Compositions and methods for enhancing the musculoskeletal benefits of one or more anabolic amino acids in bone health - Patents.com

Abstract

A method of improving and / or enhancing at least one of bone mineralization, bone strength, bone mass, and bone mineral density in an individual in need thereof, comprising administering to the individual a composition comprising one or more anabolic amino acids, the composition further comprising one or more autophagy-inducing compounds. In one embodiment, the one or more autophagy-inducing compounds are selected from the group consisting of thymol, carvacrol, spermidine, urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), 4,4'-dimethoxychalcone, and mixtures thereof.

Description

[Technical field]

[0001] Background technology

[0001] The present disclosure relates generally to compositions and methods for improving and / or enhancing at least one of bone mineralization, bone strength, bone mass, and bone mineral density in an individual in need thereof by administering to the individual a composition comprising one or more anabolic amino acids and one or more autophagy-inducing compounds. More specifically, the present disclosure relates to administering a composition comprising one or more anabolic amino acids, which further comprises one or more compounds that induce autophagy in an amount effective to render the composition at least neutral with respect to autophagy, and preferably positive with respect to autophagy, despite the negative effect of the one or more anabolic amino acids on autophagy. The composition can simultaneously promote protein synthesis and the removal of damaged cellular material. The recipient of administration may be, for example, a critically ill patient, such as a patient in an intensive care unit (ICU); an elderly patient, e.g., an aged individual; a patient suffering from bone disease or bone loss; a patient with sarcopenia or frailty; or an individual with chronic kidney disease (e.g., loss of amino acids due to dialysis) and / or an individual with acute kidney injury.

[0002]

[0002] With major advances in intensive care, critically ill patients often survive acute illnesses that were previously fatal. However, even if they survive this initial phase, the mortality rate remains high for patients who enter the critical chronic phase. Death is often due to refractory multiple organ failure, acute kidney injury and failure, severe myopathy, or less severe muscle weakness. To improve muscle myopathy and muscle weakness, treatments such as nutrition, growth hormone, or androgens have been introduced, but these interventions have been unsuccessful because they unexpectedly increase the risk of organ failure and death. Furthermore, nutritional support for trauma and surgery patients may actually have detrimental effects.

[0003]

[0003] Effective means for providing proper medical care and adequate nutrition to critically ill patients remain lacking.

[0004]

[0004] Furthermore, age-related declines in muscle mass, bone mineral density, bone mass, and function are inevitable in all individuals, but their progression varies greatly depending on genetic and environmental factors such as physical activity and nutritional intake. Sarcopenia is defined as a condition in which age-related declines in muscle mass and muscle function worsen and affect quality of life. In contrast, frailty is another classification of age-related decline in physical function, characterized by loss of muscle strength and muscle function, but not muscle mass. Sarcopenia is clinically diagnosed based on loss of muscle mass and muscle function, using cutoffs to classify individuals with pathological mobility in the aging population. Sarcopenia predicts future disability and mortality. Sarcopenia was assigned an official ICD-10 disease code in 2016 (Anker et al., 2016).

[0005]

[0005] There is a need in the art for compositions comprising anabolic amino acids and autophagy inducers, and methods of using such compositions to increase or maintain musculoskeletal effects and bone benefits (e.g., synergistic effects on bone strength and additive effects on bone mass).

[0006] Summary of the Invention

[0006] The degradation of cytoplasmic proteins is mediated by a cellular process called macroautophagy (also simply called autophagy). The autophagy process is also involved in inflammatory responses and promotes the destruction of bacteria by the immune system. Autophagy constitutes an important lysosomal degradative pathway that recycles damaged and potentially harmful cellular material, such as damaged mitochondria. In particular, autophagy counteracts the effects of cell death and extends life span in various aging models.

[0007]

[0007] As described in more detail later in this specification, the inventors have found that several amino acids known to be involved in musculoskeletal anabolism via the mTOR pathway, such as arginine, glutamine and leucine, and / or anabolic branched chain amino acids, such as leucine and isoleucine, reduce basal autophagy. However, the inventors have discovered that thymol, carvacrol, spermidine, urolithins (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), 4,4'-dimethoxychalcone, and mixtures thereof, can potently induce autophagy and thus counter the negative impact on autophagy of anabolic amino acids, such as loss of bone mass and function, muscle and neuromuscular degeneration, and loss of muscle mass and function.

[0008]

[0008] In another aspect, as detailed in the experimental data described later in this specification, the inventors have found that several amino acids known to be involved in musculoskeletal anabolism via the mTOR pathway, such as arginine, glutamine and leucine, and / or anabolic branched chain amino acids, such as leucine and isoleucine, reduce basal levels of autophagy. However, the present inventors surprisingly discovered that some autophagy inducers (such as thymol, carvacrol, spermidine, urolithins (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyric acid, ketone salts, or ketone ester derivatives), or 4,4'-dimethoxychalcone) can potently induce autophagy, thereby potentially attenuating any negative effects on autophagy by anabolic amino acids, such as bone degeneration and reduced bone mass and strength.

[0009]

[0009] Thus, in a broad embodiment, the present disclosure provides a method for enhancing the musculoskeletal and bone benefits of one or more anabolic amino acids in an individual in need of such enhanced benefits, comprising administering to the individual a composition comprising one or more anabolic amino acids, the composition further comprising one or more compounds that induce autophagy in a total amount effective for the composition to be at least neutral with respect to autophagy, and preferably positive with respect to autophagy.

[0010]

[0010] In one aspect, the present disclosure provides a method for improving and / or enhancing at least one of bone mineralization, bone strength, bone mineral density, and bone mass in an individual in need thereof, comprising administering to the individual a composition comprising one or more anabolic amino acids, the composition further comprising one or more autophagy inducers or precursors thereof.

[0011] In one embodiment, the one or more anabolic amino acids are selected from the group consisting of leucine, isoleucine, arginine, glutamine, citrulline, and mixtures thereof. The one or more anabolic amino acids may include at least one of leucine, glutamine, or arginine in an amount effective to activate mTOR in an individual.

[0012]

[0012] In one embodiment, the one or more compounds that induce autophagy are selected from the group consisting of thymol, carvacrol, spermidine, urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, thorin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), 4,4'-dimethoxychalcone, and mixtures thereof.

[0013]

[0013] In one embodiment, the composition induces autophagy in skeletal muscle.

[0014]

[0014] In one embodiment, the composition induces autophagy in bone cells.

[0015]

[0015] In one embodiment, the individual is an elderly individual.

[0016]

[0016] In one embodiment, the individual has sarcopenia or frailty or is at risk of developing sarcopenia or frailty.

[0017]

[0017] In one embodiment, the individual is critically ill.

[0018]

[0018] In one embodiment, the individual has or is at risk of developing a severe myopathy.

[0019] In one embodiment, the individual has a critical condition involving acute renal failure or is at risk of developing acute renal failure.

[0020] In one embodiment, the individual has chronic kidney disease, with or without an associated loss of bone mass or strength.

[0021] In one embodiment, the individual has a bone disease.

[0022]

[0022] In one embodiment, the individual has a condition selected from the group consisting of osteoporosis, osteomalacia (adults), and conditions of low bone mineral density or low bone mass, such as failure to thrive in children, osteitis fibrosa cysticum, Paget's disease of bone, and pyramiding (turtle).

[0023]

[0023] In one embodiment, the composition comprises a protein providing at least a portion of one or more anabolic amino acids and / or at least a portion of one or more compounds inducing autophagy, the protein being selected from the group consisting of (i) proteins from animal sources, (ii) proteins from plant sources, and (iii) mixtures thereof, preferably one or more of (a) milk proteins, (b) whey proteins, (c) caseinates, (d) casein micelles, (e) pea proteins, (f) soy proteins, and (g) mixtures thereof. In a specific, non-limiting example, at least a portion of the protein is collagen, i.e., unhydrolyzed collagen and / or hydrolyzed collagen.

[0024]

[0024] The protein may be selected from the group consisting of (i) free form amino acids, (ii) unhydrolyzed protein, (iii) partially hydrolyzed protein, (iv) extensively hydrolyzed protein, and (v) mixtures thereof. For example, partially hydrolyzed protein may include unhydrolyzed protein, amino acids, peptides, and polypeptides. Extensively hydrolyzed protein may include amino acids, peptides, and polypeptides. In one embodiment, partially hydrolyzed protein or extensively hydrolyzed protein may include peptides having a length of 2-10 amino acids. Optionally, at least a portion of the protein is hydrolyzed. For example, 5-95% of the protein may be hydrolyzed. Optionally, the protein has a composition selected from the group consisting of (i) at least 50% of the protein is a protein having a molecular weight of 1-5 kDa, (ii) at least 50% of the protein is a protein having a molecular weight of 5-10 kDa, and (iii) at least 50% of the protein is a protein having a molecular weight of 10-20 kDa.

