Compositions comprising a combination of fisetin and quercetin for the treatment of bone disorders

Abstract

The present invention relates to the use of a composition comprising an effective amount of a combination of fesoterodine and / or a derivative thereof and quercetin and / or a derivative for preventing or treating a bone disorder or maintaining bone health in an individual.

Description

Technical Field

[0001] This invention relates to bone health, and particularly to the use of compositions comprising a combination of fibrin and / or its derivatives with quercetin and / or its derivatives for the prevention or treatment of bone disorders or for the maintenance of bone health. The invention also relates to compositions and methods for enhancing bone quality, bone growth, and / or bone strength. Background Technology

[0002] Bone is a tissue that undergoes continuous remodeling due to the breakdown and de novo synthesis of bone tissue in a complex process involving two main types of cells: osteoblasts, which produce new bone tissue, and osteoclasts, which break down bone. Osteoblasts are the cells responsible for bone formation; they differentiate from precursor cells and express and secrete many enzymes and structural proteins of the bone matrix, including type I collagen, osteocalcin, osteopontin, and alkaline phosphatase. Osteoclasts are multinucleated cells that are responsible for bone loss in a process commonly known as bone resorption.

[0003] In healthy adults (humans or animals), the combined action of osteoblasts and osteoclasts enables bone mass to be maintained over time, while ensuring bone remodeling through bone resorption and de novo synthesis. However, in individuals with osteoporosis, an imbalance occurs in the process of bone remodeling, ultimately leading to bone loss at a rate faster than the rate of bone formation. Although this imbalance exists to some extent with age in most individuals, it is much more severe and appears at a younger age in individuals with osteoporosis.

[0004] Therefore, in humans and other mammals, multiple disorders are associated with abnormal metabolism of bone resorption and bone formation, leading to metabolic or bone remodeling imbalances.

[0005] Among the pathological disorders associated with bone metabolic imbalances, notable examples include osteoporosis, Paget's disease, bone loss or resorption observed near prostheses, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, and periodontal disease. Some bone metabolic disorders or diseases may result from prolonged quiescence, such as long-term hospitalization, or even after a period of weightlessness. Of the disorders associated with abnormal bone resorption, osteoporosis is the most common, most frequently observed in postmenopausal women. Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and deterioration of bone microstructure, associated with increased bone fragility and susceptibility to fractures.

[0006] Because osteoporosis (and other impairments associated with bone loss) constitute a chronic condition, its prevention and treatment must be planned for the long term.

[0007] It is now generally accepted that early treatment is the preferred approach because the two key phases of bone capital development are: the growth period, which is the time when maximum bone mass (peak bone mass) is achieved; and aging, which regulates the rate of bone loss. Therefore, the prevention of osteoporosis is no longer limited to older individuals.

[0008] Furthermore, in both humans and animals, there are numerous conditions characterized by a need to increase bone formation. For example, in the case of a fracture, it is necessary to stimulate bone growth to accelerate complete bone repair. This need also exists in periodontal disease, bone metastases, osteolytic diseases, and conditions requiring the repair of connective tissue, such as for the healing or regeneration of cartilage defects or trauma. Stimulation of bone growth is also required in primary and secondary hyperparathyroidism, as well as in osteoporosis associated with diabetes and glucocorticoid-related osteoporosis.

[0009] Although a wide variety of active compounds exist for stimulating bone formation and / or inhibiting bone resorption, the need for new active compounds remains, especially due to the limited success of current treatments.

[0010] Furthermore, given the chronic nature of some conditions caused by bone metabolic imbalances, there is a need for new active compounds that can be used long-term in humans and animals, and that may be available in the form of food additives, such as nutritional compositions.

[0011] Furthermore, in the early stages, human bone growth and development require an adequate supply of many different nutrients. Classical nutrient deficiencies are associated with stunted growth (e.g., energy, protein, Zn), rickets (e.g., vitamin D), and other bone abnormalities (e.g., Cu, Zn, vitamin C). There is evidence that childhood growth patterns and nutritional exposure influence peak bone mass and later fracture risk. However, using bone health as a standard to define dietary reference values ​​is challenging, and the question of what type of diet constitutes optimal support for bone growth and development remains unanswered (see, for example, Prentice, A. et al., 2006. Proceedings of the Nutrition Society, 65(4), pp. 348–360).

[0012] Several approaches can be taken to improve the intake of growth-limiting nutrients, including the administration of micronutrient supplements, micronutrient fortification of food, or improvement of dietary intake. However, particularly in populations with poor dietary quality, several micronutrient deficiencies may occur simultaneously, in which case growth may be affected by more than one growth-limiting nutrient (see, for example, Rivera, JA et al., 2003. The Journal of Nutrition, 133(11), pp. 4010S-4020S).

