Synergistic nutritional composition for improving ATP efficiency

Abstract

The invention disclosed herein relates to a synergistic nutritional composition for enhancing cellular ATP production. In particular, the invention relates to a synergistic and efficient nutritional composition for promoting cellular ATP production, comprising a therapeutically effective exogenous mixture of stabilized oxaloacetate and a biotin-manganese complex, together with a pharmaceutically acceptable excipient. The stabilized oxaloacetate and the biotin and manganese complex or their salts are in a weight ratio of 1:0.01 to 1:0.2. Furthermore, the synergistic nutritional composition of the present invention is useful for treating ATP deficiency disorders, age-related metabolic disorders, neurodegenerative diseases, cardiovascular diseases, bone-related disorders, central nervous system disorders, cognitive disorders, and the like.

Description

[Technical field]

[0001] The present invention relates to a synergistic nutritional composition for improving intracellular ATP production or efficiency.The present invention particularly provides a synergistic composition comprising a therapeutic exogenous mixture of stabilized oxaloacetate (OAA) and biotin-manganese (biotin-Mn) complex to increase mitochondrial ATP turnover.The synergistic nutritional composition of the present invention is further useful for treating ATP deficiency disorders, age-related metabolic disorders, neurodegenerative diseases, cardiovascular diseases, bone-related disorders, central nervous system disorders, cognitive disorders, etc. [Background technology]

[0002] Carbohydrates, lipids, and proteins are the primary components of food and serve as fuel molecules for the human body. The digestion of these nutrients in the digestive tract and the absorption of the end products of digestion (transfer into the bloodstream) enable tissues and cells to convert the potential chemical energy of food into useful work.

[0003] "Energy metabolism" is the general process by which living cells obtain and use the energy they need to survive, grow, and reproduce. In particular, energy is released by breaking chemical bonds in nutrient molecules. Nutrients are oxidized to form high-energy compounds, especially ATP, which act as the main chemical energy carrier in all cells [Andrea T. Da Poian Nature Education 3(9):11, 2010].

[0004] The breakdown and synthesis of carbohydrates, proteins, and lipids are linked to the carbohydrate catabolic pathway. Simple sugars such as glucose, galactose, fructose, glycogen, and pentoses are catabolized during glycolysis. Protein-derived amino acids participate in glucose catabolism via pyruvate, acetyl-CoA, and intermediates of the TCA cycle.

[0005] The TCA cycle is central to cellular energy metabolism, i.e., almost all pathways of energy metabolism are connected to the TCA cycle, such as ETC (electron transport chain), gluconeogenesis, transamination, oxidative phosphorylation, chemiosmosis, deamination of amino acids, or lipid biosynthesis. Moreover, it is considered as the cellular source of amphoteric metabolic pathways. The TCA cycle is important for cells to regulate the concentration of metabolites in mitochondria, where anaplerotic and cataplerotic reactions are balanced to maintain homeostasis of cellular metabolism.

[0006] The TCA cycle represents the metabolic hub of the cell, since it consumes and produces metabolites that not only generate energy but also serve as a host of other processes. It is called a "cycle" because it starts and ends with a key substrate, namely oxaloacetate. The TCA cycle takes place in the mitochondria and converts the oxidation of pyruvate into a pool of chemical energy (ATP, NADH, FADH 2 , other electron carriers).

[0007] The total number of ATP molecules produced by glycolysis and the TCA cycle, combined with the number of ATP molecules produced in the ETC, amounts to 30 ATP molecules per glucose molecule [Exercise Physology JT Millard 2013].

[0008] It has been found that ATP levels in the blood slowly decline over time due to factors such as age, exercise or injury. There are two processes that provide for the intracellular replenishment of ATP: oxidative (aerobic) phosphorylation and anaerobic phosphorylation.

[0009] Furthermore, oxidative phosphorylation is the process by which electron transfer from energy precursors from the TCA cycle leads to the phosphorylation of ADP, thereby generating ATP, the basic unit of energy for metabolic processes.

[0010] Because of the recognized importance of maintaining or restoring normal mitochondrial or cellular function and enhancing cellular respiration, much energy (ATP) production is required.

[0011] The TCA cycle is the main decomposition pathway for the production of ATP. The TCA cycle operates at a low level unless new intermediates are formed. However, to generate more ATP energy carriers, fewer intermediates are detected to be regenerated.

[0012] ATP is the main source of energy for the majority of all cellular functions. Without adequate ATP, human cells cannot function properly, so it must be constantly regenerated. Therefore, there is a need to increase the available substrates for the TCA cycle, which subsequently leads to a high yield of ATP.

[0013] Oxaloacetate is central to energy utilization in all known organisms: it is a key intermediate and substrate of the TCA cycle, which releases energy stored in carbohydrates, fats and proteins.

[0014] "Oxaloacetate" is one of the essential dicarboxylic acid organic intermediate molecules of the metabolic TCA cycle, which is part of the body's energy production and waste management system. It is essential in infant development. Genetic deficiency of the enzyme that produces oxaloacetate causes severe neurological disorders and developmental delays.

[0015] Oxaloacetate (OAA) is a bioactive agent that increases cellular energy levels. In addition, oxaloacetate has been reported to reduce hyperglycemia in type II diabetes and extend lifespan. Several researchers have examined the medicinal uses of oxaloacetate in stroke, cancer, stroke, and traumatic brain injury.

[0016] Several other useful therapeutic applications of oxaloacetate have been reported in the art.

[0017] WO2011163319A2 relates to oxaloacetate compounds that activate ATP-activated protein kinase (AMPK) and the use of such compounds in the prevention or treatment of diseases such as diabetes, metabolic syndrome, obesity, cardiovascular disease, Alzheimer's disease and cancer. WO2011148014A1 relates to the use of oxaloacetate in the treatment of ischemia.

[0018] US10016385B2 discloses compositions of oxaloacetate in a pharma- ceutical effective amount for use in the treatment of cancer.

[0019] Furthermore, WO2018 / 057737A1 relates to methods of treating symptoms of premenstrual syndrome (PMS) and premenstrual disorder (PMDD) by administering a pharmaceutical composition comprising oxaloacetate, oxaloacetate salts, oxaloacetic acid and / or anhydrous enol oxaloacetate.

[0020] Cellular studies with oxaloacetate have also shown that by providing cells with extra energy, oxaloacetate can increase muscle endurance by up to 10% and significantly extend lifespan. Additionally, it helps stabilize blood sugar levels and maintains glucose homeostasis.

[0021] Additive oxaloacetate administration shows favorable metabolic changes such as enhanced brain mitochondrial biogenesis, activation of the insulin signaling pathway, reduced neuroinflammation, and activation of hippocampal neurogenesis [Hum Mol Genet.2014 Dec 15;23(24):6528-6541].

[0022] It consists of oxaloacetate and is TM A nutritional supplement called oxaloacetate is used to improve brain health and cognitive function. Oxaloacetate has been found to help protect brain neurons and brain mitochondrial DNA, as well as provide strong antioxidant protection.

[0023] Much effort and experimentation has been undertaken in the past to establish a stable formulation of oxaloacetate suitable for oral administration.

[0024] Kiyohiko Yoshikawa [Tohoku J. Exp.Med., 1968, 96, p127-141] discloses the antidiabetic effect of sodium oxaloacetate. Krebs [Biochem. 1942, 36, p303-05] reports that polyvalent cations, such as Al, Cu, FeII, FeIII, catalyze the ketone decomposition of oxaloacetate. The cations do not stabilize α-ketone-dicarboxylic acids.

[0025] It was observed that temperature, pH, metal ions (Cu, Fe) and interactions with amines have a strong effect on the decomposition rate of oxaloacetate.

[0026] EP3056199A1 provides non-biodegradable oxaloacetate compositions comprising oxaloacetic acid in combination with one or more components selected from the group of hygroscopic pH adjusters, hygroscopic taste adjusters, hygroscopic binding agents, and hygroscopic release agents.

[0027] Furthermore, US9050306B2 discloses a method for producing anhydrous enol oxaloacetate and isolating the anhydrous enol oxaloacetate from water with less than 2% moisture content by hermetically encapsulating it to prevent the degradation of oxaloacetate. In particular, oxaloacetate not only provides the building blocks required for the TCA cycle to generate ATP energy, but also actually promotes mitochondrial biogenesis, i.e., aids in the formation of new mitochondria in cells, thus increasing energy production in the body. The important benefits of oxaloacetate can be summarized as anti-aging, increased longevity (life extension), as well as improved brain health, blood sugar regulation, and energy production.