[0025] In one embodiment, the composition comprises a carbohydrate source and / or a fat source.

[0026]

[0026] In one embodiment, the administering step uses at least one route selected from the group of oral, enteral, parenteral, and intravenous injection.

[0027] In one embodiment, the total amount of the one or more anabolic amino acids is about equal to or greater than the total amount of the one or more anabolic amino acids in the composition.

[0028]

[0028] In another embodiment, the present disclosure provides a composition comprising one or more anabolic amino acids, the composition further comprising one or more compounds that induce autophagy in a total amount effective to render the composition at least neutral with respect to autophagy, preferably positive with respect to autophagy. The composition of the present invention may be selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a powdered nutritional formulation that is reconstituted with water or milk before ingestion, a food additive, a medicine, a drink, and combinations thereof.

[0029] An advantage of one or more embodiments provided by the present disclosure is that it improves the condition of a critically ill animal, critically ill human, elderly animal, or elderly human.

[0030] Another advantage of one or more embodiments provided by the present disclosure is the prevention or treatment of excessive catabolism, for example, in critically ill patients or elderly individuals.

[0031] Yet another advantage of one or more embodiments provided by the present disclosure is reducing or preventing the risk of morbidity or mortality due to excessive catabolic effects, for example, in critically ill patients or elderly individuals.

[0032] An additional advantage of the present disclosure is the amelioration, treatment, or cure of multiple organ dysfunction syndrome in critically ill patients.

[0033]

[0033] A further advantage of the present disclosure is that it is carried out in individuals in need thereof to prevent or treat bone loss, maintain bone health, enhance or improve bone mineralization, enhance or improve bone strength, enhance or improve bone mineral density, and / or enhance or improve bone mass.

[0034]

[0034] A further advantage of the present disclosure is the prevention or treatment of bone diseases selected from the group consisting of osteoporosis, osteomalacia (adults), conditions of low bone mineral density or low bone mass, e.g., failure to thrive in children, osteitis fibrosa cysticum, Paget's disease of bone, and pyramidal deformity (turtle).

[0035]

[0035] In another aspect, as detailed in the experimental data described later in this specification, the inventors have found that some amino acids known to be involved in musculoskeletal anabolism through the mTOR pathway, such as arginine, glutamine and leucine, and / or anabolic branched-chain amino acids, such as leucine and isoleucine, reduce the basal level of autophagy.However, the inventors have surprisingly found that some autophagy inducers, such as thymol, and / or some amino acids, such as glycine, cysteine, proline, glutamic acid, valine, tyrosine, or their precursors, such as serine (as precursor of glycine), N-acetylcysteine, methionine (as precursor of cysteine), can strongly induce autophagy, and thus attenuate any negative effects on autophagy caused by anabolic amino acids, such as bone degeneration, loss of bone mass and bone strength.

[0036]

[0036] Thus, in a broad embodiment, the present disclosure provides a method for enhancing the musculoskeletal and bone benefits of one or more anabolic amino acids in an individual in need thereof. The method comprises administering to the individual a composition comprising one or more anabolic amino acids, the composition further comprising one or more autophagy inducers and / or one or more autophagy-inducing amino acids or precursors thereof in a total amount effective for the composition to be at least neutral with respect to autophagy, and preferably positive with respect to autophagy.

[0037] In one embodiment, the one or more anabolic amino acids are selected from the group consisting of leucine, isoleucine, arginine, glutamine, citrulline, lysine, and mixtures thereof. The one or more anabolic amino acids may include at least one of leucine, glutamine, or arginine in an amount effective to activate mTOR in an individual.

[0038]

[0038] In one embodiment, the one or more autophagy inducers include thymol.

[0039]

[0039] In one embodiment, the one or more autophagy-inducing amino acids are selected from the group consisting of glycine, cysteine, proline, glutamic acid, valine, tyrosine, or precursors thereof, and mixtures thereof.

[0040]

[0040] In one embodiment, the composition induces autophagy in bone.

[0041]

[0041] In one embodiment, the composition induces autophagy in bone cells.

[0042]

[0042] In one embodiment, the individual is an elderly individual.

[0043] In one embodiment, the individual is critically ill.

[0044]

[0044] In one embodiment, the individual has or is at risk of developing a severe myopathy.

[0045] In one embodiment, the individual has a critical condition involving acute renal failure or is at risk of developing acute renal failure.

[0046] In one embodiment, the individual has chronic kidney disease with or without an associated loss of bone mass or strength.

[0047] In one embodiment, the individual has a bone disease.

[0048]

[0048] In one embodiment, the individual has a condition selected from the group consisting of osteoporosis, osteomalacia (adults) and rickets (childhood), osteitis fibrosa cystica, Paget's disease of bone, and pyramidal deformity (turtle back).

[0049]

[0049] In one embodiment, the composition comprises a protein providing at least a portion of one or more anabolic amino acids and / or at least a portion of one or more autophagy inducers and / or one or more autophagy inducing amino acids or their precursors, the protein being selected from the group consisting of (i) proteins from animal sources, (ii) proteins from plant sources, and (iii) mixtures thereof, preferably one or more of (a) milk proteins, (b) whey proteins, (c) caseinates, (d) casein micelles, (e) pea proteins, (f) soy proteins, and (g) mixtures thereof. In a specific, non-limiting example, at least a portion of the protein is collagen, i.e., unhydrolyzed collagen and / or hydrolyzed collagen.

[0050] The protein may be selected from the group consisting of (i) free form amino acids, (ii) unhydrolyzed protein, (iii) partially hydrolyzed protein, (iv) extensively hydrolyzed protein, and (v) mixtures thereof. The protein may comprise peptides having a length of 2-10 amino acids. Optionally, at least a portion of the protein is 5-95% hydrolyzed. Optionally, the protein has a composition selected from the group consisting of (i) a composition in which at least 50% of the protein has a molecular weight of 1-5 kDa, (ii) a composition in which at least 50% of the protein has a molecular weight of 5-10 kDa, and (iii) a composition in which at least 50% of the protein has a molecular weight of 10-20 kDa.

[0051] In one embodiment, the composition comprises a carbohydrate source and / or a fat source.

[0052]

[0052] In one embodiment, the administering step uses at least one route selected from the group of oral, enteral, parenteral, and intravenous injection.

[0053]

[0053] In one embodiment, the total amount of the one or more anabolic amino acids is about equal to or greater than the total amount of the one or more anabolic amino acids in the composition.

[0054]

[0054] In another embodiment, the present disclosure provides a composition comprising one or more anabolic amino acids, the composition further comprising one or more autophagy-inducing amino acids or precursors thereof in a total amount effective to render the composition at least neutral with respect to autophagy, preferably positive with respect to autophagy. The composition of the present invention may be selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a powdered nutritional formulation to be reconstituted with water or milk before ingestion, a food additive, a medicine, a drink, and combinations thereof.

[0055] An advantage of one or more of the embodiments provided by the present disclosure is that it improves the condition of a critically ill animal, critically ill human, elderly animal, or elderly human.

[0056] Another advantage of one or more embodiments provided by the present disclosure is the prevention or treatment of excessive catabolism, for example, in critically ill patients or elderly individuals.

[0057] Yet another advantage of one or more embodiments provided by the present disclosure is reducing or preventing the risk of morbidity or mortality due to excessive catabolic processes, for example in critically ill patients or elderly individuals.

[0058] An additional advantage of the present disclosure is to ameliorate, treat, or cure multiple organ dysfunction syndrome in critically ill patients.

[0059]

[0059] A further advantage of the present disclosure is that it is carried out in individuals in need thereof to prevent or treat bone loss, maintain bone health, enhance or improve bone mineralization, enhance or improve bone strength, enhance or improve bone mineral density, and / or enhance or improve bone mass.

[0060]

[0060] A further advantage of the present disclosure is the prevention or treatment of bone diseases selected from the group consisting of osteoporosis, osteomalacia (adults) and rickets (children), osteitis fibrosa cystica, Paget's disease of bone, and pyramidal deformity (turtle back).

[0061]

[0061] In yet another aspect, the present disclosure provides a method for improving and / or enhancing at least one of bone mineralization, bone strength, bone mineral density, and bone mass in an individual in need thereof, comprising administering to the individual a composition comprising an anabolic amino acid, the composition further comprising an autophagy inducer or a precursor thereof.

[0062]

[0062] In one embodiment, the autophagy inducer comprises thymol.

[0063]

[0063] In one embodiment, the autophagy inducer comprises an autophagy-inducing amino acid.

