Micronutrient composition for preventing and reversing protein saccharification during oxidative stress in human body

Abstract

The mixture as the micronutrient composition is a combination of alpha-ketoglutaric acid, nicotinamide or nicotinic acid, L-proline, L-glutamine, L-citrulline, sodium pyruvate, pantothenic acid, L-cysteine or N-acetylcysteine, magnesium malate, magnesium citrate, and methionine. The mixture is effective for the treatment of diseases due to oxidative stress. More specifically, the mixtures and cores as micronutrient compositions are effective in reversing saccharification in diabetes mellitus.

Description

Technical Field

[0001] This study focused on micronutrient compositions that prevent and reverse protein glycation due to oxidative stress in humans. Background Art

[0002] Advanced glycation end products (AGEs) are modifications of proteins or lipids that form in the body in a hyperglycemic environment and during the aging process, and contribute to the pathophysiology of diabetes, cardiovascular disease, Alzheimer's disease, inflammation that accelerates overall tissue aging. Many of the negative effects of AGEs are produced by AGE activation of a cellular receptor called the receptor for advanced glycation end products (RAGE).

[0003] AGE-modified proteins or lipids are the result of non-enzymatic glycation and oxidation after they come into contact with sugars, especially fructose and glucose. The AGE formation process or Maillard reaction begins with Schiff bases and Amadori products, the latter of which are produced by the reaction of carbonyl groups of reducing sugars (such as glucose) with amino groups of proteins, lipids and nucleic acids. These highly reactive intermediate carbonyl groups, called α-dicarbonyls or oxoaldehydes, are able to react with amino, sulfhydryl and guanidine functional groups in proteins, leading to cross-linking and denaturation of proteins. In addition, α-dicarbonyls can react with lysine and arginine functional groups on proteins, leading to the formation of stable AGE compounds such as N- ε - (Carboxymethyl) lysine (CML), which is a non-fluorescent AGE. CML is also formed in vitro by incubation of LDL with copper ions and glucose and is therefore considered to be both a lipid adduct and a protein adduct. Once AGEs are formed, they are considered to be almost irreversible. Therefore, there is an urgent goal to develop effective, economical and side-effect-free methods to protect cells from oxidative stress. Summary of the invention

[0004] In the present disclosure, various combinations of micronutrients as compositions are used to treat and prevent AGE formation in patients not only with diabetes but also with other diseases in which protein turnover is inhibited. In one embodiment, the physiological dose for a mammal is calculated based on daily consumption. For ease of administration, the formulation is packaged in a pharmaceutical preparation.

[0005] In one embodiment, the micronutrient composition comprises: alpha-ketoglutarate, niacinamide or nicotinic acid, L-proline, L-glutamine, L-citrulline, sodium pyruvate, pantothenic acid, L-cysteine ​​or N-acetylcysteine, magnesium malate, magnesium citrate, methionine, S-adenosyl-methionine (SAMe), methylsulfonylmethane (MSM), and wormwood powder or extract. In one embodiment, the micronutrient composition comprises: alpha-ketoglutaric acid 10 mg-30000 mg, niacinamide or niacin 1 mg-10000 mg, L proline 10 mg-30000 mg, L glutamine 10 mg-50000 mg, L citrulline 10 mg-20000 mg, sodium pyruvate 10 mg-50000 mg, pantothenic acid 0.1 mg-200 mg, L cysteine ​​or N-acetyl cysteine ​​10 mg-20000 mg, magnesium malate 10 mg-20000 mg, magnesium citrate 10 mg-30000 mg, methionine 10 mg-20000 mg, S-adenosyl-methionine (SAMe) 1 mg-20000 mg, methylsulfonylmethane (MSM) 1 mg-20000 mg, and wormwood powder or extract 1 mg-10000 mg.

[0006] In one embodiment, the micronutrient composition shown as a mixture in the figures comprises: alpha-ketoglutarate, niacinamide or nicotinic acid, L proline, L glutamine, L citrulline, sodium pyruvate, pantothenic acid, L cysteine ​​or N-acetyl cysteine, magnesium malate, magnesium citrate and methionine. In one embodiment, the micronutrient composition comprises: alpha-ketoglutaric acid 10 mg-30000 mg, niacinamide or niacin 1 mg-10000 mg, L proline 10 mg-30000 mg, L glutamine 10 mg-50000 mg, L citrulline 10 mg-20000 mg, sodium pyruvate 10 mg-50000 mg, pantothenic acid 0.1 mg-200 mg, L cysteine ​​or N-acetyl cysteine ​​10 mg-20000 mg, magnesium malate 10 mg-20000 mg, magnesium citrate 10 mg-30000 mg, and methionine 10 mg-20000 mg.