[0028] In view of the above, oxaloacetate is a key component in generating ATP, which should be substantially replenished for better functioning of the TCA cycle and the electron transport chain.

[0029] Because oxaloacetate is excreted during cellular metabolism, additional oxaloacetate must be provided exogenously to regulate cellular function.

[0030] Interestingly, "oxaloacetate" is formed by the carboxylation of pyruvate in a reaction catalyzed by pyruvate carboxylase, a biotin-dependent enzyme that plays a key role in gluconeogenesis. It is only active in the presence of acetyl-CoA, which means there is a need for more oxaloacetate. When the energy charge is high, oxaloacetate is converted to glucose. When the energy charge is low, oxaloacetate supplements the TCA cycle [Berg JM, et al. 2002, Biochemistry, 5th Edition].

[0031] Furthermore, oxaloacetate is the main substrate for gluconeogenesis, an important end product derived from amino acid carbon skeletons when metabolism is required to maintain the supply of glucose to the central nervous system and red blood cells in the fasting state.

[0032] To provide glucose for vital functions such as red blood cell and central nervous system metabolism during the fasting period (approximately 8 hours or more after the absorption of food in humans), the body needs a way to synthesize glucose from precursors such as pyruvate and amino acids. Gluconeogenesis occurs in the liver and kidneys.

[0033] The key point here is that when citric acid cycle intermediates are withdrawn for biosynthesis, they must be replenished. Large amounts of oxaloacetate are converted to amino acids for protein synthesis, which subsequently increases the energy requirements of the cell. Since acetyl-CoA cannot enter the cycle without reacting with oxaloacetate, the citric acid cycle proceeds at a low level unless new oxaloacetate is formed. Even if oxaloacetate is recycled, a minimum level is maintained and the cycle functions.

[0034] In the metabolic pathway, oxaloacetate is formed from pyruvate by a carboxylation reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase. The synthesis of oxaloacetate by carboxylation of pyruvate is an anaplerotic reaction, which serves to replenish TCA cycle intermediates that have been withdrawn for biosynthesis.

[0035] When oxaloacetate is removed from the TCA cycle for glucose synthesis, it must be replaced. If there is not enough oxaloacetate to form citrate, the rate of metabolism of acetyl-CoA, and therefore the rate of formation of ATP, slows.

[0036] Normally, excess pyruvate is diverted to gluconeogenesis via the conversion of pyruvate to oxaloacetate, but in the event of enzyme deficiency, the excess pyruvate is converted to lactate instead.

[0037] Pyruvate carboxylase (PC) is a biotin-linked mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate when abundant acetyl-CoA is available, thus recruiting Krebs cycle intermediates to the mitochondrial matrix.

[0038] Pyruvate carboxylase (PC) is a biotin-dependent mitochondrial enzyme that catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, recruiting Krebs cycle intermediates to the mitochondrial matrix.

[0039] Furthermore, the reaction from pyruvate to oxaloacetate proceeds by the removal of a proton from pyruvate by an active site residue to generate an enolate intermediate, which is then coupled to a CO2 molecule temporarily released from pyruvate carboxylase bound to a biotin molecule. 2and releasing oxaloacetate. The biotin molecule is protonated by the active site residues and released from the active site of the pyruvate carboxylase domain to be recarboxylated.

[0040] Biotin is a biochemically essential compound that functions as a carrier of carbon dioxide in the pyruvate carboxylase reaction.

[0041] Biotin, also known as vitamin H or B7, is one of the essential vitamins for maintaining and enhancing bodily functions, including metabolism, and helping regulate good body weight. Biotin is essential for healthy bones, skin and nails in humans, and for the proper functioning of the liver and nervous system. Biotin improves ATP synthesis in pancreatic islets, leading to increased glucose-induced insulin secretion [Biochem Biophys Res Commun. 2004 Feb 13; 314(3): 824-9].

[0042] WO2014016003A1 relates to the use of biotin for treating multiple sclerosis, at effective doses of 100 mg and 300 mg.

[0043] JPH0995448A discloses a composition of oligosaccharides and biotin for preventing obesity, diabetes, dermatitis, conjunctivitis, muscle pain, and fatigue conditions.

[0044] Biotin, also known as Vitamin B7, is an essential element in healthy metabolism and the production of important enzymes used by the body to metabolize carbohydrates, fats and amino acids.

[0045] The primary biological function of biotin has been reported to be to act as a covalent coenzyme for the biological activity of mammalian biotin-dependent carboxylases. These biotin-dependent carboxylases have important roles in essential biological processes such as fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Humans cannot synthesize biotin. However, biotin is available through absorption of protein-rich foods via intestinal bacteria.

[0046] Considering the need to supplement sufficient oxaloacetate for energy production, the present inventors have conducted rigorous research and testing to develop a synergistic composition that not only increases mitochondrial oxaloacetate production, but also improves the cytoplasmic concentration of oxaloacetate without artificial or synthetic drug intervention.The present inventors have successfully demonstrated increased intracellular ATP turnover by consuming the synergistic formulation of the present invention.Furthermore, the present inventors have developed an advanced nutritional therapy to improve the synthesis of ATP as energy supplementation. Summary of the Invention [Problem to be solved by the invention]

[0047] The primary objective of the present invention is to provide a nutrient-based energy source for cellular maintenance.

[0048] Another object of the present invention is to provide a cost-effective nutritional composition consisting of organic, non-toxic, bioenergetic compounds that effectively improves the metabolic pathway of cellular respiration.

[0049] A further object of the present invention is to provide a stable nutritional composition for improving the level of the NAD pool.

[0050] Another object of the present invention is to provide a synergistic nutritional composition of bioactive compounds for enhancing intracellular ATP production.

[0051] Yet another object of the present invention is to provide a nutritional composition containing active elements that synergistically enhance cellular energy (ATP) production by establishing anaplerotic reactions.

[0052] It is a further object of the present invention to provide novel stable nutritional compositions of vitamin-mineral complexes of dicarboxylic acids for improving cellular energy production.

[0053] Another object of the present invention is to provide a novel and potent nutritional composition of vitamin-based TCA cycle substrates for treating age-related metabolic disorders or dysfunctions through site-specific action without side effects. [Means for solving the problem]

[0054] To achieve the above objectives, the inventors of the present invention have conducted extensive experiments to establish synergistic combinations of bioactive ingredients, nutritional supplements, dietary supplements, micronutrients, natural substances, metabolic intermediates, bioactive substances, biochemical substances, or TCA intermediates that improve intracellular energy efficiency in a subject in need thereof.

[0055] In a first aspect, the present invention relates to a stable, potent, therapeutically effective nutritional composition comprising an exogenous mixture of TCA intermediates and a vitamin-mineral complex, which significantly improves metabolic energy efficiency.

[0056] In a preferred embodiment, the present invention provides a stable nutritional composition that enhances cellular respiratory output by increasing therapeutic amounts of substrates or intermediates of the coenzyme-mediated TCA cycle.

[0057] In another aspect, the present invention provides a synergistic combination of an exogenous TCA cycle intermediate with a vitamin-mineral complex to improve cellular function, where the vitamin-mineral complex is a biotin-manganese (biotin-Mn) complex and the TCA cycle intermediate is oxaloacetate (OAA).

[0058] In certain embodiments, administration of an effective amount of exogenous biotin-Mn complex increases the production of oxaloacetate inside mitochondria by carboxylation of pyruvate, and the exogenous oxaloacetate improves the concentration of depleted oxaloacetate in the cytoplasm.

[0059] In another aspect, the present invention results in the compositions of the present invention not only exponentially increase ATP turnover in cells, but also enhance the metabolic process of gluconeogenesis from non-carbohydrate precursors.

[0060] In yet another particular embodiment, the present invention provides a combination therapy in which oxaloacetate or a pharma- ceutical acceptable salt thereof is administered together with a biotin-Mn complex, the combination providing a therapeutic effect significantly greater than the effect of each active ingredient administered alone.

[0061] In another aspect, the present invention results in the compositions of the present invention increasing the intracellular density of oxaloacetate through a synergistic anaplerotic effect.

[0062] In another aspect, the present invention provides nutritional compositions comprising a specific or therapeutically effective amount of oxaloacetate, wherein the oxaloacetate acts synergistically with a biotin-manganese complex to increase the capacity of multiple mitochondria in a cell to synthesize adenosine triphosphate (ATP).