[0013] Therefore, new nutritional interventions are needed to enhance bone growth and / or bone strength, especially in children with growth retardation and / or slowed growth. Summary of the Invention

[0014] The inventors have unexpectedly demonstrated that the combination of quercetin and fisetin synergistically activates mitochondrial function at the cellular level via increased mitochondrial calcium. Mitochondria are the primary source of aerobic energy production in mammalian cells and maintain a large Ca2+ gradient on their inner membrane, thereby providing a signaling potential for this molecule. Furthermore, mitochondrial Ca2+ plays a role in regulating ATP production in mitochondria and potentially contributes to the coordination of cellular metabolic homeostasis. Specifically, activation of mitochondrial Ca2+ input increases cellular energy metabolism (Glancy and Balaban, Biochemistry, Role of mitochondrial Ca2+ in the regulation of cellular energetics (2012), 246:237-247). In bone tissue and cells, mitochondria play a crucial role in cellular energy metabolism and bone formation (Sautchuk and Eliseev, Bone Report, Cell energy metabolism and bone formation (2022), 27:16:101594). Therefore, in osteoprogenitor cells, mitochondrial energy metabolism is activated during osteogenic differentiation. Furthermore, osteoblasts are heavily dependent on mitochondrial oxidative phosphorylation / mitochondrial energy production. Additionally, bone aging is associated with mitochondrial dysfunction.

[0015] Therefore, one aspect of the present invention relates to a composition comprising an effective amount of ferrous sulfate and / or its derivatives in combination with quercetin and / or its derivatives, the composition being used in an individual to prevent or treat bone disorders or to maintain bone health.

[0016] In another aspect, the present invention provides a combination of ferrous sulfate and / or its derivatives with quercetin and / or its derivatives for enhancing bone quality, bone growth and / or bone strength in an individual.

[0017] In a final aspect, the present invention relates to a method for manufacturing a composition for the stated purpose according to the invention.

[0018] Other features and advantages are described herein and will be apparent from the following figures and detailed description. Attached Figure Description

[0019] Figure 1 Indicate the chemical structures of fisetin (A) and quercetin (B).

[0020] Figure 2 This graph shows that the combination of fisetin and quercetin has a greater effect on mitochondrial activation (via increased mitochondrial Ca2+) in HeLa cells than fisetin or quercetin alone. The bars show the effects of fisetin (3 µM, black), quercetin (3 µM, gray), and the combination of 3 µM fisetin + 3 µM quercetin on the increase in integrated mitochondrial calcium induced by 100 µM histamine. Results are presented as mean + / - SEM, n = 6 experiments. *Indicates a statistically significant difference in measured mitochondrial calcium relative to the theoretical difference, P < 0.05 (one-way ANOVA test).

[0021] Figure 3 This diagram illustrates the synergistic effect of fisetin and quercetin in HeLa cells to activate mitochondria via increased mitochondrial Ca2+. To quantify the synergistic effect of the combination of quercetin and fisetin on mitochondrial activation, the expected theoretical effect (the sum of the fisetin and quercetin effects, derived from...) is... Figure 2 (Extracted from the data) and the actual measured effect of the combination (feserin + quercetin, from...) Figure 2 (Extracted from the data) for comparison. Results are expressed as mean + / - SEM, n=6 experiments. *Indicates a statistically significant difference in measured values ​​of mitochondrial calcium relative to theoretical differences, P<0.05 (Student's t-test). Detailed Implementation

[0022] Definitions

[0023] Before discussing the invention in further detail, the following terms and conventions are first defined.

[0024] In the context of this invention, percentages referred to are weight / weight percentages unless otherwise indicated.

[0025] The term “and / or” as used in the context of “X and / or Y” should be interpreted as “X” or “Y”, or “X and Y”.

[0026] The numerical ranges used herein are intended to include every numerical value and subset thereof contained within the range, whether or not specifically disclosed. Furthermore, these numerical ranges should be understood to support claims relating to any numerical value or subset thereof within the range. For example, disclosures 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.

[0027] The term "prevention" means applying the composition disclosed herein to an individual who does not exhibit any symptoms of the condition in order to reduce or prevent the development of at least one symptom associated with the condition. Furthermore, "prevention" includes reducing the risk, morbidity, and / or severity of the condition or disorder.

[0028] As used in this article, "effective amount" is the amount in an individual that treats or prevents defects, diseases or medical conditions, or more generally, the amount that reduces symptoms, manages disease progression, or provides nutritional, physiological or medical benefits to an individual.

[0029] "Animals" 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 using "animals," "mammals," or their plural forms, these terms also apply to any animal capable of having an effect manifested or intended to be manifested by the context of the paragraph, such as an animal benefiting from improved mitochondrial calcium input. While the terms "individual" or "subject" are commonly used herein to refer to humans, this disclosure is not limited thereto. Therefore, the terms "individual" or "subject" refer to any animal, mammal, or human who may benefit from the methods and compositions disclosed herein.

[0030] The term "pet" means any animal that can benefit from or enjoy the compositions provided in this disclosure. For example, a pet can be a bird, a bovine, a canine, a horse, a feline, a goat, a wolf, a rodent, a sheep, or a pig, but it can also be any suitable animal. The term "companion animal" means a dog or a cat.

[0031] The term "subject" or "individual" is a mammal, preferably a human. In the case of humans, the term "elderly person" means someone who is at least 60 years old from birth, preferably 63 years or older, more preferably 65 years or older, and most preferably 70 years or older. In the context of humans, the term "middle-aged or elderly person" means someone who is at least 45 years old from birth, preferably 50 years or older, more preferably 55 years or older, and includes elderly individuals. In one embodiment, the individual is a postmenopausal or perimenopausal individual, particularly a postmenopausal or perimenopausal woman.