[0007] In one embodiment, a method of treating a person suffering from a disease manifested by oxidative stress by administering a micronutrient composition is disclosed. In another embodiment, the micronutrient composition (mixture) comprises: alpha-ketoglutaric acid 10 mg-30000 mg, niacinamide or niacin 1 mg-10000 mg, L-proline 10 mg-30000 mg, L-glutamine 10 mg-50000 mg, L-citrulline 10 mg-20000 mg, sodium pyruvate 10 mg-50000 mg, pantothenic acid 0.1 mg-200 mg, L-cysteine ​​or N-acetylcysteine ​​10 mg-20000 mg, magnesium malate 10 mg-20000 mg, magnesium citrate 10 mg-30000 mg, and methionine 10 mg-20000 mg.

[0008] In one embodiment, a method of treating a human suffering from a disease manifested due to oxidative stress by administering a micronutrient composition is disclosed. In another embodiment, the micronutrient composition (core mixture) consists of: alpha-ketoglutarate 10 mg-30000 mg, niacinamide or niacin 1 mg-10000 mg, L-proline 10 mg-30000 mg, L-glutamine 10 mg-50000 mg, L-citrulline 10 mg-20000 mg, and sodium pyruvate 10 mg-50000 mg.

[0009] In one embodiment, the micronutrient compositions, mixtures and cores are used as pharmaceutical compositions and are administered to humans suffering from specific diseases caused by oxidative stress, such as diabetes, cardiovascular disease, Alzheimer's disease, and inflammation that accelerates the aging of tissues as a whole. The micronutrient compositions, mixtures and core mixtures are administered to humans in various forms to treat specific diseases, such as diabetes, cardiovascular disease, Alzheimer's disease, and inflammation that accelerates the aging of tissues as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Exemplary embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings in which like reference numerals indicate similar elements, and in which:

[0011] FIG1 shows a micronutrient composition as a mixture and core with simultaneous pleiotropic beneficial effects on diabetic metabolism.

[0012] FIG. 2 illustrates a prior art showing the chemical reaction of a glycation reversal assay.

[0013] Figure 3 The reversion of carboxymethyl-lysine (CML) to native lysine in the presence of individual compounds and their combinations (core and mixtures) is shown.

[0014] Figure 4 The effects of individual natural compounds and their combinations (as mixtures and cores) on AGE-BSA formation in the presence of glucose are shown.

[0015] Figure 5 The effect of individual natural compounds and their combinations (as mixtures and cores) on AGE-BSA formation in the presence of fructose is shown.

[0016] Figure 6 Shown are the effects of individual components and their combinations (core and mixture) on succinate dehydrogenase (SDHA) levels in cardiomyocytes.

[0017] Figure 7 Shown are the effects of individual components and their combinations (nuclear and mixtures) on Cox-1 levels in cardiomyocytes.

[0018] Figure 8 Shown are the protection of microglia by individual compounds and their combinations (core and mixtures) against oxidative stress generated by hydrogen peroxide.

[0019] Other features of the present embodiments will be apparent from the accompanying drawings and from the detailed description that follows. DETAILED DESCRIPTION

[0020] The present disclosure shows various combinations of individual components of micronutrient compositions, mixtures and cores, and their effects on preventing and treating oxidative stress-related functions in specific diseases such as diabetes, cardiovascular disease, Alzheimer's disease, and inflammation that accelerates overall aging of tissues. The micronutrient composition as a mixture includes: alpha-ketoglutarate, niacinamide or nicotinic acid, L-proline, L-glutamine, L-citrulline, sodium pyruvate, pantothenic acid, L-cysteine ​​or N-acetylcysteine, magnesium malate, magnesium citrate, and methionine. The physiological range of the micronutrient composition for the mixture is: alpha-ketoglutarate 10 mg-30000 mg, niacinamide or niacin 1 mg-10000 mg, L proline 10 mg-30000 mg, L glutamine 10 mg-50000 mg, L citrulline 10 mg-20000 mg, sodium pyruvate 10 mg-50000 mg, pantothenic acid 0.1 mg-200 mg, L cysteine ​​or N-acetyl cysteine ​​10 mg-20000 mg, magnesium malate 10 mg-20000 mg, magnesium citrate 10 mg-30000 mg, and methionine 10 mg-20000 mg.

[0021] The micronutrient composition as the core comprises: α-ketoglutaric acid, nicotinamide or nicotinic acid, L proline, L glutamine, L citrulline, sodium pyruvate, and its physiological dosage is included in the following range: α-ketoglutaric acid 10 mg-30000 mg, nicotinamide or nicotinic acid 1 mg-10000 mg, L proline 10 mg-30000 mg, L glutamine 10 mg-50000 mg, L citrulline 10 mg-20000 mg, and sodium pyruvate 10 mg-50000 mg.

[0022] In our study, we tested several natural compounds individually and as mixtures to investigate their efficacy in both preventing and reversing AGE formation in vitro. The AGE reversal assay is based on the induction of N-terminal peptides, which are known physiological ligands for AGE production and RAGE. ε -(carboxymethyl)lysine (CML) conversion to L-lysine. Therefore, the conversion of CML to L-lysine would represent a reversal of the glycation modification. This assay was adapted from a publication by Kim et al., in which they used a modified E. coli enzyme to cleave CML. We applied natural compounds, amino acids, and Krebs cycle components to obtain the same effect.

