Compositions for enhancing metabolic oxidation of fatty acids

Abstract

The present disclosure relates to amino acid compositions for enhancing the metabolic oxidation of fatty acids obtained from endogenous sources, such as triglycerides stored in adipose tissue and other sources within the body.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 449,866, entitled "COMPOSITIONS FOR ENHANCED METABOLIC OXIDATION OF FATTY ACIDS," filed March 3, 2023, which is incorporated herein in its entirety.

[0002] The present invention relates to an amino acid composition for enhancing the metabolic oxidation of fatty acids obtained from endogenous sources, such as triglycerides stored in adipose tissue, muscle tissue, and other sources in the body.The composition also enhances the metabolic oxidation of fatty acids from dietary sources.Fatty acid oxidation is enhanced in healthy individuals of all ages at rest, during and after exercise, and during rehabilitation from trauma or major surgery, or long periods of inactivity, such as bed rest. [Background technology]

[0003] Fatty Acid Oxidation and Energy Metabolism Most metabolic reactions in the body require energy in the form of adenosine triphosphate (ATP). The majority of ATP in the body is produced by the β-oxidation of fatty acids. β-oxidation is a catabolic process in which fatty acid molecules are broken down to produce acetyl-CoA, NADH, and FADH2, which are used in the electron transport chain in mitochondria to produce ATP. Fatty acid oxidation plays a key role in maintaining metabolic balance. Impaired fatty acid oxidation leads to elevated plasma levels of circulating very-low-density lipoproteins and the deposition of fatty acids in the form of triglycerides in the liver, which are associated with the development of metabolic syndrome and diabetes (1). Increasing the capacity for fatty acid oxidation at rest is beneficial to metabolic balance. When energy expenditure increases, for example, during exercise, accelerated fatty acid oxidation is essential for ATP production. Summary of the Invention [Problem to be solved by the invention]

[0004] Under normal resting conditions, fatty acid oxidation accounts for 75–85% of ATP production in the postabsorptive state (2). When ATP demand increases with exercise, the extent of fatty acid oxidation increases dramatically. Fat oxidation is responsible for the majority of the ATP required to perform moderate exercise (3). As exercise intensity increases, the absolute amount of fatty acid oxidation remains similar to that oxidized at lower intensities but decreases as a percentage of total substrate oxidation (3). Fatty acids released from adipose tissue and taken up by muscle cells from plasma are the primary source of fatty acids for oxidative metabolism at low exercise intensities, whereas intracellular triglyceride breakdown in muscle tissue is the primary source of fatty acids for oxidative metabolism at higher exercise intensities (3). The increase in capacity for fatty acid oxidation both at rest and during exercise is a major adaptation to endurance exercise training and plays a central role in improving endurance capacity (4). [Means for solving the problem]

[0005] Fatty acids are hydrophobic. Therefore, fatty acids are bound to proteins and transported throughout the body. Fatty acids released from adipose tissue are bound to albumin for transport to muscles and ultimately oxidized. Once inside cells, fatty acid-binding proteins (FABPs) are involved in the transport of fatty acids within cells, including transport to mitochondria for oxidation. There are many FABPs in the FABP family. FABP3 is specific to muscle and cardiac tissue. Therefore, the appropriate availability of FABP3 is central to the process of delivering fatty acids to mitochondria. The compositions described herein enhance fatty acid oxidation at rest and during exercise by increasing the availability of FABP3 through activation of the corresponding gene expression. DETAILED DESCRIPTION OF THE INVENTION

[0006] The present application relates to a method for stimulating metabolic oxidation of long-chain fatty acids containing 16 or more carbons, comprising administering a composition comprising an amino acid mixture containing one or more of the following: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine, wherein fatty acid oxidation is increased compared to a subject not administered the amino acid composition. In certain embodiments, the administered composition comprises the following concentrations of amino acids, expressed as % w / w: about 1-2% histidine, about 9-11% isoleucine, about 35-38% leucine, about 14-17% lysine, about 2-4% methionine, about 5-7% phenylalanine, about 8-9% threonine, about 9-11% valine, about 0.05-0.8% tryptophan, and about 8-11% arginine, and fatty acid oxidation is increased compared to a subject not administered the amino acid composition.