[0063] In yet another aspect, the present invention relates to a synergistic nutritional composition comprising a combination of 1-500 mg of oxaloacetate salt (equivalent to oxaloacetic acid), 10-10000 mcg of biotin, and 1-100 mg of cofactor Mn in the form of an amino acid chelate, together with a pharma- ceutically acceptable excipient / carrier.

[0064] In another embodiment, the synergistic combination of oxaloacetate and biotin-manganese complex promotes the oxidation of NADH to NAD+, resulting in increased production of ATP.

[0065] In further aspects, the nutritional compositions of the present invention are useful for treating age-related metabolic disorders, neurodegenerative disorders, cardiovascular diseases, bone-related disorders, central nervous system disorders, insulin restriction, obesity, premenstrual syndrome, cognitive disorders, cancer, insulin resistance, diabetes, etc.

[0066] Abbreviation OAA: Oxaloacetate ATP: Adenosine triphosphate ADP: adenosine diphosphate AMP: adenosine monophosphate NAD+: Nicotinamide adenine dinucleotide (oxidized form) NADH: Nicotinamide adenine dinucleotide (reduced form) TCA: Tricarboxylic acid cycle Mn-biotin: Manganese-biotin PC: pyruvate carboxylase NADPH + : Nicotinamide adenine dinucleotide phosphate CNS: central nervous system [Brief description of the drawings]

[0067] [Figure 1] 1 shows the pyruvate-oxaloacetate metabolic pathway. [Diagram 2] 1 shows transport of oxaloacetate via the shuttle pathway. [Diagram 3] Effect of test substances on cellular ATP concentration (mM ATP concentration) in different test groups {G1: normal control, G2: positive control (H2O2, 20 μM), G3: standard (resveratrol) 100 μg / ml, G4: stabilized oxaloacetate (500 μg / ml), G5: D-biotin (500 μg / ml), G6: manganese sulfate monohydrate (500 μg / ml), G7: oxaloacetate + biotin + manganese sulfate monohydrate (500 μg / ml) [1:0.05:0.01]}. [Figure 4]The ATP increase rate of the test groups compared to the control is shown {G1: normal control, G2: positive control (H2O2, 20 μM), G3: standard (resveratrol) 100 μg / ml, G4: stabilized oxaloacetate (500 μg / ml), G5: D-biotin (500 μg / ml), G6: manganese sulfate monohydrate (500 μg / ml), G7: oxaloacetate + biotin + manganese sulfate monohydrate (500 μg / ml) [1:0.05:0.01]}. [Diagram 5] The ATP increase rate of the test substrates compared to the controls is shown {G1: normal control, G2: positive control (H2O2, 20 μM), G3: standard (resveratrol) 100 μg / ml, G4: stabilized oxaloacetate (500 μg / ml), G5: D-biotin (500 μg / ml), G6: manganese sulfate monohydrate (500 μg / ml), G7: oxaloacetate + biotin + manganese sulfate monohydrate (500 μg / ml) [1:0.05:0.01]}. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The present invention has been described in detail with respect to several preferred and optional embodiments so that various aspects of the invention may be more fully appreciated and understood, although those skilled in the art will appreciate the extent to which such embodiments may be generalized in practice.

[0069] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope.

[0070] Unless otherwise defined, all technical and scientific terms or terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain.

[0071] In describing and claiming the embodiments of the present invention, the following terminology is used in accordance with definitions known in the art.

[0072] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates to be limited to the singular only. Also, the term "composition" does not limit the scope of the invention but may include multiple compositions to establish the best mode of the invention.

[0073] As used herein, the term "pharmaceutical / nutritional acceptable salt" refers to a salt that, in medical judgment, is suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, etc., and which represents a reasonable balance of benefits and risks. In particular, the term "pharmaceutical acceptable salt" refers to relatively non-toxic inorganic and organic acid addition salts, alkali or alkaline earth metal salts, as well as solvates, co-crystals, polymorphs, isomers, enantiomers, and the like, of a compound.

[0074] The term "tricarboxylic acid cycle" (TCA cycle) as used herein is interchangeable with the Krebs cycle, the citric acid cycle (CAC), and metabolic pathways without distinction.

[0075] In one preferred embodiment, the present invention provides novel, stable, potent, therapeutically active nutritional compositions comprising vitamin-mineral complexes mediated TCA intermediates for enhancing cellular energy output.

[0076] Additionally, the nutritional compositions of the present invention can increase the supply of exogenous substrates and vitamins available for the TCA cycle, resulting in significant energy output, where the supply is TCA intermediates, i.e., dicarboxylates and vitamin-mineral complexes, and the output is energy in the form of adenosine triphosphate (ATP).

[0077] Furthermore, the present invention provides a non-toxic and safe nutritional composition of bioenergetic compounds to restore the quality of aged or damaged cells that survive anaplerotic reactions.

[0078] In another embodiment, the present invention provides bioenergetic intervention involving metabolic and enzymatic processes that lead to the production of energy in the form of adenosine triphosphate (ATP) molecules.

[0079] In another embodiment, the present invention relates to a nutritional composition comprising a synergistic combination of a TCA cycle substrate and a vitamin-mineral complex in specific amounts, together with a pharma- ceutical acceptable salt or carrier.

[0080] In another embodiment, the present invention relates to a nutritional composition comprising a combination of bioenergetic substances, the bioenergetic substances being a TCA cycle substrate and a vitamin-mineral complex, the TCA cycle substrate being oxaloacetate (OAA) and the vitamin-mineral complex being a biotin-manganese complex, which synergistically improves intracellular energy (ATP) pools via anaplerotic reactions.

[0081] In accordance with the present invention, administration of exogenous oxaloacetate provides a substrate for regulating the citric acid cycle. The ability to regulate the cycle keeps cells in a stable state and avoids the loss of metabolic energy.

[0082] "Oxaloacetic acid" has the chemical formula C 4 H 4 O 5 This acid is also known as "2-oxosuccinic acid", "ketosuccinic acid", "oxobutanedioic acid", or "3-carboxy-3-oxopropanoic acid". Oxaloacetic acid, also known as keto-oxaloacetate or 2-oxobutanoic acid, belongs to a class of organic compounds known as short-chain keto acids and derivatives and is represented by formula I: [ka]

[0083] Oxaloacetic acid in the form of its conjugate base is called oxaloacetate and is represented by formula II above. Oxaloacetate is a metabolic intermediate in many processes occurring in the animal body. Oxaloacetic acid can be obtained in low concentrations from oranges, apples, bananas, peas, potatoes, and spinach. Oxaloacetic acid is highly water-soluble, and oxaloacetate is the water-soluble ion of oxaloacetic acid. Oxaloacetate improves metabolic pathways in a variety of ways.

[0084] According to the present invention, a therapeutically effective amount of oxaloacetate present in the composition improves depleted oxaloacetate concentrations in cells, thereby increasing cellular NAD+ levels.

[0085] The conversion of oxaloacetate to malate is an energetically favorable reaction in cells that drives the conversion of NADH to NAD+. In mitochondria, the electron transport chain is the main link for the oxidation of NADH to NAD+ and participates in this reaction in ATP synthesis.

[0086] In another embodiment, the present invention provides the regulation of intracellular NAD pools, which are important for mitochondrial function, cell viability, and ATP levels, by additional or exogenous oxaloacetate.NAD functions through the activation of transcriptional cascades that cause increased expression of mitochondrial proteins involved in ATP production.In particular, the concentration of OAA, which is important, regulates the concentration of TCA cycle metabolites in mitochondria through synergistic anaplerotic reactions.

[0087] In another embodiment, the oxaloacetate used in the composition is in a stabilized form. Here, the oxaloacetate is in the form of the corresponding acid, alkyl-protected oxaloacetate, thermally stable oxaloacetate, or their metal ion salts. The metal ion is selected from Na, K, Li. To further improve the stability of OAA, a small amount of antioxidant or stabilizer is optionally added to the composition. In a preferred embodiment, the antioxidant is selected from the group consisting of ascorbic acid, n-propyl gallate, α-tocopherol, etc.

[0088] In yet another embodiment, the present invention provides a synergistic nutritional composition containing a therapeutically effective amount of oxaloacetate or a salt thereof equivalent to 1-500 mg of oxaloacetate in the total composition.

[0089] In another preferred embodiment, the present invention provides a synergistic composition for increasing ATP through mitochondrial anaplerotic reaction, wherein the mitochondrial inducer is a vitamin-mineral complex. In one embodiment, the vitamin-mineral complex is a biotin-manganese complex.