[0064]

[0064] In one embodiment, the autophagy-inducing amino acid is selected from the group consisting of glycine, cysteine, proline, glutamic acid, valine, tyrosine, or precursors thereof, and mixtures thereof.

[0065] In one embodiment, the precursor is selected from the group consisting of serine, N-acetylcysteine ​​(NAC), methionine, and mixtures thereof.

[0066] In one embodiment, the one or more anabolic amino acids are selected from the group consisting of leucine, isoleucine, arginine, glutamine, citrulline, and mixtures thereof.

[0067]

[0067] In one embodiment, the anabolic amino acid is selected from the group consisting of leucine, arginine, glutamine, and mixtures thereof, and the composition is administered in an amount effective to activate mTOR in an individual.

[0068]

[0068] In one embodiment, the composition is administered in an amount effective to induce autophagy in bone.

[0069]

[0069] In one embodiment, the individual is an elderly individual.

[0070] In one embodiment, the individual has a bone disease.

[0071]

[0071] In one embodiment, the individual has a condition selected from the group consisting of osteoporosis, osteomalacia, rickets, osteitis fibrosa cysticum, Paget's disease of bone, pyramidal deformity, and combinations thereof.

[0072]

[0072] In one embodiment, the composition comprises a protein that provides at least a portion of the anabolic amino acids and / or at least a portion of the autophagy inducer or a precursor thereof, wherein at least a portion of the protein is selected from the group consisting of proteins derived from animal sources, proteins derived from plant sources, and mixtures thereof.

[0073] In one embodiment, the composition comprises a protein selected from the group consisting of milk protein, whey protein, caseinate, casein micelles, pea protein, soy protein, and mixtures thereof.

[0074] In one embodiment, the composition comprises a component selected from the group consisting of amino acids in free form, unhydrolyzed proteins, partially hydrolyzed proteins, extensively hydrolyzed proteins, and mixtures thereof.

[0075]

[0075] In one embodiment, at least a portion of the protein is hydrolyzed.

[0076] In one embodiment, the composition comprises at least partially hydrolyzed proteins, including peptides having between 2 and 10 amino acids.

[0077]

[0077] In one embodiment, the protein has a composition selected from the group consisting of: (i) at least 50% of the protein has a molecular weight of 1-5 kDa, (ii) at least 50% of the protein has a molecular weight of 5-10 kDa, and (iii) at least 50% of the protein has a molecular weight of 10-20 kDa.

[0078]

[0078] In one embodiment, the composition further comprises a carbohydrate source and / or a fat source.

[0079]

[0079] In one embodiment, the composition is administered by at least one route selected from the group of oral administration, enteral administration, parenteral administration, and intravenous injection.

[0080]

[0080] In one embodiment, the composition comprises an anabolic amino acid in an amount approximately equal to or greater than the amount of an anabolic amino acid.

[0081]

[0081] Additional features and advantages are described in, and will be apparent from, the following detailed description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] [Figure 1] A diagram showing an example of autophagy activation by individual amino acids in the experimental examples disclosed herein. As shown in Figure 1, amino acids such as glycine, cysteine, proline, glutamic acid, valine, tyrosine, arginine, asparagine and phenylalanine all show at least some autophagy activation effect. Preferably, glycine, cysteine, proline, glutamic acid, valine and tyrosine all show autophagy activation effect. [Figure 2] Another exemplary set of graphs showing autophagy activation by individual amino acids in the experimental examples disclosed herein. As shown in Figure 2, cysteine, glycine, and valine all exhibit an autophagy activation effect, but leucine does not exhibit an autophagy activation effect. [Figure 3] Another set of exemplary graphs showing autophagy activation by individual amino acids in the experimental examples disclosed herein. As shown in Figure 3, proline and glutamic acid show obvious autophagy activation effects, but arginine shows less autophagy activation effects compared to proline and glutamic acid. [Figure 4] Effect of a specific amino acid mixture (AA) on the force frequency response in aged (old) rats and comparison with adult mice fed a normal diet. 20-month-old rats were fed either a control diet or the same diet supplemented with the specific amino acid mixture for 3 months. 6-month-old adult mice were fed a control diet for 3 months. Results are expressed as mean ± standard error. Adult: 6-month-old mice fed a control diet. Aged: 20-month-old mice fed a control diet. Aged + AA: 20-month-old mice fed a control diet supplemented with the specific amino acid mixture. [Figure 5] Results of evaluating the long-term (3 months) effects of thymol (an exemplary autophagy inducer) and an amino acid mix known as an autophagy inducer, either alone or in combination, on bone health (bone mass and / or bone strength) in aged mice. [Figure 6] A series of graphs showing the additive effect of a combination of thymol and an amino acid mix (AA) on bone mass (Figure 6a) and the synergistic effect of a combination of thymol and an amino acid mix (AA) on bone strength (Figure 6b). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083]

[0088] definition

[0089] Some definitions are provided below, however definitions may be found in the "Embodiments" section below, and the heading "Definitions" above does not imply that such disclosure in the "Embodiments" section is not a definition.

[0084]

[0090] All percentages are by weight relative to the total weight of the composition unless otherwise stated. Similarly, all amounts and all ratios are by weight unless otherwise stated. When reference is made to pH, the value corresponds to the pH measured at 25°C by standard equipment. As used herein, "about", "approximately" and "substantially" are understood to refer to a number within a certain range of values, for example, within the range of -10% to +10% of the reference number, preferably within the range of -5% to +5% of the reference number, more preferably within the range of -1% to +1% of the reference number, and most preferably within the range of -0.1% to +0.1% of the reference number.

[0085]

[0091] Moreover, all numerical ranges herein should be understood to include all integers, whole or fractional, within that range. Moreover, these numerical ranges should be interpreted to support claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted to correspond to ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0086]

[0092] As used herein and in the appended claims, singular words include the plural unless the context clearly indicates otherwise. Thus, references to "a," "an," and "the" generally include the plural of the respective terms. For example, reference to "an amino acid" or "the amino acid" includes a plurality of such "amino acids." The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y." Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "both X and Y."

[0087]

[0093] Similarly, the terms "comprise", "comprises", and "comprising" should be interpreted as inclusive rather than exclusive. Similarly, the terms "include", "including", and "or" should all be interpreted as inclusive unless such interpretation is clearly prevented by the context. However, embodiments provided by the present disclosure may not include any element not specifically disclosed herein. Thus, disclosure of an embodiment defined using the term "comprising" is also a disclosure of an embodiment "consisting essentially of" and "consisting of" the disclosed components.

[0088]

[0094] As used herein, the term "example," particularly when followed by a list of terms, is merely exemplary and illustrative and should not be considered as exclusive or comprehensive. All embodiments disclosed herein can be combined with any other embodiment disclosed herein, unless expressly stated otherwise.

[0089]

[0095] "Animal" includes, but is not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, livestock such as sheep, pigs, cows and horses, and mammals including, but not limited to, humans. When "animal", "mammal", or plurals thereof are used, these terms also apply to any animal capable of the effect indicated or intended to be indicated by the context of the text, for example, animals capable of autophagy. As used herein, the term "patient" is understood to include animals, for example, mammals, preferably humans, that are undergoing or will undergo treatment as defined herein. As used herein, the terms "individual" and "patient" are often used to refer to humans, but the present disclosure is not limited to humans.

[0090]

[0096] Thus, the terms "individual" and "patient" refer to any animal, mammal or human that may benefit from the methods and compositions disclosed herein. Indeed, non-human animals experience long-term severe illnesses that closely resemble the human condition. These severely ill animals experience the same metabolic, immune and endocrine disorders as their human counterparts, as well as the onset of organ failure, bone loss and muscle wasting. Additionally, animals also experience the effects of aging.

[0091]

[0097] The term "elderly" in the human context means an age of at least 55 years, preferably greater than 63 years, more preferably greater than 65 years, and most preferably greater than 70 years. The term "older adult" or "ageing individual" in the human context means an age of at least 45 years, preferably greater than 50 years, and more preferably greater than 55 years, and includes geriatric individuals.

[0092]

[0098] For other animals, "elderly adults" or "senior individuals" refer to those that have exceeded 50% of the average lifespan of their respective species and / or genus within a species. An animal is considered "elderly" if it has exceeded 66% of the average expected lifespan, preferably if it has exceeded 75% of the average expected lifespan, and more preferably if it has exceeded 80% of the average expected lifespan. An elderly cat or dog is at least about 5 years old. An elderly cat or dog is at least about 7 years old.

[0093]

[0099] Sarcopenia is defined as the age-related decline in muscle mass and muscle function (including muscle strength and walking speed). Sarcopenia can be assessed by one or more of the following: decreased muscle mass, decreased muscle strength, and decreased physical performance.