[0007] In certain embodiments, administration of the composition increases the metabolic oxidation of dietary long-chain fatty acids, whether in free form or as components of dietary fats in other forms, including triacylglycerides and phospholipids, compared to subjects not administered the amino acid composition. In another embodiment, the metabolic oxidation of endogenous long-chain fatty acids, whether in free form or as components of triacylglycerides, is increased compared to subjects not administered the amino acid composition. In another embodiment, the metabolic oxidation of long-chain fatty acids during exercise is increased compared to subjects not administered the amino acid composition. In another embodiment, the metabolic oxidation of long-chain fatty acids in obese individuals or individuals with diabetes or metabolic syndrome is increased compared to subjects not administered the amino acid composition. In yet another embodiment, the metabolic oxidation of endogenous long-chain fatty acids, whether in free form or as components of triacylglycerides, is increased compared to subjects not administered the amino acid composition by inducing a similar increase of more than 1000% in FAB3 gene expression.

[0008] The composition may contain a mixture of amino acids. In some embodiments, the composition may include the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, and tryptophan, hi other embodiments, the composition may include the amino acid arginine in addition to the essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, and tryptophan.

[0009] Generally, amino acids may be L-amino acids, D-amino acids, or mixtures thereof. In another embodiment, the amino acids are L-amino acids. Those skilled in the art will recognize that the amino acids of the present compositions may be free amino acids or amino acid salts. The amino acid compositions of the present invention may be in the form of an intact protein or peptide, provided that the protein or peptide contains the amino acids of the present invention at the correct concentrations relative to each other. In other embodiments, the amino acids of the present invention are free amino acids or amino acid salts. If not commercially available, individual amino acids may be produced by methods well known in the art, including chemical synthesis or the use of recombinant microorganisms.

[0010] The amino acids may be the standard amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine, or may be non-standard amino acid derivatives (e.g., amino acid precursors). Non-limiting examples of non-standard amino acids that can be used in the present invention include L-lysine acetate, derivatives of L-tyrosine, ornithine, ketoacid analogs, hydrochloride salts (L-cysteine ​​HCl.H2O), and N-acetyl derivatives of various amino acids. In another embodiment, the amino acids used in the compositions and solutions of the present invention may be in free form or salt form.

[0011] In one aspect, the amino acid composition of the present disclosure comprises one or more essential amino acids. Essential amino acids (EAA) are amino acids that cannot be synthesized de novo by the subject and therefore must be provided through diet. The amino acids considered essential for humans are phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine and histidine. In some embodiments, the combination of EEA comprises phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine and histidine. In certain embodiments, the amino acid composition also comprises at least one conditionally essential amino acid.

[0012] In each of the above embodiments, amino acids and nutrients (if present) can be formulated for animal or human use. In some embodiments, each amino acid and nutrient (if present) is formulated separately. In other embodiments, two or more amino acids and nutrients (if present) are formulated together. In still other embodiments, all amino acids and nutrients comprising the combination of the present invention are formulated together. One or more preparations can then be processed into one or more dosage forms that can be administered together, sequentially, or over a period of time (for example, 1 minute, 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours or longer).

[0013] Administration can be carried out using standard, effective techniques, including oral, parenteral (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), buccal, sublingual, or suppository administration. As used herein, the term "oral" refers to any form of oral administration, including adding the composition to animal feed or other food. The formulation of pharmaceutical compositions is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975) and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY (1980). Methods for preparing compositions for animal or human use are well known to those skilled in the art. For example, compositions can generally be formulated as liquid compositions, solid compositions, or semisolid compositions. Liquid compositions include, but are not limited to, aqueous suspensions, solutions, emulsions, elixirs, or syrups. Liquid compositions will generally include a solvent carrier selected from polar solvents, nonpolar solvents, or a combination of both. The choice of solvent will be influenced by the properties of the components of the composition. For example, if the components are water-soluble, a polar solvent may be used. Alternatively, if the components of the composition are lipid-soluble, a non-polar solvent may be used.

