WO2023027859A1 - Méthodes et compositions permettant d'améliorer la cicatrisation de plaies - Google Patents

Méthodes et compositions permettant d'améliorer la cicatrisation de plaies Download PDF

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Publication number
WO2023027859A1
WO2023027859A1 PCT/US2022/038620 US2022038620W WO2023027859A1 WO 2023027859 A1 WO2023027859 A1 WO 2023027859A1 US 2022038620 W US2022038620 W US 2022038620W WO 2023027859 A1 WO2023027859 A1 WO 2023027859A1
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hmb
exosome
enriched product
nutritional composition
bovine milk
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PCT/US2022/038620
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English (en)
Inventor
José María LÓPEZ PEDROSA
Ricardo Rueda Cabrera
Jorge GARCÍA MARTÍNEZ
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Abbott Laboratories
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Publication of WO2023027859A1 publication Critical patent/WO2023027859A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A23L33/28—Substances of animal origin, e.g. gelatin or collagen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to methods for improving wound healing in a subject.
  • the present invention also relates to nutritional compositions which employ beta- hydroxy- betamethylbutyrate (HMB), arginine, glutamine, and an exosome-enriched product comprising intact bovine milk-derived exosomes.
  • HMB beta- hydroxy- betamethylbutyrate
  • arginine arginine
  • glutamine glutamine
  • exosome-enriched product comprising intact bovine milk-derived exosomes.
  • Skin the body’s largest organ, acts as a barrier between the internal organs and the external environment. Its function is critical for maintaining health, as it prevents the penetration of pathogens and dehydration. Not only does skin represent a physical barrier, but it also has a regulatory role. Due to its permanent exposure to the external environment, skin is subjected to a continuous process of damage and renewal. In response to the different types of skin lesions which frequently occur, the human body has developed an efficient mechanism aimed at restoring the skin barrier function in order to prevent further damage and infection.
  • Wound healing is an extremely complex process that relies on the interaction of many cell types and mediators in a tightly regulated temporal and spatial sequence.
  • the interaction of platelets, macrophages, fibroblasts, epithelial cells, and endothelial cells with the extracellular matrix, growth inhibiting factors, cytokines, and chemokines occurs in a highly synchronized manner throughout four phases: hemostasis, inflammation, proliferation/migration and tissue remodeling.
  • Hemostasis occurs immediately after an injury. As the mechanism which leads to the cessation of bleeding from a damaged blood vessel, hemostasis involves multiple interlinked steps. In the initial stage of wound healing, vasoconstriction, platelet activation, and aggregation occur. During hemostasis, platelets release several signaling molecules, including cyclic AMP (cAMP), transforming growth factor-p (TGF-P), epidermal growth factor (EGF), and platelet- derived growth factor (PDGF). The role of platelets in hemostasis is crucial. Platelets comprise the majority of the initial clot and also release signaling molecules responsible for the activation and recruitment of fibroblasts, macrophages, and endothelial cells. PDGF is required for the activation and recruitment of fibroblasts which migrate to the wound site to begin the production of collagen and glycosaminoglycans.
  • cAMP cyclic AMP
  • TGF-P transforming growth factor-p
  • EGF epidermal growth factor
  • the next stage of wound healing is inflammation, which occurs within the first 24 hours after skin damage.
  • the inflammatory phase is characterized by the generation of a highly inflammatory environment through the secretion of several compounds, including pro- inflammatory cytokines, proteases, reactive oxygen species and growth factors. Macrophages play a crucial role during the inflammatory phase, as they are attracted to the wounded area and remove bacteria and dead tissue. As important as their phagocytic role is, macrophages also mediate the transition to the next phase in would healing. Activated macrophages release soluble mediators that initiate angiogenic processes and recruit and activate fibroblasts which will synthesize the new tissue matrix.
  • the proliferative phase begins.
  • soluble mediators released during the inflammatory phase promote migration of epithelial cells and fibroblasts to the damaged region, thereby supporting capillary growth and new tissue synthesis.
  • Fibroblasts are the key cells in the proliferative phase of wound healing.
  • fibroblasts settle down and begin to proliferate and synthesize collagen, fibronectin, proteoglycans, and glycosaminoglycans, which are the major constituents of the extracellular matrix (ECM).
  • ECM extracellular matrix
  • Fibroblasts not only produce ECM, but, together with macrophages, vascular endothelial cells, and epidermal cells, also secrete angiogenic signals.
  • granulation tissue a transitional replacement for normal dermis called granulation tissue is formed.
  • the newly synthesized, highly vascularized, tissue has significantly less tensile strength than unwounded tissue.
  • the granulation tissue is remodeled until its vascularization degree, matrix composition, and tissue tensile strength resemble those of healthy tissue. However, it has been reported that tensile strength is normally increased to a maximum of about 80% of normal tissue.
  • Acute wounds normally heal efficiently as they progress throughout the four overlapping phases described above.
  • the skin natural ability to self-regenerate after injury can be seriously compromised under certain circumstances.
  • altered wound healing capacity has been reported in patients suffering from deep burns, massive skin loss, non-healing ulcers, surgical interventions, and prolonged immobilization.
  • Altered wound healing capacity has also been reported in patients suffering from conditions such as diabetes, neuropathy, and/or vascular disease. In such patients, the healing process is uncoordinated, incomplete, and prolonged, which results in a poor functional outcome.
  • skin lesions can enter into a chronic state.
