Compositions for use in stimulating bone growth

Abstract

To provide a composition for maintaining bone and / or cartilage health, or for preventing, alleviating and / or treating bone and / or cartilage disorders.SOLUTION: There is provided a composition comprising Vitamin D and oleuropein for use to enhance the stimulation of bone formation by oleuropein, wherein the composition comprises between 800 and 1200IU of Vitamin D and between 0. 01mg and 1g of oleuropein. The composition may further comprise one or more bioactive molecules selected from the group consisting of antioxidants, anti-inflammatory compounds, fatty acids, prebiotic fibers, probiotics, glucosamine, chondroitin sulfate, minerals, trace elements and vitamins.SELECTED DRAWING: None

Description

Technical field of the invention

[0001] The present invention relates to the use of compositions for the maintenance of bone and / or cartilage health or for the prevention, alleviation and / or treatment of bone and / or cartilage disorders.

[0002] Bone mass develops throughout life and is regulated by genetic, mechanical, and hormonal mechanisms. Bone mineral acquisition occurs during childhood, with peak bone mass achieved around age 20. During this period, bone formation exceeds bone resorption. Later in life, particularly around menopause or in older adults, bone mass and quality are compromised by higher bone turnover with excessive bone resorption, leading to a gradual loss of bone mass, bone microarchitecture, structure, and strength.

[0003] Restoring the balance between bone formation and bone resorption is crucial for bone maintenance. This bone remodeling process is regulated at the osteocyte level, involving the close interaction between bone-forming cells (osteoblasts) and bone-resorbing cells (osteoclasts).

[0004] In healthy, adult humans or animals, the coordinated actions of osteoblasts and osteoclasts allow the maintenance of bone mass over time, ensuring simultaneous bone resorption and remodeling of bone tissue through de novo bone synthesis.

[0005] In healthy adults, the rates of osteoclast and osteoblast formation influence the balance between bone formation and bone resorption. However, in osteoporotic individuals, an imbalance in the bone remodeling process is created, leading to bone loss that outpaces bone formation. While this imbalance exists to some degree in most individuals with aging, it is much more severe and occurs at a younger age in osteoporotic individuals.

[0006] Thus, in humans and other mammals, a wide variety of diseases are associated with metabolic abnormalities in bone resorption and bone formation, leading to an imbalance in metabolism or bone remodeling.

[0007] Furthermore, there are many conditions in humans and animals that are characterized by the need to enhance bone formation. For example, in the case of fractures, it is necessary to stimulate bone growth to stimulate complete bone repair. This need also exists in periodontal disease, bone metastatic disease, osteolytic disease, and conditions where connective tissue repair is required (e.g., for scarring or regeneration of cartilage defects or traumatic injuries). Stimulation of bone growth is also required in cases of primary and secondary hyperparathyroidism, as well as diabetes-related osteoporosis and glucocorticoid-related osteoporosis.

[0008] Although today there is a wide variety of active compounds for stimulating bone formation and / or inhibiting bone resorption, there is a constant need for new active compounds, particularly due to the limited success of current treatments.

[0009] Another joint disease is osteoarthritis, the leading cause of disability in the elderly. Currently, there is no cure for osteoarthritis, and treatments are palliative, aimed at improving symptoms. Summary of the Invention

[0010] In a surprising manner, the applicant has shown that a combination of vitamin D and the olive tree-derived polyphenols hydroxytyrosol and / or oleuropein can act synergistically to stimulate bone formation and / or cartilage anabolism. This combination can stimulate bone formation as well as cartilage anabolism through stimulation of osteoblast differentiation and activity and collagen synthesis. The addition of vitamin D in combination with either or both of oleuropein and hydroxytyrosol results in osteoblast stimulation that is increased in intensity and shifts forward in time (i.e., stimulation is induced earlier than with either component by itself). In the context of this application, the effect of the addition of vitamin D is said to "potentiate" the effect of polyphenols on bone formation and / or cartilage anabolism.

[0011] Thus, in a first aspect, the present invention relates to a composition comprising vitamin D for use in enhancing the effect of one or more polyphenols on bone formation and / or cartilage anabolism.

[0012] In a further aspect, the present invention relates to a composition comprising vitamin D and one or more polyphenols for use in enhancing polyphenol stimulation of bone formation and / or cartilage anabolism.

[0013] In a further aspect, the present invention relates to a composition for use according to the invention comprising vitamin D and one or more polyphenols, to be administered once per day for at least two months, preferably at least three months.

[0014] In a further aspect, the present invention relates to a pharmaceutical composition comprising vitamin D and one or more polyphenols.

[0015] In a still further aspect, the present invention relates to a method for treating, alleviating and / or preventing bone and / or cartilage disorders comprising administering to an individual in need thereof an effective amount of vitamin D together with one or more polyphenols.

[0016] In all figures, 1,25(OH)2D3 is 10 -9 M 1,25-dihydroxyvitamin D3; OLP 1 μM oleuropein; HTy 1 μM hydroxytyrosol; C+ indicates the use of optimized medium as a positive control; C- indicates the use of minimal medium as a negative control.