[0014] Suitable polar and non-polar solvents are known to those skilled in the art. Semi-solid compositions include vaginal douches, suppositories, creams, and topicals. Dry compositions include, but are not limited to, reconstitutable powders, chewable tablets, fast-dissolving tablets, effervescent tablets, multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatin capsules, caplets, lozenges, chewable lozenges, beads, powders, granules, particles, microparticles, and dispersible granules. Formulations may contain the combination of the present invention together with excipients. Non-limiting examples of excipients include binders, diluents (fillers), disintegrants, effervescent disintegrants, preservatives (antioxidants), flavor modifiers, lubricants and flow agents, dispersants, colorants, pH adjusters, chelating agents, antimicrobial agents, controlled-release polymers, and any combination of these agents.

[0015] Non-limiting examples of binders suitable for the formulation of various embodiments include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxazolidine, polyvinyl alcohol, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, sugars, oligosaccharides, polypeptides, oligopeptides, and combinations thereof. Polypeptides can be amino acids of any configuration ranging from about 100 to about 300,000 daltons. In one embodiment, the binder can be incorporated into the mixture to be granulated into a solid form, including, but not limited to, crystals, particles, powders, or any other finely divided solid form known to those skilled in the art. In another embodiment, the binder can be dissolved or suspended in a solvent and sprayed onto the mixture in the granulation device as a binding liquid during granulation.

[0016] Non-limiting examples of diluents (also referred to as "fillers" or "thinners") include carbohydrates, inorganic compounds, and biocompatible polymers such as polyvinylpyrrolidone (PVP). Other non-limiting examples of diluents include dibasic calcium sulfate, tribasic calcium sulfate, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, calcium hydrogen phosphate, tribasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, sugars such as sucrose, dextrose, lactose, microcrystalline cellulose, fructose, xylitol, and sorbitol, polyols; starches; pre-fabricated direct compression diluents; and mixtures of any of the foregoing.

[0017] Disintegrants can be effervescent or non-effervescent.Non-limiting examples of non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, agar, guar gum, locust bean gum, karaya gum, pectin and gum tragacanth.Suitable effervescent disintegrants include, but are not limited to, sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0018] Non-limiting examples of preservatives include ascorbic acid and its salts, ascorbyl palmitate, ascorbyl stearate, anoxomer, N-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid, o-aminobenzoic acid, p-aminobenzoic acid (PABA), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid, canthaxanthin, alpha carotene, beta carotene, beta carotene, beta apocarotenoic acid, carnosol, carvacrol, catechin, cetyl gallate, chlorogenic acid, citric acid and its salts, clove extract, coffee bean extract, p-coumaric acid, 3,4-dihydroxybenzoic acid, N,N'-diphenyl -p-phenylenediamine (DPPD), dilauryl thiodipropionate, distearyl thiobispropionate, 2,6-di-t-butylphenol, dodecyl gallate, edetic acid, ellagic acid, erythorbic acid, sodium erythorbate, esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethyl gallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA), eucalyptus extract, eugenol, ferulic acid, flavonoids [e.g., catechin, epicatechin, epicatechin gallate, epigallocatechin (EGC), epigallocatechin gallate (EGCG), epigallocatechin gallate], polyphenols epigallocatechin-3-gallate)], flavones (e.g., apigenin, chrysin, luteolin), flavonols [e.g., datiscetin, myricetin, daemfero], lavanones, fraxetin, fumaric acid, gallic acid, gentian extract, gluconic acid, glycine, guaiac resin, hesperitin, alpha-hydroxybenzylphosphinic acid, hydroxycinnamic acid, hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid, hydroxytyrosol, hydroxyurea, rice bran extract, lactic acid and its salts, lecithin, lecithin citrate; R-alpha-lipoic acid, lutein, lycopene, malic acid, maltol, 5-methoxytryptamine, methyl gallate, monoglyceride citrate; monoisopropyl citrate;Morin, beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate, oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine, phosphoric acid, phosphate salts, phytic acid, phytylubichromel, pimento extract, propyl gallate, polyphosphate salts, quercetin, trans-resveratrol, rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin, sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaric acid, thymol, tocopherols (i.e., alpha, beta, gamma, and delta tocopherol), tocotrienols (i.e., Examples of preservatives include, but are not limited to, tocotrienols (e.g., alpha, beta, gamma, and delta tocotrienols), tyrosol, vanillic acid, 2,6-di-t-butyl-4-hydroxymethylphenol (e.g., Ionox 100), 2,4-(tris-3',5'-di-t-butyl-4'-hydroxybenzyl)-mesitylene (e.g., Ionox 330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxybutyrophenone, tryptamine, tyramine, uric acid, vitamin K and derivatives, vitamin Q10, wheat germ oil, zeaxanthin, or combinations thereof. In exemplary embodiments, the preservative is an antioxidant, such as α-tocopherol or ascorbic acid, and an antimicrobial, such as paraben, chlorobutanol, or phenol.