  • Chronic wounds are characterized by the disruption of the normal regeneration process, with increased risk of infection and complicated, slow healing.
  • fibroblasts-focused therapies constitute an area of increased research interest.
  • Current approaches include delivering viable fibroblasts directly to the wound site or the application of growth factors (e.g., PDGF, TGF-P) to stimulate fibroblast division or activity.
  • growth factors e.g., PDGF, TGF-P
  • Chronic wounds are particularly common in the elderly, with up to 3% of the population over the age of 65 being affected. Thus, chronic wounds not only represent a painful condition profoundly affecting patients’ mobility and quality of life, but also a substantial economic burden on the healthcare system.
  • methods for improving wound healing are desirable, including, but not limited to, methods for healing wounds such as deep burns, massive skin loss, non-healing ulcers, surgical interventions, radiotherapy-induced injuries, and/or other acute or chronic wounds.
  • methods for healing wounds such as deep burns, massive skin loss, non-healing ulcers, surgical interventions, radiotherapy-induced injuries, and/or other acute or chronic wounds.
  • a nutritional intervention that can help improve wound healing is also desirable.
  • the present invention is directed to a nutritional composition, comprising HMB, arginine, glutamine, and an exosome-enriched product comprising intact bovine milk-derived exosomes.
  • the invention is directed to a method of improving wound healing in a subject, comprising administering HMB, arginine, glutamine, and an exosome- enriched product comprising intact bovine milk-derived exosomes to the subject in need thereof.
  • the invention is also directed to a method of improving wound healing in a subject, comprising administering a nutritional composition comprising HMB, arginine, glutamine, and an exosome- enriched product comprising intact bovine milk-derived exosomes to the subject in need thereof.
  • FIG. 1 illustrates the proliferation activity of CCD1064Sk human fibroblast cells incubated with, respectively, (1) increasing amounts of an exosome-enriched product containing intact bovine milk-derived exosomes, (2) HMB, arginine, and glutamine, and (3) increasing amounts of an exosome-enriched product containing intact bovine milk-derived exosomes, combined with HMB, arginine, and glutamine, as described in Example 2.
  • FIG. 2 illustrates the results of an in vitro wound closure simulation for evaluating the migration capacity of CCD1064Sk human fibroblast cells incubated with, respectively, (1) an exosome-enriched product containing intact bovine milk-derived exosomes, (2) an exosome- enriched product containing sonicated bovine milk-derived exosomes, (3) HMB, arginine, and glutamine, (4) a combination of an exosome-enriched product containing intact bovine milk- derived exosomes with HMB, arginine, and glutamine, and (5) a combination of sonicated bovine milk-derived exosomes with HMB, arginine, and glutamine, as described in Example 3.
  • FIG. 2 illustrates the results of an in vitro wound closure simulation for evaluating the migration capacity of CCD1064Sk human fibroblast cells incubated with, respectively, (1) an exosome-enriched product containing intact bovine milk-derived exosomes, (2) an exosome- enriched product containing sonicated
  • FIG. 3 illustrates representative micrographs of a wound healing assay for evaluating the migration capacity of CCD1064Sk human fibroblast cells incubated with, respectively, (1) HMB, arginine, and glutamine, (2) an exosome-enriched product comprising intact bovine milk- derived exosomes, and (3) a combination of an exosome-enriched product comprising intact bovine milk-derived exosomes with HMB, arginine, and glutamine, as described in Example 3.
  • compositions described in the present disclosure can comprise, consist of, or consist essentially of any of the elements and steps as described herein.
  • nutritional product and “nutritional composition” as used herein, unless otherwise specified, refer to nutritional liquids and nutritional powders, the latter of which may be reconstituted or otherwise mixed with a liquid in order to form a nutritional liquid, and are suitable for oral consumption by a human.
  • improved wound healing refers to obtaining an improved wound healing metric as compared with wound healing in the absence of the present methods and/or compositions.
  • improved wound healing comprises improving fibroblast proliferation and/or improving fibroblast migration.
  • improved wound healing comprises increased wound closure and/or a reduction in the amount of time needed to complete wound healing.
  • a method for promoting wound healing in a subject comprises administering HMB, arginine, glutamine, and an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject in need thereof.
  • the nutritional composition comprises HMB, arginine, glutamine, and an exosome-enriched product comprising intact bovine milk-derived exosomes.
  • a method for promoting wound healing in a subject comprises administering a nutritional composition comprising HMB, arginine, glutamine, and an exosome-enriched product comprising intact bovine milk-derived exosomes, as described herein, to the subject in need thereof.
  • HMB refers to beta- hydroxy- betamethyl butyrate (also referred to as beta-hydroxyl-3-methyl butyric acid, beta-hydroxy isovaleric acid) and sources thereof. All weights, percentages, and concentrations as used herein to characterize HMB are based on the weight of HMB, regardless of source, unless otherwise specified.
  • calcium HMB refers to the calcium salt of beta-hydroxy-beta-methylbutyrate (also referred to as beta-hydroxyl-3-methyl butyric acid, beta-hydroxy isovaleric acid, or HMB), which is most typically in a monohydrate form. All weights, percentages, and concentrations as used herein to characterize calcium HMB are based on the weight of calcium HMB monohydrate, unless otherwise specified.
  • HMB is a naturally occurring amino acid metabolite that is known for use in a variety of nutritional products and supplements.
