mixture thereof. In a general embodiment, 0.1 g to 1.0 g of calcium is administered to an individual per day, preferably 125 mg to 950 g of calcium per day, more preferably 150 mg to 900 mg of calcium per day, more preferably 175 mg to 850 mg of calcium per day, and most preferably 200 mg to 800 mg of calcium per day.

[0050] In an alternative embodiment, the combination of oleuropein and fisetin can be administered sequentially with calcium in separate compositions. The term "sequentially" means that oleuropein or at least one of its metabolites and calcium are administered in a sequential manner, such that oleuropein or at least one of its metabolites is administered at a first time without calcium, and calcium is administered at a second time (before or after the first time) without the combination of oleuropein and fisetin. The time between sequential administrations can be, for example, one second or seconds, one minute or minutes, or one hour or hours in the same day; one day or days or one week or weeks in the same month; or one or more months in the same year.

[0051] At least one oleuropein or a 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, and each of these ratios may be fisetin:oleuropein in some embodiments and oleuropein:fisetin in other embodiments. Preferably, the oleuropein:fisetin ratio is 1:1 to 1:10.

[0052] In some embodiments, oleuropein or a metabolite thereof and fisetin or a derivative thereof are the only polyphenols in the composition administered to the individual and / or the only polyphenols administered to the individual.

[0053] The composition may include an effective amount of oleuropein or at least one of its metabolites. For example, a single serving or dose of the composition may include an effective amount, and the package may contain one or more servings, or one or more doses. Optionally, the composition may also include calcium.

[0054] 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 (each entitled “Bioconversion of oleuropein” and “Method of selecting a probiotic”) and WO 2019 / 092069 (each entitled “Homovanillyl alcohol (HVA), HVA isomers, methods of making compositions comprising such compounds. and methods of using such compounds”), each of which is incorporated herein by reference in its entirety.

[0055] Ingredients - Additional Bioactive Compounds

[0056] The composition for use according to the invention may also comprise at least one further biologically active compound chosen from antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibers, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

[0057] In the context of this application, the term "biologically active" means that the compound contributes to the health of an individual, or has an effect on the human body, other than satisfying basic nutritional requirements.

[0058] The at least one additional biologically active compound may be derived from a natural source. Thus, the compound may be derived from plants, animals, fish, fungi, algae, extracts from microbial fermentations. Minerals are considered to be derived from natural sources and are also within this definition.

[0059] In a preferred embodiment, the enzyme may be a protease such as trypsin, or an enzyme extract such as, for example, bromelain.

[0060] Nutritional composition

[0061] The composition for use according to the invention may be a nutritional composition or a pharmaceutical composition and may be for human or veterinary use.

[0062] Therefore, in a preferred embodiment, the composition for use according to the invention is a nutritional composition.

[0063] In the context of this application, by "nutritional composition" is meant a composition that is a source of nutrition for an individual.

[0064] The nutritional products or compositions of the present invention may be a complete nutritional source, or may be an incomplete nutritional source.

[0065] As used herein, "complete nutrition" includes nutritional products and compositions containing a complete variety of macronutrients (protein, fat and carbohydrates) and micronutrients in sufficient amounts to be administered to an animal as the sole source of nutrition. Patients can get 100% of their nutritional requirements from such complete nutritional compositions.

[0066] As used herein, "incomplete nutrition" includes nutritional products and compositions that do not contain sufficient amounts of macronutrients (protein, fat, and carbohydrates) or micronutrients to be administered to an animal as a sole source of nutrition. Partial or incomplete nutritional compositions can be used as nutritional supplements.

[0067] The combination of oleuropein and fisetin can be administered in any composition suitable for human and / or animal consumption. In a preferred embodiment, it is administered orally or enterally (e.g., by tube feeding). For example, it can be administered to an individual in the form of a beverage, food product, capsule, tablet, powder, or suspension.

[0068] Non-limiting examples of suitable compositions include food compositions, dietary supplements, dietary supplements (e.g., liquid ONS), complete nutritional compositions, beverages, pharmaceuticals, oral nutritional supplements, medical foods, nutraceuticals, foods for special medical purposes (FSMPs), powdered nutritional products to be reconstituted with water or milk before consumption, food additives, medicaments, drinks, pet foods, and combinations thereof.