[0019] Suitable flavor modifiers include flavoring agents, flavor masking agents, sweeteners, etc. Flavoring agents include, but are not limited to, synthetic flavor oils and flavoring aromatics and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof. Other non-limiting examples of flavoring agents include cinnamon oil, wintergreen oil, peppermint oil, clover oil, hay oil, anise oil, eucalyptus oil, vanilla, citrus oils such as lemon oil, orange oil, grape and grapefruit oil, fruit essential oils including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.

[0020] Flavoring agents include, but are not limited to, cellulose hydroxypropyl ethers (HPC) such as Klucel®, Nisswo HPC, and PrimaFlo HP22; low-substituted hydroxypropyl ethers (L-HPC); cellulose hydroxypropyl methyl ethers (HPMC) such as Seppifilm-LC, Pharmacoat®, Metolose SR, Opadry YS, PrimaFlo, MP3295A, Benecel MP824, and Benecel MP 843; methylcellulose polymers such as Methocel® and Metolose®; ethylcellulose (EC) and mixtures thereof such as E461, Ethocel®, Aqualon®-EC, Surelease; polyvinyl alcohol (PVA) such as Opadry AMB; hydroxyethylcellulose such as Natrosol®; carboxymethylcellulose (CMC) and salts of carboxymethylcellulose such as Aualon®-CMC; Kollicoat Copolymers of polyvinyl alcohol and polyethylene glycol, such as IR®; monoglycerides (Myverol), triglycerides (KLX), polyethylene glycol, modified food starch, acrylic polymers and mixtures of acrylic polymers and cellulose ethers, such as Eudragit® EPO, Eudragit® RD100, and Eudragit® E100; cellulose acetate phthalate; Sepifilm, such as a mixture of HPMC and stearic acid, cyclodextrin, and mixtures of these materials. In other embodiments, additional flavor masking agents contemplated are those described in U.S. Patent Nos. 4,851,226, 5,075,114, and 5,876,759, each of which is incorporated herein by reference in its entirety.

[0021] Non-limiting examples of sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as the sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia rebaudiana (stevioside); chloro derivatives of sucrose, such as sucralose; sugar alcohols, such as sorbitol, mannitol, and sylitol, hydrogenated starch hydrolysates, and the synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly its potassium salt (acesulfame K) and sodium and calcium salts. Lubricants can be utilized to lubricate the ingredients forming the compositions of the present invention. As flow agents, lubricants facilitate the movement of solid dosage forms during the manufacturing process. Non-limiting examples of lubricants and flow agents include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, Sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

[0022] The compositions will generally contain from about 0.01% to about 10% by weight of lubricant. In some embodiments, the compositions will contain from about 0.1% to about 5% by weight of lubricant. In further embodiments, the compositions will contain from about 0.5% to about 2% by weight of lubricant.

[0023] Dispersing agents can include, but are not limited to, high hydrophile-lipophile balance (HLB) emulsifier surfactants such as starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silica, and microcrystalline cellulose.

[0024] In some embodiments, it may be desirable to include a colorant. Suitable color additives include, but are not limited to, Food, Drug, and Cosmetic Colors (FD&C), Drug and Cosmetic Colors (D&C), or External Drug and Cosmetic Colors (Ext. D&C). These colorants or dyes, as well as corresponding mordants, and certain natural and derived colorants may be suitable for use in various embodiments.