[0017] Data represent the mean ± SEM (three replicates). Statistics: Kruskal Wallis test (two-tailed) followed by Mann Whitney U test. *P<0.05; **P<0.01; ***P<0.001 vs. negative control (C-). [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1 shows collagen synthesis (measured as micrograms of collagen produced per milligram of total protein) by osteoblasts after 9 days in culture in response to stimulation with a combination of 1,25(OH)2D3, OLP, and HTy. [Figure 2] Figure 2 shows alkaline phosphatase (ALP) activity (expressed as micromoles of ALP per milligram of protein per hour) in response to a combination of 1,25(OH)2D3, OLP, and HTy after 9 days in culture. [Figure 3] Figure 3 shows alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of 1,25(OH)2D3 after 5, 9, 12 or 15 days of treatment. [Figure 4] Figure 4A shows the alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of oleuropein after 5, 9, 12, or 15 days of treatment. Figure 4B shows the alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of 1,25(OH)2D3 and oleuropein after 5, 9, 12, or 15 days of treatment. [Figure 5] Figure 5A shows the alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of hydroxytyrosol after 5, 9, 12, or 15 days of treatment. Figure 5B shows the alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of 1,25(OH)2D3 and hydroxytyrosol after 5, 9, 12, or 15 days of treatment. [Figure 6] Figure 6A shows the alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of oleuropein and hydroxytyrosol after 5, 9, 12, or 15 days of treatment. Figure 6B shows the alkaline phosphatase (ALP) activity of osteoblasts cultured in the presence of 1,25(OH)2D3, oleuropein, and hydroxytyrosol after 5, 9, 12, or 15 days of treatment. Detailed Description of the Invention

[0019] Definition: Before discussing the present invention in further detail, the following terms and conventions will first be defined.

[0020] In the context of the present invention, unless otherwise stated, the percentages stated are weight / weight percentages.

[0021] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y".

[0022] Numerical ranges used herein are intended to include all numbers and subsets of numbers within that range, whether or not specifically disclosed. Moreover, these numerical ranges should be construed as providing support for claims directed to any number or any subset of numbers within that range. For example, a disclosure of 1 to 10 should be construed as supporting ranges from 1 to 8, 3 to 7, 4 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0023] Any reference to a singular feature or limitation of the invention is intended to include the corresponding plural feature or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context of the reference.

[0024] 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.

[0025] Compositions containing vitamin D for use in: Polyphenols have previously been shown to have beneficial effects on inflammation and bone metabolism.

[0026] The inventors have surprisingly shown that combining one or more polyphenols (such as one or more of HTy and OLP) with vitamin D enhances the stimulatory effect of those polyphenols on bone formation and / or cartilage anabolism (see Example 2 and its discussion and Figure 2).

[0027] "Stimulation of bone formation and / or cartilage anabolism" refers to the ability of the combination of vitamin D, oleuropein and hydroxytyrosol to stimulate osteoblast activity and thus the synthesis of bone and / or cartilage protein networks, which leads to stimulation of mineral deposition, especially calcium, in these protein networks, i.e., bone mineralization, also called osteogenesis, and to stimulation of cartilage anabolism.

[0028] By "enhancing the stimulatory effect of polyphenols" is meant that the stimulatory effect of one or more polyphenols is both increased in intensity and advanced in time (i.e., induced earlier than in the absence of vitamin D) by the addition of vitamin D.

[0029] Thus, in a first aspect, the present invention relates to a composition comprising vitamin D for use in enhancing the effect of one or more polyphenols on bone formation and / or cartilage anabolism, wherein the polyphenols may be, for example, one or more of oleuropein and hydroxytyrosol.

[0030] Vitamin D can be present in any suitable form. For example, various useful forms of vitamin D are available, including cholecalciferol (D3), ergocalciferol (D2) and their biologically active metabolites, and precursors such as 1-α-hydroxyvitamin D, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D.

[0031] In one embodiment of this aspect of the invention, vitamin D can be provided simultaneously with or sequentially to one or more polyphenols. Vitamin D can be provided in the same composition as one or more polyphenols, or in separate compositions, for example, as part of a kit or in a series of interacting products. When vitamin D and one or more polyphenols are provided in one or more separate compositions, they can be mixed before administration, or can be administered simultaneously or sequentially.

[0032] The enhancing effect of vitamin D is already evident in combination with single polyphenols, such as in combination with oleuropein or hydroxytyrosol.

[0033] The potentiating effect is further increased when vitamin D is administered in combination with both oleuropein and hydroxytyrosol.

[0034] In a further aspect, the present invention relates to the use of vitamin D in the manufacture of a composition or medicament for enhancing the effect of one or more polyphenols on bone formation and / or cartilage anabolism.

[0035] Composition for use: In a further aspect, the present invention relates to a composition comprising vitamin D and one or more polyphenols for use in enhancing the stimulation of bone formation and / or cartilage anabolism by those polyphenols.

[0036] The polyphenol may be an olive polyphenol, such as a polyphenol selected from the group consisting of oleuropein, ligstroside, hydroxytyrosol, and / or tyrosol. In a preferred embodiment, the olive polyphenol comprises one or more of oleuropein and hydroxytyrosol, and more preferably the polyphenol comprises oleuropein and hydroxytyrosol.