[0023] In addition, using the established AGE formation scheme, we show that the combination of natural compounds (mixture and core) and individual test ingredients effectively prevent the saccharification of bovine serum albumin (BSA) caused by both glucose and fructose within a 5-day incubation period. We also show the beneficial effects of micronutrients on the important aspects related to hyperglycemia and diabetes (including oxidative stress, nerve damage and mitochondrial dysfunction) at the cellular level. Our results show that each component acts synergistically to promote mitochondrial biogenesis in muscle cells and protect microglia from death caused by oxidative stress. The mitochondrial biogenesis assay specifically detects Cox-1 activity (in respiratory chain complex IV) and SDH (succinate dehydrogenase) subunit A as another mitochondrial membrane protein and tumor suppressor. Fig. 1 shows a general view of a micronutrient composition, which has a pleiotropic beneficial effect in the metabolism of diabetes.

[0024] Materials: N-carboxymethyl lysine (CML) was purchased from Cayman Chemical Company (Michigan, USA). The sources of other test compounds are listed in Tables 1 and 2:

[0025] Table 1. Test ingredients combined in core (bold) and mixtures (bold and regular font)

[0026]

[0027] Table 2. Additional ingredients evaluated in the study

[0028]

[0029] All ingredients are dissolved in dimethyl sulfoxide (DMSO) obtained from MilliporeSigma (Massachusetts, USA). The micronutrient composition comprises 14 ingredients. A mixture is formed by combining all 11 ingredients listed in Table 1 in equal amounts. The subgroup of the mixture ingredients represented as the core is formed by 6 ingredients. Other compounds not included in the mixture and the core that were tested in the study are listed in Table 2.

[0030] Other reagents used were: 2,4-dinitrophenylhydrazine (2,4-DNPH) from MilliporeSigma (MA, USA), hydrochloric acid from Thermo Fisher Scientific (MA, USA), phosphate buffered saline (PBS), hypoxanthine and xanthine oxidase from MilliporeSigma (MA, USA), KH2PO4, K2HPO4, ethylenediaminetetraacetic acid (EDTA), ferric chloride, bovine serum albumin (BSA), glucose, fructose and sodium hydroxide (NaOH) from Thermo Fisher Scientific (MA, USA).

[0031] Cell lines: Rat cardiomyocytes (H9c2) were obtained from ATCC (Virginia, USA). They were derived from embryonic BD1X rat heart tissue that exhibits a variety of skeletal muscle properties. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (obtained from Thermofisher (MA, USA)) supplemented with 10% fetal bovine serum (FBS) (obtained from Thermofisher (MA, USA)) and 1% penicillin-streptomycin (PS) (obtained from MilliporeSigma (MA, USA)). Microglia (IMG) were obtained from Kerafast (MA, USA). It is an immortalized microglial cell line isolated from the brain of adult mice. The cells were maintained in DMEM supplemented with 10% FBS and 1% PS.

[0032] Glycation reversal test: For CML cleavage reaction, 90 μl of 100mM CML in PBS was incubated with 10 μl of ingredients diluted in PBS. Incubation was carried out at 37°C and 300 rpm for 4 hours. After 4 hours, 50 μL of 2,4-DNPH solution (1 mm in 1N HCl) was then added to the mixture and incubated with 350 μL of 0.6N NaOH at 37°C for 10 minutes. The optical density of 150 μl volume at 445 nm was measured. The reading of the CML-dissolution test is based on the cleavage of CML, which leads to the formation of L-lysine, hydrogen peroxide and pyruvic acid. Using the described α-keto-acid derivatization test, sodium pyruvate reacts with 2,4-DNPH to form an adduct, which can be modified by a base and detected by measuring optical density. The chemical reaction shown by Kim et al. is shown in Figure 2 taken from their publication.

[0033] Glycation prevention test: The glycation prevention reaction buffer contains the following components: 500mL of phosphate buffer (KH2PO4+K2HPO4, 0.1M), EDTA (73mg), FeCl36H2O (33mg), hypoxanthine (20.5mg). The following reaction mixture was added to an Eppendorf microcentrifuge tube (1.5mL): 94μL BSA (16mg / ml in buffer), 25μL glucose (1.67M in buffer), 10μL xanthine oxidase (XO-18mU in buffer), 10μL test component (40mg / ml stock solution in DMSO), 882μL buffer. The control was set to lack BSA, glucose and xanthine oxidase (XO). The positive control lacked the test component. Each reaction was set to be repeated. The tube was incubated at 37°C in the dark for 5 days with shaking at 450rpm. After incubation, aliquots were transferred to black 96-well plates. Each reaction tube was aliquoted into 3 wells, 290 μL per well, and the analysis was repeated three times. Total fluorescence intensity was measured using excitation and maximum emission at 360 nm and 460 nm, respectively.