[0069] Nutritional composition ingredients

[0070] Protein Source

[0071] In one embodiment, the composition for use according to the invention comprises a protein source. The protein source may be a dietary 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 a combination thereof. In one embodiment, the protein is selected from whey, chicken, corn, caseinate, wheat, flax, soy, carob, pea or a combination thereof.

[0072] Carbohydrate sources

[0073] In one embodiment, the composition comprises a carbohydrate source. Any suitable carbohydrate can 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, tapioca starch, corn starch, xylitol, sorbitol or a combination thereof.

[0074] Fat sources

[0075] In one embodiment, the composition comprises a fat source. The fat source may include any suitable fat or fat mixture. For example, the fat source may include, but is not limited to, vegetable fat (such as, 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, lecithin, etc.), animal fat (such as, milk fat) or a combination thereof. The fat source may also be a less refined form of the fat listed above (e.g., olive oil containing polyphenols).

[0076] Flavoring agents, etc.

[0077] Furthermore, the composition for use according to the invention may also comprise natural or artificial flavourings, for example fruit flavourings such as banana, orange, peach, pineapple or raspberry or other plant flavourings such as vanilla, cocoa, coffee and the like.

[0078] Nutritional composition form

[0079] In addition to the main bioactive component and any additional bioactive components and optionally one or more of a protein, carbohydrate and fat source, the nutritional composition may also contain any number of optional additional food ingredients, including conventional (synthetic or natural) food additives, such as one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavoring agents, minerals, osmotic agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturants and / or vitamins. The optional ingredients may be added in any suitable amount.

[0080] The nutritional composition may be provided in any suitable form.

[0081] Examples of nutritional composition forms in which the composition for use according to the present invention can be provided include solutions, ready-to-use compositions (e.g. ready-to-drink compositions or instant beverages), liquid foods, soft drinks, juices, sports drinks, dairy drinks, milkshakes, yogurt drinks, soups, etc.

[0082] In other embodiments, the nutritional composition may be provided in the form of a concentrate, powder, or granules (eg, effervescent granules) that are diluted with water or other liquids (such as milk or juice) to produce a ready-to-use composition.

[0083] Other forms of nutritional compositions include baked products, dairy products, desserts, confectionery products, cereal bars and breakfast cereals. Examples of dairy products include milk and dairy drinks, yogurt and other sour milk products, ice cream and cheese. Examples of baked products include bread, biscuits and cakes.

[0084] In one embodiment, the composition for use according to the invention can also be designed in various forms of animal food, in particular animal food for dogs or cats, whether obtained in wet, semi-moist or dry form, in particular in the form of biscuits.

[0085] Route of administration

[0086] The nutritional composition of the present disclosure can be administered by any means suitable for human administration, and in particular, in any part of the gastrointestinal tract. Enteral administration, oral administration, and administration through a tube or catheter are all encompassed by the present invention. The nutritional composition can also be administered by a means selected from oral, rectal, sublingual, sublabial, intraoral, topical, and the like.

[0087] The nutritional composition can be administered in any known form, including, for example, convenient dosage forms of tablets, capsules, liquids, chewable tablets, soft gels, sachets, powders, syrups, liquid suspensions, emulsions and solutions. In soft capsules, the active ingredient is preferably dissolved or suspended in a suitable liquid, such as a fatty oil, paraffin oil or liquid polyethylene glycol. A stabilizer may be optionally added.

[0088] If the nutritional composition is administered by tube feeding, the nutritional composition may be used for short-term or long-term tube feeding.

[0089] Inhibit or reduce cartilage degeneration

[0090] Cartilage degradation may be the result of pathology (chronic or acute), trauma, or a combination thereof.

[0091] Cartilage degradation occurs both in conditions where inflammation is predominant, such as rheumatoid arthritis, and in conditions where inflammation is less prominent, such as osteoarthritis.

[0092] Trauma can also cause the cartilage degeneration process to be initiated. For example, tearing the ligaments in the knee causes instability of the knee joint and will initiate the degeneration process.

[0093] In the context of this application, trauma refers to a physiological injury caused by an external source, such as for example by a fall or being hit by a car etc. Trauma can also be the accumulation of small injuries over time, so-called "wear and tear".

[0094] Although surgical treatment of wounds is generally preferred, in one embodiment, the invention relates to a method of treatment in which the wound is treated both surgically and by administering a composition of the invention.