[0090] Biotin, also known as vitamin H or vitamin B7, belongs to the B vitamin family. Biotin is a water-soluble vitamin that helps metabolize substances including fatty acids and glucose, converting food into caloric energy. Biotin is stable at room temperature. Foods that contain biotin are peanuts, yeast, egg yolks, raspberries, bananas, pork, avocados, broccoli, sweet potatoes, cauliflower, spinach, milk, bananas, nuts, grains, and liver.

[0091] Biotin is important in several essential metabolic reactions in humans, such as catalyzing the synthesis of fatty acids, the metabolism of the amino acid leucine, and gluconeogenesis. Biotin is important in cell growth and plays a role in the Krebs cycle.

[0092] Biotin has the chemical formula C 10 H16 N 2 O 3 Biotin is a five-membered organic compound having a ureido (tetrahydroimidizalone) ring fused to a tetrahydrothiophene ring, the tetrahydrothiophene ring containing four carbon atoms and one sulfur atom. [ka]

[0093] According to the present invention, administration of exogenous biotin-manganese complex enhances OAA production in mitochondria through pyruvate carboxylase-catalyzed carboxylation of pyruvate.

[0094] In another embodiment, the present invention provides an ATP enhancing composition comprising biotin, wherein the biotin is CO 2 It acts as a carrier or transporter of CO2. It is a major cofactor involved in carbon dioxide metabolism.

[0095] In particular, the biotin moiety acts as a swinging arm to transport carbon dioxide to the catalytic site of pyruvate carboxylase, ultimately increasing the expression of pyruvate carboxylase for the production of oxaloacetate in the mitochondria. The reaction catalyzed by the active pyruvate carboxylase enzyme provides a supply of intermediates to the citric acid cycle when they are removed for different biosynthetic purposes (basically providing oxaloacetate). In short, pyruvate carboxylase plays a role in an anaplerotic reaction. By producing oxaloacetate, this enzyme helps to replenish important TCA intermediates and also opens the pathway for gluconeogenesis from pyruvate and other compounds that are converted to pyruvate, such as non-carbohydrate precursors.

[0096] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising a therapeutically effective amount of biotin, wherein the amount is 10-10,000 mcg in the total composition.

[0097] In another embodiment, the present invention provides a nutritional composition comprising a manganese salt chelate, wherein an effective amount of the manganese salt chelate acts as a catalyst to increase the enzymatic activity or kinetics of pyruvate carboxylase, thereby increasing the rate of anaplerotic reactions.

[0098] In the present invention, a manganese salt chelating cofactor is a non-protein chemical compound, metal ion catalyst, or substance that enhances the rate of a chemical reaction for the production of oxaloacetate. More specifically, it is an ion that provides benefits that aid in the biochemical conversion of pyruvate to oxaloacetate.

[0099] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising a therapeutically effective amount of a manganese salt, the amount being 0.5-100 mg of the total composition, the manganese salt being used as a chelating agent.

[0100] Additionally, manganese exists in many different forms, including, but not limited to, amino acid chelates such as manganese bisglycinate chelate, manganese glycinate chelate, and manganese aspartate, and non-amino acid chelate forms such as manganese gluconate, manganese picolinate, manganese sulfate, manganese citrate, and manganese chloride.

[0101] In another embodiment, the present invention provides a synergistic anaplerotic reaction by administering exogenous biotin-manganese complex and oxaloacetate, where administration of exogenous biotin-manganese complex enhances mitochondrial oxaloacetate production and cytoplasmic oxoacetate levels are balanced by an effective amount of exogenous oxaloacetate.

[0102] According to the present invention, administration of an effective amount of exogenous biotin-manganese complex enhances the production of oxaloacetate in mitochondria via carboxylation of pyruvate, and the exogenous oxaloacetate improves the depleted oxaloacetate concentration in the cytoplasm.

[0103] In particular, oxaloacetate formed in the cytosol is transported back to the mitochondria, where the inner mitochondrial membrane is impermeable to oxaloacetate, requiring a bypass reaction called the shuttle or cleavage pathway that substantially improves NAD levels.

[0104] First, oxaloacetate is reduced to malate by NADH in the cytoplasm. OAA + NADH + H ⇔ Malic acid + NAD +

[0105] Next, malic acid is converted to NADP + It is oxidatively decarboxylated by bound malic enzyme to form pyruvate. Malic acid + NADP + ⇔ Pyruvate + CO 2 +NADPH

[0106] Pyruvate formed in this reaction readily enters mitochondria and is carboxylated to oxaloacetate by pyruvate carboxylase, catalyzed by the biotin-Mn complex (Figure 1). Pyruvate + CO 2 +ATP+H 2 O(biotin + Mn)⇔OAA + ADP + Pi + 2H +

[0107] Here, Pi is inorganic phosphate, i.e., the phosphate released when ATP is hydrolyzed to ADP.

[0108] Combining these three reactions, we get: NADPH + +NADH+ATP+H 2 O⇔NADPH+NAD ++ADP+Pi+H+

[0109] Similarly, exogenous OAA enters the Krebs cycle via the citrate shuttle and provides electrons for ATP formation in the ETC (Figure 2).

[0110] In view of the above, the overall reaction is an anaplerotic reaction, where the total amount of oxaloacetate is increased via mitochondrial and cytoplasmic repletion pathways, resulting in increased levels of NAD+ and the generation of ATP molecules via the ETC.

[0111] In yet another embodiment, the present invention relates to a synergistic nutritional composition comprising a combination of 1-500 mg of oxaloacetate salt (equivalent to oxaloacetic acid) in the total composition, 10-10,000 mcg of biotin in the total composition, and 1-100 mg of cofactor Mn in the form of a chelate in the total composition, together with a pharma- ceutically acceptable excipient / carrier.

[0112] In some preferred embodiments, the present invention provides a synergistic nutritional composition for promoting intracellular ATP production in a subject in need thereof, comprising a therapeutically active exogenous combination of stabilized oxaloacetate and a biotin-manganese complex together with a pharma- ceutically acceptable excipient.

[0113] In another preferred embodiment, the present invention provides a synergistic nutritional composition comprising a therapeutically active exogenous combination of stabilized oxaloacetate and biotin-manganese complex, together with a pharma- ceutically acceptable excipient, wherein the stabilized oxaloacetate and biotin-manganese complex, or salts thereof, are in a weight ratio of 1:0.01 to 1:0.2.

[0114] In another preferred embodiment, the present invention provides a method for increasing cellular ATP efficiency in a subject in need thereof, comprising orally administering a therapeutically effective amount of a nutritional composition comprising an exogenous synergistic mixture of stabilized oxaloacetate and biotin-manganese complex or salts thereof, together with a pharma- ceutically acceptable excipient, wherein the stabilized oxaloacetate and biotin-manganese complex or salts thereof are in a weight ratio of 1:0.01 to 1:0.2.

[0115] In another preferred embodiment, the present invention provides a synergistic nutritional composition comprising stabilized oxaloacetate and biotin-manganese complex, wherein the stabilized oxaloacetate, biotin and manganese are in a weight ratio of 1:0.01:0.01 to 1:0.5:0.05.

[0116] In another embodiment, the present invention provides a potent synergistic nutritional composition comprising 1-500 mg of stabilized oxaloacetate in the total composition.

[0117] In another embodiment, the stabilized oxaloacetate comprises a homogenous premix of crystalline organic oxaloacetate and standardized ascorbic acid or vitamin C as antioxidants, the weight ratio of crystalline organic oxaloacetate to standardized ascorbic acid being 1:1 to 1:2. In a preferred embodiment, the weight ratio of oxaloacetate to standardized ascorbic acid is 1:1.2 to 1:1.8.

[0118] The term "standardized ascorbic acid" refers to a supplement that provides a therapeutically effective concentration of ascorbic acid. Further, standardized ascorbic acid contains 1-250 mg of ascorbic acid.

[0119] In another embodiment, the present invention provides a potent synergistic nutritional composition comprising a synergistic combination of stabilized oxaloacetate and biotin-manganese complex, where biotin is a water soluble vitamin B complex, at 1-10 mg in the total composition.

[0120] In yet another embodiment, the manganese (Mn) chelate is a manganese sulfate monohydrate salt.

[0121] In another embodiment, the manganese salt is a crystalline form of manganese(II) sulfate monohydrate, containing 10% manganese atoms and present at 1-10 mg.

[0122] In a further embodiment, the present invention provides a synergistic nutritional composition of a biotin-manganese complex, wherein the biotin-manganese complex comprises a homogenous premix of a crystalline form of the D-isomer of biotin and crystalline manganese(II) sulfate monohydrate in a weight ratio of 1:0.1 to 1:1.