[0025] Non-limiting examples of pH adjusters include citric acid, acetic acid, tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid, sorbic acid, benzoic acid, sodium carbonate, and sodium bicarbonate.

[0026] Chelating agents may be included as excipients to immobilize oxidizing groups, including but not limited to metal ions, and inhibit oxidative degradation of the morphinan by these oxidizing groups. Non-limiting examples of chelating agents include lysine, methionine, glycine, gluconate, polysaccharides, glutamic acid, aspartate, and disodium ethylenediaminetetraacetic acid (NaEDTA).

[0027] Antimicrobial agents may be included as excipients to minimize degradation of the compounds described herein by microbial agents, including but not limited to bacteria and fungi. Non-limiting examples of antimicrobial agents include parabens, chlorobutanol, phenol, calcium propionate, sodium nitrate, sodium nitrite, Na2EDTA, and sulfites, including but not limited to sulfur dioxide, sodium bisulfite, and potassium hydrogen sulfite.

[0028] A release-controlling polymer can be included in various embodiments of the solid dosage composition incorporating the compounds described in the present disclosure. In one embodiment, the release-controlling polymer can be used as a tablet coating. In other embodiments, including but not limited to bilayer tablets, the release-controlling polymer can be mixed with granules and other excipients before forming tablets by known processes, including but not limited to, compression in a tablet die. Suitable release-controlling polymers include, but are not limited to, hydrophilic polymers and hydrophobic polymers.

[0029] Suitable hydrophilic controlled-release polymers include cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose ethers, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, nitrocellulose, crosslinked starch, agar, casein, chitin, collagen, gelatin, maltose, mannitol, maltodextrin, pectin, pullulan, sorbitol, xylitol, polysaccharides, ammonium alginate, sodium alginate, calcium alginate, potassium alginate, propylene glycol alginate, sodium carmellose alginate, calcium carmellose, carrageenan, fucoidan, furcellaran, gum arabic, carrageenan gum, ghatti gum, guar gum, karaya gum, locust bean gum, and oat flour. These include, but are not limited to, curcuma gum, tragacanth gum, scleroglucan gum, xanthan gum, brambleweed, laminaran, acrylic polymers, acrylate polymers, carboxyvinyl polymers, copolymers of maleic anhydride and styrene, copolymers of maleic anhydride and ethylene, copolymers of propylene maleic anhydride or copolymers of isobutylene maleic anhydride, crosslinked polyvinyl alcohol and poly N-vinyl-2-pyrrolidone, diesters of polyglucan, polyacrylamide, polyacrylic acid, polyamide, polyethylene glycol, polyethylene oxide, poly(hydroxyalkyl methacrylate), polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polystyrene, polyvinylpyrrolidone, anionic and cationic hydrogels and combinations thereof.

[0030] The amino acid compositions disclosed herein can also include compositions that can be created as powders and concentrates that can be added to foodstuffs such as baked goods (e.g., cookies and brownies). The concentrates can be added to water or other ingestible liquids to create nutritional drinks. Nutritional supplements are generally contained in single- or multi-serving containers such as packets, boxes, cardboard boxes, wrappers, bottles, or cans. When the nutritional supplement is prepared in the form of a concentrate that can be added to and mixed with a beverage, a bottle or can is used to package the concentrate. The nutritional supplement can also contain water. The amino acid mixture can be balanced using a protein source.

[0031] Amino acid dosage As will be recognized by those skilled in the art, the dosage of the amino acid composition of the present invention can and will vary depending on the subject's weight, sex, age, and / or medical condition, the intensity of physical exercise performed by the subject, and the method of administration. Non-limiting examples of species include humans, companion animals, laboratory animals, zoo animals, and agricultural animals. The required dosage can be easily established through routine experimentation. A typical amino acid dosage for oral administration can be about 7 g per dose. In some embodiments, an amino acid dosage of about 3, 4, 5, 10, or 12 g of the amino acid composition can be administered. In other embodiments, an amino acid dosage of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 g of the amino acid composition can be administered. In a typical embodiment, the amino acid dosage can be about 7 g per dose. Multiple doses of the amino acid composition can be administered per day as needed to provide the desired stimulation of muscle protein synthesis. For example, 1, 2, 3, 4, or more doses of the amino acid composition can be administered per day. In certain embodiments, one dose of the amino acid composition may be administered per day. In other embodiments, two doses of the amino acid composition may be administered per day. In other embodiments, three doses of the amino acid composition may be administered per day.