[0095] Thus, embodiments of the use according to the invention include use for inhibiting or reducing cartilage degradation, wherein the cartilage degradation is a result of a pathology or trauma.

[0096] Examples of conditions involving cartilage degradation 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-Schönlein purpura ( purpura), hyperimmunoglobulinemia D with recurrent fevers, sarcoidosis, TNF receptor-associated periodic syndromes, Wegener's granulomatosis (and many other vasculitic syndromes), familial Mediterranean fever, and systemic lupus erythematosus.

[0097] In a preferred embodiment, the compositions of the present invention are used to inhibit or reduce cartilage degradation in RA and / or OA.

[0098] In another preferred embodiment, the composition of the present invention is used to inhibit or reduce cartilage degradation in OA.

[0099] Additionally, without wishing to be bound by theory, it has been observed that while inflammation generally leads to cartilage degradation in joints, cartilage degradation also occurs in situations where the inflammatory component is less pronounced, and perhaps even negligible.

[0100] For example, joint trauma may readily induce cartilage degradation, but without a significant inflammatory component such as is present in RA. The trauma may, for example, involve a torn ligament, or impact trauma to a joint such as a knee or finger.

[0101] In another example, OA is primarily a degenerative disease of the joints, with a minor inflammatory component.

[0102] Thus, in one embodiment the invention relates to a composition for use according to the invention for inhibiting or reducing cartilage degradation, and wherein the cartilage degradation occurs in the context of a pathology with little or no inflammatory component, such as trauma or, for example, OA.

[0103] Use to counteract early degenerative events

[0104] Hypertrophy indicates catabolic activity of chondrocytes that is not a normal phenotype.

[0105] Therefore, in one embodiment, the present invention relates to a composition comprising oleuropein and / or its metabolites in combination with fisetin and / or derivatives according to the present invention for use in inhibiting or reducing chondrocyte hypertrophy, which is an early event indicative of cartilage degradation.

[0106] The composition for use according to the invention has been shown to inhibit or reduce proteolytic activity.

[0107] Aging causes cartilage degeneration.

[0108] Therefore, the present invention relates to the composition of the present invention for inhibiting or reducing cartilage degeneration associated with aging.

[0109] In another embodiment, the invention relates to a composition of the invention for use in inhibiting or reducing collagen degradation in cartilage degradation associated with aging, such as for use in inhibiting or preventing collagen II degradation in cartilage associated with aging.

[0110] Cartilage degeneration can lead to joint stiffness and joint pain, resulting in reduced mobility for the patient.

[0111] In other embodiments, the composition for use according to the invention may be used to i) maintain or improve joint function during aging, including cartilage function, ii) reduce joint pain, including inflammatory pain and / or nociceptive pain.

[0112] In another embodiment, the invention relates to a composition for use according to the invention for improving motility in a subject, such as in an adult or elderly mammal.

[0113] Thus, in a preferred embodiment, the composition according to the invention may be used to improve the mobility and / or mobility of an individual, for example by preventing or treating osteoarthritis, and / or by inhibiting or reducing cartilage degradation.

[0114] Other preferred embodiments relate to compositions for use according to the invention, wherein the use is to prevent cartilage degradation and thus maintain healthy joints, or to maintain or improve mobility, prevent or reduce joint pain (inflammatory and / or nociceptive pain). In another embodiment, the composition of the invention can be used to maintain the state of cartilage.

[0115] Target Group

[0116] The target population of the composition of purposes according to the present invention can be any mammal that shows cartilage degradation, for example because they suffer from one or more conditions related to cartilage degradation mentioned herein. Cartilage degradation can be detected by visual means, such as by radiography. Alternatively, the detection of cartilage degradation products can be detected in body fluids. For example, such as one or more collagen II epitopes (Coll2-1, Col112-1NO2, CTX-II) can be detected in, for example, samples (such as blood plasma or urine samples).

[0117] Another target group may be any mammal that does not yet exhibit cartilage degradation, but is at risk of cartilage degradation, for example at risk of OA, RA or any of the conditions mentioned herein involving cartilage degradation. In a preferred embodiment, the present invention relates to administering to the target group a composition according to the present invention, the composition comprising a combination of oleuropein or a metabolite thereof and fisetin or a derivative, the composition being used for inhibiting or reducing early degeneration of cartilage.