[0123] In yet another embodiment, the present invention provides a method for improving cellular ATP efficiency in a subject in need thereof, the method comprising orally administering a therapeutically effective amount of a nutritional composition comprising an exogenous synergistic mixture of stabilized oxaloacetate and biotin-manganese complex or a salt thereof together with a pharma- ceutically acceptable excipient, wherein the stabilized oxaloacetate, biotin, and the manganese salt thereof are in a weight ratio of 1:0.01:0.01 to 1:0.5:0.05.

[0124] In yet another embodiment, the present invention provides a method of increasing cellular ATP efficiency in a subject in need thereof, the method comprising orally administering a therapeutically effective amount of a nutritional composition comprising an exogenous synergistic mixture of stabilized oxaloacetate, a crystalline form of the D-isomer of biotin, and crystalline manganese(II) sulfate monohydrate, together with a pharmaceutically acceptable excipient, wherein the stabilized oxaloacetate, the crystalline form of the D-isomer of biotin, and the crystalline manganese(II) sulfate monohydrate are in a weight ratio of 1:0.01:0.01 to 1:0.5:0.05.

[0125] In a further embodiment, the present invention provides a synergistic nutritional composition comprising 50-96% stabilized oxaloacetate by weight of the total composition.

[0126] In yet another embodiment, the stabilized oxaloacetate comprises a homogenous premix of crystalline organic oxaloacetic acid and standardized ascorbic acid, with the crystalline organic oxaloacetic acid being 25-50% by weight and the standardized ascorbic acid being 20-60% by weight of the total homogenous premix.

[0127] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising a crystalline form of the D-isomer of biotin, wherein the D-isomer of biotin is in an amount of 0.1-10% by weight of the total composition.

[0128] In a further embodiment, the present invention provides a synergistic nutritional composition comprising 0.1-5% crystalline manganese(II) sulfate monohydrate by weight of the total composition.

[0129] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising stabilized oxaloacetate, a crystalline form of the D-isomer of biotin, and crystalline manganese(II) sulfate monohydrate in a weight ratio of 1:0.01:0.01 to 1:0.5:0.05.

[0130] In one embodiment, the present invention provides a nutritional composition comprising a synergistic combination of crystalline oxaloacetate, standardized ascorbic acid, D-biotin, and manganese sulfate in a weight ratio of 1:1:0.1:0.01 to 1:2:0.5:0.1, where the D-isomer of biotin is also referred to as D-biotin.

[0131] In another embodiment, the present invention provides a method of improving cellular ATP efficiency by administering a nutritional composition comprising a synergistic combination of crystalline organooxaloacetic acid, standardized ascorbic acid, a crystalline form of the D-isomer of biotin, and crystalline manganese(II) sulfate monohydrate, together with a pharma- ceutical acceptable excipient, wherein the crystalline organooxaloacetic acid, standardized ascorbic acid, a crystalline form of the D-isomer of biotin, and crystalline manganese(II) sulfate monohydrate are in a weight ratio of 1:1:0.1:0.01 to 1:2:0.5:0.1.

[0132] In another embodiment, the present invention provides a method for increasing intracellular ATP levels in a subject in need thereof, the method comprising orally administering a therapeutically effective amount of a nutritional composition comprising a synergistic combination of crystalline biotin-manganese and stabilized oxaloacetate together with a pharma- ceutically acceptable excipient, wherein the stabilized oxaloacetate is 50-96% by weight, the crystalline form of the D-isomer of biotin is 0.1-20% by weight, and the crystalline manganese(II) sulfate monohydrate is 0.1-10% by weight of the total composition.

[0133] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising stabilized oxaloacetate and biotin-manganese complex, wherein the stabilized oxaloacetate is 80-96% by weight and the biotin-manganese complex is 0.5-10% by weight of the total composition.

[0134] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising a biotin-manganese complex, wherein the biotin-manganese complex contains a homogenous premix of a crystalline form of the D-isomer of biotin and crystalline manganese(II) sulfate monohydrate in a weight ratio of 1:0.1 to 1:1.

[0135] In the present invention, the term "composite therapy" or "combination therapy" refers to a feature of the composition of the present invention or a feature of the present invention, in which two active ingredients function simultaneously in a systematic pathway without deviation or overlap of mechanisms, resulting in improved brain function.

[0136] In another embodiment, the present invention provides an additional bioenhancer to enhance the bioavailability of the compositions of the present invention by facilitating the absorption of the active ingredient in the body.

[0137] As used herein, the term "therapeutically effective amount" is intended to mean an amount of an active compound of the invention effective to increase intracellular ATP levels.

[0138] In particular, ATP-improving therapy, i.e., improving energy production, is useful for subjects suffering from mitochondrial deficiency, disorder, or disease.Mitochondrial disease is caused by mitochondrial dysfunction.When mitochondria do not function, the energy produced in cells gradually decreases, and cell injury or even cell death may result.When this process is repeated throughout the body, the lifespan of patients suffering from such dysfunction is severely impaired, and it is highly likely to cause disorders related to muscle, brain, liver, and heart.

[0139] It is noteworthy that the enhancement of ATP through metabolic pathways improves the pathological conditions of diseases in which a decrease in ATP is associated with pathological conditions such as cellular senescence, cell injury, loss of cellular function, apoptosis, necrosis, cell death, etc. ATP enhances the immune system, thereby improving the host's self-defense mechanisms to eradicate virus-infected cells and restore normal immune function.

[0140] According to the present invention, mitochondrial disorders are most frequently manifested in highly energy demanding organs such as the brain, heart, liver, skeletal muscles, kidneys, and endocrine and respiratory systems. Symptoms of mitochondrial disease include loss of muscle control, muscle pain, seizures, visual / hearing disorders, liver and kidney disease, eye disorders, cardiac disease, liver disease, gastrointestinal disorders, swallowing disorders, etc.

[0141] In another embodiment, the synergistic compositions of the present invention are non-toxic, cost-effective, and rich in nutrients or biomolecules that protect against mitochondrial damage caused by harmful toxins without causing side effects.

[0142] Additionally, the inventive synergistic combination of oxaloacetate and biotin-manganese complex is used to treat metabolic disorders, disorders of mitochondrial dysfunction, mitochondrial diseases, and to stimulate mitochondrial energy production.

[0143] The synergistic combination of bioactive compounds of the present invention are used in the treatment of cancer and the following hypoxia, ischemia, stroke, myocardial infarction, acute angina, acute kidney injury, coronary artery occlusion and atrial fibrillation, or to avoid or offset reperfusion injury.

[0144] In some embodiments, the present invention provides a method for improving cellular ATP levels in subjects suffering from ATP deficiency disorders, age-related metabolic disorders, mitochondrial dysfunction, metabolic disorders, neurodegenerative diseases, cardiovascular diseases, bone-related disorders, or central nervous system diseases.The age-related decline in ATP levels reduces the ability to induce apoptosis and promotes necroinflammation, which may lead to some age-dependent disorders.

[0145] In another embodiment, the present invention provides a stable nutritional composition that improves or enhances "energy metabolism" useful for treating ATP deficiency disorders such as metabolic disorders or malformations, including, but not limited to, insulin restriction, diabetes, obesity, female hormone imbalance, premenstrual syndrome (PMS), premenstrual disorder (PMDD), dementia, endocrine disorders, traumatic brain injury, stroke, subarachnoid hemorrhage (SAH), ischemia, Alzheimer's and Parkinson's diseases, traumatic spinal cord or traumatic brain injury (TBI), brain inflammation, insulin resistance, brain cancer, brain injury, heart and kidney disease, neurodegenerative diseases such as caloric restriction inflammation, cardiovascular disease, bone disease, mental or central nervous system disorders.

[0146] In another embodiment, the metabolic disorder is selected from the group consisting of metabolic syndrome, insulin deficiency or insulin resistance related disorders, diabetes, glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, cardiac conditions, stroke, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, immune system dysfunction, obesity, cardiovascular disease, high cholesterol, high triglycerides, asthma, heart attack, osteoarthritis, neurodegeneration, gallbladder disease, syndrome X, inflammatory and immune disorders, atherogenic dyslipidemia and cancer.

[0147] In yet another embodiment, the present invention provides a method for treating a subject suffering from a disease of mitochondrial dysfunction or a disease associated with mitochondrial dysfunction, comprising administering to the subject an effective amount of the synergistic nutritional composition of the present invention to enhance mitochondrial function.