[0034] Cell protection from oxidative stress: IMG cells were grown to confluence and pretreated with test compounds at 37°C for 24 hours. The culture medium was removed and the cells were exposed to hydrogen peroxide for 1 hour at 37°C. After one hour, the H2O2 was removed and the cells were incubated for another 24 hours at 37°C in DMEM supplemented with 1% BSA to stabilize them. Subsequently, Alamar Blue cell viability reagent (obtained from Thermofisher (MA, USA)) was used to evaluate cell viability. Alamar Blue reagent is the oxidized form of a blue redox indicator. When incubated with live cells, the color of the reagent changes from blue to red and can be measured by absorbance at 570nm.

[0035] Mitochondrial biogenesis: MitoBiogenesis TM In-Cell ELISA kit (Colorimetric) was purchased from Abcam (Cambridge, UK). H9c2 cells were grown to fusion in 96-well plates and treated with test components at 37°C for 24 hours. The culture medium was removed and the cells were washed with phosphate buffered saline provided in the kit. The cells were then fixed and treated according to the protocol provided in the kit. The experiment was used to quantify two mitochondrial proteins: subunit I (COX-I) of complex IV and the 70kDa subunit (SDH-A) of complex II. No treatment control was used to show the relative protein levels in each case.

[0036] Natural compounds promote reversal of glycation: To date, there are no available therapies that can break down or repair fully formed AGEs. Our research focuses on a fundamental biological process in protein glycation involving Nε-(carboxymethyl)lysine (CML) as an AGE derived from lysine. The presence of CML is associated with the progression of various diabetic complications and some neurodegenerative diseases. CML is one of the most abundant AGEs found in the renal compartment and is associated with loss of renal function in chronic kidney disease.

[0037] Previous studies have shown that MnmC, an enzyme involved in the bacterial tRNA-modification pathway, is able to revert carboxymethyl-lysine (CML) back to the native lysine structure. Here, we applied various natural compounds and their mixtures to study their efficacy in reverting CML to native lysine. Figure 3 Reversion of CML to native lysine in the presence of individual compounds and their combinations (mixture and core) is shown.

[0038] Figure 3The results in show that the selection of two different concentrations of the natural compounds tested individually had no significant effect on the release of free lysine from CML. However, their combination in a mixture significantly increased the process in a concentration-dependent manner. Compared with the control, the application of a mixture with a concentration of 22 mg / ml (containing each individual compound at a dose of 2 mg / ml) resulted in about 4 times higher release of free lysine. In the presence of a mixture of 440 mg / ml (containing each individual compound at a dose of 40 mg / ml), the increase was even higher, increasing by about 10 times. The compound subset (core) administered at 12 mg / ml (with a dose of 2 mg / ml of each component) also effectively increased the CME reversal process by about 500% compared to the control group.

[0039] Natural compounds prevent protein glycation: Figure 4 and Figure 5 The results in 2014 showed that incubation of BSA in the presence of glucose and fructose increased the formation of AGE-BSA, with glucose ( Figure 4 ) compared to fructose ( Figure 5 ) caused more protein glycation. The individual ingredients showed different efficacy in preventing AGE-BSA formation. The highest efficacy in preventing the glycation process was observed when the ingredients were applied as a mixture or core combination. Thus, in glucose-driven glycation, the core combination effectively prevented 54% of AGE-BSA, and the mixture effectively prevented 64% ( Figure 4 ). Fructose-promoted BSA glycation was inhibited by 57% by the core combination and by 73% by the mixture ( Figure 5 ).

[0040] Natural compounds promote mitochondrial biogenesis: Mitochondrial metabolism is the main source of bioenergy, as most of the cellular ATP is produced in the mitochondria. The cellular mitochondrial pool is inherited from our mothers and can only be increased by the growth and fission of pre-existing mitochondria. Both high mitochondrial activity and its number are important in our body metabolism, in supporting heart and skeletal muscle function, controlling diabetes, aging and many chronic diseases. Mitochondria are the master regulators of insulin secretion, and their dysfunction is associated with the pathogenesis of insulin resistance, a hallmark of type 2 diabetes.

[0041] Mitochondrial biogenesis can be assessed by changes in two mitochondrial enzymes: succinate dehydrogenase (SDH), which forms part of mitochondrial complex IIA, and cyclooxygenase-1 (Cox-1), which is present in mitochondrial complex IV. We evaluated the effects of various natural compounds and their combinations (as mixtures and cores) on SDH and COX-1 levels as a measure of mitochondrial biogenesis in cardiomyocytes. Figure 6 and Figure 7 Available in.

[0042] Figure 6 The results in the study showed that among the individual compounds, sodium pyruvate, pantothenic acid, magnesium citrate and magnesium malate had the most pronounced stimulatory effect on SDH in cardiomyocytes, increasing its levels from 54% (pyruvate) to 86% (magnesium malate) compared to the control. The highest stimulatory effect on SDH was observed when all tested ingredients were combined as a mixture, with an increase of 118%. The nuclear component stimulated SDH by 68% compared to the control. Cox-1 levels in cardiomyocytes were increased by 52-58% in the presence of nicotinamide, magnesium malate and methionine, and by 41% in the presence of cysteine ​​compared to the control. Figure 7 ). The highest stimulation of Cox1 levels was achieved in the presence of the mixture (79%), whereas the nucleus had a more moderate effect of 42% increase.