[0037] Compositions according to the present invention may contain, for example, 800-1200 IU of vitamin D per serving. The polyphenol(s) may each be present in an amount of 0.01 mg to about 1 g, preferably 0.1 mg to 1 g, and even more preferably 1 mg to about 1 g, per serving.

[0038] The effective amount of the compositions of the present invention required to achieve a therapeutic effect will vary depending on the particular composition, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated.

[0039] As an example, a ready to drink (RTD) supplemented with a combination of vitamin D, hydroxytyrosol, and oleuropein may contain 0.01 mg to 500 mg of each active ingredient per serving.

[0040] In one embodiment, the composition of the present invention comprises, for example, 800 to 1200 IU, for example, 800 to 1100, 800 to 1000, 850 to 1000, 850 to 950, 900 to 950 IU, for example, about 900 or about 950 IU of vitamin D. In a further embodiment, the composition of the present invention comprises, in addition to vitamin D, an amount of 0.01 mg to 1 g, for example, 0.01 mg to 500 mg (for example, 0.1 mg to 500 mg, 0.5 mg to 500 mg, 1 mg to 500 mg, 10 mg to 500 mg, 20 mg to 500 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 300 mg to 500 mg). mg, 350mg to 500mg, 400mg to 500mg), or 400mg to 1g (e.g., 500mg to 1g, 550mg to 1g, 600mg to 1g, 650mg to 1g, 750mg to 1g, 800mg to 1g, 900mg to 1g), or 550mg to 950mg (e.g., 600mg to 900mg, 700mg to 800mg). In this embodiment, for example, an amount of 0.01 mg to 1 g, for example, 0.01 mg to 500 mg (for example, 0.1 mg to 500 mg, 0.5 mg to 500 mg, 1 mg to 500 mg, 10 mg to 500 mg, 20 mg to 500 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 300 mg to 500 mg, 350 mg to 500 mg) g, 400 mg to 500 mg), or 400 mg to 1 g (e.g., 500 mg to 1 g, 550 mg to 1 g, 600 mg to 1 g, 650 mg to 1 g, 750 mg to 1 g, 800 mg to 1 g, 900 mg to 1 g), or 550 mg to 950 mg (e.g., 600 mg to 900 mg, 700 mg to 800 mg).

[0041] In a preferred embodiment, the composition of the present invention comprises 800 to 1200 IU of vitamin D, 0.01 mg to 1 g of oleuropein, and 0.01 mg to 1 g of hydroxytyrosol.

[0042] 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.

[0043] In one embodiment, the composition for use according to the invention is a nutritional composition.

[0044] "Nutritional composition" means, according to the present invention, a composition containing a combination of vitamin D and one or more polyphenols, such as one or more olive polyphenols (e.g. one or more of oleuropein, ligstroside, hydroxytyrosol and / or tyrosol), preferably oleuropein and / or hydroxytyrosol, constituting a dietary composition without pharmaceutical properties.

[0045] It is understood that the nutritional compositions may contain any number of optional additional ingredients, including, for example, one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, additives, flavorings, minerals, osmotic agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, and / or conventional food additives (synthetic or natural), such as vitamins. These optional ingredients may be added in any suitable amount. The nutritional products or compositions of the present invention may be complete or incomplete sources of nutrition.

[0046] As used herein, "complete nutrition" includes nutritional products and compositions that contain sufficient types and levels of macronutrients (protein, fat, and carbohydrates) and micronutrients to be the sole source of nutrition for the animal to which they are administered. A patient can receive 100% of their nutritional requirements from such a complete nutritional composition.

[0047] As used herein, "incomplete nutrition" includes nutritional products or compositions that do not contain sufficient levels of macronutrients (protein, fat, and carbohydrates) or micronutrients to be the sole source of nutrition for the animal to which they are administered. Partial or incomplete nutritional compositions can be used as nutritional supplements.

[0048] The nutritional compositions of the present invention can be medical foods. Medical foods are a special class of nutritional compositions designed to provide dietary management of specific conditions. Medical foods meet certain criteria established and regulated by Section 5 [360ee](b)(2)(D) of the Orphan Drug Act of 1983.

[0049] component: The compositions of the present invention may include one or more additional bioactive molecules such as antioxidants, anti-inflammatory compounds, fatty acids, prebiotic fibers, probiotics, glucosamine, chondroitin sulfate, minerals, trace elements and / or vitamins.

[0050] Examples of minerals and trace elements that may be included in the nutritional compositions of the present invention include mineral elements and trace elements such as calcium, magnesium, sodium, potassium, phosphorus, copper, zinc, iron, selenium, chromium and molybdenum.

[0051] In a preferred embodiment, the compositions of the present invention comprise the mineral calcium, which may be, for example, one or more organic and / or inorganic physiologically acceptable compounds, such as, for example, one or more inorganic salts of calcium (such as calcium chloride, calcium phosphate, calcium sulfate, calcium oxide, calcium hydroxide and / or calcium carbonate), organic salts of calcium (such as calcium citrate, calcium maleate or mixtures thereof), and / or calcium-containing organic ingredients such as powdered skim milk or calcium casein.