[0148] A "subject in need thereof" is preferably a mammal, more preferably a human, and the subject may be an infant and / or an adult human. The subject may also include a patient with symptoms or signs that precede metabolic disorders such as cardiovascular disease and neurodegenerative disease. Furthermore, the subject may be a healthy individual to whom the composition of the present invention is administered as a preventive therapy.

[0149] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids, bases, metal ions, minerals, chelates, complexes, esters, sulfates, hydrates, oxides, and amines that are well known in the art.

[0150] In a further embodiment, the nutritional composition of the present invention is used to normalize metabolic function in a subject in need thereof. In particular, the therapeutic agent is administered to prevent either the occurrence or the cause of the development of a metabolic disorder.

[0151] As used herein, the term "specific or effective amount" is intended to mean a therapeutically effective dose of the bioactive compounds of the present invention, i.e., biotin, manganese and oxaloacetate, in combination with one another to provide the composition with a synergistic effect not obtainable by using the single components of the composition.

[0152] In particular, a "therapeutically effective amount" is an amount that reduces the risk, future potential, possibility or onset of a disease or disorder, or that causes a significant alleviation, reduction, and / or decrease or reversal of at least one indicator / biomarker (e.g., blood or serum CRP levels) and / or minimizes at least one clinical symptom of an age-related metabolic disorder.

[0153] In describing the present invention, the term "treat" and the like means to reduce, promote, sedate, prevent, attenuate, manage, regulate, mitigate, optimize, suppress, inhibit, stabilize, improve, reverse, boost, normalize, induce, trigger cure, or cure of the decline in cellular ATP due to aging.

[0154] Notably, the synergistic composition of the present invention is free of side effects, non-hazardous, non-toxic and safe for human consumption, therefore, the composition of the present invention can also be used for preventive therapy in healthy subjects.

[0155] In another embodiment, the present invention relates to a synergistic composition that is prepared by a method well known in the pharmaceutical field and administered by various routes depending on whether local or systemic treatment is desired and the area to be treated.Preferred routes of administration include, but are not limited to, sublingual, rectal, topical, parenteral, nasal, or oral.Therapeutic (prescription) supplements are generally administered by oral, parenteral, or nasal routes to treat mitochondrial disease.Therapeutic administration of the composition of the present invention may be combined with other treatments.

[0156] In one embodiment, the synergistic nutritional composition of the present invention is administered to a subject in a form suitable for oral use, such as tablets, capsules (in delayed release, extended release, sustained release, enteric coated release forms), hard gelatin capsules, soft gelatin capsules in oily vehicles, granules for sublingual use, effervescent tablets, aqueous or oily solutions, suspensions or emulsions, encapsulations, matrices, coatings, beadlets, nanoparticles, caplets, granules, particulates, agglomerates, sustained release tablets, chewables, lozenges, troches, solutions, suspensions, fast dissolving films, elixirs, gels, tablets, pellets, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays, or in a reconstituted dry powder form with a liquid medium or syrup. In another embodiment, the composition is formulated for parenteral use, including intravenous, subcutaneous, intramuscular, intravascular, infusion, intraperitoneal, intracerebral, intracerebral ventricular, or intradermal routes. Additionally, the synergistic compositions of the present invention are useful for nasal administration via ionic liquid spray devices, nasal sprays, intranasal spray devices, nano nasal sprays, saline sprays, and the like.

[0157] In another embodiment, the pharma- ceutically acceptable carrier, diluent, or excipient is selected from the group consisting of adjuvants, carriers, excipients, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonicity agents, solvents, emulsifiers, or encapsulating agents such as liposomes, cyclodextrins, encapsulating polymer delivery systems, or polyethylene glycol matrices that are acceptable for use in subjects, preferably humans. Excipients also include anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, glidants (glidants), lubricants, preservatives, adsorbents, suspending or dispersing agents, sweeteners, surfactants, anti-caking agents, food additives, or water of hydration.

[0158] In another embodiment of the invention, the diluent is selected from the group consisting of starch, hydrolyzed starch, partially pregelatinized starch, anhydrous lactose, cellulose powder, lactose monohydrate, sugar alcohols such as sorbitol, xylitol and mannitol, silicified microcrystalline cellulose, ammonium alginate, calcium carbonate, calcium lactate, dibasic calcium phosphate (anhydrous / dibasic dehydrated / tribasic), calcium silicate, calcium sulfate, cellulose acetate, corn starch, pregelatinized starch, dextrin, β-silicic acid, cellulose acetate ... The sugars are selected from clodextrin, dextrin, dextrose, erythritol, ethyl cellulose, fructose, fumaric acid, glyceryl palmitostearate, magnesium carbonate, magnesium oxide, maltodextrin, maltose, medium chain triglycerides, polydextrose, polymethacrylates, sodium alginate, sodium chloride, sterilizable corn, sucrose, sugar spheres, talc, trehalose, xylitol, vehicles like petrolatum, dimethyl sulfoxide, mineral oil, and the like.

[0159] In a preferred embodiment of the present invention, the diluent in the composition / formulation is from 1 to 30% by weight of the total composition / formulation.

[0160] In yet another embodiment of the invention the binder is selected from the group consisting of disaccharides such as sucrose, lactose, polysaccharides and derivatives thereof such as starch, cellulose, modified cellulose such as microcrystalline cellulose or cellulose esters such as hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), sugar alcohols such as xylitol, sorbitol or mannitol, proteins such as gelatin, synthetic resins such as polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), starch, acacia, agar, alginic acid, calcium carbonate, calcium lactate, carbomer, sodium carboxymethylcellulose, carrageenan, cellulose acetate phthalate, chitosan, copovidone, corn starch, pregelatinized starch, etc. The preferred oils are selected from among cellulose, cottonseed oil, dextrose, dextrin, dextrose, ethylcellulose, guar gum, hydrogenated vegetable oils, mineral oil, hydroxyethylcellulose, hydroxymethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, inulin, cellulose, methylcellulose, polyvinylpyrrolidone and polyethylene glycols, lactose, liquid glucose, hypromellose, magnesium aluminum silicate, maltodextrin, maltose, methylcellulose, microcrystalline cellulose, pectin, poloxamer, polydextrose, polymethacrylates, povidone, sodium alginate, stearic acid, sucrose, sunflower oil, various animal and vegetable oils, and white soft paraffin, paraffin, flavors, colorants and waxes.

[0161] In a preferred embodiment of the present invention, the binder in the composition / formulation is 0.1-40% by weight of the composition / formulation.

[0162] In a further embodiment of the invention, the lubricant is selected from magnesium stearate, zinc stearate, calcium stearate, glyceryl monostearate, glyceryl behenate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil, light mineral oil, magnesium lauryl sulfate, medium chain triglycerides, mineral oil, myristic acid, palmitic acid, poloxamer, polyethylene glycol, sodium benzoate, sodium chloride, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, potassium benzoate, sodium benzoate, and the like.

[0163] In a preferred embodiment of the present invention, the lubricant in the composition / formulation is present in an amount of 0.1 to 5.0% by weight of the total composition / formulation.

[0164] In another embodiment of the invention, the solubilizer is selected from the group consisting of polysorbate 80, sodium lauryl sulfate, anionic emulsifying wax, nonionic emulsifying wax, glyceryl monooleate, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sorbitan esters, triethyl citrate, vitamin E, polyethylene glycol succinate, microcrystalline cellulose, carboxymethylcellulose sodium salt, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, hypromellose, hypromellose acetate succinate, lecithin, polyethylene alkyl ethers, aluminum oxide, poly(methyl vinyl ether / maleic anhydride), calcium carbonate, crospovidone, cyclodextrin, fructose, hydroxypropyl betadex, oleyl alcohol, povidone, benzalkonium chloride, benzethonium chloride, benzyl alcohol, benzyl benzoate, cetylpyridinium chloride, inulin, meglumine, poloxamer, pyrrolidone, sodium bicarbonate, starch, stearic acid, sulfobutyl ether beta cyclodextrin, tricaprylin, triolein, docusate sodium, glycine, alcohol, self-emulsifying glyceryl monooleate, cationic benzethonium chloride, cetrimide, xanthan gum, lauric acid, myristyl alcohol, butylparaben, ethylparaben, methylparaben, propylparaben, sorbic acid, and the like.

[0165] In one embodiment of the invention, the amount of solubilizer or surfactant in the composition / formulation is 0.1-10% by weight of the composition / formulation. In a preferred embodiment of the invention, the amount of solubilizer or surfactant is 0.1-5.0% by weight of the composition / formulation.