  • HMB is a metabolite of the essential amino acid leucine and has been shown to modulate protein turnover and inhibit proteolysis. While HMB is commonly used in nutritional products to help build or maintain healthy muscle in selected individuals, and a combination of HMB, arginine and glutamine have been found to improve wound healing in certain individuals, the present inventors have surprisingly discovered that HMB, in combination with arginine, glutamine, and an exosome-enriched product comprising intact bovine milk-derived exosomes, further promote wound healing to a surprising extent.
  • HMB any source of HMB is suitable for use in the methods and nutritional compositions of the invention.
  • examples include HMB as the free acid, a salt, including an anhydrous salt or a hydrate salt, an ester, a lactone, or other product forms that otherwise provide a bioavailable form of HMB.
  • the source of HMB is selected from the group consisting of alkali metal HMB, alkaline earth metal HMB, HMB free acid, HMB lactone and combinations of two or more thereof.
  • the source of HMB is selected from the group consisting of sodium HMB, potassium HMB, magnesium HMB, chromium HMB, calcium HMB and combinations of two or more thereof, or, more specifically, the HMB is calcium HMB monohydrate.
  • the methods and nutritional compositions of the present invention also employ an exosome-enriched product comprising intact bovine milk-derived exosomes.
  • an exosome-enriched product comprising intact bovine milk-derived exosomes.
  • exosome-enriched product comprising intact bovine milk-derived exosomes refers to a product in which intact exosomes have been substantially separated from other bovine milk components such as lipids, cells, and debris, and are concentrated in an amount higher than that found in bovine milk. Exosomes are small, extracellular vesicles and account for a minor percentage of milk’s total content.
  • the exosome-enriched product is provided in a liquid form or a powdered form and also contains co-isolated milk solids.
  • intact exosomes refers to exosomes in which the vesicle membrane is not ruptured and/or otherwise degraded and the endogenous cargo, i.e. , the bioactive agents, therapeutics (e.g. miRNA), and/or other biomolecules which are inherently present in a bovine milk-derived exosome, are retained therein in active form.
  • the endogenous cargo i.e. , the bioactive agents, therapeutics (e.g. miRNA), and/or other biomolecules which are inherently present in a bovine milk-derived exosome, are retained therein in active form.
  • the exosome-enriched product comprises at least 0.001 wt % exosomes. In other specific embodiments of the methods and nutritional compositions of the invention, the exosome- enriched product comprises at least about 0.001 wt %, 0.01 wt %, 1 wt %, 5 wt %, 10 wt %, 15 wt %, 20 wt %, 25 wt %, 30 wt %, 35 wt %, 40 wt %, 45 wt %, or 50 wt % exosomes.
  • the exosome-enriched product comprising intact bovine milk-derived exosomes comprises at least 10 wt % exosomes. In further embodiments, the exosome-enriched product comprises at least about 10 8 exosomes per gram of the exosome-enriched product as measured by a nanotracking procedure.
  • nanoparticle tracking analysis can be used to determine exosome diameter and concentration. The principle of NTA is based on the characteristic movement of nanosized particles in solution according to the Brownian motion. The trajectory of the particles in a defined volume is recorded by a camera that is used to capture the scatter light upon illumination of the particles with a laser. The Stokes-Einstein equation is used to determine the size of each tracked particle. In addition to particle size, this technique also allows determination of particle concentration.
  • the exosome-enriched product comprises from about 10 8 to about 10 14 exosomes per gram of the exosome-enriched product. In yet a more specific embodiment, the exosome-enriched product comprises from about 10 9 to about 10 13 exosomes per gram of the exosome-enriched product. In another specific embodiment, the exosome- enriched product contains at least about a three-fold increase in the number of exosomes, as compared to a raw whey-containing bovine milk fraction. In a more specific embodiment, the exosome-enriched product contains a 3-fold to 50-fold increase in the number of exosomes, as compared to a raw whey-containing bovine milk fraction, for example cheese whey.
  • At least about 50 wt % of the exosomes in the exosome-enriched product are intact. In further specific embodiments of the methods and nutritional compositions of the invention, at least about 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt % of the exosomes in the exosome-enriched product are intact.
  • the intact bovine milk-derived exosomes are sourced from a whey-containing bovine milk fraction.
  • the whey-containing bovine milk fraction is cheese whey.
  • the exosomes are obtained from a whey-containing bovine milk fraction using gentle procedures which do not disrupt the exosome vesicle membrane, thereby leaving the exosomes intact and active bioactive agents contained within the exosome structure.
  • Various methods may be employed to isolate exosomes with care being exercised to avoid disruption of the lipid membrane.
  • Fresh bovine milk, refrigerated bovine milk, thawed frozen bovine milk, or otherwise preserved bovine milk, or any bovine milk fraction containing exosomes, for example, cheese whey, may be employed as a source of exosomes.
  • Isolating the exosomes may comprise performing the isolation immediately upon obtaining milk from a bovine.
  • isolating the exosomes may comprise performing the isolation within about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days or about 6 days, or about 7 days from the time of obtaining the milk from a bovine.
  • the exosomes may be isolated within about 10 days, or within about 14 days from the time of obtaining milk from a bovine. Additionally, the bovine milk may be frozen and then thawed for processing for isolating exosomes, with the bovine milk preferably having been frozen within about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days or about 6 days, or about 7 days from the time of obtaining the milk from a bovine. Thawed milk is preferably processed immediately upon thawing.