[0094]

[0100] Low muscle mass can be roughly measured by a reduced appendicular lean mass index (ALM index) normalized to the square of height, which is 7.00 kg / m2 for men. 2 less than 5.40 kg / m for women 2 The ALM index may be based on less than 0.8 m / s. The decline in physical performance may be based on less than 0.8 m / s. The decline in muscle strength may be based on less than 26 kg for men and less than 18 kg for women.

[0095]

[0101] Additionally or alternatively, sarcopenia can be diagnosed in a subject based on the definition of EWGSOP (European Working Group for Sarcopenia in Older People), e.g., as described in Crutz-Jentoft et al. (2010). Muscle loss is generally characterized by a low appendicular lean mass normalized to height squared (ALM index), specifically, 7.23 kg / m for men and 7.28 kg / m for women. 2 less than 5.67 kg / m 2The ALM index may be based on less than 0.8 m / s. The decline in physical performance may be based on walking speed, specifically less than 0.8 m / s. The decline in muscle strength may be based on grip strength, specifically less than 30 kg for men and less than 20 kg for women.

[0096]

[0102] Additionally or alternatively, sarcopenia can be diagnosed in a subject based on the Foundation for the National Institutes of Health (FNIH) definition, for example as described in Studenski et al. (2014). Low muscle mass is generally associated with a body mass index (BMI: kg / m 2 The assessment may be based on low appendicular lean mass (ALM) adjusted for BMI, specifically less than 0.789 for men and less than 0.512 for women when ALM is adjusted for BMI. The decline in physical performance may generally be based on walking speed, specifically less than 0.8 m / s. The decline in muscle strength may generally be based on reduced grip strength, specifically less than 26 kg for men and less than 16 kg for women. The decline in muscle strength may also generally be based on reduced grip strength to body mass index, specifically less than 1.00 for men and less than 0.56 for women.

[0097]

[0103] As used herein, "frailty" is defined as a clinically discernible state of heightened vulnerability resulting from age-related declines in capacity and function across multiple biological systems, resulting in impaired ability to cope with routine or acute stressors. In the absence of established quantitative standards, frailty has been defined functionally by Fried et al. as meeting three of the following five phenotypic criteria indicative of impaired energetics: (1) muscle weakness (grip strength in the bottom 20% of the population at baseline, adjusted for sex and body mass index), (2) poor endurance and energy (self-reported fatigue related to VO2 max), (3) bradykinesia (baseline in the bottom 20% of the population based on 15-foot walking time, adjusted for sex and height), (4) reduced physical activity (weighted score of kilocalories expended per week at baseline, lowest quintile of physical activity identified for each sex; e.g., <383 kcal / week for men, <270 kcal / week for women), and / or unintentional weight loss (10 pounds in the past year). Fried LP, Tangen CM, Walston J et al., "Frailty in older adults: evidence for a phenotype." J. Gerontol. A. Biol. Sci. Med. Sci. 56(3):M146-M156 (2001). The presence of one or two of these criteria identifies the pre-frailty stage as being at high risk for progression to frailty.

[0098]

[0104] "Cachexia" is a complex metabolic syndrome associated with underlying disease and characterised by loss of muscle mass with or without loss of fat mass. The hallmark clinical feature of cachexia is weight loss (corrected for fluid retention) in adults or growth retardation in children (except for endocrine disorders).

[0099]

[0105] Cachexia is often seen in patients with diseases such as cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis of the liver, anorexia, chronic pancreatitis, and / or metabolic acidosis and neurodegenerative diseases.

[0100]

[0106] Cachexia is particularly common in certain types of cancer, such as pancreatic, esophageal, gastric, intestinal, lung, and / or liver cancer.

[0101]

[0107] The internationally accepted diagnostic criteria for cachexia are a weight loss of more than 5% over a limited period of time, e.g., 6 months, or a reduction in current weight and height (body mass index [BMI] of 20 kg / m 2 The most common pathological features of cachexia are a weight loss of more than 2% in individuals already showing wasting by total body mass (less than 100 mg / kg) or skeletal muscle mass (measured by DXA, MRI, CT, or bioimpedance). Cachexia can progress gradually through different stages, from pre-cachexia to cachexia to refractory cachexia. Severity can be classified according to the depletion of energy stores and body protein (BMI), in combination with the degree of ongoing weight loss.

[0102]

[0108] In particular, cancer cachexia is defined as a weight loss of >5% in the past 6 months (without simple starvation); or a BMI of <20 and a weight loss of >2%; or a lean appendicular mass consistent with a loss of muscle mass (7.26 kg / m2 in men). 2 less than 5.45 kg / m for women 2 weight loss is defined as any weight loss of more than 2% (Fearon et al., 2011).

[0103]

[0109] "Pre-cachexia" can be defined as weight loss of 5% or less combined with anorexia and metabolic changes. Currently, there are no robust biomarkers to identify pre-cachexia patients likely to progress further or the rate at which pre-cachexia progresses. Refractory cachexia is defined essentially based on the clinical characteristics and circumstances of the patient.

[0104]

[0110] The terms "treatment" and "treating" include any effect that results in the improvement of a condition or disease (disorder), such as alleviating, reducing, controlling, or eliminating the condition or disease. The term does not necessarily imply that the subject is treated to a cure. Non-limiting examples of "treating" or "treatment of" a condition or disease include (1) inhibiting the condition or disease, i.e., arresting the development of the condition or disease or its clinical symptoms, and (2) relieving the condition or disease, i.e., causing a temporary or permanent relief of the condition or disease or its clinical symptoms. Treatment may be patient-related or physician-related.

[0105]

[0111] The term "prevention" or "preventing" means not causing the development of clinical symptoms of the referenced condition or disease in an individual who may be exposed to or susceptible to the condition or disease, but who has not yet exhibited or manifested symptoms of the condition or disease. The terms "condition" and "disease / disorder" refer to any disease, condition, symptom, or manifestation.

[0106]

[0112] The relative terms "improved," "increased," and "enhanced," etc., refer to the effect of a composition that includes both one or more anabolic amino acids and one or more compounds that induce autophagy compared to an otherwise identical composition that does not include or has less of the one or more compounds that induce autophagy.

[0107]

[0113] As used herein, the term "autophagy" refers to a cellular catabolic mechanism that involves cellular degradation of unnecessary or dysfunctional cellular components, such as intracellular proteins, protein aggregates, organelles, cell membranes, organelle membranes, and other cellular components, by the action of lysosomes. The mechanism of autophagy may include (i) separation of contents from the rest of the cytoplasm by membrane formation around the targeted cellular region, (ii) fusion of the resulting vesicles with lysosomes or endosomes, and subsequent degradation of the vesicle contents. The term autophagy may also refer to one of the mechanisms by which starving cells reallocate nutrients from less essential processes to more essential processes.

[0108]

[0114] In one embodiment, autophagy is a survival pathway in which non-functional proteins and organelles are degraded in lysosomes for recycling and energy production. For example, in vitro and animal and human studies have shown that autophagy dysfunction in bone cells is associated with the development of bone diseases such as osteoporosis. Autophagy has been identified as playing an important role in both physiological processes and the development and progression of bone-related diseases (Pierrefite-Carle, V., Santucci-Darmanin, S., Breuil, V., Camuzard, O. & Carle, GFAutophagy in bone: self-eating to stay in balance. Ageing Res Rev. 24 (Pt B), 206-217 (2015)). Autophagy controls the energy and chemical homeostasis of individual cells and various types of tissues, including bone (Smith, M. & Wilkinson, S. ER homeostasis and autophagy. Essays Biochem. 61, 625-635 (2017)).

[0109]

[0115] As used herein, the term "bone disease" refers to any bone-related disease or any metabolic bone disorder.For example, metabolic bone disease can include osteoporosis, osteomalacia (adults), and conditions with low bone mineral density or low bone mass, such as failure to thrive in children, osteitis fibrosa cystica, Paget's disease of bone, pyramidal deformity (turtle), and renal osteodystrophy.