[0166] In one embodiment of the present invention, the lubricant is selected from colloidal silicon dioxide, magnesium stearate, fumed silica (colloidal silicon dioxide), starch, talc, calcium phosphate tribasic, cellulose powder, hydrophobic colloidal silica, magnesium oxide, zinc stearate, magnesium silicate, magnesium trisilicate, silicon dioxide, and the like.

[0167] In another embodiment of the invention, the lubricant in the composition / formulation is 0.1-5.0% by weight of the total composition / formulation.

[0168] In one embodiment of the present invention, the stabilizer is selected from the group consisting of alginate, agar, carrageen, gelatin, guar gum, gum arabic, locust bean gum, pectin, starch, xanthan gum, trehalose, and the like.

[0169] In a preferred embodiment of the present invention, the stabilizer in the composition / formulation is 0.1-8.0% by weight of the total composition / formulation.

[0170] In one embodiment of the present invention, the solvent is selected from water, alcohol, isopropyl alcohol, propylene glycol, mineral oil, benzyl alcohol, benzyl benzoate, flavor glycols, carbon dioxide, castor oil, corn oil (maize), cottonseed oil, dimethyl ether, albumin, dimethylacetamide, ethyl acetate, ethyl lactate, medium chain triglycerides, methyl lactate, olive oil, peanut oil, polyethylene glycol, polyoxyl castor oil, propylene carbonate, pyrrolidone, safflower oil, sesame oil, soybean oil, sunflower oil, water miscible solvents, organic polar solvents, non-polar solvents, or mixtures thereof.

[0171] In a preferred embodiment of the invention, the solvent in the composition / formulation is used in an amount sufficient to bring the weight of the composition / formulation to 100% by weight.

[0172] Additional additives include polymers, plasticizers, sweeteners, powdered flavors, preservatives, colorants, surfactants, and other excipients. Powdered flavor compositions include flavors supported on solid carriers. Coating materials such as synthetic polymers, shellac, corn protein (zein), other polysaccharides, gelatin, fatty acids, waxes, shellac, plastics, and vegetable fibers are used. In a preferred embodiment of the invention, additives are used at 1-20 w / w% of the unit dose.

[0173] In another embodiment, the present invention provides a synergistic nutritional composition comprising an exogenous mixture of biotin-Mn complex and stabilized OAA together with a pharmaceutical excipient, wherein the pharmaceutical excipient is selected from a diluent, binder, surfactant, lubricant, glidant, additive, stabilizer, or mixtures thereof.

[0174] In yet another embodiment, the present invention provides a synergistic nutritional composition comprising a therapeutic mixture of biotin-manganese complex and stabilized oxaloacetate together with a pharmaceutical excipient, wherein the pharmaceutical excipient is selected from diluents, binders, lubricants, glidants, additives, surfactants, stabilizers or mixtures thereof. In a preferred embodiment, the diluent is 1-30% by weight, the binder is 0.1-25% by weight, the lubricant is 0.1-5.0% by weight, the glidant is 0.1-5.0% by weight, the additive is 1-10% by weight, the surfactant is 0.1-5.0% by weight, and the stabilizer is 0.1-5.0% by weight based on the total composition.

[0175] In another embodiment, the compositions of the present invention are formulated in age appropriate pediatric oral dosage forms such as syrups, minitablets, chewable formulations, orodispersible films, and orodispersible tablets.

[0176] In a preferred embodiment, the pharmaceutical composition / formulation of the present invention is formulated for oral administration. Specifically, the solid pharmaceutical composition is in the form of a tablet, capsule, pill, liquid- or solid-filled hard capsule, soft capsule, sachet, powder, granule, suspension, solution, or modified release formulation. The formulation of the present invention suitable for oral administration is provided in separate forms such as capsules (e.g., soft gel capsules, hard gel capsules), sachets, or tablets, each containing a predetermined amount of active ingredient, as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, as a syrup, as an oil-in-water emulsion, or as a water-in-oil emulsion.

[0177] The prophylactic or therapeutic dose will usually vary depending on the nature and severity of the condition to be treated and the route of administration. The dose, and often the frequency of administration, will vary according to the age, weight and response of the individual patient. In general, the total daily dose (single or multiple doses) is about 1-5000 mg / day. In a preferred embodiment, the total daily dose is about 10-1000 mg / day.

[0178] In a further embodiment, the present invention provides a synergistic nutritional composition comprising a particular combination of stabilized oxaloacetate and biotin-manganese complex together with a pharma- ceutically acceptable excipient or carrier, wherein an effective unit dose for oral administration is 50-800 mg.A particularly effective unit dose of the composition is 20-500 mg.

[0179] In one embodiment, oral administration of an effective amount of the composition increases intracellular ATP levels by 46% over normal controls.

[0180] It is further recommended that children and patients over 60 years of age use low doses initially and adjust the dose based on individual physiological response and / or pharmacokinetics. As will be clear to those skilled in the art, in some cases it may be necessary to use dosages outside the above range. The composition of the present invention can be used as an infant formulation as well as an adult formulation by adjusting the concentration of the active ingredient. In addition, it should be noted that a nutritionist, dietitian, or certified physician knows how and when to interrupt, adjust, or terminate treatment depending on the response of each individual patient.

[0181] The use of any and all examples or exemplary language (e.g., "such as") described herein is intended merely to make the invention more clear and does not limit the scope of the invention unless and until such time as the claims are limited thereto.

[0182] While the present invention has been described in relation to specific embodiments thereof and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is capable of alternative embodiments and that some of the details described herein may be modified considerably without departing from the underlying principles of the invention.

[0183] The present invention can be further described by the following examples, which are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. The present invention is not limited to the terms of the specific embodiments described in this application, which are intended to be illustrative of one of the individual aspects of the present invention. In addition to those recited herein, functionally equivalent compositions and procedures within the scope of the present invention will be apparent to those skilled in the art from the foregoing description and examples. Such modifications and variations are intended to fall within the scope of the appended claims. The contents of each document, patent, and patent application cited in this application are incorporated herein by reference in their entirety. EXAMPLES

[0184] Example 1 I. Composition 1: Synergistic Mixture TIFF0007680443000003.tif39130

[0185] II. Composition 2: Tablets / Capsules TIFF0007680443000004.tif62129

[0186] III. Composition 3: Tablets / Capsules TIFF0007680443000005.tif99131

[0187] IV Composition 4: Tablets / Capsules TIFF0007680443000006.tif126154

[0188] V Composition 5: Tablets / Capsules TIFF0007680443000007.tif133154

[0189] IV Composition 6: Tablets / Capsules TIFF0007680443000008.tif134155

[0190] VII Composition 7: Tablets / Capsules TIFF0007680443000009.tif126153

[0191] VIII Composition 8: Tablets / Capsules TIFF0007680443000010.tif126154

[0192] Example 2: Cell line analysis To evaluate the potential of test substances to increase intracellular ATP levels in human hepatic cell lines in vitro The in vitro effects of test substances on cellular energetics were evaluated by measuring intracellular ATP levels in a human hepatic cell line (HepG2). Under the given experimental conditions, treatment with test substances improved in vitro intracellular ATP levels (J. Immunol Methods;1986;89:271-277), (Proc Natl Acad Sci USA;2009;106:15651-15656).

[0193] procedure I. Method Overview In vitro cytotoxicity against HepG2 (human hepatocyte) line was evaluated by MTT assay to find the non-toxic concentration of the test substances, and the potential of the test substances to increase intracellular ATP levels was evaluated by the kit method.

[0194] II. Preparation of test solutions For test substance ABC, the test substances were combined in a weight ratio of 1:0.05:0.01. Approximately 10 mg of ABC was dissolved in 100 μl of DMSO and made up to 1 ml with DMEM-HG (Dulbecco's Modified Eagle Medium High Glucose). For individual test substances A, B and C, approximately 10 mg of the total test substance was weighed and dissolved separately in 100 μl of DMSO and DMEM-HG containing 2% inactivated FBS was added to obtain a stock solution with a concentration of 1 mg / ml. The samples were further sterilized by 0.22 μ syringe filtration. Serial two-fold dilutions were prepared from the stock to perform further studies.

[0195] III Cell lines and media Cell lines were incubated at 37°C with 5% CO 2 Cells were cultured to confluence in DMEM-HG supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU / ml), streptomycin (10 μg / ml) and amphotericin B (5 μg / ml) in a humidified atmosphere at 4 °C. Cells were detached with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). Stock cultures were plated on 25 cm plates. 2All cultures were grown in 96-well culture flasks and all experiments were transferred to 96-well microtiter plates (Tarsons India Pvt. Ltd., Kolkata, India).