[0032] "Oral nutritional supplements" or "ONS" are compositions containing at least one macronutrient and / or at least one micronutrient, in the form of sterile liquid, semi-solid, or powder, and are intended to supplement other nutrient intakes, such as those from food. Non-limiting examples of commercially available ONS products include MERITENE. ® BOOST ® NUTREN ® and SUSTAGEN ®In some embodiments, the ONS can be a beverage in liquid form that can be consumed without further addition of liquid, such as a portion of liquid in a composition.

[0033] "Pillbox" means a unit in which the multiple components of a pillbox are physically associated in or with one or more containers and are regarded as a unit of manufacture, packaging, sale or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, outer packaging, shrink wrap, attachments (e.g., binding components, adhesive components, etc.), any type of packaging, any design or material, or combinations thereof.

[0034] All singular features or limitations mentioned in this invention shall include the corresponding plural features or limitations, and vice versa, unless the context in which these contents are mentioned otherwise indicates or explicitly implies the contrary.

[0035] 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.

[0036] Composition for use

[0037] The inventors have demonstrated that providing a combination of ferrous sulfate and / or its derivatives with quercetin and / or its derivatives synergistically improves, for example, altered mitochondrial function in bone disorders.

[0038] In one embodiment, the composition is used to stimulate bone formation and / or inhibit bone resorption; ii) improve bone quality; iii) prevent or treat disorders associated with an imbalance in the relationship between bone formation and bone resorption.

[0039] According to the present invention, the term "stimulates bone formation" means the ability of the combination of quercetin and fisetin of the present invention to induce an increase in bone mineral density at a given dose.

[0040] Those skilled in the art may also resort to any other tests also described in the embodiments, such as measurements of bone fracture resistance or, further, measurements of alkaline phosphatase activity and measurements of calcium accumulation in osteoblasts.

[0041] The term "inhibition of bone resorption" according to this invention refers to the inhibition of osteoclast-mediated destructive activity on bone tissue. To demonstrate that the composition inhibits bone resorption in humans or animals, those skilled in the art can measure urinary excretion of deoxypyridinium phosphate as described in the examples; a reduction in deoxypyridinium phosphate expression reflects inhibition of bone resorption.

[0042] Supplying an animal organism with the composition according to the invention simultaneously induces stimulation of bone formation and inhibition of bone resorption; the overall increase in bone mineralization and thus the overall increase in bone density are the result of the induction of these two mechanisms.

[0043] In order to determine whether a subject is in a state of osteopenia and therefore requires the supply of the composition according to the invention, those skilled in the art will be able to refer in particular to the World Health Organization (WHO) report entitled “Assessment of fracture risk and its use in postmenopausal osteoporosis screening” (WHO Technical Series-843) of 1994.

[0044] The compositions according to the invention are also designed for individuals exhibiting or potentially suffering from bone defects, i.e., an imbalance in the relationship between bone formation and bone resorption, which, if this imbalance persists, induces bone loss. The compositions according to the invention are also designed for individuals exhibiting symptoms of bone defects caused by fractures, surgery, or dental conditions.

[0045] The composition for the stated use according to any one of the preceding claims is characterized in that the composition is designed for treating bone defects caused by fractures.

[0046] Specifically, the composition is designed to prevent or treat diseases selected from the following: osteoporosis, Paget's disease, bone loss or osteolysis observed near a prosthesis, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, periodontitis, or osteoarthritis.

[0047] As mentioned above, many disorders related to bone metabolism imbalances (such as osteoporosis) develop gradually over a long period and require long-term treatment. Therefore, their prevention or treatment can be carried out by regularly supplying the compositions according to the invention, preferably in the form of nutritional compositions.

[0048] Similarly, the composition according to the invention, when regularly supplied with nutrients to young growing individuals, humans, or animals, makes it possible to produce high bone density and elevated peak bone mass by stimulating bone formation when these individuals reach adulthood.

[0049] Regular nutritional supply of the compositions according to the invention can also be used to prevent bone loss that occurs with aging and / or to protect osteocytes during bone aging.

[0050] In another embodiment, the composition is designed to stimulate bone formation in young individuals during their growth period.

[0051] The compositions as defined above are also designed to promote bone quality and bone growth in young individuals in order to obtain individuals with high bone density and, if possible, high peak bone mass. In particular, the compositions according to the invention are useful during the growth period of humans and other mammals, especially domestic dogs and racehorses.

[0052] As used herein, “bone quality” can refer to aspects of bone composition and structure that contribute to bone strength, independent of bone mineral density. These include bone turnover, microstructure, mineralization, micro-damage, and the composition of the bone matrix and minerals. Methods for measuring bone quality are known in the art.

[0053] As used herein, “promoting bone growth and / or strength” can refer to supporting normal bone growth and / or strength, for example, during childhood and adolescence. Supporting normal bone growth and / or strength results in normal bone anatomy and physiology. Appropriate methods and parameters for determining bone growth and strength will be known to those skilled in the art (see, for example, Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469(8), pp. 2128-2138). Suitablely, one or more bone parameters selected from the following can be used to determine normal bone growth and / or strength: trabecular bone volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), medial and lateral diameters, anteroposterior diameters, bone ultimate force (FMax), and bone stiffness. In some embodiments, one or more bone parameters selected from the following are used to determine normal bone growth and / or strength: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax). Those skilled in the art can obtain suitable methods for determining these parameters.