[0052] In a preferred embodiment, the compositions of the present invention comprise the mineral magnesium, which may be, for example, one or more organic and / or inorganic physiologically acceptable compounds, such as, for example, one or more inorganic magnesium salts (such as magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium oxide, magnesium hydroxide, and / or magnesium carbonate), organic magnesium salts (such as magnesium citrate, magnesium maleate, or mixtures thereof), and / or organic components containing magnesium.

[0053] Therefore, preferred embodiments of the compositions of the present invention include the minerals calcium and / or magnesium.

[0054] One or more antioxidants may also be present in the nutritional composition. Non-limiting examples of antioxidants include astaxanthin, carotenoids, coenzyme Q10 ("CoQ10"), flavonoids, glutathione, wolfberry (wolfberry), hesperidin, lactowberry, lignans, lutein, lycopene, polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinations thereof.

[0055] In embodiments, the nutritional composition comprises one or more prebiotics, non-limiting examples of which include acacia gum, alpha-glucan, arabinogalactan, beta-glucan, dextran, fructooligosaccharides, fucosyllactose, galactooligosaccharides, galactomannan, gentiooligosaccharides, glucooligosaccharides, guar gum, inulin, isomaltooligosaccharides, lactoneotetraose, lactosucrose, lactulose, levan, maltodextrin, milk oligosaccharides, partially hydrolyzed guar gum, pectin-derived oligosaccharides, resistant starch, retrograded starch, sialooligosaccharides, sialyllactose, soybean oligosaccharides, sugar alcohols, xylooligosaccharides, hydrolysates thereof, or combinations thereof.

[0056] The nutritional compositions may include one or more probiotics. Non-limiting examples of probiotics include Aerococcus, Aspergillus, Bacteroides, Bifidobacterium, Candida, Clostridium, Debaromyces, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Mucor, and the like. Mucor, Oenococcus, Pediococcus, Penicillium, Peptostrepococcus, Pichia, Propionibacterium, Pseudocatenulatum, Rhizopus, Saccharomyces, Staphylococcus, Streptococcus, Torulopsis, Weissella, non-replicating microorganisms, or combinations thereof.

[0057] The nutritional composition may contain fiber or a blend of various types of fiber. The fiber blend may contain a mixture of soluble and insoluble fiber. Soluble fibers may include, for example, fructooligosaccharides, acacia gum, inulin, agar, alginate, carob, carrageenan, gum arabic, guar gum, karaya gum, pectin, or xanthan gum, and these soluble fibers may be in hydrolyzed or non-hydrolyzed form. Insoluble fibers may include, for example, pea outer fiber.

[0058] The nutritional compositions of the present invention may also include one or more additional vitamins selected from vitamin A, vitamin E, vitamin K, vitamin C, folic acid, thiamine, riboflavin, vitamin B6, vitamin B12, niacin, biotin, and pantothenic acid.

[0059] In one embodiment, the nutritional composition includes 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 an embodiment, the protein is selected from the group consisting of whey, chicken, corn, casein, wheat, flax, soy, carob, pea, or a combination thereof.

[0060] In one embodiment, the nutritional composition comprises a carbohydrate source. Any suitable carbohydrate may be used in the nutritional compositions 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.

[0061] In one embodiment, the nutritional composition includes a fat source. The fat source can include any suitable fat or fat mixture. For example, the fat source can include, but is not limited to, vegetable fats (such as olive oil, corn oil, sunflower oil, high oleic sunflower oil, rapeseed oil, canola oil, hazelnut oil, soybean oil, palm oil, coconut oil, black currant seed oil, borage oil, lecithin, etc.), animal fats (such as dairy fat), or combinations thereof. The fat source can also be a less refined version of the fats listed above (e.g., polyphenol-containing olive oil).

[0062] Additionally, the nutritional compositions of the present invention may also contain natural or artificial flavors, such as fruit flavors such as banana, orange, peach, pineapple, or raspberry, or other botanical flavors such as vanilla, cocoa, coffee, etc.

[0063] Nutritional compositions according to the present invention may comprise an amount of vitamin D and one or more of oleuropein and hydroxytyrosol suitable for daily oral administration, comprising between 0.01 mg and 500 mg of each component.

[0064] The amount of calcium contained in the nutritional composition of the present invention is adapted to the daily dose provided by said composition, which contains 100 mg to 1000 mg of calcium, preferably 200 mg to 700 mg, more preferably 300 mg to 600 mg.

[0065] Nutritional composition forms: Suitable nutritional composition forms according to the present disclosure include, for example, infant formula, solutions, ready-for-consumption compositions (e.g., ready-to-drink compositions or instant drinks), liquid foodstuffs, soft drinks, juices, sports drinks, milk drinks, milkshakes, yogurt drinks, soups, etc. In further embodiments, the nutritional compositions can be manufactured and sold in the form of concentrates, powders, or granules (e.g., effervescent granules) that can be diluted with water or other liquids (e.g., milk or fruit juice) to obtain ready-to-consume compositions (e.g., ready-to-drink compositions or instant drinks).

[0066] Further forms of nutritional compositions include dairy products, baked products, dairy desserts, confectionery products, cereal bars, and breakfast cereals. Examples of dairy products include milk and milk drinks, yogurt and other fermented dairy products, ice cream, and cheese. Examples of baked products include bread, biscuits, and cakes. Examples of formula include nutritionally complete formula.