[0043] Natural compounds protect glial cells from oxidative damage: Oxidative stress plays a key role in the development of diabetic complications, including diabetes-specific pathologies in the peripheral nerves, retina, glomeruli, and accelerated atherosclerosis affecting the arteries that supply blood to the heart, brain, and lower extremities. Oxidative stress is also associated with the development of cancer, neurodegenerative diseases (Alzheimer's and Parkinson's) ALS, lung diseases, and various allergies.

[0044] We tested the protective effects of individual natural compounds and their combinations in microglia exposed to oxidative stress generated by hydrogen peroxide.

[0045] Figure 8 The results in show that the survival rate of cells exposed to hydrogen peroxide is increased in the presence of the individual components tested. Glial cells exposed to H2O2 in the presence of magnesium citrate, magnesium malate and methionine survived about 50%. Other test compounds can protect cells in the range of 5% to 30%. When these nutrients are combined as cores and mixtures, the highest cell protection against oxidative stress (about 70% of viable cells) is achieved. The control is a cell that is not treated with any component and is not exposed to hydrogen peroxide, and H2O2 represents the survival rate of cells exposed to hydrogen peroxide.

[0046] Pharmaceutical formulations suitable for these routes of administration can be prepared in the following manner: one or more pharmaceutically acceptable carriers are added to the reagent, and then the micronutrient composition is processed by conventional processes known to those skilled in the art. Modes of administration include, but are not limited to, non-invasive oral, topical (e.g., transdermal), enteral, transmucosal, targeted delivery, sustained release delivery, delayed release, pulse release, and parenteral methods. Oral administration can be administered in both liquid and dry states. In one embodiment, the micronutrient composition will more specifically be a mixture and a core. The pharmaceutical composition comprises: alpha-ketoglutaric acid 10mg-30000mg, nicotinamide or nicotinic acid 1mg-10000mg, L proline 10mg-30000mg, L glutamine 10mg-50000mg, L citrulline 10mg-20000mg, sodium pyruvate 10mg-50000mg, pantothenic acid 0.1mg-200mg, L cysteine ​​or N-acetyl cysteine ​​10mg-20000mg, magnesium malate 10mg-20000mg, magnesium citrate 10mg-30000mg, and methionine 10mg-20000mg, and is used as a nutritional supplement composition or a pharmaceutical composition. There is a method for preparing a micronutrient composition mixture and a core, the method comprising the steps of mixing the ingredients of the micronutrient composition and optionally formulating the micronutrient composition.

[0047] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base such as gelatin and glycerin or sucrose and acacia), each containing a predetermined amount of the subject composition as the active ingredient. The subject composition may also be administered as a pill, ointment or paste.

[0048] When preparing oral solid pharmaceutical products, the micronutrient composition is mixed with an excipient (and, if necessary, one or more additives, such as a binder, a disintegrant, a lubricant, a colorant, a sweetener and a flavoring agent), and the resulting mixture is processed by conventional methods to produce an oral solid pharmaceutical product, such as a tablet, a coated tablet, a granule, a powder or a capsule. The additive can be those commonly used in the art. Examples of excipients include lactate, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose and silicic acid. Binders include water, ethanol, propanol, simple syrup, glucose solution, starch solution, liquefied gelatin, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate and polyvinyl pyrrolidone. Disintegrants include dry starch, sodium arginate, powdered agar, sodium bicarbonate (sodium hydroxy carbonate), calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride and lactose. Lubricants include refined talc, stearates, borax and polyethylene glycol. Sweeteners include sucrose, orange peel, citric acid and tartaric acid.

[0049] When preparing a liquid drug product for oral administration, the micronutrient composition is mixed with an additive (such as a sweetener, a buffer, a stabilizer or a flavoring agent), and the resulting mixture is processed by a conventional method to produce a liquid drug product for oral administration, such as an internal solution, a syrup or an elixir. Examples of sweeteners include vanillin; examples of buffers include sodium citrate; and examples of stabilizers include tragacanth, gum arabic and gelatin.

[0050] For transdermal (eg, topical) administration, dilute sterile aqueous or partially aqueous solutions (generally about 0.1% to 5% concentration) may be prepared with the micronutrient compositions, otherwise similar to the parenteral solutions described above.

[0051] Formulations containing the micronutrient composition for rectal or vaginal administration can be provided as suppositories, which can be prepared by mixing the subject composition with one or more suitable non-irritating carriers (including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate), which are solid at room temperature but liquid at body temperature, and thus melt in a suitable body cavity and release the encapsulated one or more compounds and one or more compositions. Formulations suitable for vaginal administration also include vaginal suppositories, tampons, creams, gels, ointments, foams or spray formulations containing such carriers as are known in the art.