[0054] The combinations of the present invention can promote catch-up growth in individuals with, for example, stunted and / or slowed growth. As used herein, “catch-up growth” can refer to a high rate of growth exceeding the normal age limit for at least one year after a brief period of growth inhibition, and can be complete or incomplete (see, for example, Wit, JM and Boersma, B., 2002. Journal of Pediatric Endocrinology and Metabolism, 15, pp. 1229-1242).

[0055] The appropriate methods and parameters for determining catch-up growth are known to those skilled in the art. Appropriately, catch-up growth can be determined using high-velocity or high-standard-deviation scores (see, for example, Frongillo, EA, Leroy, JL and Lapping, K., 2019. Advances in Nutrition, 10(3), pp. 372-379 and Desmond, C. and Casale, D., 2017. PLOS ONE, 12(12), p. e0189135).

[0056] In some implementations, catch-up growth is measured as an absolute term of linear growth (i.e., a reduction in high-level defects from the mean of a healthy reference group). In some implementations, catch-up growth is measured as a relative linear growth (i.e., an improvement in age-high z-score and / or passing the -2SD or -1SD cutoff).

[0057] Individual

[0058] The individual can be any suitable individual. Appropriately, the individual can be a mammal. In a preferred embodiment, the individual is a human. In other embodiments, the individual is an animal, preferably a pet. The pet can be an animal selected from dogs, cats, birds, fish, rodents (such as mice, rats, and guinea pigs), rabbits, etc. In some embodiments, the pet is a small dog breed.

[0059] In some implementations, the individual is an older or middle-aged person. In another implementation, the individual is a postmenopausal or perimenopausal individual, particularly a postmenopausal or perimenopausal woman.

[0060] In the context of humans, the term "elderly person" means someone who is at least 60 years old from birth, preferably 63 years or older, more preferably 65 years or older, and most preferably 70 years or older. In the human context, the term "middle-aged or older person" means someone who is at least 45 years old from birth, preferably 50 years or older, more preferably 55 years or older, and includes elderly individuals.

[0061] In some implementations, individuals are referred to as adolescents, teenagers, or children. The term "adolescent" may refer to an individual who has not yet reached adulthood. The term "teenager" may refer to an individual from the onset of puberty to adulthood. The term "child" may refer to an individual between birth and puberty.

[0062] In a preferred embodiment, the individual is about 3 years old or older. In some embodiments, the individual is about 4 years old or older, or about 5 years old or older. In a preferred embodiment, the individual is about 10 years old or younger. In some embodiments, the individual is about 9 years old or younger, about 8 years old or younger, about 7 years old or younger, about 6 years old or younger, or about 5 years old or younger.

[0063] In some implementations, the individual is approximately 3 to 10 years old, approximately 3 to 9 years old, approximately 3 to 8 years old, approximately 3 to 7 years old, approximately 3 to 6 years old, or approximately 3 to 5 years old.

[0064] This invention is particularly applicable to children who are premature or have low birth weight or experience intrauterine growth retardation, or infants and young children who suffer from growth retardation due to malnutrition or disease (such as Crohn's disease and / or celiac disease and / or cancer), or infants and young children treated with medications (such as chemotherapy drugs and / or corticosteroids) that cause malabsorption, anorexia, and / or metabolic bone disease. This invention is particularly preferred for children who are premature or have low birth weight or experience intrauterine growth retardation or suffer from intrauterine malnutrition or growth retardation. This invention is also applicable to children at risk of bone disease, with a family history of bone disease, or who have experienced at least one, preferably several, fractures.

[0065] In some implementations, individuals suffer from and / or are suffering from growth retardation. Growth retardation can be defined as an age-related height value that is less than two standard deviations from the median of the WHO child growth standards (see, for example, De Onis, M. and Branca, F., 2016. Maternal & child nutrition, 12, pp. 12-26).

[0066] In some implementations, the subject suffers from and / or is suffering from growth retardation. The term “growth retardation” can describe a pattern of weight gain that is slower than expected for age and sex in children and other adolescents (see, for example, King, C. and Davis, T., 2010. European journal of clinical nutrition, 64(1), pp. S11–S13). In some implementations, the individual suffers from and / or is suffering from growth retardation and / or growth slowing due to malnutrition or experiencing conditions such as anorexia, Crohn's disease, and / or celiac disease. In some implementations, the individual suffers from and / or is suffering from growth retardation and / or growth slowing due to treatment with medications that cause malabsorption, anorexia, and / or metabolic bone disease, such as chemotherapy drugs and / or corticosteroids.

[0067] In some implementations, an individual is considered preterm, has low birth weight, or experiences intrauterine growth restriction. The term "preterm infant" can refer to an infant born at a gestational age of less than 37 weeks. The term "low birth weight infant" can refer to an infant born with a live birth weight of less than 2,500g.

[0068] Ingredient - primary bioactive compound

[0069] Fertility and quercetin are the main bioactive molecules according to the present invention.

[0070] Feutherin (7,3′,4′-flavon-3-ol) (see...) Figure 1It is a polyphenol found in many plants, where it is used as a yellow / ochre coloring agent. It is also present in many fruits and vegetables, such as strawberries, apples, persimmons, grapes, onions, and cucumbers.