[0067] In one embodiment, the composition for use according to the invention may also be available in a wide variety of forms designed as animal food, in particular for dogs or cats, whether in moist, semi-moist or dry form, in particular in the form of biscuits.

[0068] Administration route: The nutritional compositions of the present disclosure can be administered by any means suitable for administration to humans, particularly in any part of the gastrointestinal tract. Enteral administration, oral administration, and administration through a tube or catheter are all covered by the present disclosure. The nutritional compositions can also be administered by a means selected from oral, rectal, sublingual, sublabial, buccal, topical, etc.

[0069] When the nutritional composition is formulated for oral administration, the composition may be a liquid oral nutritional supplement (e.g., an incomplete feeding) or a complete feeding. In this manner, the nutritional composition may be administered in any known form, including, for example, tablets, capsules, liquids, chewable tablets, soft gels, sachets, powders, syrups, liquid suspensions, emulsions, and liquids in convenient dosage forms. In soft capsules, the active ingredient is preferably dissolved or suspended in a suitable liquid, such as, for example, fatty oils, paraffin oil, or liquid polyethylene glycol. Optionally, a stabilizer may be added.

[0070] The nutritional compositions of the present disclosure may be either a source of incomplete or complete nutrition. The nutritional compositions may be administered orally or via tube feeding. When formulated for oral administration, the nutritional compositions may be liquid oral nutritional supplements or supplements. The nutritional compositions may also be used for short-term or long-term tube feeding.

[0071] Compositions for use (disorders and target groups): The inventors have shown that the compositions of the present invention result in increased stimulation of osteoblasts, which exhibit increased bone formation and / or cartilage anabolism.

[0072] In one embodiment, the use according to the present invention is for the prevention, alleviation and / or prophylaxis of disorders associated with an imbalance between bone formation and bone resorption and / or associated with cartilage turnover by enhancing stimulation of bone formation and / or cartilage anabolism.

[0073] The composition for use according to the invention may, for example, be for use in maintaining bone and / or cartilage health in an individual to whom the composition according to the invention is administered.

[0074] The compositions of the present invention may be useful, for example, to stimulate bone formation and cartilage anabolism in young, growing individuals, to increase bone mineral density achieved at the beginning of adulthood and to increase maximum bone mass (peak bone mass) at the beginning of adulthood.

[0075] The compositions may further be useful for stimulating bone formation and / or cartilage anabolism in adults to increase peak bone mass. Thus, the compositions may be useful for stimulating bone formation and / or cartilage anabolism in healthy adults and / or healthy aging adults, such as perimenopausal women, healthy adults and / or healthy aging adults who are pre-osteoarthritic, or adults diagnosed with bone and / or cartilage disorders, such as osteoarthritis, osteoporosis, and / or fracture events.

[0076] In other embodiments, compositions of the present invention may be useful, for example, in preventing bone loss that occurs with aging.

[0077] Compositions of the present invention may, in another embodiment, be useful for maintaining and improving joint function, including bone and cartilage function, during aging.

[0078] Compositions of the present invention may, in some embodiments, be useful in improving bone strength.

[0079] In a further embodiment, the composition for use according to the invention relates to use in the treatment of disorders of bone formation and / or cartilage anabolism, such as osteoporosis, Paget's disease, bone loss or osteolysis observed near prosthetic devices, metastatic bone disease, hyperthyroidism, hypocalcemia due to cancer, multiple myeloma, periodontal disease, osteoarthritis, bone defects due to fracture or fracture healing.

[0080] In a preferred embodiment, the invention relates to a composition for use according to the invention, wherein the disorder is osteoporosis and / or osteoarthritis.

[0081] In a further embodiment, the composition for use according to the present invention relates to use for treating bone defects resulting from bone fractures.

[0082] The compositions for use according to the invention may result in improved bone formation and / or regeneration, and / or cartilage anabolism during fracture healing, which may be useful for reducing the risk of osteoporosis and improving recovery after fracture.

[0083] The compositions of the present invention may be useful in preventing bone loss, particularly bone loss associated with aging in mammals or associated with prolonged hospitalization.

[0084] Additionally, it can help build cartilage in mammals and prevent osteoarthritis in pets and humans, leading to better activity or mobility for individuals (eg, pets and / or humans).

[0085] Thus, in particular, the compositions of the present invention may be for use to improve the activity and / or mobility of an individual, for example by treating, alleviating or preventing osteoarthritis and / or by improving bone strength.

[0086] Certain embodiments of the present invention relate to compositions for use to improve the activity and / or mobility of an individual, for example, by treating, alleviating or preventing osteoarthritis and / or improving bone strength in elderly or aging individuals.

[0087] 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.

[0088] Although the present invention may be useful in many different age groups, in preferred embodiments, compositions for use according to the present invention are targeted to aging populations, particularly elderly patients.

[0089] Method for producing the nutritional composition of the present invention: The present invention relates in a further aspect to a method for producing the nutritional composition of the present invention, said method comprising: Providing a nutritional composition; and adding one or more vitamin Ds and one or more polyphenols to the nutritional composition so that the prepared nutritional composition contains a combination of vitamin D and one or more polyphenols. Includes.