[0052] The targeted release portion for capsules containing the micronutrient composition can be incorporated into the delayed release system by applying an immediate release layer over the delayed release core; using a coating or compression process, or in a multi-unit system, such as a capsule containing delayed release beads and immediate release beads.

[0053] When used in relation to micronutrient compositions, the term "slow release" is art-recognized. For example, a therapeutic composition that releases a substance over time may exhibit a slow release profile compared to a bolus administration in which the entire amount of the substance is made bioavailable at once. In specific embodiments, upon contact with body fluids (including blood, cerebrospinal fluid, mucous secretions, lymph, etc.), one or more pharmaceutically acceptable excipients may undergo gradual or delayed degradation (e.g., by hydrolysis), thereby concomitantly releasing any material incorporated therein, such as therapeutic and / or biologically active salts and / or compositions, over a sustained or extended period of time (compared to release from a bolus). This release may result in extended delivery of a therapeutically effective amount of any therapeutic agent disclosed herein.

[0054] Current efforts in the field of drug delivery include developing targeted delivery, in which the drug is active only in target areas of the body (e.g., mucous membranes, such as in the nasal cavity), and sustained-release formulations, in which the micronutrient composition is released from the formulation in a controlled manner over a period of time. Types of sustained-release formulations include liposomes, drug-loaded biodegradable microspheres, and micronutrient composition polymer conjugates.

[0055] Delayed release dosing formulations are produced by coating solid dosage forms with a film of a polymer, which is insoluble in the acidic environment of the stomach, but soluble in the neutral environment of the small intestine. Delayed release dosage units can be prepared, for example, by coating micronutrient compositions with selected coating materials. Micronutrient compositions can be tablets for incorporation into capsules, tablets for use as kernels in "coated core" dosage forms, or multiple drug-containing beads, particles or granules for incorporation into tablets or capsules. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble and / or enzymatically degradable polymers, and can be conventional "enteric" polymers. As will be appreciated by those skilled in the art, enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract, or slowly erode when the dosage form passes through the gastrointestinal tract, and enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Alternatively, delayed release tablets can be prepared by dispersing the drug in a matrix of a suitable material (such as a hydrophilic polymer or a fatty compound). Suitable hydrophilic polymers include, but are not limited to, cellulosic polymers or copolymers, cellulose esters, acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate and vinyl or enzyme-degradable polymers or copolymers as described above. These hydrophilic polymers are particularly useful for providing a delayed release matrix. The fatty compound used as matrix material includes, but is not limited to, wax (e.g., carnauba wax) and tristearin. Once the active ingredient is mixed with the matrix material, the mixture can be compressed into tablets.

[0056] Pulsatile release dosage forms are dosage forms that simulate multiple dosing profiles without the need for repeated dosing and typically allow for at least a two-fold reduction in dosing frequency compared to drugs provided as conventional dosage forms (e.g., as solutions or conventional solid dosage forms for immediate drug release). Pulsatile release profiles are characterized by periods of no release (lag time) or reduced release followed by rapid drug release. These can be formulated for critically ill patients using the micronutrient compositions of the present invention.

[0057] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical, such as injection, and include, but are not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.

[0058] Certain micronutrient compositions suitable for parenteral administration disclosed herein comprise a combination of one or more of the subject compositions with one or more pharmaceutically acceptable sterile, isotonic, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which may be reconstituted into a sterile injectable solution or dispersion just prior to use, and which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic in the blood of the intended recipient, or suspending or thickening agents.

[0059] When preparing an injection product, the micronutrient composition is mixed with an additive (such as a pH adjuster, a buffer, a stabilizer, an isotonic agent or a local anesthetic), and the resulting mixture is treated by a conventional method, thereby producing an injection for subcutaneous injection, intramuscular injection or intravenous injection. Examples of pH adjusters or buffers include sodium citrate, sodium acetate and sodium phosphate; examples of stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid; examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride; and examples of isotonic agents include sodium chloride and glucose.

[0060] Adjuvants can be used to enhance the immune response. Various types of adjuvants are available. Haptens and Freund's adjuvants can also be used to produce water-in-oil emulsions of the immunogen.

[0061] The phrase "pharmaceutically acceptable" is recognized in the art. In some embodiments, the term includes compositions, polymers and other materials and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals (both humans and animals) without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit-risk ratio.

[0062] The phrase "pharmaceutically acceptable carrier" is art-recognized and includes, for example, a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material, which is involved in carrying or transporting any subject composition from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient. In some embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution solution); (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0063] In certain embodiments, the micronutrient compositions described herein are formulated such that the composition will be delivered to a mammal in a therapeutically effective amount as part of a prophylactic, preventative, or therapeutic treatment to overcome an infection caused by a coronavirus (regardless of the type).

[0064] In certain embodiments, the dosage of the micronutrient composition (which may be referred to as the therapeutic composition provided herein) can be determined by reference to the plasma concentration of the therapeutic composition or other encapsulated materials. For example, blood samples can be tested to learn their immune response to their corresponding viral load or its lack of viral load.