  • the fresh bovine milk may be subjected to the processing within about 5 days of obtaining the milk from a bovine, or thawed bovine milk which is subjected to processing is thawed from bovine milk that was frozen within about 5 days of obtaining the milk from a bovine.
  • a whey-containing bovine milk fraction or, specifically, cheese whey may serve as a source of exosomes.
  • Cheese whey is the liquid by-product of milk after the formation of curd during the cheese-making or casein manufacturing process. Since cheese whey has already been separated from the casein fraction during the cheese manufacture process, cheese whey has very low casein content. Furthermore, cheese whey advantageously retains more than 50% of milk nutrients, including lactose, fat, proteins, mineral salts, and, surprisingly, a significant number of exosomes that were originally present in the milk in intact form.
  • cheese whey is less expensive than raw milk, and thus using cheese whey as a starting material significantly reduces costs for production of an exosome- enriched product.
  • cheese whey is a novel and promising source for isolating milk exosomes and producing exosome-enriched products.
  • the cheese whey is obtained by applying an enzyme or enzyme mixture, and more specifically a protease enzyme, for example chymosin, to milk to hydrolyze casein peptide bonds, thus allowing for enzymatic coagulation of casein in the milk.
  • a protease enzyme for example chymosin
  • the protease enzyme cleaves the protein, it causes the casein in the milk to coagulate and form a gel structure.
  • the casein protein gel network and milk fat then contract together and form curd.
  • the resulting liquid that is separated from the curd is often referred to as sweet whey or cheese whey, typically has a pH from about 6.0 to about 6.5, and comprises whey proteins, lactose, minerals, water, fat and other low level components.
  • the enzyme or enzyme mixture is capable of destabilizing the casein protein in the milk fraction by cleaving peptides which stabilize the casein protein in the milk. Therefore, any proteolytic enzyme suitable for this purpose may be used to obtain cheese whey.
  • the cheese whey is provided by adding rennet enzyme to bovine milk, resulting in enzymatic coagulation of casein.
  • Rennet enzyme is commonly used in the cheese making process and comprises a set of enzymes which are produced in the stomachs of ruminant mammals. These enzymes normally include chymosin, pepsin, and lipase.
  • the rennet enzyme mix destabilizes the casein protein in the bovine milk fraction by proteolytically cleaving peptides which stabilize the protein in the milk.
  • the casein in the milk coagulates and contracts with milk fat to form the cheese curd.
  • the remaining liquid, i.e. , the sweet cheese whey comprises whey proteins, lactose, minerals, water, fat, and other low level components.
  • a gentle procedure of obtaining an exosome-enriched product containing intact bovine milk-derived exosomes may comprise physical methods and/or chemical methods.
  • an exosome-enriched product is obtained by cascade membrane filtration.
  • the exosome-enriched product is lactose-free.
  • sweet cheese whey which may be obtained as described in the preceding paragraph, is processed using tandem multiple ceramic filtration steps.
  • a multiple filtration process employs, successively, membranes with cut offs which gradually decrease in size.
  • the method of processing sweet cheese whey is subjected to microfiltration (MF), ultrafiltration (UF) and diafiltration (DF).
  • the process employs, successively, MF, UF and DF membranes with cut offs of about 1.4 pm, 0.14 pm and 10 kDa, respectively, to provide an exosome-enriched product.
  • a first MF step employs a first membrane with a molecular weight cut off of, for example, about 1.4 pm and yields a first retentate R1 and a first permeate P1.
  • the first permeate P1 is then subjected to a an UF step employing a second membrane with a molecular weight cut off of, for example, about 0.14 pm, which yields a second retentate R2 and second permeate P2.
  • the second retentate R2 may be re-suspended in water and again passed through the second membrane to remove additional lactose, minerals and the like, if desired. For example, in one embodiment, about 5 volumes of water may be added to one volume of the second retentate R2 and the resulting suspension is then passed through the 0.14 pm MF membrane. The resulting third retentate R3 is then subjected to a DF step using a 10 kDa membrane.
  • the third retentate is first diluted with an approximately equal volume of water and diafiltered to obtain a fourth retentate R4, and then the fourth retentate R4 is again diluted with water, for example with a volume of water five times that of the fourth retentate R4 and then diafiltered to yield a concentrated retentate R5.
  • This exosome- enriched product may be used in the form of the concentrated retentate R5, or the concentrated retentate R5 may be further processed.
  • the exosome-enriched product resulting from successive filtration steps may be pasteurized to provide storage stability.
  • the exosome- enriched product may be heated, for example, at about 70°C for about 15 seconds, to ensure microbiological stability in order to yield a pasteurized fraction, R6.
  • Other pasteurization conditions will be apparent to those skilled in the art and may be employed.
  • the exosome-enriched product may be used as is or subjected to additional processing steps to provide a desired physical form.
  • the exosome-enriched product optionally pasteurized, may be converted to a powder form.
  • the exosome-enriched product can be spray-dried, freeze dried, or otherwise converted to powder form.
  • the exosome-enriched product may be spray dried, for example, at 185°C/85°C, to obtain an exosome-enriched product in the form of a spray-dried powder (SP).
  • SP spray-dried powder
  • the exosome-enriched product Prior to spray drying, the exosome-enriched product may be subjected to an optional evaporation step to increase the solids content of the product and therefore reduce the time and/or energy demand for the spray drying process.
  • an optional evaporation step to increase the solids content of the product and therefore reduce the time and/or energy demand for the spray drying process.