[0071] In one embodiment, at least a portion of the fisetin is obtained by known means, such as extraction from plant / vegetable / fruit sources of fisetin. Additionally or alternatively, at least a portion of the fisetin and / or its derivatives may be obtained by chemical synthesis.

[0072] Non-limiting examples of suitable derivatives of ferrous sulfate include its glucuroninated form, its sulfated form, its derivatives, and mixtures thereof. In a preferred embodiment, the derivative is gerardol.

[0073] Quercetin is the glycosidic form of many other flavonoid glycosides found in citrus fruits, buckwheat, and onions, such as rutin and quercitrin. Quercetin is derived from the glycosides quercitrin and rutin, along with rhamnose and rutin.

[0074] Similarly, guavaside is a 3-O-arabinoside, hyperoside is a 3-O-galactoside, isoquercetin is a 3-O-glucoside, and meadowsweet is a 4'-O-glucoside. Miquelianin is quercetin 3-O-β-D-glucuronopyranoside.

[0075] In a preferred embodiment, the quercetin derivative may be selected from quercetin 3-O-galactoside, quercetin 3-O-glucoside (isoquercetin), quercetin 3-O-xyloside, quercetin 3-O-rhamnoside (quercetin), quercetin 3-O-glucuronide, quercetin 7-O-glucoside, quercetin 3-O-diglucoside, quercetin 3,4'-diglucoside, quercetin 3-O-rhamnoside-7O-glucoside, quercetin 3-O-rutin (rutin), quercetin 3-O-6''-acetylglucoside, quercetin 3-methyl ether, quercetin 3,3'-dimethyl ether, isorhamnetin, and mixtures thereof.

[0076] Quercetin can be derived from any suitable source and can be isolated and / or chemically synthesized.

[0077] In a preferred embodiment, fisetin and quercetin and their derivatives are obtained from plant sources. For example, fisetin can be obtained from strawberries, apples, persimmons, grapes, onions, cucumbers, etc. For example, quercetin can be obtained from onions, green tea, apples, berries, ginkgo leaf extract, St. John's wort, elderberry, buckwheat tea, etc.

[0078] The effective amount of each of fisetin and / or its derivatives, and quercetin and / or its derivatives, varies depending on the specific composition, the recipient's age and condition, and the specific disorder or disease being treated. However, in a general embodiment, an individual may be administered 0.001 mg to 1.0 g daily, preferably 0.01 mg to 0.9 g daily, more preferably 0.1 mg to 750 mg daily, even more preferably 0.5 mg to 500 mg daily, and most preferably 1.0 mg to 200 mg daily. Furthermore, the inventors have found that the active dose of fisetin or its derivatives in the combination can be reduced to obtain equivalent efficacy.

[0079] In some embodiments, a combination of ferrous sulfate or its derivatives with quercetin or its derivatives is administered in the form of a composition also comprising calcium. At least a portion of the calcium may be one or more calcium salts, such as calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium lactate, or mixtures thereof. In general embodiments, an individual is administered 0.1 g to 1.0 g of calcium daily, preferably 125 mg to 950 mg daily, more preferably 150 mg to 900 mg daily, even more preferably 175 mg to 850 mg daily, and most preferably 200 mg to 800 mg daily.

[0080] In an alternative embodiment, the combination of fisetin and quercetin may be administered sequentially with calcium in a separate composition. The term "sequentially" means that at least one of fisetin or its derivatives is administered with calcium in a sequential manner, such that at least one of fisetin or its derivatives 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 fisetin and quercetin. The time between sequential administrations may be, for example, a second or several seconds, a minute or several minutes, or an hour or several hours within the same day; a day or several days, or a week or several weeks within the same month; or one or several months within the same year.

[0081] Feserin or its derivatives and quercetin or its derivatives can be formulated in specific ratios. In some embodiments, the formulation may comprise 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 feserin:quercetin in some embodiments and quercetin:feserin in others. Preferably, the ratio is 1:1 to 1:10.

[0082] In some embodiments, fipronil or its derivatives and quercetin or its derivatives are the only polyphenols in the composition and / or the only polyphenols administered to an individual.

[0083] The composition may contain an effective amount of at least one of ferrous sulfate or its derivatives. For example, a single serving or dose of the composition may contain an effective amount, while a package may contain one or more servings, or one or more doses. Optionally, the composition may also contain calcium.

[0084] Ingredient - additional bioactive compound

[0085] The composition for the stated purpose according to the present invention may further comprise at least one additional bioactive compound selected from antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fiber, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

[0086] In the context of this application, the term "bioactive" means that, in addition to meeting basic nutritional needs, the compound contributes to an individual's health or has an effect on the human body.

[0087] At least one additional bioactive compound may be derived from a natural source. Therefore, this compound may be derived from extracts of plants, animals, fish, fungi, algae, or microbial fermentation. Minerals, considered to be of natural origin, are also included in this definition.

[0088] In a preferred embodiment, the enzyme may be a protease such as trypsin, or an enzyme extract such as, for example, bromelain.

[0089] Nutritional composition

[0090] The composition for the stated purpose according to the present invention can be a nutritional composition or a pharmaceutical composition, and can be used for human or veterinary purposes. Therefore, in a preferred embodiment, the composition for the stated purpose according to the present invention is a nutritional composition.