[0118] One particular embodiment of the invention relates to compositions for improving mobility and / or motility in an individual, for example by preventing or treating osteoarthritis, and / or by inhibiting or reducing cartilage degeneration in elderly or aging individuals.

[0119] In other embodiments, the composition for use according to the present invention can be used for mammals, such as humans or pets. Examples of pets include cats, dogs and horses.

[0120] While the present invention is useful for many different age groups, in a preferred embodiment, the compositions for increasing motility according to the present invention are targeted to the aging population, particularly healthy aging and / or elderly mammals.

[0121] Method for producing the nutritional composition of the present invention

[0122] In another aspect, the present invention relates to a method for producing a nutritional composition for use according to the present invention, said method comprising the following steps:

[0123] - Providing an ingredient for a nutritional composition, the ingredient comprising a combination of oleuropein and / or its metabolites and fisetin and / or its derivatives; and mixing so that the nutritional composition comprises the combination of oleuropein and / or its metabolites and fisetin and / or its derivatives.

[0124] Pharmaceutical compositions for use .

[0125] In another embodiment, the present invention relates to a composition for inhibiting or preventing cartilage degradation according to the present invention, wherein the composition is a pharmaceutical composition.

[0126] By pharmaceutical composition is meant a composition other than a nutritional composition, wherein the substance is used on or in the body to medically prevent, diagnose, alleviate, treat or cure a disease in a human or animal. According to the present invention, the medicament can be used to inhibit or reduce cartilage degradation.

[0127] The medicament may be for human use. Alternatively, it may be a veterinary composition, for example suitable for dogs, cats or horses, in particular thoroughbred horses.

[0128] In a preferred embodiment, the pharmaceutical composition of the present invention comprises a combination of oleuropein or a metabolite thereof and fisetin or a derivative thereof.

[0129] In another preferred embodiment, the pharmaceutical composition of the present invention comprises oleuropein or its metabolites and fisetin or its derivatives.

[0130] The present invention also relates to the use of a medicament according to the present invention, such as the use of a composition of the present invention as described herein.

[0131] The pharmaceutical composition for use according to the present invention comprises a combination of oleuropein or its metabolites and fisetin or derivatives in combination with at least one excipient, the excipient being selected from the group consisting of pharmaceutically acceptable excipients. The procedure for preparing the pharmaceutical composition according to the present 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 excipients, vehicles and adjuvants are also described in the handbook entitled "Handbook of Pharmaceutical Excipients" (Second Edition, American Pharmaceutical Association, 1994). In order to prepare the pharmaceutical composition according to the present invention, those skilled in the art will advantageously be able to refer to the latest edition of the European Pharmacopoeia or the United States Pharmacopoeia (USP). Specifically, those skilled in the art can advantageously refer to the fourth edition "2002" of the European Pharmacopoeia or also to the USP 25-NF 20 edition of the United States Pharmacopoeia.

[0132] Advantageously, the pharmaceutical composition as defined above is suitable for oral, parenteral or intravenous administration. When the pharmaceutical composition according to the use of the invention comprises at least one pharmaceutically or physiologically acceptable excipient, it is in particular an excipient suitable for administering the composition by the oral route or an excipient suitable for administering the composition by the parenteral route.

[0133] The pharmaceutical composition for use according to the invention can indifferently be obtained in solid or liquid form.For oral administration, solid pharmaceutical compositions in the form of tablets, capsules or gelatin capsules will be preferred.

[0134] Among liquid forms, the pharmaceutical composition will preferably be in the form of an aqueous or non-aqueous suspension, or also preferably in the form of a water-in-oil or oil-in-water emulsion.

[0135] Solid pharmaceutical dosage forms may contain at least one diluent as a vehicle, adjuvant or excipient, a flavoring agent, a solubilizer, a lubricant, a suspending agent, a binder, a disintegrant and an encapsulating agent. Such compounds are, for example, magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, gelatin, cellulosic materials, cocoa butter, etc. Compositions in liquid form may also contain water, possibly as a mixture with propylene glycol or polyethylene glycol, and may also contain colorants, flavoring agents, stabilizers and thickeners.

[0136] Combination with known treatments

[0137] In the absence of disease-modifying OA drugs (DMOADs), nutrient replacement therapy can be used to treat and prevent OA.