  • Other spray drying conditions will be apparent to those skilled in the art and may be employed.
  • the exosome-enriched product may be freeze-dried, for example at -50°C and 0.5 mbar vacuum to obtain an exosome-enriched freeze-dried powder (FP).
  • FP exosome-enriched freeze-dried powder
  • Other freeze drying conditions will be apparent to those skilled in the art and may be employed.
  • the exosome-enriched product is administered in the form of an exosome-enriched powder.
  • the exosome-enriched product is administered in the form of an exosome- enriched liquid.
  • the exosome enriched product can be administered to the subject in either form.
  • the HMB, arginine, glutamine, and the exosome-enriched product comprising intact bovine milk-derived exosomes are administered to the subject orally.
  • the HMB, arginine, glutamine, and the exosome-enriched product comprising intact bovine milk-derived exosomes are provided in a nutritional composition, which can be administered to the subject orally.
  • the nutritional compositions may be formulated and administered in any known or otherwise suitable oral product form, so long as they include HMB, glutamine, arginine, and the exosome-enriched product comprising intact bovine milk-derived exosomes.
  • Any solid, liquid, semi-solid, semi-liquid or powder form, including combinations or variations thereof, are suitable for use in the methods described herein, provided that such forms allow for safe and effective oral delivery to the subject of the ingredients as also defined herein.
  • the nutritional composition is in the form of a nutritional powder.
  • nutritional powder refers to nutritional powders that are generally flowable particulates and that, in certain embodiments, are reconstitutable with an aqueous liquid, and which are suitable for oral administration to a human.
  • a serving size is from about 20 g to about 60 g, from about 25 g to about 55 g, or from about 45 g to about 50 g, to be administered as a powder or to be mixed or reconstituted in from about 1 ml to about 500 ml of liquid, for example water, juice, coffee or tea.
  • the nutritional composition is in the form of a nutritional liquid.
  • the HMB, arginine, glutamine, and exosome-enriched product comprising intact bovine milk-derived exosomes are provided as a nutritional powder and the nutritional powder is mixed in a liquid to form the nutritional liquid.
  • nutritional liquid refers to nutritional products made in ready-to-drink liquid form and to nutritional liquids made by reconstituting nutritional powders described herein prior to use.
  • a serving ranges from about 1 ml to about 500 ml, including from about 110 ml to about 500 ml, from about 110 ml to about 417 ml, from about 120 ml to about 500 ml, from about 120 ml to about 417 ml, from about 177 ml to about 417 ml, from about 207 ml to about 296 ml, from about 230 m to about 245 ml, from about 110 ml to about 237 ml, from about 120 ml to about 245 ml, from about 110 ml to about 150 ml, and from about 120 ml to about 150 ml.
  • the serving is about 1 ml, or about 100 ml, or about 225 ml, or about 237 ml
  • HMB, arginine, glutamine, and exosome-enriched product comprising intact bovine milk-derived exosomes may be administered to the subject for a time period of at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks, or at least 9 weeks, or at least 10 weeks, or at least 11 weeks, or at least 12 weeks, or at least 14 weeks, or at least 16 weeks, or at least 18 weeks, or at least 24 weeks or longer.
  • the HMB, arginine, glutamine, and exosome-enriched product comprising intact bovine milk-derived exosomes are administered to a subject once or multiple times daily or weekly.
  • the HMB, arginine, glutamine, and exosome-enriched product comprising intact bovine milk-derived exosomes are administered to the subject from about 1 to about 6 times per day or per week, or from about 1 to about 5 times per day or per week, or from about 1 to about 4 times per day or per week, or from about 1 to about 3 times per day or per week.
  • the HMB, arginine, glutamine, and exosome-enriched product comprising intact bovine milk- derived exosomes are administered once or twice daily for a period of at least one week, at least two weeks, at least three weeks, or at least four weeks.
  • the HMB, arginine, glutamine, and the exosome-enriched product comprising intact bovine milk-derived exosomes are administered to the subject at least once per day for a period of at least 2 weeks.
  • the desired amounts of HMB, glutamine, arginine, and the exosome-enriched product comprising intact bovine milk-derived exosomes may be administered in more than one serving or, more specifically, in two servings.
  • Each serving is desirably administered as a single, undivided dose, although the serving may also be divided into two or more partial or divided servings to be taken at two or more times during the day.
  • the methods of the present invention include continuous day after day administration, as well as periodic or limited administration, although continuous day after day administration is generally desirable.
  • the subject is administered from about 1 grams to about 20 grams per day of arginine.
  • the subject is administered from about 1 grams to about 10 grams, or from about 1 grams to about 5 grams, or from about 3 grams to about 20 grams, or from about 4 grams to about 20 grams, or from about 5 grams to about 20 grams, or from about 10 grams to about 20 grams, or from about 10 grams to about 15 grams, or from about 10 grams to about 14 grams per day of arginine.
  • the subject is administered from about 1 grams to about 20 grams per day of glutamine.
  • the subject is administered from about 1 grams to about 10 grams, or from about 1 grams to about 5 grams, or from about 3 grams to about 20 grams, or from about 4 grams to about 20 grams, or from about 5 grams to about 20 grams, or from about 10 grams to about 20 grams, or from about 10 grams to about 15 grams, or from about 10 grams to about 14 grams per day of glutamine.