[0091] In the context of this application, the term "nutritional composition" means a composition that serves as an individual's source of nutrition.

[0092] The nutritional products or compositions of the present invention may be complete or incomplete sources of nutrition. As used herein, "complete nutrition" includes nutritional products and compositions containing a full range of macronutrients (proteins, fats, and carbohydrates) and micronutrients sufficient to serve as the sole source of nutrition for animals administered to them. Patients can obtain 100% of their nutritional needs from such complete nutritional compositions.

[0093] As used herein, "incomplete nutrition" includes nutritional products and compositions that do not contain sufficient amounts of macronutrients (proteins, fats, and carbohydrates) or micronutrients to serve as the sole source of nutrition for animals to which they are administered. Partial or incomplete nutritional compositions may be used as nutritional supplements.

[0094] The combination of fisetin and quercetin can be administered in any compositional form suitable for human and / or animal consumption. In a preferred embodiment, it is administered orally or enterally (e.g., via tube feeding). For example, it can be administered to an individual in the form of a beverage, food product, capsule, tablet, powder, or suspension.

[0095] 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, nutritional products, foods for special medical purposes (FSMP), powdered nutritional products reconstituted in water or milk before consumption, food additives, pharmaceuticals, beverages, pet foods, and combinations thereof.

[0096] Nutritional composition ingredients

[0097] Protein source

[0098] In one embodiment, the composition for the stated use according to the invention comprises a protein source. The protein source may be a dietary protein, including but not limited to animal proteins (such as milk protein, meat protein, or egg protein), plant proteins (such as soy protein, wheat protein, rice protein, and pea protein), or combinations thereof. In one embodiment, the protein is selected from whey, chicken, corn, caseinate, wheat, flax, soybean, carob, pea, or combinations thereof.

[0099] Carbohydrate source

[0100] In one embodiment, the composition comprises a carbohydrate source. Any suitable carbohydrate may be used in the compositions 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 combinations thereof.

[0101] Fat source

[0102] In one embodiment, the composition comprises a fat source. The fat source may include any suitable fat or mixture of fats. For example, the fat source may include, but is not limited to, vegetable fats (such as olive oil, corn oil, sunflower oil, high-oleic sunflower oil, rapeseed oil, low-erucic acid rapeseed oil, hazelnut oil, soybean oil, palm oil, coconut oil, blackcurrant seed oil, borage oil, lecithin, etc.), animal fats (such as milk fat), or combinations thereof. The fat source may also be a less refined form of the fats listed above (e.g., olive oil containing polyphenols).

[0103] Flavourings etc.

[0104] Furthermore, the composition for the stated purpose according to the present invention may also contain natural or artificial flavorings, such as fruit flavorings like banana, orange, peach, pineapple or raspberry, or other plant flavorings like vanilla, cocoa, coffee, etc.

[0105] Form of the nutritional composition

[0106] In addition to the primary bioactive component and any other bioactive components, and optionally one or more sources of protein, carbohydrates, and fats, the nutritional composition may contain any number of optional additional food ingredients, including conventional (synthetic or natural) food additives such as one or more acidifiers, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavoring agents, minerals, penetrants, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texture agents, and / or vitamins. Optional ingredients may be added in any suitable amount.

[0107] The nutritional composition may be provided in any suitable form. Examples of nutritional composition forms available for use according to the invention include solutions, ready-to-drink compositions (e.g., ready-to-drink compositions or instant beverages), liquid foods, soft drinks, fruit juices, sports drinks, dairy drinks, milkshakes, yogurt drinks, soups, etc.

[0108] In another embodiment, the nutritional composition may be provided in the form of a concentrate, powder, or granules (e.g., effervescent granules), which are diluted with water or other liquids (e.g., milk or juice) to produce a ready-to-use composition. Other forms of the nutritional composition include baked goods, dairy products, desserts, confectionery products, cereal bars, and breakfast cereals. Examples of dairy products include milk and dairy drinks, yogurt and other yogurt products, ice cream, and cheese. Examples of baked goods include bread, cookies, and cakes.

[0109] In one embodiment, the composition for the stated purpose according to the invention can also be formulated in various forms of animal food (specifically, animal food for dogs or cats), whether obtained in a wet, semi-wet, or dry form, specifically in the form of biscuits.

[0110] Route of administration

[0111] The nutritional composition disclosed herein can be administered in any manner suitable for human use, and specifically in any part of the gastrointestinal tract. In a preferred embodiment, the composition is for oral administration, preferably wherein the combination is administered simultaneously.

[0112] Enteral, oral, and tube- or catheter-based administration are all covered by this disclosure. The nutritional composition can also be administered via a route selected from oral, rectal, sublingual, sublipal, intraoral, and topical administration.

[0113] This nutritional composition can be administered in any known form, including, for example, convenient dosage forms such as tablets, capsules, liquids, chewable tablets, soft gels, capsules, powders, syrups, liquid suspensions, emulsions, and solutions. In soft capsules, the active ingredient is preferably dissolved or suspended in a suitable liquid, such as fatty oils, paraffin oils, or liquid polyethylene glycol. Stabilizers may optionally be added.