[0090] In an embodiment of this aspect of the invention, the polyphenol may be, for example, an olive polyphenol, such as one or more selected from oleuropein, ligstroside, hydroxytyrosol and / or tyrosol, preferably one or more of oleuropein and hydroxytyrosol, even more preferably both oleuropein and hydroxytyrosol.

[0091] Pharmaceutical Composition: In a further aspect, the present invention relates to a pharmaceutical comprising vitamin D and one or more polyphenols, such as olive polyphenols (e.g., selected from oleuropein, ligstroside, hydroxytyrosol, and / or tyrosol), preferably oleuropein and / or hydroxytyrosol. The pharmaceutical may be a human composition. Alternatively, it may be a veterinary composition suitable for, for example, dogs, cats, or horses, particularly thoroughbreds.

[0092] Pharmaceutical product means a composition, other than a nutritional composition, for use in contact with or within the body to medically prevent, diagnose, mitigate, treat or cure disease in humans or animals.

[0093] In one preferred embodiment, the pharmaceutical composition of the present invention comprises vitamin D and oleuropein and / or hydroxytyrosol.

[0094] The present invention further relates to the use of a pharmaceutical agent according to the invention, as described above for the use of a composition according to the invention.

[0095] The pharmaceutical composition of the present invention comprises a combination of vitamin D and one or more polyphenols with at least one additive selected from the group consisting of pharmaceutically acceptable additives. Procedures for preparing the pharmaceutical composition of 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 additives, vehicles, and adjuvants are also described in the handbook "Handbook of Pharmaceutical Excipients, 2nd Edition, American Pharmaceutical Association, 1994." To formulate the pharmaceutical composition of the present invention, those skilled in the art can advantageously refer to the latest edition of the European Pharmacopoeia or the United States Pharmacopoeia (USP). In particular, those skilled in the art can advantageously refer to the European Pharmacopoeia, 4th Edition "2002," and to the USP25-NF20 (United States Pharmacopoeia) edition of the United States Pharmacopoeia.

[0096] Advantageously, the pharmaceutical composition as defined above is suitable for oral, parenteral or intravenous administration. If the pharmaceutical composition according to the invention comprises at least one pharmaceutically or physiologically acceptable additive, it is in particular an additive suitable for administering the composition by the oral route or an additive suitable for administering the composition by the parenteral route.

[0097] Pharmaceutical compositions containing vitamin D and one or more polyphenols according to the present invention are generally available in solid or liquid form. For oral administration, solid pharmaceutical compositions in the form of tablets, capsules or gelatin capsules may be preferred.

[0098] Liquid pharmaceutical compositions may be preferably in the form of an aqueous or non-aqueous suspension, or alternatively in the form of a water-in-oil or oil-in-water emulsion. Solid pharmaceutical forms may contain, as vehicles, adjuvants, or additives, at least one diluent, one flavoring agent, one stabilizer, 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, cellulose-based materials, cocoa butter, etc. Liquid compositions may also contain water, possibly in admixture with propylene glycol or polyethylene glycol, and possibly also with colorants, flavorings, stabilizers, and thickeners.

[0099] Treatment: The present invention also relates to a method for treating, alleviating and / or preventing disorders associated with an imbalance in bone and / or cartilage metabolism, in particular disorders associated with bone mass loss and / or cartilage loss, comprising administering to an individual in need thereof an effective amount of a composition of the present invention. For example, the method comprises administering an effective amount of vitamin D in combination with one or more polyphenols.

[0100] 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, reduces symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0101] The effective amount of a composition of the present invention required to achieve a therapeutic effect will vary depending on the particular composition, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated.

[0102] The present invention further provides methods for enhancing bone formation and / or cartilage anabolism, increasing bone mineral density during growth, and optimizing peak bone mass, treating or preventing osteoarthritis, and / or stimulating bone formation and / or cartilage anabolism during fracture healing, comprising administering to an individual an effective amount of a composition of the present invention.

[0103] In one embodiment, the present invention relates to a method for the treatment, alleviation and / or prevention of disorders associated with an imbalance in bone metabolism, in particular disorders associated with bone mass loss, comprising administering to an individual in need thereof a therapeutically effective amount of vitamin D and one or more polyphenols, or a pharmaceutical composition containing vitamin D and one or more polyphenols.

[0104] In one embodiment, the method of treatment according to the present invention relates to the treatment, alleviation and / or prevention of osteoarthritis.

[0105] The method of treatment according to the invention may be in mammals such as humans or pets, for example dogs, cats and / or horses.

[0106] In certain embodiments, the compositions of the present invention administered in the treatment method may be one or more nutritional compositions of the present invention and / or pharmaceutical compositions of the present invention.

[0107] Dosage regimen: The present invention relates to the surprising finding that a combination of vitamin D with one or more polyphenols, such as hydroxytyrosol and / or oleuropein, enhances the osteogenic and / or cartilage anabolic effects of those polyphenols.

[0108] The combination can both enhance the effects of polyphenols and induce an early response from osteoblasts, and thus the compositions of the present invention may be useful for shorter-term treatment, as opposed to the long-term treatments used in conventional treatments for, for example, osteoarthritis.