  • the subject is administered from about 0.1 grams to about 10 grams per day of HMB. In further specific embodiments of the methods of the invention, the subject is administered from about 0.5 grams to about 3 grams, or from about 0.5 grams to about 1.5 grams, or from about 1 gram to about 5 grams, or from about 2 grams to about 9 grams, or from about 2 grams to about 8 grams, or from about 2 grams to about 7 grams, or from about 2 grams to about 6 grams, or from about 2 grams to about 5 grams, or from about 2 grams to about 4 grams, or from about 2 grams to about 3 grams per day of HMB.
  • the subject is administered from about 0.1 grams to about 30 grams per day of the exosome-enriched product comprising intact bovine milk-derived exosomes.
  • the subject is administered from about 0.1 grams to about 20 grams, or from about 0.1 grams to about 10 grams, or from about 0.5 grams to about 10 grams, or from about 0.5 grams to about 5 grams, or from about 5 grams to about 30 grams, or from about 10 grams to about 25 grams per day of the exosome-enriched product comprising intact bovine milk-derived exosomes.
  • the subject is administered from about 1 gram to about 20 grams per day of arginine, from about 1 gram to about 20 grams per day of glutamine, from about 0.1 to about 30 g per day of the exosome-enriched product comprising intact bovine milk-derived exosomes, and/or from about 0.1 gram to about 10 grams per day of HMB.
  • the subject is administered from about 5 grams to about 15 grams per day of arginine, from about 5 grams to about 15 grams per day of glutamine, from about 0.1 to about 10 grams per day of the exosome- enriched product comprising intact bovine milk-derived exosomes, and/or from about 1 gram to about 5 grams per day of HMB.
  • the nutritional composition comprises from about 0.1 to about 15 wt %, from about 1 to about 10 wt %, from about 1 to about 8 wt %, or from about 1 to about 5 wt % of HMB, based on the weight of the nutritional composition. In additional specific embodiments, the nutritional composition comprises from about 1 to about 6 wt % of HMB, based on the weight of the nutritional composition.
  • the nutritional composition comprises from about 0.1 to about 50 wt %, from about 0.1 to about 30 wt %, from about 10 to about 50 wt %, or from about 10 to about 30 wt % of arginine, based on the weight of the nutritional composition.
  • the nutritional composition is a liquid and comprises from about 10 to about 30 wt % of arginine, based on the weight of the nutritional composition.
  • the nutritional composition is a powder and comprises from about 20 to about 30 wt % of arginine, based on the weight of the nutritional composition.
  • the nutritional composition comprises from about 0.1 to about 50 wt %, from about 0.1 to about 30 wt %, from about 10 to about 50 wt %, or from about 10 to about 30 wt % of glutamine, based on the weight of the nutritional composition.
  • the nutritional composition is a liquid and comprises from about 10 to about 30 wt % of glutamine, based on the weight of the nutritional composition.
  • the nutritional composition is a powder and comprises from about 20 to about 30 wt % of glutamine, based on the weight of the nutritional composition.
  • the nutritional composition comprises from about 0.001 to about 30 wt %, from about 0.01 to about 30 wt %, from about 0.01 to about 20 wt %, from about 0.01 to about 10 wt %, from about 0.01 to about 5 wt %, from about 0.1 to about 30 wt %, from about 0.1 to about 20 wt %, from about 0.1 to about 10 wt %, from about 0.1 to about 5 wt %, from about 1 to about 30 wt %, from about 1 to about 20 wt %, from about 1 to about 10 wt %, or from about 1 to about 5 wt % of the exosome-enriched product comprising intact bovine milk-derived exosomes, based on the weight of the nutritional composition.
  • the nutritional composition comprises from about 0.01 to about 10 wt % of the exosome-enriched product comprising the intact bovine milk-derived exosomes, based on the weight of the nutritional composition.
  • the nutritional composition comprises from about 0.1 to about 15 wt %, or from about 1 to about 6 wt %, of HMB; from about 0.1 to about 50 wt %, or from about 10 to about 30 wt %, of arginine; from about 0.1 to about 50 wt %, or from about 10 to about 30 wt %, of glutamine; and from about 0.01 to about 20 wt %, or from about 0.1 to about 10 wt % of the exosome- enriched product comprising intact bovine milk-derived exosomes, all based on the weight of the nutritional composition.
  • the composition comprises from about 0.05 to about 7.5 g, or from about 0.5 g to about 3 g HMB; from about 0.05 g to about 25 g, or from about 5 g to about 15 g arginine; from about 0.05 g to about 25 g, or from about 5 g to about 15 g glutamine; and from about 0.005 to about 5 g, or from about 0.05 g to about 5 g of the exosome-enriched product comprising intact bovine milk-derived de exosomes.
  • such a powder serving of 50 g is added to about
  • the nutritional compositions further comprise one or more vitamins and/or trace minerals.
  • vitamins include vitamin A, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, niacin, folic acid, pantothenic acid, biotin, choline, inositol, and/or salts and derivatives thereof, and combinations thereof.
  • minerals and trace minerals include calcium, phosphorus, magnesium, zinc, manganese, sodium, potassium, molybdenum, chromium, iron, copper, and/or chloride, and combinations thereof.
  • the nutritional composition comprises one or more vitamins selected from the group consisting of vitamin B12, vitamin C, vitamin E, and combinations of two or more thereof.
  • Any vitamin E is suitable for use in the methods and compositions of the present invention, including, inter alia, d-alpha-tocopherol, dl-alpha-tocopherol, and gamma-tocopherol, and salts or esters thereof.