[0110]

[0116] As used herein, the term "bone loss" refers to bone loss resulting from conditions in which bone turnover is abnormally increased, such as osteoporosis (including postmenopausal osteoporosis, male osteoporosis, glucocorticosteroid-induced osteoporosis and juvenile osteoporosis), osteopenia, hormone deficiency (vitamin D deficiency, tnalc and female hypogonadism), hormone excess (hyperprolactinemia, excess glucocorticoids, hyperthyroidism, hyperparathyroidism), nutritional factors (digestive disorders, malabsorption syndromes), Paget's disease, other forms of bone loss due to medications, osteoarthritis, renal bone disease, inflammatory disorders (Crohn's disease, rheumatoid arthritis), immobilisation, space travel, neoplastic bone marrow disorders, and the like. Osteoporosis refers to any loss of bone mass resulting from osteoporosis caused through osteoporosis, osteogenesis imperfecta, hypophosphatasia, osteoporosis after hormone replacement therapy, and genetic diseases and inborn errors of metabolism causing bone fragility (familial hypophosphatasia, Gaucher disease, osteogenesis imperfecta, collagen disorders). Other dysostoses and disorders that can be treated by the combination of the present invention include achondroplasia, cleidocranial dysplasia, enchondromatosis, fibrous dysplasia, hypophosphatemic rickets, Marfan syndrome, multiple hereditary exoioses, neurofibromatosis, osteopetrosis, bone porphyria, sclerotic lesions, fractures, periodontal disease, pseudoarthrosis, pyogenic spondylitis, periodontal disease, antiepileptic drug-induced bone loss (primary and secondary), renal osteodystrophy, infiltrative diseases of bone, oral bone loss, osteoporosis ... loss, osteonecrosis of the jaw, juvenile Paget's disease, osteoporosis, metabolic bone disease, mastocytosis, sickle cell anemia / disease, organ transplant-associated bone loss, kidney transplant-associated bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidoses, Fabry's disease, Turner's syndrome, Down's syndrome, Klinefelter's syndrome, leprosy, Perthes' disease, adolescent idiopathic scoliosis, infantile onset multisystem inflammatory disease, Winchester syndrome, Menkes' disease, Wilson's disease, ischemic bone diseasebone disease (e.g. Legg-Calve-Perthes disease, regional migratory osteoporosis), anemic conditions, heparin-induced bone loss, bone marrow disease, scurvy, malnutrition, calcium deficiency, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chrome liver disease, postmenopause, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory bowel disease, oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus, Cushing's disease, acromegaly, hypogonadism, reflex sympathetic dystrophy syndrome, regional osteoporosis osteoporosis, osteomalacia, bone loss associated with joint replacement, bone loss associated with HIV, bone loss associated with reduced growth hormone, bone loss associated with cystic fibrosis, fibrous dysplasia, bone loss associated with chemotherapy, tumor-induced bone loss, cancer-related bone loss, bone loss associated with hormone ablative, multiple myeloma, anorexia nervosa, facial bone loss associated with disease, skull bone loss associated with disease, jaw bone loss associated with disease, and skull bone loss associated with disease. Further conditions relate to bone loss associated with age, including facial bone loss associated with age, skull bone loss associated with age, jaw bone loss associated with age, and skull bone loss associated with age.

[0111]

[0117] In one embodiment, bone density or bone mineral density (BMD) can be measured to follow bone loss or the effectiveness of any treatment for bone loss.

[0112]

[0118] As used herein, the term "bone mineral density" or "bone mineral density" or "BMD" refers to the amount of bone mineral in bone tissue. Bone mineral density measurements can be used in clinical medicine as an indirect indicator of osteoporosis and fracture risk. BMD test results can be reported in three different terms: 1) Measured areal density (g / cm) 22) Z-score: the number of standard deviations above or below the mean for the patient's age, sex, and ethnicity, or 3) T-score: the number of standard deviations above or below the mean for a healthy 30-year-old adult of the same sex and ethnicity as the patient. Many non-invasive tests can be used to measure BMD. Examples of tests may include Dual-energy X-ray absorptiometry (DXA or DEXA), Dual X-ray absorptiometry and Laser (DXL), Quantitative computed tomography (QCT), Quantitative ultrasound (QUS), Single photon absorptiometry (SPA), Dual photon absorptiometry (DPA), Digital X-ray radiogrammetry (DXR), and Single energy X-ray absorptiometry (SEXA). DXA is currently the most widely used, but quantitative ultrasound (QUS) has been reported as a more cost-effective approach to measuring bone density.

[0113]

[0119] The terms "bone mineralization" and "bone mass augmentation" are used interchangeably within this application. Thus, within the scope of this specification or claims, they should be considered synonymous. "Bone mineralization" should also be considered synonymous with increasing, enhancing or improving "bone strength," "bone mineral density," "bone mineral content," "bone mass," "bone growth," and the like.

[0114]

[0120] As used herein, the terms "autophagy inducer," "autophagy inducer," or "autophagy enhancer" refer to any agent or compound that can increase or stimulate autophagy in a cell by any pathway.

[0115]

[0121] As used herein, the term "anabolic amino acid" refers to the amino acid that does not induce autophagy in the composition of the present invention.Non-limiting examples of suitable anabolic amino acids can include leucine, isoleucine, arginine, glutamine, citrulline, and mixtures thereof.Preferably, anabolic amino acids can include leucine and isoleucine.

[0116]

[0122] As used herein, the term "branched chain amino acids" or "BCAA" refers to the group related to three essential amino acids: leucine, isoleucine, and valine.

[0117]

[0123] As used herein, the term "precursor" refers to a compound or substance (or compounds or substances) that can produce an autophagy inducer.

[0118]

[0124] The terms "food," "food product," and "food composition" refer to a product or composition intended for ingestion by an individual, such as a human, and providing at least one nutrient to such an individual. The compositions of the present disclosure, including the many embodiments described herein, may comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in a dietary regimen.

[0119]

[0125] As used herein, "complete nutrition" refers to a composition that contains sufficient variety and quantity of macronutrients (proteins, lipids, and carbohydrates) and micronutrients to be the sole source of nutrition for the animal to which the composition is administered, such that the individual can receive 100% of the individual's nutritional needs from such a complete nutritional composition.

[0120]

[0126] As used herein, the term "critically ill patient" refers to an individual who is experiencing an acute, life-threatening condition or has been diagnosed as being at imminent risk of such development. Critically ill patients are medically unstable and, if untreated, have a high likelihood of dying (e.g., greater than a 50% chance of dying).

[0121]

[0127] Non-limiting examples of critically ill patients include patients who have sustained or are at risk of sustaining acute, life-threatening single or multiple organ system failure due to disease or injury, patients who have undergone surgery and are experiencing complications, and patients who have undergone surgery on a vital organ within the last week or who have undergone major surgery within the last week.

[0122]

[0128] More specific non-limiting examples of critically ill patients include patients who have sustained or are at risk of sustaining acute, life-threatening single or multiple organ system failure due to disease or injury, and patients undergoing surgery and experiencing complications. Further specific non-limiting examples of critically ill patients include patients who require one or more of cardiac surgery, cerebral surgery, thoracic surgery, abdominal surgery, vascular surgery, or transplantation, and patients suffering from one or more of neurological disease, cerebral trauma, respiratory failure, peritonitis, multiple trauma, severe burns, or severe polyneuropathy.

[0123]

[0129] The term "intensive care unit (ICU)" refers to a section of a hospital where critically ill patients are treated. The term "intensive care unit" also includes nursing homes, or clinics, such as private physician's offices, if the therapeutic activities performed in these locations are the same or similar to those in an ICU. An "ICU patient" is included in the term "critically ill patient."

[0124]

[0130] The term "multiple organ dysfunction" refers to a condition resulting from infection, reduced blood flow, hypermetabolism, or injury such as accident or surgery. "Multiple organ failure" leading to the death of critically ill patients is considered to be a descriptive clinical syndrome defined as the dysfunction or failure of at least two vital organ systems. The vital organ systems uniformly and most specifically affected are the liver, kidneys, lungs, and the cardiovascular, nervous, and hematological systems. Non-limiting examples of multiple organ dysfunction include acute respiratory distress syndrome, heart failure, liver failure, renal failure, respiratory failure, intensive care, shock, extensive burns, sepsis (e.g., systemic inflammatory response syndrome), and stroke.

[0125]

[0131] The term "enteral administration" includes oral administration (including oral gavage) and rectal administration, although oral administration is preferred. The term "parenteral administration" refers to the delivery of a substance given by a route other than the digestive tract and covers such routes of administration as intravenous, intraarterial, intramuscular, intraventricular, intraosseous, intradermal, intrathecal, and also intraperitoneal administration, intravesical instillation, and intracavernous injection.

[0126]

[0132] A preferred parenteral administration is intravenous administration. A particular form of parenteral administration is intravenous delivery of nutrition. Parenteral nutrition is "total parenteral nutrition" when food is not provided by other routes. "Parenteral nutrition" is preferably an isotonic or hypertonic aqueous solution (or a solid composition that is dissolved, or a liquid concentrate that is diluted to obtain an isotonic or hypertonic solution) that contains sugars such as glucose, and further contains one or more of lipids, amino acids, and vitamins.