[0065] The therapeutic micronutrient compositions provided herein can be administered to a subject in need of treatment by various conventional routes of administration, including oral, topical, parenteral, such as intravenous, subcutaneous or intramedullary. In addition, the therapeutic compositions can be administered intranasally, as rectal suppositories, or using a "flash" formulation (i.e., dissolving the drug in the mouth without the use of water). In addition, the compositions can be administered to a subject in need of treatment by controlled release formulations, site-specific drug delivery, transdermal drug delivery, patch-mediated drug delivery (active / passive), by stereotactic injection, or in nanoparticles.

[0066] The active ingredient may be present in the therapeutic compositions of the present invention in concentrations intended for topical administration to the skin, intranasally, pharynx, bronchially, intravaginally, rectally or to the eye.

[0067] For use as an aerosol, the active ingredient can be packaged in a pressurized aerosol container together with a gaseous or liquefied propellant (e.g., dichlorodifluoromethane, carbon dioxide, nitrogen, propane, etc.) and conventional adjuvants (e.g., co-solvents and wetting agents) as may be desired or required. The most commonly used routes of administration also include preferred transmucosal (nasal, oral / sublingual, vaginal, ocular and rectal) and inhalation routes.

[0068] In addition, in certain embodiments, the subject micronutrient composition of the present application can be lyophilized or subjected to other suitable drying techniques such as spray drying. The subject composition can be administered once, or can be divided into multiple smaller doses to be administered at different time intervals, which depends in part on the release rate of the composition and the desired dosage.

[0069] Preparations that can be used for the methods provided herein include those suitable for oral, nasal, topical (including oral and sublingual), rectal, vaginal, aerosol and / or parenteral administration. Preparations can be conveniently provided in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of the subject micronutrient composition that can be combined with a carrier material to produce a single dose can vary according to the subject being treated and the specific mode of administration. The physiological dosage level consumed by mammals is calculated based on various factors, including the type of administration, species dependence and mode of action, such as transdermal relative to oral. The disclosed range includes those factors and scientific calculations. Depending on the formulation and type, the range may also vary within the range. Pharmaceutical preparations suitable for these routes of administration can be prepared in the following manner: one or more pharmaceutically acceptable carriers are added to the reagent, and then the micronutrient composition is processed by conventional processes known to those skilled in the art. Modes of administration include, but are not limited to: non-invasive oral, topical (e.g., transdermal), enteral, transmucosal, targeted delivery, sustained release delivery, delayed release, pulse release and parenteral methods. Oral administration can be administered in both liquid and dry states.

[0070] The acceptable amount of treatment described herein can be administered in an inhalation or aerosol formulation. An inhalation or aerosol formulation can include one or more agents, such as adjuvants, diagnostic agents, imaging agents, or therapeutic agents that can be used for inhalation therapy. Relative to the gross weight of the formulation, the final aerosol formulation can, for example, contain 0.005-90% w / w, for example 0.005-50%, 0.005-5% w / w, or 0.01-1.0% w / w of the drug.

[0071] Examples of suitable aqueous and non-aqueous carriers that can be used in the micronutrient compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

Claims

1. A micronutrient composition, comprising: comprising or consisting of at least 6 ingredients, such as alpha-ketoglutarate, niacinamide and / or niacin, L-proline, L-glutamine, L-citrulline and sodium pyruvate; Preferably, it comprises or consists of at least 11 ingredients, such as alpha-ketoglutarate, nicotinamide and / or nicotinic acid, L-proline, L-glutamine, L-citrulline, sodium pyruvate, pantothenic acid, L-cysteine ​​and / or N-acetylcysteine, magnesium malate, magnesium citrate and methionine; More preferably, it comprises or consists of all of the following ingredients, wherein the all ingredients are selected from the group consisting of alpha-ketoglutaric acid, nicotinamide and / or nicotinic acid, L-proline, L-glutamine, L-citrulline, sodium pyruvate, pantothenic acid, L-cysteine ​​and / or N-acetylcysteine, magnesium malate and / or magnesium citrate, methionine and / or S-adenosyl-methionine (SAMe), methylsulfonylmethane (MSM) and wormwood powder and / or extract, The micronutrient composition optionally further contains one or a combination of a pharmaceutically acceptable carrier, or an excipient, or a liquefied propellant, or a buffer, or a pH adjuster, or a stabilizer, or a coating, or a flavoring agent, or a formulation adjuvant. The micronutrient composition is optionally formulated as a tablet, coated tablet, capsule, pill, intranasal, lozenge, emulsion, troche, suppository, ointment or injectable solution.