[0196] IV Cytotoxicity Analysis The monolayer cultured cells were trypsinized and the cells were numbered at 1.0 × 10 using DMEM-HG containing 10% FBS. 5 The cells were adjusted to 0.1 ml / ml. 0.1 ml of the diluted cell suspension was added to each well of a 96-well microtiter plate. After 24 hours, when a partial monolayer had formed, the supernatant was removed, the monolayer was washed once with medium, and 100 μl of different concentrations of the test substances were added. The plates were then incubated at 4°C for 24 h at 5% CO 2 The plates were incubated at 37°C in ambient air for 72 hours and examined microscopically at 24 hour intervals.

[0197] IV MTT assay After 72 hours of incubation, the drug solutions in the wells were discarded, and 50 μl of MTT in PBS was added to each well. The plate was gently shaken and incubated in 5% CO 2 The plates were incubated at 37°C for 3 hours in an ambient atmosphere. The supernatant was removed, 100 ml of 2-propanol was added, and the plate was gently shaken to solubilize the formazan formed. The absorbance was measured at a wavelength of 540 nm using a microplate reader. The percent growth inhibition was calculated using the standard formula. The concentration of test substance required to inhibit cell proliferation by 50%, i.e., CTC, was calculated as 50 Values ​​were generated from dose-response curves.

[0198] V. Estimation of Cellular Respiration by Measuring Intracellular ATP Levels Trypsinize HepG2 cells from a stock culture flask and count the cells to 1.0 x 10 5The cell suspension was adjusted to 100 / ml and seeded in a 6-well plate. After 24 hours, the cultured cells had reached 70-80% confluence and were treated with different non-toxic concentrations of the test substances. After 2 hours of treatment, the plates were washed with phosphate-buffered saline. The cellular ATP levels were measured based on the method described in the kit manual (SIGMA, #MAK190). At the end of the experiment, the optical density was read at 570 nm using a microplate reader. From the absorbance, the cellular ATP levels were estimated using the kit protocol, and the concentration of cellular ATP in the treated group was compared with the control group.

[0199] VI Research Design Table 1: Groups, doses and treatments TIFF0007680443000011.tif104165

[0200] Table 2: Cytotoxicity of test substances against HepG2 cell line TIFF0007680443000012.tif140165

[0201] Table 3: Effect of test substances on intracellular ATP levels in HepG2 cells TIFF0007680443000013.tif69165

[0202] VII Discussion Test substances A, B, C and ABC were evaluated for their respective cytotoxicity at different concentrations ranging from 1000μg / ml to 62.5μg / ml. Non-toxic concentrations of test substances were determined for further study. The cellular ATP levels of test substances were 5.42±0.25mM for A, 6.05±0.33mM for B, 4.92±0.13mM for C, and 7.21±0.27mM for ABC (1:0.05:0.01) at the respective test concentrations. Test substances A, B and C at a concentration of 500μg / ml did not show a significant increase in intracellular ATP levels compared to the control. However, the combination of A, B and C at a specific weight ratio of 1:0.05:0.01 showed a significant increase in cellular ATP levels, with the increase in cellular ATP levels being 46% compared to the control. Furthermore, the increase in intracellular ATP level relative to the control was 1.90 for A, 1.11 for B, 1.00 for C, and 1.50 for ABC (1:0.05:0.01) at each test concentration.

[0203] Conclusion: The results show that the test substances increase cellular respiration in HepG2 cells. The combination of test substances ABC at a weight ratio of 1:0.05:0.01 shows a significant increase in cellular ATP levels at the concentrations tested compared to untreated controls. The synergistic nutritional composition of the present invention increases cellular ATP levels by 46% when administered orally at an effective dose.

Claims

1. 1. A synergistic nutritional composition that enhances cellular ATP efficiency, comprising: containing a therapeutically effective exogenous mixture of stabilized oxaloacetate and a biotin-manganese complex together with a pharma- ceutically acceptable excipient; A synergistic nutritional composition, wherein the weight ratio of the stabilized oxaloacetate or its salt to the biotin-manganese complex or its salt is 1:0.01 to 1:0.

2.

2. 2. The synergistic nutritional composition of claim 1, A synergistic nutritional composition, wherein the stabilized oxaloacetate is 50-96% by weight of the total composition.

3. 3. A synergistic nutritional composition according to claim 1 or 2, comprising: Stabilized Oxaloacetate is a synergistic nutritional composition containing a homogenous premix of the crystalline organic compound oxaloacetate and standardized ascorbic acid in a 1:1 to 1:2 weight ratio.

4. 2. The synergistic nutritional composition of claim 1, Biotin-manganese complex is a synergistic nutritional composition containing a homogenous premix of a crystalline form of the D-isomer of biotin and crystalline manganese(II) sulfate monohydrate in a weight ratio of 1:0.1 to 1:

1.

5. 5. The synergistic nutritional composition of claim 4, A synergistic nutritional composition, wherein the crystalline form of the D-isomer of biotin is 0.1-10% by weight of the total composition.

6. 5. The synergistic nutritional composition of claim 4, A synergistic nutritional composition, wherein the crystalline manganese(II) sulfate monohydrate is 0.1-5% by weight of the total composition.

7. A synergistic nutritional composition according to any one of claims 1 to 6, comprising: The present invention comprises a synergistic mixture of the crystalline organic compounds oxaloacetic acid, stabilized oxaloacetate, a crystalline form of the D-isomer of biotin, and crystalline manganese(II) sulfate monohydrate, together with pharma- ceutically acceptable excipients; A synergistic nutritional composition comprising a weight ratio of the crystalline organic compounds oxaloacetic acid, stabilized oxaloacetate, crystalline forms of the D-isomer of biotin, and crystalline manganese(II) sulfate monohydrate of 1:1:0.1:0.01 to 1:2:0.5:0.

1.

8. 2. The synergistic nutritional composition of claim 1, A synergistic nutritional composition, wherein the pharma- ceutically acceptable excipient is selected from the group consisting of 1-30% by weight of a diluent, 0.1-25% by weight of a binder, 0.1-5.0% by weight of a lubricant, 0.1-5.0% by weight of a glidant, 1-10% by weight of an additive, 0.1-5.0% by weight of a surfactant, and 0.1-5.0% by weight of a stabilizer, based on the total composition.

9. 2. The synergistic nutritional composition of claim 1, A synergistic nutritional composition, wherein an effective unit dose for oral administration of the synergistic nutritional composition is 20-500 mg.

10. 2. The synergistic nutritional composition of claim 1, The synergistic nutritional composition is useful in the treatment of insulin restriction, diabetes, obesity, overweight, female hormone imbalance, premenstrual syndrome (PMS), premenstrual disorder (PMDD), dementia, endocrine disorders, traumatic brain injury, ischemia, Alzheimer's disease, Parkinson's disease, traumatic spinal cord, traumatic brain injury (TBI), brain inflammation, brain injury, bone disease, caloric restriction, heart disease, liver disease, and kidney disease.

11. 1. A synergistic nutritional composition for enhancing cellular ATP production in a subject in need thereof, comprising: A synergistic nutritional composition comprising an exogenous therapeutic mixture of stabilized oxaloacetate and biotin-manganese complex in a weight ratio of 1:0.01 to 1:0.

2.

12. The synergistic nutritional composition of claim 11, comprising: A synergistic nutritional composition, wherein the stabilized oxaloacetate is 80-96% by weight of the total composition.

13. The synergistic nutritional composition of claim 11, comprising: A synergistic nutritional composition, wherein the biotin-manganese complex is 0.5-10% by weight of the total composition.

14. The synergistic nutritional composition of claim 11, comprising: Stabilized Oxaloacetate is a synergistic nutritional composition containing a homogenous premix of the crystalline organic compound oxaloacetic acid and standardized ascorbic acid in a 1:1 to 1:2 weight ratio.

15. The synergistic nutritional composition of claim 11, comprising: Biotin-manganese complex is a synergistic nutritional composition containing a homogenous premix of a crystalline form of the D-isomer of biotin and crystalline manganese(II) sulfate monohydrate in a weight ratio of 1:0.1 to 1:

1.

16. The synergistic nutritional composition of claim 11, comprising: A synergistic nutritional composition, wherein oral administration of an effective amount of the synergistic nutritional composition enhances intracellular ATP production.

17. The synergistic nutritional composition of claim 11, comprising: A synergistic nutritional composition, wherein an effective unit dose of the synergistic nutritional composition is 50-800 mg.

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