  • the vitamin E is dl-alpha-tocopheryl acetate.
  • the nutritional composition includes one or more minerals selected from the group consisting of zinc, calcium (to the extent the HMB source does not provide sufficient calcium), and combinations thereof.
  • the nutritional composition comprises vitamin C, vitamin, E, vitamin, B12, zinc, calcium, or a combination of two or more thereof.
  • the nutritional compositions further comprise collagen.
  • Any collagen protein that is suitable for use in nutritional compositions is contemplated for use in the present invention, including, inter alia, collagen from bones, skin, and connective tissue of animals, including cattle, fish, horses, pigs, or rabbits.
  • the nutritional compositions further comprise beef collagen, or, more specifically, hydrolyzed beef collagen.
  • the nutritional composition in addition to the protein, carbohydrate and fat contents provided by the HMB, amino acids, exosome-enriched product comprising intact bovine milk-derived exosomes or other components mentioned above, the nutritional composition further comprises additional sources of protein, carbohydrate, and/or fat.
  • additional sources of protein, carbohydrate, and/or fat can be used in embodiments of nutritional compositions described herein.
  • the nutritional composition includes additional sources of protein, carbohydrate and fat.
  • an additional source of protein in the nutritional composition comprises whey protein concentrate, whey protein isolate, whey protein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, organic milk protein concentrate, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, acid casein, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, nonfat dry milk, condensed skim milk, L-Carnitine, L-Lysine, taurine, lutein, rice protein concentrate, rice protein isolate, rice protein hydrolysate, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, meat protein, potato protein, chickpea protein, canola protein, mung protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax seed protein, earthworm protein, insect protein, or combinations of two or more thereof.
  • the nutritional composition may comprise additional protein source(s) in an amount from about 1 wt % to about 30 wt % of the nutritional composition. More specifically, the additional protein source(s) may be present in an amount from about 1 wt % to about 25 wt % of the nutritional composition, including from about 1 wt % to about 20 wt %, from about 2 wt % to about 20 wt %, from about 1 wt % to about 15 wt %, from about 1 wt % to about 10 wt %, from about 5 wt % to about 10 wt %, from about 10 wt % to about 25 wt %, or from about 10 wt % to about 20 wt % of the nutritional composition. Even more specifically, the additional protein source(s) comprise from about 1 wt % to about 5 wt % of the nutritional composition, or from about 20 wt % to about 30 wt % of the nutritional composition
  • the additional carbohydrate source(s) in the nutritional composition comprise fiber, maltodextrin, corn maltodextrin, corn syrup, organic corn maltodextrin, corn syrup, corn syrup solids, sucralose, cellulose gel, cellulose gum, gellan gum, carrageenan, fructooligosaccharides, inositol, maltodextrin, hydrolyzed starch, glucose polymers, rice-derived carbohydrates, sucrose, glucose, lactose, honey, sugar alcohols, isomaltulose, sucromalt, pullulan, potato starch, galactooligosaccharides, oat fiber, soy fiber, corn fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinoglactins, glucomanna
  • the nutritional composition may comprise additional carbohydrate source(s) in an amount from about 0.5 wt % to about 75 wt % of the nutritional composition. More specifically, the additional carbohydrate source(s) may be present in an amount from about 1 wt % to about 70 wt % of the nutritional composition, including from about 5 wt % to about 70 wt %, from about 5 wt % to about 65 wt %, from about 5 wt % to about 50 wt %, from about 5 wt % to about 40 wt %, from about 5 wt % to about 30 wt %, from about 5 wt % to about 25 wt %, from about 10 wt % to about 65 wt %, from about 20 wt % to about 65 wt %, from about 30 wt % to about 65 wt %, from about 40 wt % to about 65 wt %, from about 40 wt
  • fat and oil as used herein, unless otherwise specified, are used interchangeably to refer to lipid materials derived or processed from plants or animals. These terms also include synthetic lipid materials so long as such synthetic materials are suitable for oral administration to humans.
  • the additional fat source(s) comprise coconut oil, fractionated coconut oil, soy oil, soy lecithin, corn oil, safflower oil sunflower oil, palm olein, canola oil monoglycerides, lecithin, canola oil, medium chain triglycerides, one or more fatty acids such as linoleic acid, alpha-linolenic acid, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, medium chain triglyceride oil (MCT oil), high gamma linolenic (GLA) safflower oil, palm oil, palm kernel oil, marine oil, fish oil, algal oil, borage oil, cottonseed oil, fungal oil, interesterified oil, transesterified oil, structured lipid, omega-3 fatty acid, or combinations of two or more thereof.
  • fatty acids such as linoleic acid, alpha-linolenic acid, fractionated coconut oil, soy oil, corn oil,
  • the omega-3 fatty acid is selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, and alpha-linolenic acid, and combinations of two or more thereof.
  • concentration and relative amounts of the sources of protein, carbohydrate, and fat in the exemplary nutritional compositions can vary considerably depending upon, for example, the specific dietary needs of the intended user.
  • the nutritional composition may also comprise one or more components to modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition or serve as additional nutritional components.
  • additional components include preservatives, emulsifying agents (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharine, aspartame, acesulfame K, sucralose), colorants, flavorants, thickening agents, stabilizers, and so forth.