[0127]

[0133] Embodiment

[0134] One aspect of the present disclosure is a method of preventing or treating bone loss, enhancing or improving bone mineralization, enhancing or improving bone strength, enhancing or improving bone mineral density, and / or enhancing or improving bone mineral density in an individual in need thereof. The method includes administering to the individual a composition comprising one or more anabolic amino acids, the composition further comprising one or more components that induce autophagy, e.g., in bone, in an amount effective to render the composition at least autophagy neutral, preferably autophagy positive. The compositions of the present invention can be administered parenterally, enterally, or intravenously.

[0128]

[0135] As used herein, "enhancing the musculoskeletal effect" means that (i) the positive (anabolic) effect of one or more anabolic amino acids is greater than would be the case in an otherwise identically formulated composition in the absence or in lesser amounts of one or more compounds that induce autophagy, and / or (ii) the positive (anabolic) effect of one or more anabolic amino acids lasts longer than would be the case in an otherwise identically formulated composition in the absence or in lesser amounts of one or more compounds that induce autophagy.

[0129]

[0136] Non-limiting examples of suitable anabolic amino acids include leucine, isoleucine, arginine, glutamine, citrulline, and mixtures thereof.The composition may comprise one or more of leucine, isoleucine, or arginine in free form and / or in bound form as peptide and / or protein (such as milk protein, animal protein, or vegetable protein).Preferably, any leucine or arginine is present in the composition in an amount that is effective for activating mTOR. A daily dose of the composition may comprise one or more of: 0.175-142.85 mg leucine per kg body weight, preferably 0.35-71.425 mg leucine per kg body weight; 0.175-71.425 mg isoleucine per kg body weight; 20-300 mg arginine per kg body weight, preferably 50-200 mg arginine per kg body weight, and / or 20-300 mg citrulline per kg body weight, preferably 100-200 mg citrulline per kg body weight. The daily dose of one or more anabolic amino acids may be provided by a single or multiple servings of the composition per day.

[0130]

[0137] Non-limiting examples of suitable compounds that induce autophagy include thymol, carvacrol, spermidine, urolithins (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), 4,4'-dimethoxychalcone, and mixtures thereof. Non-limiting examples of suitable forms of spermidine include spermidine trihydrochloride, spermidine phosphate hexahydrate, spermidine phosphate hexahydrate, and L-arginyl-3,4-spermidine.

[0131]

[0138] The composition includes one or more compounds that induce autophagy in an amount effective to render the composition at least neutral with respect to autophagy, and preferably positive with respect to autophagy, despite the one or more anabolic amino acids having a negative effect on autophagy.

[0132]

[0139] The composition may comprise a pharmacologically effective amount of an autophagy inducer in a pharma- ceutical suitable carrier. In the aqueous liquid composition, the concentration is preferably in the range of about 0.05% to about 4% by weight, or about 0.5% to about 2% by weight, or about 1.0% to about 1.5% by weight of the aqueous liquid composition.

[0133]

[0140] In a particular embodiment, the method is a treatment for increasing the plasma autophagy inducer level in an individual to, for example, a level of 50-6000 nmol per L of plasma, preferably 100-6000 nmol per L of plasma. The method may include a step of daily administering an autophagy inducer in a weight range of 0.05 mg-1 g per kg of body weight, preferably 1 mg-200 mg per kg of body weight, more preferably 5 mg-150 mg per kg of body weight, even more preferably 10 mg-120 mg per kg of body weight, or most preferably 40 mg-80 mg per kg of body weight.

[0134]

[0141] Typically, 50 μg to 10 g of an autophagy inducer is administered to an individual per day in one or more divided doses.

[0135]

[0142] Thymol (10-64%) is one of the main constituents of the essential oil of thyme (Thymus vulgaris L, Lamiaceae). Carvacrol is present in the essential oil of oregano (Origanum vulgare), in thyme oil, in oil obtained from cress, and in monarda. The essential oil of the thyme subspecies contains 5%-75% carvacrol, whereas the Satureja subspecies has a content of 1%-45%. Marjoram (origanum mamaorana) and Dittany of Crete are rich in carvacrol, with 50% and 60-80%, respectively. Thus, some embodiments of the composition of the present invention comprise plant extracts and / or concentrated plant extracts, essential oils or fractions, in particular from thyme and oregano, that provide at least a portion of the thymol and / or carvacrol in the composition.

[0136]

[0143] Wheat germ is rich in spermidine. Thus, some embodiments of the compositions of the invention include wheat germ and / or concentrated wheat germ extract, which provides at least a portion of the autophagy inducers in the compositions of the invention.

[0137]

[0144] Whey protein is rich in BCAAs such as leucine and isoleucine. Thus, some embodiments of the composition include whey protein, which provides at least a portion of the anabolic amino acids in the composition.

[0138]

[0145] The composition of the present invention can induce autophagy in muscle, for example, skeletal muscle.Non-limiting examples of such muscles include one or more of the following: vastus lateralis, gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, extraocular muscles, facial muscles, or diaphragm.

[0139]

[0146] An individual in need of induction of autophagy can be an elderly individual, such as an elderly animal or an elderly human, hi some embodiments, an individual in need of induction of autophagy is an elderly animal or an elderly human.

[0140]

[0147] The composition can induce autophagy in bone.For example, the present inventors have surprisingly found that the composition of the present invention can prevent or treat bone loss, enhance or improve bone mineralization, enhance or improve bone strength, enhance or improve bone mineral density, and / or enhance or improve bone mineral density in individuals in need.

[0141]

[0148] For example, Figure 6b and the Examples demonstrate a synergistic effect of the compositions of the present invention on improving or enhancing bone strength and bone mineral density in a subject. Figure 6b and the Examples demonstrate a significant effect of the compositions of the present invention on improving or enhancing bone strength and bone mineral density in a subject.

[0142]

[0149] In some embodiments, the individual in need of induction of autophagy has at least one bone disease selected from the group consisting of osteoporosis, osteomalacia (adults), low bone mineral density or low bone mass conditions (e.g., failure to thrive in children), osteitis fibrosa cysticis, Paget's disease of bone, pyramidal deformity (turtle), and renal osteodystrophy.

[0143]

[0150] In some embodiments, an individual in need of induction of autophagy may have osteoporosis due to conditions of abnormally increased bone turnover, such as osteoporosis (including postmenopausal osteoporosis, male osteoporosis, glucocorticosteroid-induced osteoporosis, and juvenile osteoporosis), osteopenia, hormone deficiency (vitamin D deficiency, tnalc, and female hypogonadism), hormone excess (hyperprolactinemia, excess glucocorticoids, hyperthyroidism, hyperparathyroidism), nutritional factors ( Suffer from bone loss due to digestive disorders, malabsorption syndromes), Paget's disease, other forms of bone loss due to medications, osteoarthritis, renal bone disease, inflammatory disorders (Crohn's disease, rheumatoid arthritis), osteoporosis caused through immobilization, space travel, tumor-induced bone marrow damage, osteogenesis imperfecta, hypophosphatasia, osteoporosis after hormone replacement therapy, and genetic and inborn errors of metabolism that cause bone fragility (familial hypophosphatasia, Gaucher disease, osteogenesis imperfecta, collagen diseases).

[0144]

[0151] The individual who needs to induce autophagy may be a critically ill patient. In various embodiments, the method can treat or prevent multiple organ dysfunction in critically ill patients, for example, when homeostasis is lost or prevented by the patient receiving parenteral nutrition; can protect critically ill patients from multiple organ dysfunction; can treat or prevent the development of lactic acidosis, for example, parenteral nutrition-induced lactic acidosis; can treat or prevent muscle weakness in critically ill patients; can reduce or prevent morbidity or mortality due to nutritional deterioration caused by parenteral nutrition; and / or can prevent the collapse of body systems.

[0145]

[0152] In some embodiments, the critically ill patient has at least one life-threatening condition selected from the group consisting of lactic acidosis, muscle weakness, hyperglycemia, multiple organ failure, failure of homeostasis, and disturbance of homeostasis. In one embodiment, the critically ill patient has a bone disease or bone-related disorder. In one embodiment, the critically ill patient has a bone disease or bone-related disorder selected from the group consisting of osteoporosis, osteomalacia (adults), low bone mineral density or low bone mass conditions, such as failure to thrive in children, osteitis fibrosa cysticis, Paget's disease of bone, pyramidal deformity (turtle), and renal osteodystrophy. In one embodiment, the critically ill patient has a non-infectious disease. In one embodiment, the critically ill patient has multiple organ dysfunction not caused by or associated with sepsis. Multiple organ dysfunction and muscle weakness are common in critical care settings and can be caused or exacerbated by uncoordinated parenteral nutrition delivery or relative or absolute nutritional overload delivered parenterally.