[0109] Therefore, the present invention also relates to the use according to the invention, wherein the composition of the invention is administered at least once a day for a period of at least one month, such as at least two months, preferably at least three months.

[0110] In another embodiment, the invention relates to a use according to the invention, wherein the composition is administered at least once a day for at least one month.

[0111] 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.

[0112] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety. [Example]

[0113] The invention is described in further detail in the following non-limiting examples.

[0114] Example 1 (Collagen synthesis by osteoblasts): Primary osteoblasts were isolated from the calvaria of newborn Wistar rats by enzymatic digestion as described by Declercq et al. (2004). During all experiments, cells were maintained in α-minimum essential medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin at 37°C with 5% CO2. Cells were plated onto Col-1-coated 96-well plates at a density of 3500 cells / well or 3 × 10 4 Cells were seeded in 24-well plates at a density of 100 cells / well and cultured in α-MEM for 2 days until confluence was reached. Then, cells were cultured in various conditions: minimal medium (C-), minimal medium containing 50 μg / ml ascorbic acid and 5 mM β-glycerophosphate (optimized medium C+), and minimal medium containing 1 μM oleuropein (OLP) or / and 1 μM hydroxytyrosol (HTy) or / and 10 μM hydroxytyrosol (HTy). -9Cells were exposed to minimal medium supplemented with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). OLP and HTy were dissolved in dimethyl sulfoxide (DMSO), and 1,25(OH)2D3 was dissolved in ethanol. The final concentrations of DMSO and ethanol in the medium were 0.1%, respectively. The medium was changed every 2 days.

[0115] After 9 days, the collagen content of the cells was measured using the Sircol assay from Biocolor. The Sircol collagen assay is a quantitative dye-binding method designed for the analysis of collagen extracted from mammalian tissues and cells in in vitro culture. The dye reagent specifically binds to the [Gly-XY]n helical structure found in mammalian collagen (type IV).

[0116] FIG. 1 shows collagen synthesis (measured by micrograms of collagen produced per milligram of total protein) by osteoblasts after 9 days of culture in response to stimulation with a combination of 1,25(OH)2D3, OLP, and HTy.

[0117] 1,25(OH)2D3 is 10 -9 M 1,25-dihydroxyvitamin D3; OLP 1 μM oleuropein; HTy 1 μM hydroxytyrosol; C+ indicates the use of optimized medium as a positive control.

[0118] Data represent the mean ± SEM (three replicates). Statistics: Kruskal Wallis test (two-tailed) followed by Mann Whitney U test. * P<0.05; ** P<0.01; *** P<0.001 vs. negative control (C-).

[0119] As shown in Figure 1, the combination of 1,25(OH)2D3, OLP and HTy significantly stimulates cellular collagen synthesis in osteoblasts after 9 days of culture.

[0120] Example 2 (Osteoblast alkaline phosphatase activity): Primary osteoblasts were isolated from the calvaria of newborn Wistar rats by enzymatic digestion as described by Declercq et al. (2004). During all experiments, cells were maintained in α-minimum essential medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin at 37°C with 5% CO2. Cells were plated onto Col-1-coated 96-well plates at a density of 3500 cells / well or 3 × 10 4 Cells were seeded in 24-well plates at a density of 1000 cells / well and cultured in α-MEM for 2 days until confluence was reached. Then, cells were cultured in various conditions: minimal medium (C-), minimal medium containing 50 μg / ml ascorbic acid, 5 mM β-glycerophosphate (optimized medium C+), and 1 μM oleuropein (OLP) or / and 1 μM hydroxytyrosol (HTy) or / and 10 μM hydroxytyrosol (HTy). -9 Cells were exposed to minimal medium supplemented with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). OLP and HTy were dissolved in dimethyl sulfoxide (DMSO), and 1,25(OH)2D3 was dissolved in ethanol. The final concentrations of DMSO and ethanol in the medium were 0.1%, respectively. The medium was changed every 2 days.

[0121] Alkaline phosphatase (ALP) enzyme activity was measured kinetically on days 0, 5, 9, 12, and 15 of treatment. Osteoblasts were washed twice with PBS before freezing on dry ice. Cells were then lysed by freeze-thaw cycles and homogenization in 200 μl of 1 M diethanolamine and 0.24 mM magnesium chloride buffer, pH 9.8 (ALP buffer). Cell lysate (10 μl) was added to 200 μl of a 6 mg / ml solution of p-nitrophenyl phosphate (p-NPP) in ALP buffer. Absorbance was measured at 405 nm every 2.5 minutes for 30 minutes at 30°C using a VarioSklan flash microplate reader. ALP activity was expressed as micromoles of p-NPP per hour per mg of protein. Protein measurements were performed using the Quick Start BioRad protein assay according to the method of Bradford.

[0122] FIG. 2 shows alkaline phosphatase (ALP) activity (expressed as micromoles of ALP per milligram of protein per hour) after 9 days in response to a combination of 1,25(OH)2D3, OLP, and HTy.

[0123] C- indicates the use of minimal medium as a negative control; 1,25(OH)2D3 is 10 -9 M 1,25-dihydroxyvitamin D3; OLP 1 μM oleuropein; HTy 1 μM hydroxytyrosol; C+ indicates the use of optimized medium as a positive control.