[0032] The timing and administration period of the compositions of the present invention can be and will be variable.For example, when the composition is administered to stimulate muscle protein synthesis with or without exercise, the composition can be administered without exercise routine, before starting exercise routine, during exercise routine or after exercise routine.Alternatively, when the composition is administered to improve muscle protein synthesis in subjects who are prone to muscle problems, such as elderly human subjects, the composition can be administered periodically.

[0033] In an exemplary embodiment, if the subject is a human, three doses of 3 g of the amino acid composition may be administered per day. In another exemplary embodiment, two doses of 7 g of the amino acid composition may be administered per day. In another embodiment, a single dose of 7 g of the amino acid composition may be administered per day. Dosages may range from 1 gram to 15 grams per dose.

[0034] Administration The compositions of the present invention may be administered by intravenous, intramuscular, subcutaneous injection or parenteral route. In some embodiments, the compositions may be formulated for administration by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). Therefore, these compositions are preferably combined with a pharmaceutically acceptable vehicle such as saline, Ringer's solution, dextrose solution, etc.

[0035] The amino acid composition of the present invention may contain a pharmaceutical carrier (or excipient). Such a carrier is non-toxic and may be any solvent or solid material for encapsulation. The carrier can provide form or consistency or act as a diluent. Suitable pharmaceutical carriers include liquid carriers such as saline and other non-toxic salts at or near physiological concentrations, as well as solid carriers not suitable for human use, such as talc or sucrose, or animal feed. Carriers may also include stabilizers, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, buffers, and epidermal penetration enhancers. Carriers and excipients and formulations for parenteral and non-parenteral drug delivery are described in Remington's Pharmaceutical Sciences, 19th Ed., Mack Publishing (1995).

[0036] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal), the composition may be an aqueous or oily solution. Aqueous solutions may contain sterile diluents such as water, saline, pharmaceutically acceptable polyols such as glycerin, propylene glycol, or other synthetic solvents; antibacterial and / or antifungal agents such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and / or tonicity adjusters such as sodium chloride, dextrose, or polyalcohols such as mannitol or sorbitol. The pH of aqueous solutions may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oily solutions or suspensions may further contain sesame, peanut, olive, or mineral oil.

[0037] In certain embodiments, the amino acid composition of the present invention can be administered orally.Non-limiting examples of oral preparations that can be used to administer the amino acid composition of the present invention can be nutritional preparations, medical foods, medical drinks, or can be in the form of pharmaceutical preparations such as complete nutritional foods, partial nutritional foods, food additives such as powders for dissolution, tablets, pills, sachets or capsules, or tube feeding methods such as nasogastric, nasoduodenal, esophagostomy, gastrostomy or jejunostomy tube, or peripheral or total parenteral nutrition.In typical embodiments, the composition of the present invention can be administered orally as a nutritional supplement.

[0038] Compositions for oral administration generally contain inert excipients in addition to the amino acid component of the composition. Oral preparations may be enclosed in gelatin capsules or compressed into tablets. Typical excipients used in these preparations include pharmaceutically acceptable fillers / diluents such as microcrystalline cellulose, hydroxypropylmethylcellulose, starch, lactose, sucrose, glucose, mannitol, sorbitol, calcium hydrogen phosphate, or calcium carbonate; binders such as alginic acid, carboxymethylcellulose, microcrystalline cellulose, gelatin, tragacanth, or polyvinylpyrrolidone; disintegrating agents such as alginic acid, cellulose, starch, or polyvinylpyrrolidone; lubricants such as calcium stearate, magnesium stearate, talc, silica, or sodium stearyl fumarate; flow agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or citrus flavorings; coloring agents; and preservatives such as antioxidants (e.g., vitamin A, vitamin C, vitamin E, or retinyl palmitate), citric acid, or sodium citrate. Oral preparations may be administered as aqueous suspensions, elixirs, or syrups. In this case, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, if necessary, emulsifying and / or suspending agents, and diluents such as water, ethanol, glycerin, and combinations thereof.