[0146]

[0153] In some embodiments, the critically ill patient has at least one disease selected from the group consisting of severe trauma, multiple trauma, high-risk surgery, extensive surgery, cerebral trauma, cerebral hemorrhage, respiratory failure, peritonitis, acute kidney injury, acute liver injury, severe burns, severe polyneuropathy, severe myopathy, and ICU-acquired muscle weakness.

[0147]

[0154] In some embodiments, the critically ill patient is receiving enteral or parenteral nutrition. In some embodiments, the compositions of the invention treat or prevent mitochondrial dysfunction, for example, mitochondrial dysfunction induced by inadequate or inconsistent parenteral nutrition to the critically ill patient.

[0148]

[0155] In another aspect of the disclosure, a method overcomes one or more negative effects of one or more anabolic amino acids by preventing degenerative processes associated with impaired autophagy. The method includes administering to an individual a composition comprising one or more anabolic amino acids, the composition further comprising one or more compounds that induce autophagy, such as thymol, carvacrol, spermidine, urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, thorin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), 4,4'-dimethoxychalcone, and mixtures thereof, in an amount effective to render the composition at least neutral with respect to autophagy, preferably positive with respect to autophagy, e.g., in bone.

[0149]

[0156] As used herein, the term "protein" includes amino acids in free form, molecules of 2-20 amino acids (herein referred to as "peptides"), as well as longer amino acid chains. Small peptides, i.e., chains of 2-10 amino acids, are suitable for the compositions of the invention, either alone or in combination with other proteins. The "free form" of an amino acid is the monomeric form of an amino acid. Suitable amino acids include both natural and unnatural amino acids. The compositions of the invention may include a mixture of one or more proteins, e.g., a mixture of one or more of (i) peptides, (ii) longer amino acid chains, or (iii) amino acids in free form; the mixture is preferably formulated to obtain a desired amino acid profile / content.

[0150]

[0157] The compositions of the invention may include a protein that provides at least a portion of one or more anabolic amino acids and / or at least a portion of one or more compounds that induce autophagy, where at least a portion of the protein may be of animal or vegetable origin, such as a dairy protein, such as a milk protein concentrate or milk protein isolate; a caseinate or casein, such as a casein micelle concentrate or casein micelle isolate; or a whey protein, such as a whey protein concentrate or whey protein isolate. Additionally or alternatively, at least a portion of the protein may be a vegetable protein, such as one or more of soy protein or pea protein.

[0151]

[0158] Mixtures of these proteins are also suitable, such as mixtures in which casein is the majority but not all of the protein, whey protein is the majority but not all of the protein, pea protein is the majority but not all of the protein, and soy protein is the majority but not all of the protein. In one embodiment, at least 10% by weight of the protein is whey protein, preferably at least 20% by weight, more preferably at least 30% by weight. In one embodiment, at least 10% by weight of the protein is casein, preferably at least 20% by weight, more preferably at least 30% by weight. In one embodiment, at least 10% by weight of the protein is vegetable protein, preferably at least 20% by weight, more preferably at least 30% by weight.

[0152]

[0159] The whey protein can be, for example, any whey protein selected from the group consisting of whey protein concentrate, whey protein isolate, whey protein micelles, whey protein hydrolysate, acid whey, sweet whey, denatured sweet whey (sweet whey from which the caseinoglycomacropeptide has been removed), a fraction of whey protein, and any combination thereof.

[0153]

[0160] The casein may be obtained from any mammal, but is preferably obtained from cow's milk, and preferably as casein micelles.

[0154]

[0161] The proteins may be non-hydrolyzed, partially hydrolyzed (i.e., peptides with molecular weights between 3 kDa and 10 kDa and an average molecular weight less than 5 kDa), or extensively hydrolyzed (i.e., 90% of the peptides have a molecular weight less than 3 kDa), for example, in the range of 5% to 95% hydrolyzed. In some embodiments, the peptide profile of the hydrolyzed protein may be in different molecular weight ranges. For example, the majority of the peptides (greater than 50 mol% or greater than 50 wt%) may have a molecular weight within 1-5 kDa, or 5-10 kDa, or 10-20 kDa.

[0155]

[0162] In one embodiment, the compositions of the present invention include a carbohydrate source. Any suitable carbohydrate may be used in the compositions of the present invention, including, but not limited to, starch (e.g., modified starch, amylose starch, tapioca starch, corn starch), sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, xylitol, sorbitol, or combinations thereof.

[0156]

[0163] The carbohydrate source preferably represents no more than 50% of the energy of the compositions of the present invention, more preferably no more than 36% of the energy of the compositions of the present invention, and most preferably no more than 30% of the energy of the compositions of the present invention.

[0157]

[0164] In one embodiment, the composition of the present invention comprises a fat source. The fat source can comprise any suitable fat or fat mixture. Non-limiting examples of suitable fat sources include vegetable fats (e.g., 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 (e.g., dairy fats); or combinations thereof.

[0158]

[0165] The compositions of the invention can be administered to an individual, such as a human, for example an elderly or critically ill individual, in a therapeutically effective dose. The therapeutically effective dose can be determined by one of skill in the art and can vary depending on a number of factors known to those of skill in the art, such as the severity of the condition and the weight and general condition of the individual.

[0159]

[0166] The compositions of the present invention are preferably administered to an individual at least 2 days per week, more preferably at least 3 days per week, most preferably 7 days per week; at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or even longer. In some embodiments, the compositions of the present invention are administered to an individual continuously for several days, for example, at least until a therapeutic effect is achieved. In some embodiments, the compositions of the present invention can be administered to an individual daily for at least 30, 60, or 90 consecutive days.

[0160]

[0167] The above dosing examples do not require uninterrupted daily administration. Rather, there may be several short interruptions in administration, such as a 2-4 day break during the administration period. The ideal duration of administration of the compositions of the present invention can be determined by one of skill in the art.

[0161]

[0168] In a preferred embodiment, the compositions of the invention are administered orally or enterally (e.g., by tube feeding) to an individual. For example, the compositions of the invention can be administered to an individual as a drink, capsule, tablet, powder, or suspension.

[0162]

[0169] The composition of the present invention may be any type of composition suitable for human and / or animal consumption. For example, the composition of the present invention may be selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional formulations that are reconstituted with water or milk before ingestion, food additives, medicines, beverages and drinks. In one embodiment, the composition of the present invention is an oral nutritional supplement (ONS), a complete nutritional formula, a medicine, a drug or a food product. In a preferred embodiment, the composition of the present invention is administered to an individual as a drink. The composition of the present invention may be stored as a powder in a sachet and then suspended in a liquid such as water for use.

[0163]

[0170] In some cases where oral or enteral administration is not possible or advisable, the compositions of the invention may also be administered parenterally.

[0164]

[0171] In some embodiments, the compositions of the invention are administered to an individual in a single dosage form, i.e., all compounds are present in one product given to the individual in combination with a meal. In other embodiments, the compositions of the invention are co-administered in separate dosage forms, e.g., at least one component is co-administered separately from one or more of the other components of the compositions of the invention. EXAMPLES

[0165]

[0173] The following non-limiting examples present scientific data that develop and support the concept of inducing autophagy in an individual in need thereof by administering a composition comprising one or more anabolic amino acids, which composition further comprises one or more autophagy inducers and / or one or more autophagy-inducing amino acids in an amount effective to render the composition at least neutral with respect to autophagy, and preferably positive with respect to autophagy, despite any negative effects on autophagy of the one or more anabolic amino acids.

[0166]

[0174] Example 1

[0175] Experimental Protocol

[0176] A zebrafish autophagy reporter was created by stable expression of LC3 protein fused to ZsGreen under the control of a skeletal muscle-specific promoter. Outcrossed transgenic zebrafish larvae were reared at 28 °C under standard laboratory conditions and treated with the indicated amino acids at concentrations ranging from 0.25 to l0 mM for 48 h post-fertilization in 96-well plates (n = 24). After 16 h of treatment, larvae were anesthetized with 0.016% tricaine and imaged at 20x magnification using an ImageXpress confocal system (Molecular Devices). Z-stack images of each larva were captured and maximum projection images were made. The number of LC3 granules was calculated with MetaXpress software (Moleculare Devices) and normalized by fish area.

[0177] 1 to 3 show the experimental results.

[0167]

[0178] Example 2

[0179] Materials and Methods

[0180] Aged mice are a good model for evaluating the effect of nutritional therapy on age-related decline. This model allows the observation of age-related decline in muscle function, similar to that in humans. In this model, 20-month-old mice were fed either a normal diet or the same diet supplemented with a specific amino acid mixture (AA) for 3 months (see Table 1). The dose of the AA mixture was 1.65 mg / kg BW / day.