[0124] Data represent mean ± SEM. Statistics: Kruskal Wallis test (two-tailed) followed by Mann Whitney U test. *P<0.05; **P<0.01; ***P<0.001 vs. negative control (C-).

[0125] FIG. 2 further shows that the addition of a combination of 1,25(OH)2D3, OLP, and HTy significantly stimulated cellular collagen synthesis in osteoblasts after 9 days of culture.

[0126] Figures 3-6 show alkaline phosphatase (ALP) activity in osteoblasts cultured in the presence of 1,25(OH)2D3, oleuropein (OLP), and hydroxytyrosol (HTy), separately or in combination, in a time-course model.

[0127] C- indicates the use of minimal medium as a negative control; 1,25(OH)2D3 is 10 -9 M 1,25-dihydroxyvitamin D3; OLP 1 μM oleuropein; HTy 1 μM hydroxytyrosol; C+ indicates the use of optimized medium as a positive control.

[0128] Data represent mean ± SEM. Statistics: Kruskal Wallis test (two-tailed) followed by Mann Whitney U test. *P<0.05; **P<0.01; ***P<0.001 vs. negative control (C-).

[0129] Conclusion: As can be seen from the data shown in Figure 3, addition of vitamin D3 by itself induces a small but statistically significant increase in osteoblast activity and AP activity, indicative of bone formation and / or cartilage anabolism, at day 15. Similarly, addition of either HTy by itself or OLP by itself also results in a small increase at day 15 that is comparable to the effect of vitamin D3 (see respective figures).

[0130] The combination of both HTy and OLP also increases osteoblast activity (see day 15), but not to the same extent as the combination of vitamin D with either HTy or OLP. The only points that are statistically significantly different from the control are days 12 and 15. Early measurements on days 5 and 9 are not statistically different from the negative control.

[0131] Thus, the present data illustrate two effects of vitamin D supplementation: First, the addition of vitamin D along with either or both HTy and OLP increases the osteoblast response, as seen, for example, by comparing levels at day 15.

[0132] Second, the addition of vitamin D with either or both of HTy and OLP induces osteoblast activity more quickly. Consequently, the addition of vitamin D results in an earlier and stronger response. It is concluded that the addition of vitamin D enhances the osteogenic and / or chondroanabolic responses induced by OLP and / or HTy.

Claims

1. A composition comprising vitamin D for use in enhancing the effect of one or more polyphenols on bone formation and / or cartilage anabolism.

2. 2. The use according to claim 1, wherein the polyphenol is one or more of oleuropein and hydroxytyrosol.

3. A composition comprising vitamin D and one or more polyphenols for use in enhancing the stimulation of bone formation and / or cartilage anabolism by said polyphenols.

4. 4. The composition for use according to claim 3, wherein the polyphenols comprise one or more of oleuropein and hydroxytyrosol.

5. 5. The composition for use according to claim 3 or 4, wherein the polyphenols include oleuropein and hydroxytyrosol.

6. 6. The composition for use according to claim 5, comprising 800 to 1200 IU of vitamin D, 0.01 mg to 1 g of oleuropein, and 0.01 mg to 1 g of hydroxytyrosol.

7. 7. The composition for use according to any one of claims 1, 2 or 3 to 6, comprising one or more further biologically active molecules such as antioxidants, anti-inflammatory compounds, fatty acids, prebiotic fibres, probiotics, glucosamine, chondroitin sulphate, minerals, trace elements and / or vitamins.

8. 8. A composition for use according to claim 7, comprising the minerals calcium and / or magnesium.

9. The composition for use according to any one of claims 1 to 8, wherein said use is aimed at an ageing population, in particular elderly patients.

10. 10. The composition for use according to any one of claims 1 to 9, wherein said use is in the treatment, alleviation and / or prevention of disorders associated with an imbalance between bone formation and / or cartilage anabolism and bone resorption by enhancing the stimulation of bone formation and / or cartilage anabolism.

11. The composition for use according to any one of claims 1 to 10, wherein said use is for maintaining and / or improving mobility.

12. The composition for use according to any one of claims 1 to 11, wherein said use is for improving bone strength.

13. 13. The composition for use according to any one of claims 1 to 12, wherein the disorder is selected from osteoporosis, Paget's disease, bone loss or osteolysis observed near a prosthetic device, metastatic bone disease, hyperthyroidism, hypercalcemia due to cancer, multiple myeloma, periodontal disease, osteoarthritis, bone defects due to fracture or fracture healing.

14. 14. The composition for use according to claim 13, wherein the disorder is osteoporosis.

15. The composition for use according to any one of claims 1 to 14, which is a nutritional composition.

16. The composition for use according to any one of claims 1 to 15, which is administered at least once a day for a period of at least 2 months, preferably at least 3 months.

17. A pharmaceutical composition comprising vitamin D and one or more polyphenols.

18. A method for the treatment, alleviation and / or prevention of disorders associated with an imbalance in bone and / or cartilage metabolism, comprising administering to an individual in need thereof an effective amount of vitamin D in combination with one or more polyphenols.

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