[0039] The compositions according to the invention may be nutritionally complete, i.e., may contain vitamins, minerals, trace elements, and nitrogen, carbohydrates, and fat and / or fatty acid sources so that they may be used as the sole source of nutrients that provide essentially all of the required daily amounts of vitamins, minerals, carbohydrates, fat and / or fatty acids, protein, etc. [Example]

[0040] The effects of placebo (Compound A), essential amino acids (EAA, Compound B), and their combination were evaluated on gene expression profiles of normal human dermal fibroblasts (NHDF). The effects of these compounds were assessed using RT-qPCR technology. Extracted mRNA was analyzed using a PCR array ("mQPA-NHDF AGING-64") designed by Bioalternatives for the analysis of target genes (including three housekeeping genes).

[0041] Prior to this assay, a cytotoxicity assay was performed using a standard WST-8 reduction assay to determine the concentrations to be tested in this study.

[0042] Biological Model - Cell type: Normal human dermal fibroblasts (NHDF), Bioalternatives reference PF2, were used at passage 8. - Culture conditions: 37°C, 5% CO2 - Culture medium: Assay-optimized DMEM supplemented with 10% fetal calf serum (FCS) Assay medium: Assay-optimized DMEM supplemented with 1% FCS Test concentration: Compound A, 33%; Compound B, 10%.

[0043] Preliminary cytotoxicity assay - Cell type: NHDF in assay medium - Incubation time: 48 hours - Evaluation parameters: WST-8 reduction assay and microscopic morphological observation

[0044] After treatment, cells were incubated with WST-8 (a highly water-soluble tetrazolium salt) which is reduced to a water-soluble orange product (formazan) by succinate dehydrogenase (a mitochondrial enzyme). This conversion is proportional to the number of viable cells and their metabolic activity. The optical density (OD) of the extracts at 450 nm was recorded using a spectrophotometer (VERSAmax, Molecular Devices).

[0045] Cultures and treatments The cells were seeded into 24-well plates and incubated in culture medium for 24 hours and in assay medium for another 24 hours. The medium was then replaced with assay medium containing or not containing (control) test compounds or their combinations, and the cells were incubated for 48 hours. All experimental conditions were performed in triplicate. At the end of the incubation period, the cells were washed in phosphate-buffered saline (PBS) solution and immediately frozen at -80°C.

[0046] Differential expression analysis Marker expression was analyzed using RT-qPCR on total RNA extracted from cell monolayers from each experimental condition (replicates of the same experimental condition were pooled before RNA extraction). Transcript analysis was performed in duplicate using a PCR array ("mQPA-NHDF AGING-64"), adapted for a research-specific "screening" format (Marker qPCR array or "mQPA" designed by Bioalternatives) and targeting 64 genes selected for their importance in fibroblast physiology and skin structure.

[0047] RNA extraction and reverse transcription Total RNA was extracted from each sample using TriPure Isolation Reagent® according to the supplier's instructions. RNA quality was assessed using capillary electrophoresis (Bioanalyzer 2100, Agilent technologies). RNA quantity was assessed using a spectrophotometer (Synergy H1, BioTek Instruments). RNA quality control is shown in the appendix (Figures 1 and 2). Complementary DNA (cDNA) was synthesized by reverse transcription of total RNA in the presence of oligo(dT) and "Transcriptor Reverse Transcriptase" (Roche). The cDNA quantity was then adjusted before the PCR step.

[0048] quantitative PCR PCR (polymerase chain reaction) was carried out using the "LightCycler®" system (Roche Molecular Systems Inc.) according to the supplier's instructions.

[0049] The reaction mixture (final 10 μL) was prepared as follows: - 2.5 μL of cDNA, - primers (forward and reverse), - A reagent mix containing taq DNA polymerase, SYBR Green I and MgCl2 (Ozyme).

[0050] Data Management The raw data was analyzed using Microsoft Excel software.