[0168] [Table 1]

[0169]

[0182] To assess the effects of aging, adult mice (6 months old) were also fed a normal diet for 3 months. The age-related decline in the force-frequency response in vivo was measured with the 305C muscle lever system. Figure 4 shows the effect of a specific amino acid mixture (AA) on the force-frequency response in aged rats (aged) and compared with adult mice fed a normal diet. The force-frequency response was significantly improved by the addition of AA, suggesting improved muscle function.

[0170]

[0183] Example 3

[0184] Assessment of the long-term (3 months) effects of thymol (an example of an autophagy inducer) and an amino acid mixture (known autophagy inducers), alone or in combination, on muscle function in aged mice.

[0171]

[0185] As shown in Figure 5, young mice (24-26 weeks old, n=12 / group, control) were fed a control diet containing alanine to balance the nitrogen content. Old mice (78 weeks old, n=12 / group, present invention) were fed 1) thymol (compound 1), 2) amino acid (AA) mix and thymol (compound 2 and compound 1), or 3) AA mix (compound 2). In the experiment, thymol was given by gavage. Young mice (adult) were fed AA mix in water and controls were fed alanine to balance the nitrogen content. Old untreated mice were also used as another control (old untreated).

[0172]

[0186] The AA mix contains 28.67% leucine, 14.29% isoleucine, 14.29% valine, 12.21% proline, 12.21% glycine, 14.84% lysine, and 3.48% cysteine ​​(see Table 1).

[0173]

[0187] FIG. 6a illustrates the unexpected results of the present invention on improving or enhancing bone mineral density (BMD) in subjects.

[0174]

[0188] For example, as shown in Figure 6a, old untreated subjects showed very low bone mineral density compared to adult controls fed alanine to match nitrogen. Thymol-fed mice (compound 1, old thymol) unexpectedly showed increased bone mineral density compared to untreated adult and old controls. Specifically, thymol-fed mice (compound 1, old thymol) unexpectedly showed a 1.6% increase in bone mineral density compared to untreated old controls.

[0175]

[0189] As shown in Figure 6a, mice fed the AA mix (Compound 2, Aged AA) unexpectedly exhibited an increase in bone mineral density compared to untreated adult and aged controls. Specifically, mice fed the AA mix (Compound 2, Aged AA) unexpectedly exhibited a 0.8% increase in bone mineral density compared to untreated aged controls.

[0176]

[0190] Furthermore, as shown in Figure 6a, mice fed both the amino acid (AA) mix and thymol (Compound 2 and Compound 1) unexpectedly showed a higher level of increase in bone mineral density compared to untreated adult and aged controls. Specifically, mice fed both the amino acid (AA) mix and thymol (Compound 2 and Compound 1) showed a 2.5% increase in bone mineral density compared to untreated aged controls.

[0177]

[0191] Thus, the exemplary combination of the amino acid (AA) mix and thymol of the claimed invention demonstrated an additive effect of both the amino acid (AA) mix and thymol on bone mineral density.

[0178]

[0192] FIG. 6b shows the unexpected results of the synergistic effect of the present invention on improving or enhancing bone strength and bone mineral density in subjects.

[0179]

[0193] For example, as shown in FIG. 6b, old untreated subjects (OldUntreated) exhibited much lower bone stiffness compared to adult controls (Adult) fed alanine to match nitrogen content.

[0180]

[0194] Furthermore, as shown in Figure 6b, mice fed thymol (Aged Thymol) unexpectedly exhibited increased bone stiffness compared to old untreated (Aged Untreated) controls. Specifically, mice fed thymol (Compound 1, Aged Thymol) unexpectedly exhibited a 12% increase in bone stiffness compared to untreated old controls (Aged Untreated).

[0181]

[0195] In contrast, mice fed the AA mix (Compound 2, Aged AA) showed no increase in bone stiffness compared to untreated aged controls (Aged Untreated).

[0182]

[0196] Unexpectedly, as shown in Figure 6b, mice fed both the amino acid (AA) mix and thymol (Compound 2 and Compound 1) showed a much higher level of increase in bone stiffness compared to adult controls (Adult) and aged controls (Aged Untreated). Specifically, mice fed both the amino acid (AA) mix and thymol (Compound 2 and Compound 1) showed a 20% increase in bone stiffness compared to untreated aged controls (Aged Untreated).

[0183]

[0197] Thus, the experimental results in FIG. 6b demonstrated a synergistic effect of the amino acid (AA) mixture and thymol (compound 2 and compound 1).

[0184]

[0198] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be encompassed by the appended claims. [Brief description of the drawings]

[0185] [Figure 1]FIG. 1 shows the results of evaluating the long-term (3 months) effects of thymol (an exemplary autophagy inducer) and an amino acid mix known to be an autophagy inducer, alone or in combination, on bone health (bone mass and / or bone strength) in aged mice. [Diagram 2] FIG. 6 is a series of graphs showing the additive effect of a combination of thymol and an amino acid mix (AA) on bone mass (FIG. 6a) and the synergistic effect of a combination of thymol and an amino acid mix (AA) on bone strength (FIG. 6b).

Claims

1. 1. A method of improving and / or enhancing at least one of bone mineralization, bone strength, bone mass, and bone mineral density in an individual in need thereof, comprising administering to said individual a composition comprising one or more anabolic amino acids, said composition further comprising one or more autophagy-inducing compounds.

2. 2. The method of claim 1, wherein the one or more autophagy-inducing compounds are selected from the group consisting of thymol, carvacrol, spermidine, urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), 4,4'-dimethoxychalcone, and mixtures thereof.

3. 2. The method of claim 1, wherein the one or more anabolic amino acids are selected from the group consisting of leucine, isoleucine, arginine, glutamine, citrulline, and mixtures thereof.

4. 2. The method of claim 1, wherein the one or more anabolic amino acids are selected from the group consisting of leucine, isoleucine, arginine, glutamine, citrulline, and mixtures thereof.

5. 2. The method of claim 1, wherein the anabolic amino acid is selected from the group consisting of leucine, arginine, glutamine, and mixtures thereof, and the composition is administered in an amount effective to activate mTOR in the individual.

6. The method of claim 1 , wherein the composition is administered in an amount effective to induce autophagy in bone.

7. The method of claim 1 , wherein the individual is an elderly individual.

8. The method of claim 1 , wherein the individual has a bone disease.

9. 10. The method of claim 1, wherein the individual has a condition selected from the group consisting of osteoporosis, osteomalacia, rickets, osteitis fibrosa cysticis, Paget's disease of bone, pyramidal deformity, and combinations thereof.

10. 2. The method of claim 1, wherein the composition comprises a protein that provides at least a portion of the anabolic amino acids and / or at least a portion of the autophagy-inducing compounds, and at least a portion of the protein is selected from the group consisting of proteins from animal sources, proteins from plant sources, and mixtures thereof.

11. 10. The method of claim 1, wherein the composition comprises a protein selected from the group consisting of milk protein, whey protein, caseinate, casein micelles, pea protein, soy protein, and mixtures thereof.

12. 2. The method of claim 1, wherein the composition comprises a component selected from the group consisting of amino acids in free form, non-hydrolyzed proteins, partially hydrolyzed proteins, extensively hydrolyzed proteins, and mixtures thereof.

13. The method of claim 12, wherein at least a portion of the protein is hydrolyzed.

14. 10. The method of claim 1, wherein the composition comprises at least partially hydrolyzed proteins, including peptides having 2 to 10 amino acids.

15. 13. The method of claim 12, wherein the protein has a composition selected from the group consisting of: (i) at least 50% of the protein is a protein having a molecular weight of 1-5 kDa, (ii) at least 50% of the protein is a protein having a molecular weight of 5-10 kDa, and (iii) at least 50% of the protein is a protein having a molecular weight of 10-20 kDa.

16. The method of claim 1 , wherein the composition further comprises a carbohydrate source and / or a fat source.

17. 10. The method of claim 1, wherein the composition is administered by at least one route selected from the group of oral administration, enteral administration, parenteral administration, and intravenous injection.

18. 2. The method of claim 1, wherein the composition comprises an amount of the anabolic amino acid that is about equal to or greater than the amount of the anabolic amino acid.

19. 2. The method of claim 1, wherein the one or more autophagy-inducing compounds comprise one or more autophagy-inducing amino acids or precursors thereof.

20. 20. The method of claim 19, wherein the one or more autophagy-inducing amino acids are selected from the group consisting of glycine, cysteine, proline, glutamic acid, valine, tyrosine, or precursors thereof, and mixtures thereof.

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