2. The micronutrient composition according to claim 1, wherein If present, the following amounts of the ingredients are contained: alpha-ketoglutaric acid 10 mg-30,000 mg, niacinamide and / or niacin 1 mg-10,000 mg, L-proline 10 mg-30,000 mg, L-glutamine 10 mg-50,000 mg, L-citrulline 10 mg-20,000 mg, sodium pyruvate 10 mg-50,000 mg, pantothenic acid 0.1 mg-200 mg, L-cysteine ​​and / or N-acetylcysteine ​​10 mg-20,000 mg, magnesium malate 10 mg-20,000 mg, magnesium citrate 10 mg-30,000 mg, methionine 10 mg-20,000 mg, S-adenosyl-methionine (SAMe) 1 mg-20,000 mg, methylsulfonylmethane (MSM) 1 mg-20,000 mg, and wormwood powder and / or extract 1 mg-10,000 mg.

3. The micronutrient composition according to claim 1, consisting of: α-ketoglutaric acid, nicotinamide and / or nicotinic acid, L-proline, L-glutamine, L-citrulline, sodium pyruvate, pantothenic acid, L-cysteine ​​and / or N-acetylcysteine, magnesium malate and / or magnesium citrate and methionine, and optionally one or a combination of additional pharmaceutically acceptable carriers, or excipients, or liquefied propellants, or buffers, or pH adjusters, or stabilizers, or coatings, or flavoring agents.

4. The micronutrient composition according to one of claims 1 to 3, wherein If present, the following amounts of the ingredients are contained: alpha-ketoglutaric acid 10 mg-30,000 mg, niacinamide and / or niacin 1 mg-10,000 mg, L-proline 10 mg-30,000 mg, L-glutamine 10 mg-50,000 mg, L-citrulline 10 mg-20,000 mg, sodium pyruvate 10 mg-50,000 mg, pantothenic acid 0.1 mg-200 mg, L-cysteine ​​and / or N-acetylcysteine ​​10 mg-20,000 mg, magnesium malate 10 mg-20,000 mg, magnesium citrate 10 mg-30,000 mg, and methionine 10 mg-20,000 mg.

5. The micronutrient composition according to one of claims 1 to 3, wherein the dosage form is one oral capsule once a day, twice a day or three times a day.

6. The micronutrient composition according to one of claims 1 to 3, wherein the formulation adjuvant is selected from the group consisting of binders, disintegrants, lubricants, colorants, sweeteners, flavoring agents and mixtures thereof.

7. Use of the micronutrient composition according to one of claims 1 to 3 in a pharmaceutical composition.

8. A pharmaceutical composition comprising the micronutrient composition according to one of claims 1 to 3 and optionally a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, which is used for preventing or treating diabetes, cardiovascular disease, Alzheimer's disease or inflammation.

10. A process for preparing a micronutrient composition according to one of claims 1 to 3 by mixing the ingredients of the micronutrient composition according to one of claims 1 to 3 and optionally bringing the mixture into the desired dosage form.

11. The method according to claim 10, comprising: If present, the following amounts of the ingredients are mixed: alpha-ketoglutaric acid 10 mg-30,000 mg, niacinamide and / or niacin 1 mg-10,000 mg, L-proline 10 mg-30,000 mg, L-glutamine 10 mg-50,000 mg, L-citrulline 10 mg-20,000 mg, sodium pyruvate 10 mg-50,000 mg, pantothenic acid 0.1 mg-200 mg, L-cysteine ​​and / or N-acetylcysteine ​​10 mg-20,000 mg, magnesium malate 10 mg-20,000 mg, magnesium citrate 10 mg-30,000 mg, methionine 10 mg-20,000 mg, S-adenosyl-methionine (SAMe) 1 mg-20,000 mg, methylsulfonylmethane (MSM) 1 mg-20,000 mg, and wormwood powder and / or extract 1 mg-10,000 mg.

12. The method according to claim 10, comprising: The micronutrient composition is formulated as a tablet, coated tablet, capsule, pill, intranasal agent, lozenge, emulsion, troche, suppository, ointment or injectable solution.

13. A method for the pharmaceutical composition of claim 8, said method being carried out by mixing the ingredients of the pharmaceutical composition of claim 8, and optionally bringing the mixture into a desired dosage form.

14. The method according to claim 13, comprising: If present, the following amounts of the ingredients are mixed: alpha-ketoglutaric acid 10 mg-30,000 mg, niacinamide and / or niacin 1 mg-10,000 mg, L-proline 10 mg-30,000 mg, L-glutamine 10 mg-50,000 mg, L-citrulline 10 mg-20,000 mg, sodium pyruvate 10 mg-50,000 mg, pantothenic acid 0.1 mg-200 mg, L-cysteine ​​and / or N-acetylcysteine ​​10 mg-20,000 mg, magnesium malate 10 mg-20,000 mg, magnesium citrate 10 mg-30,000 mg, methionine 10 mg-20,000 mg, S-adenosyl-methionine (SAMe) 1 mg-20,000 mg, methylsulfonylmethane (MSM) 1 mg-20,000 mg, and wormwood powder and / or extract 1 mg-10,000 mg.

15. The method according to claim 13, comprising: The micronutrient composition is formulated as a tablet, coated tablet, capsule, pill, intranasal agent, lozenge, emulsion, troche, suppository, ointment or injectable solution.

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