[0051] Preliminary cytotoxicity assay The formula used in this report is: Standard error of the mean: sem = Sd / √n

[0052] The standard error of the mean (sem) is a measure of how far a sample mean is likely to be from the true population mean. The sem is calculated as the sd divided by the square root of the sample size. Percentage of survival: Viability (%) = (OD sample / OD control) x 100

[0053] quantitative PCR Fluorescence incorporation in the amplified DNA was measured continuously during the PCR cycles, resulting in a "fluorescence intensity" versus "PCR cycle" plot that allowed assessment of the relative expression (RE) value of each marker.

[0054] The value chosen for RE calculation is the "output point" (Ct) of the fluorescence curve. For the markers considered, the highest is the cycle number; the lowest is the mRNA amount. RE values ​​were calculated using the formula: (1 / 2 cycle number) x 10.

[0055] The PCR array used in this study contained three reference genes (RPS28, GAPDH, and ACTB). These housekeeping genes were used to normalize the data because their expression is constitutive and theoretically stable. Therefore, the level of expression of the target markers was compared to the average expression level of these three markers across all test conditions.

[0056] result Of the 64 genes tested, expression of the FAB3 gene was stimulated by compound B (EAA) to a greater extent (1212% greater than control) than any other gene. The combination of compounds A and B caused a similar increase in FAB3 gene expression (1309% greater than control), meaning that the combined effect could be entirely attributable to compound B.

Claims

1. A method for stimulating metabolic oxidation of long-chain fatty acids containing 16 or more carbons, comprising administering a composition having an amino acid mixture containing one or more of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine, wherein fatty acid oxidation is increased compared to a subject not administered the amino acid composition.

2. 1. A method for stimulating metabolic oxidation of long-chain fatty acids containing 16 or more carbons, comprising administering a composition having a mixture of the amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, tryptophan, and arginine, wherein fatty acid oxidation is increased compared to a subject not administered the amino acid composition.

3. 1. A method for stimulating metabolic oxidation of long chain fatty acids containing 16 or more carbons, comprising administering a composition having the following concentrations, expressed as % w / w: about 1-2% histidine, about 9-11% isoleucine, about 35-38% leucine, about 14-17% lysine, about 2-4% methionine, about 5-7% phenylalanine, about 8-9% threonine, about 9-11% valine, about 0.05-0.8% tryptophan, and about 8-11% arginine, wherein fatty acid oxidation is increased compared to a subject not administered the amino acid composition.

4. The method of claims 1 to 3, wherein metabolic oxidation of dietary long-chain fatty acids, whether in free form or as components of other forms of dietary fat, including triacylglycerides and phospholipids, is increased compared to a subject not administered the amino acid composition.

5. The method of claims 1 to 3, wherein the metabolic oxidation of endogenous long-chain fatty acids, whether in free form or as components of triacylglycerides, is increased compared to a subject not administered the amino acid composition.

6. The method of claims 1 to 3, wherein the metabolic oxidation of long-chain fatty acids during exercise is increased compared to a subject not administered the amino acid composition.

7. The method of claims 1 to 3, wherein the metabolic oxidation of long-chain fatty acids during physical exercise is increased compared to a subject not administered the amino acid composition.

8. The method of claims 1 to 3, wherein the metabolic oxidation of long-chain fatty acids in obese individuals is increased compared to subjects not administered the amino acid composition.

9. The method of claims 1 to 3, wherein the metabolic oxidation of long-chain fatty acids in an individual with diabetes or metabolic syndrome is increased compared to a subject not administered the amino acid composition.

10. The method of claims 1 to 3, wherein the metabolic oxidation of endogenous long-chain fatty acids, whether in free form or as components of triacylglycerides, is increased compared to a subject not administered the amino acid composition by inducing a similar increase of more than 1000 percent in FAB3 gene expression.

11. The following concentrations expressed as % w / w: approximately 1-2% histidine; Approximately 9-11% isoleucine; approximately 35-38% leucine; approximately 14-17% lysine; Approximately 2-4% methionine; Approximately 5-7% phenylalanine; Approximately 8-9% threonine; approximately 9-11% valine; about 0.05-0.8% tryptophan; and Approximately 8-11% arginine 1. A composition for stimulating metabolic oxidation of long-chain fatty acids containing 16 or more carbons, comprising an amino acid of:

Images