[0138] From the histological data, it can be seen that oleuropein has a greater effect on OA scores than compounds that primarily affect degeneration. Therefore, the efficacy of oleuropein may be due to a combined effect on inflammation and degeneration. Therefore, in a preferred embodiment, the compositions of the present invention that have been shown to inhibit or reduce degeneration can be combined with a treatment for inhibiting or reducing inflammation.

[0139] Treatment

[0140] The present invention also relates to a method for preventing or treating cartilage degradation, such as a condition in which cartilage degradation occurs or a trauma associated with cartilage degradation, the method 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.

[0141] As used herein, an "effective amount" is an amount that prevents a defect, treats a disease or medical condition in an individual, or more generally, reduces symptoms, controls disease progression, or provides a nutritional, physiological or medical benefit to the individual.

[0142] The effective amount of the composition according to the invention required to achieve a therapeutic effect will of course vary with the particular composition, the route of administration, the age and condition of the recipient and the particular disorder or disease being treated.

[0143] The present invention also provides the following methods: preventing or treating conditions involving cartilage degradation, such as, for example, OA or RA; inhibiting or reducing cartilage degradation; inhibiting or reducing collagen degradation in cartilage; inhibiting or reducing collagen II degradation in cartilage; the method comprises administering to an individual an effective amount of a composition according to the use of the present invention.

[0144] In one embodiment, the methods of treatment according to the invention relate to the prevention or treatment of osteoarthritis.

[0145] The treatment methods according to the invention may be carried out in mammals such as humans or pets (eg dogs, cats and / or horses).

[0146] In certain embodiments, the composition of the present invention to be administered in a method of treatment may be one or more nutritional compositions of the present invention and / or pharmaceutical compositions of the present invention.

[0147] Pill Box

[0148] The present disclosure also provides a kit comprising a combination of oleuropein and / or its metabolites and fisetin and / or derivatives in one or more containers. In one embodiment of the kit, the one or more containers comprise at least one first container storing oleuropein and / or metabolites separated from fisetin and / or derivatives, the fisetin and / or derivatives being stored in at least one second container, and the kit further comprises instructions for mixing oleuropein with fisetin into a unit dosage form.

[0149] In one embodiment of the kit, the combination may be provided together in one or more prepackaged unit dosage forms, for example in separate containers each containing a dry powder, such that each container contains one prepackaged unit dosage form.

[0150] In another embodiment, the kit may contain a plurality of compositions for mixing together to form one or more compositions disclosed herein. For example, the kit may contain two or more dry powders in separate containers relative to each other, each of the separate powders containing a portion of the final unit dosage form. As a non-limiting example of such an embodiment, the kit may contain one or more first containers containing oleuropein, and may also contain one or more second containers containing fisetin. The contents of one of the first containers may be mixed with the contents of one of the second containers to form at least a portion of the unit dosage form of the composition.

[0151] The above application examples do not require continuous daily application without interruption. Instead, there may be some brief interruptions in the application, such as two to four days of interruption during the application. The ideal duration of application of the composition can be determined by those skilled in the art.

[0152] Combination of public content

[0153] It should be noted that embodiments and features described in the context of one aspect of the invention are also applicable to the other aspects of the invention.

[0154] The composition according to the invention for the use described herein is described in different parameters, such as ingredients, nutritional composition form, use, target group, etc. It should be noted that, unless explicitly stated otherwise, embodiments and features described in the context of one of the parameters of the composition according to the invention for the use described may also be combined with other embodiments and features described in the context of another parameter.

[0155] All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety.

[0156] The present invention will now be described in further detail in the following non-limiting examples.

[0157] Example

[0158] The following non-limiting examples provide experimental data supporting the compositions and methods disclosed herein.

[0159] Example 1

[0160] In order to test the effect of oleuropein (or oleuropein aglycone), fisetin and their combination in living cells, the inventors measured the increase of mitochondrial calcium in HeLa cells.HeLa cells were purchased from ATCC.HeLa cells were seeded in 96-well plates with a density of 50,000 cells / well in the basic essential medium (DMEM, Gibco) (high glucose + 10% fetal bovine serum).Mitochondrial calcium measurements were performed using HeLa cells infected with adenovirus (from Sirion biotech) expressing mitochondrial-targeted calcium sensor mitochondrial mutation aequorin (Montero et al., 2004).For aequorin reconstruction, 24 hours after infection, cells were incubated at room temperature (22 ± ℃ ℃) in standard medium (145mM NaCl, 5mM KCl, 1mM MgCl2, 1mM MgCl2, 10mM glucose and 10mM Hepes, pH7.4) with 1μM wild-type coelenterazine for 2 hours. For treatment, the compound was added directly to the cell culture or myotube culture 2 hours before measurement. Luminescence was measured on FLIPR Tetra Aequorin (Molecular Devices). Mitochondrial calcium was obtained by stimulating cells with 100 μM histamine to raise. The luminescence data was calibrated to calcium concentration (Alvarez & Montero, 2002) using the algorithm as described above. The custom module analysis based on Excel (Microsoft) and GraphPad Prism 7.02 (GraphPad) software was used for quantitative analysis.