[0114] If the nutritional composition is administered via tube feeding, it can be used for short-term or long-term tube feeding.

[0115] Method of manufacturing the nutritional composition of the invention

[0116] In another aspect, the present invention relates to a method for manufacturing a nutritional composition for the stated purpose according to the invention, the method comprising the following steps:

[0117] - Provide an ingredient for a nutritional composition comprising a combination of fisetin and / or its derivatives with quercetin and / or its derivatives; and mix such that the nutritional composition comprises the combination of fisetin and / or its derivatives with quercetin and / or its derivatives.

[0118] Pharmaceutical composition for use .

[0119] In another embodiment, the present invention relates to a composition according to the invention for inhibiting or preventing cartilage degeneration, wherein the composition is a pharmaceutical composition. A pharmaceutical composition means a composition that is not 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 invention, the pharmaceutical composition can be used to inhibit or reduce cartilage degeneration.

[0120] This drug is intended for human use. Alternatively, it may be a veterinary composition, for example, suitable for dogs, cats, or horses, specifically thoroughbred horses.

[0121] In a preferred embodiment, the pharmaceutical composition of the present invention comprises a combination of fisetin or a derivative thereof with quercetin or a derivative thereof.

[0122] This invention also relates to the use of pharmaceuticals according to the invention, and the use of the compositions of the invention as described herein.

[0123] The pharmaceutical composition for the stated use according to the invention comprises a combination of ferrous sulfate or a derivative thereof with quercetin or a derivative thereof and at least one excipient selected from the group consisting of pharmaceutically acceptable excipients. Procedures for preparing the pharmaceutical composition according to the invention can be readily found by those skilled in the art, for example, in the Remington's Pharmaceutical Sciences, Mid. Publishing Co, Easton, Pa., USA. Physiologically acceptable excipients, mediators, and adjuvants are also described in the handbook entitled "Handbook of Pharmaceutical Excipients" (Second Edition, American Pharmaceutical Association, 1994). To formulate the pharmaceutical composition according to the invention, those skilled in the art will advantageously be able to refer to the latest edition of the European Pharmacopoeia or the United States Pharmacopeia (USP). Specifically, those skilled in the art can advantageously refer to the fourth edition "2002" of the European Pharmacopoeia or also to USP 25-NF 20.

[0124] Advantageously, the pharmaceutical compositions as defined above are suitable for oral, parenteral, or intravenous administration. When a pharmaceutical composition for the stated purpose according to the invention comprises at least one pharmaceutically or physiologically acceptable excipient, it is specifically an excipient suitable for oral administration or an excipient suitable for parenteral administration.

[0125] The pharmaceutical compositions for the stated uses according to the present invention can be obtained in either solid or liquid form. For oral administration, solid pharmaceutical compositions in the form of tablets, capsules, or gelatin capsules are preferred.

[0126] In liquid form, the pharmaceutical composition is preferably in the form of an aqueous or non-aqueous suspension, or even more preferably in the form of an oil-in-water or water-in-oil emulsion.

[0127] Solid drug dosage forms may contain at least one diluent as a medium, adjuvant, or excipient; a flavoring agent; a solubilizer; a lubricant; a suspending agent; a binder; a disintegrant; and an encapsulating agent. Examples of such compounds include magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, gelatin, cellulose materials, and cocoa butter. Liquid compositions may also contain water, possibly as a mixture with propylene glycol or polyethylene glycol, and may also contain colorants, flavoring agents, stabilizers, and thickeners.

[0128] This disclosure also provides a kit containing a combination of fisetin and / or its derivatives with quercetin and / or its derivatives in one or more containers. In one embodiment of the kit, the one or more containers include at least one first container storing fisetin and / or its derivatives separately from quercetin and / or its derivatives, which are stored in at least one second container, and the kit further includes instructions for mixing fisetin and quercetin into a unit dosage form.

[0129] In one embodiment of the pillbox, the combination may be provided together in one or more pre-packaged unit dosage forms, for example in separate containers each containing dry powder, such that each container contains one pre-packaged unit dosage form.

[0130] In another embodiment, the pillbox may include a plurality of compositions for mixing together to form one or more of the compositions disclosed herein. For example, the pillbox may include two or more dry powders in separate containers relative to each other, each powder comprising a portion of a final unit dosage form. As a non-limiting example of such embodiments, the pillbox may include one or more first containers containing fipronil and may also include one or more second containers containing quercetin. 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 a unit dosage form of the composition.

[0131] The above application examples do not require uninterrupted, continuous daily application. Instead, brief interruptions may be allowed during application, such as interruptions of two to four days during the application period. The ideal duration of application of the composition can be determined by those skilled in the art.

[0132] Combination of the disclosure

[0133] It should be noted that the embodiments and features described in the context of one aspect of the invention are also applicable to other aspects of the invention.

[0134] The compositions for the stated uses according to the present invention are described herein with different parameters, such as ingredients, nutritional composition form, uses, target population, etc. It should be noted that, unless otherwise explicitly stated, embodiments and features described in the context of one of the parameters of the compositions for the stated uses according to the present invention may also be combined with other embodiments and features described in the context of another parameter.

[0135] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0136] The invention will now be described in further detail in the following non-limiting embodiments.

[0137] Examples

[0138] The following non-limiting examples provide experimental data to support the compositions and methods disclosed herein.

[0139] To test the effects of fisetin, quercetin, and their combinations in living cells, the inventors measured the increase in mitochondrial calcium in HeLa cells. HeLa cells were purchased from ATCC. HeLa cells were seeded at a density of 50,000 cells / well in 96-well plates in essential medium (DMEM, Gibco) (high glucose + 10% fetal bovine serum). Mitochondrial calcium measurements were performed using HeLa cells infected with an adenovirus expressing a mitochondrial mutant of the mitochondrial-targeting calcium sensor luminescent protein (from Sirion Biotech) (Monange et al., 2004). For luminescent protein remodeling, cells were incubated at room temperature (22±°C) for 2 hours at 24 hours post-infection in standard medium containing 1 μM wild-type coelenterate (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM Hepes, pH 7.4). For processing, the compound was added directly to cell or myotube cultures 2 hours prior to measurement. Luminescence was measured on a FLIPRTetra Aequorin (Molecular Devices). Mitochondrial calcium elevation was obtained by stimulating cells with 100 mM histamine. Luminescence data were calibrated to calcium concentration using the algorithm described previously (Alvarez & Montero, 2002). Quantification was performed using a custom module analysis based on Excel (Microsoft) and GhaphPad Prism 7.02 (GraphPad) software.

[0140] like Figure 2 As shown, in HeLa cells, the combination of fisetin and quercetin had a greater effect on mitochondrial activation (via increased mitochondrial Ca2+) than fisetin or quercetin alone. Figure 3 As shown, in HeLa cells, fisetin and quercetin work synergistically to activate mitochondria via mitochondrial Ca2+ elevation.

Claims

1. A composition comprising an effective amount of a combination of ferrous sulfate and / or its derivatives with quercetin and / or its derivatives, the composition being used in an individual to prevent or treat bone disorders or to maintain bone health.

2. The composition according to claim 1, characterized in that... The composition is suitable for oral administration.

3. The combination for the purpose according to any of the preceding claims, wherein the individual is a human or an animal, preferably wherein the animal is a pet.

4. The composition for the said use according to any of the preceding claims, wherein the derivative of the ferrous sulfate is selected from its glucuronidated form, its sulfated form, its derivatives, and mixtures thereof, preferably gerardol.

5. The composition for the stated use according to any of the preceding claims, wherein the quercetin derivative is selected from quercetin 3-O-galactoside, quercetin 3-O-glucoside (isoquercetin), quercetin 3-O-xyloside, quercetin 3-O-rhamnoside (quercetin), quercetin 3-O-glucuronide, quercetin 7-O-glucoside, quercetin 3-O-diglucoside, quercetin 3,4'-diglucoside, quercetin 3-O-rhamnoside-7O-glucoside, quercetin 3-O-rutin (rutin), quercetin 3-O-6''-acetylglucoside, quercetin 3-methyl ether, quercetin 3,3'-dimethyl ether, isorhamnetin, and mixtures thereof.

6. The composition for the stated use according to any of the preceding claims, wherein the composition further comprises calcium.

7. The composition for the purpose according to any preceding claim, wherein the composition further comprises at least one compound selected from: antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fiber, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

8. A composition for the said use according to any of the preceding claims, wherein the composition is selected from food compositions, dietary supplements, nutritional compositions, oral nutritional supplements, medical foods, nutritional products, beverages, powdered nutritional products reconstituted in water or milk before consumption, food additives, foods for special medical purposes (FSMP), pharmaceuticals, beverages, pet foods, and combinations thereof.

9. The composition for the said use according to any of the preceding claims, wherein the composition is in the form of a solid powder, powder rod, capsule or solution.

10. The composition for the use according to any of the preceding claims, wherein the use is for: i) stimulating bone formation and / or inhibiting bone resorption; ii) improving bone quality; iii) preventing or treating disorders related to an imbalance in the relationship between bone formation and bone resorption.

11. The composition for the said use according to any of the preceding claims, characterized in that, The composition is designed to prevent bone loss that occurs with aging and / or to protect bone cells during bone aging.

12. The composition for the said use according to any of the preceding claims, characterized in that, The composition is designed to treat bone defects caused by fractures.

13. The composition for the said use according to any of the preceding claims, characterized in that, The composition is designed to prevent or treat diseases selected from: osteoporosis, Paget's disease, bone loss or osteolysis observed near a prosthesis, metastatic bone disease, hypercalcemia due to cancer, multiple myeloma, periodontitis, or osteoarthritis.

14. The composition for the said use according to any of the preceding claims, characterized in that, The composition is designed to: i) stimulate bone formation in young individuals during their growth period; ii) enhance bone quality, bone growth, and / or bone strength in children or adolescents.

15. The combination of any of the preceding claims for the purpose, wherein the individual suffers from and / or is suffering from growth retardation and / or growth slowdown.

16. The combination for the purpose according to any of the preceding claims, wherein the combination promotes catch-up growth, preferably wherein catch-up growth is determined using altitude velocity.

17. A method for manufacturing a nutritional composition for the stated purpose according to any one of the preceding claims, the method comprising the steps of: Provide one or more ingredients for use in a nutritional composition, fibrin or its derivatives and quercetin and / or its derivatives, and optionally additional calcium; And mixing.

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