[0161] like Figure 2 As shown, in HeLa cells, the effect of the combination of oleuropein and fisetin on mitochondrial activation was greater than the effect of fisetin or oleuropein alone on mitochondrial activation through the increase of mitochondrial Ca2+. Figure 3 Fisetin was shown to act synergistically with oleuropein in HeLa cells to activate mitochondria through mitochondrial Ca2+ elevation.

[0162] Example 2

[0163] In order to verify the synergistic effect of the combination of oleuropein (aglycone form) and fisetin on the cell model of osteoarthritis chondrocytes (chondrocytes), the inventors measured mitochondrial calcium in SW1353 cells treated with the proinflammatory cytokine interleukin-1β. SW1353 cells were purchased from ATCC. SW1 353 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) with 10% fetal bovine serum and 1% penicillin-streptomycin. SW1 353 cells were treated with 10 ng / ml of the proinflammatory cytokine interleukin-1β as an osteoarthritis mimetic for 48 hours. Using the same procedure as for HeLa cells, mitochondrial calcium in SW1353 cells was measured. The synergistic effect of oleuropein aglycone and fisetin in the osteoarthritis chondrocyte model was quantified as described for HeLa cells.

[0164] result

[0165] like Figure 4 As shown, in the cell model of osteoarthritis chondrocytes (SW1353 cells treated with IL-1β), oleuropein aglycone and fisetin act synergistically to activate Ca2+ via mitochondrial Ca2+. 2+ Increased to activate mitochondria.

Claims

1. A composition comprising an effective amount of oleuropein and / or its metabolites in combination with fisetin and / or derivatives, for use in preventing or treating cartilage degeneration in an individual.

2. The composition for use according to claim 1, wherein the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, olive acid, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

3. The composition for use according to claim 1 or 2, wherein the metabolite of fisetin is selected from the group consisting of its glucuronidated form, its sulfated form, its derivatives and mixtures thereof, preferably gerardol.

4. A composition for use according to any one of claims 1 to 3, further comprising calcium.

5. The composition for use according to any one of claims 1 to 4, wherein the composition further comprises at least one compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibers, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

6. The composition for use according to any one of claims 1 to 5, wherein the composition is selected from the group consisting of food compositions, dietary supplements, nutritional compositions, oral nutritional supplements, medical foods, nutraceuticals, beverages, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, foods for special medical purposes (FSMP), medicaments, drinks, pet foods, and combinations thereof.

7. The composition for use according to any one of claims 1 to 6, wherein the composition is in the form of a solid powder, a powdered stick, a capsule or a solution.

8. The composition for use according to any one of claims 1 to 7, wherein the use is for i) maintaining or improving joint function during aging, including cartilage function, ii) alleviating joint pain, including inflammatory pain and / or nociceptive pain.

9. Composition for use according to any one of claims 1 to 8, wherein the use is to improve the mobility and / or motility of the individual.

10. A composition for use according to any one of the preceding claims, wherein the use is to inhibit or reduce cartilage degradation in osteoarthritis.

11. The composition for use according to any one of the preceding claims, wherein the individual is middle-aged or elderly.

12. A method for producing a nutritional composition for use according to any one of the preceding claims, said method comprising the steps of: One or more ingredients for a nutritional composition, oleuropein or a metabolite thereof and fisetin and / or a derivative and optionally additional calcium, and admixture are provided.

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

14. The kit of claim 13, wherein the one or more containers comprises at least one first container storing oleuropein and / or metabolites separated from the fisetin and / or derivatives, the fisetin and / or derivatives being stored in at least one second container, and the kit further comprising instructions for mixing the oleuropein with the fisetin into a unit dosage form.

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

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