  • the nutritional composition has a neutral pH, i.e., a pH of from about 6 to 8 or, more specifically, from about 6 to 7.5. In more specific embodiments, the nutritional composition has a pH of from about 6.5 to 7.2 or, more specifically, from about 6.8 to 7.1.
  • the nutritional compositions may be formed using any techniques known in the art.
  • a powder product can be formed by dry blending ingredients.
  • the nutritional compositions may be formed by preparing an aqueous solution comprising protein and carbohydrate, preparing an oil blend comprising fat and oil-soluble components, and mixing together the aqueous solution and the oil blend to form an emulsified liquid nutritional composition.
  • the HMB, arginine, glutamine and exosome-enriched product comprising intact bovine milk-derived exosomes may be added at any time as desired in the process, for example, to the aqueous solution or to the emulsified blend.
  • the compositions may be spray-dried or otherwise dried, if a powder product is desired.
  • the nutritional compositions are subjected to a heat treatment which provides sterilization sufficient to maintain microbiological stability of the compositions over a desired shelf-life.
  • Example 1 Preparation and Characterization of Exosome-enriched Products
  • This example describes a method of preparing an exosome-enriched product from cheese whey.
  • the cheese whey was provided by adding rennet enzyme to bovine milk, resulting in enzymatic coagulation of casein and production of sweet cheese whey, as described above.
  • An exosome-enriched product containing from about 10 8 to about 10 14 intact bovine milk- derived exosomes per gram of the exosome-enriched product was prepared by cascade membrane filtration.
  • First, 1 ,000 L of sweet cheese whey was processed using tandem multiple ceramic filtration steps.
  • the first microfiltration MF step employed a membrane with a molecular weight cut off of 1.4 pm, which yielded a first retentate R1 and a first permeate P1.
  • the first permeate P1 was then subjected to a ultrafiltration step UF with a molecular weight cut off of 0.14 pm, which yielded a second retentate R2 and second permeate P2.
  • the lactose-free exosome-enriched product R5 was pasteurized at 70°C for 15 seconds to ensure microbiological stability in order to yield a pasteurized exosome-enriched product R6.
  • the pasteurized exosome-enriched product R6 was subjected to evaporation at about 65°C to increase the solids content up to 17-18% and then spray-dried at 185°C/85°C to obtain a exosome-enriched spray-dried product, SP.
  • Example 2 MTT Assay for Fibroblast Proliferation and Viability
  • This example demonstrates that a combination of an exosome-enriched product containing intact bovine milk-derived exosomes with HMB, arginine, and glutamine exhibits a synergistic improvement on human fibroblast proliferation over the administration of either the exosome-enriched product alone or HMB, arginine and glutamine in the absence of exosome- enriched product. This was shown by evaluating fibroblast proliferation through MTT assay of human dermal fibroblasts derived from normal male skin tissue.
  • MTT assay is a colorimetric method which measures the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide, MTT) to formazan, an insoluble crystalline product with a deep purple color. Only metabolically active cells are able to convert MTT to formazan. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means. Absorbance values greater than the control indicate cell proliferation and increased viability, while lower values suggest cell death or inhibition of proliferation.
  • MTT yellow tetrazolium salt
  • the CCD1064Sk human fibroblast cells were grown at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine and antibiotics (0.1 mg/mL penicillin) in 5% CO2 and 95% air.
  • DMEM Modified Eagle’s Medium
  • FBS fetal bovine serum
  • GABA fetal bovine serum
  • antibiotics 0.1 mg/mL penicillin
  • the CCD1064Sk human fibroblast cells were seeded in 48-well plates at a density of 15x10 3 cells/well. After 24 hours of incubation, the cells were treated, respectively, with (1) increasing concentrations (10 pg/mL, 12.5 pg/mL, 15 pg /mL, or 20 pg /mL) of the exosome- enriched product comprising intact bovine milk-derived exosomes, (2) HMB, arginine, and glutamine (Abound®, 5 pM HMB, 19.1 pM Arg, 22.7 pM Gin), or (3) a combination of the exosome- enriched product comprising bovine milk-derived exosomes (10 pg/mL, 12.5 pg/mL, 15 pg /mL, or 20 pg /mL) and HMB, arginine, and glutamine (Abound®, 5 pM HMB, 19.1 pM Arg, 22.7 pM Gin),
  • MTT reagent was diluted (1 :100) in DMEM without FBS and 200 pL were added to each well. The cells were then incubated at 37°C for 45 minutes. Following incubation, the MTT-containing medium was discarded and acidic isopropanol (40 mM HCI in isopropanol) was added to dissolve the formazan crystals. The optical densities (OD) were measured at 595 nm using an absorbance microplate reader Multiskan Spectrum (Thermo Labsystems). Absorbance values from both treated and control cells were calculated and expressed as percentage of cell viability.
  • This example demonstrates that a combination of an exosome-enriched product containing intact bovine milk-derived exosomes with HMB, arginine, and glutamine exhibits a synergistic improvement of human fibroblast migration over the administration of each of the individual components. This was shown by evaluating fibroblast migration through in vitro scratch wound healing assay using CCD1064Sk (ATCC® CRL2076TM) human dermal fibroblasts derived from normal male skin tissue.
  • the CCD1064Sk human fibroblast cells were grown at 37°C in DMEM supplemented with 10% FBS, 2 mmol/L glutamine and antibiotics (0.1 mg/mL penicillin) in 5% CO2 and 95% air.
  • the cells were seeded in 12-well plates at a density of 15x10 5 cells/well and were grown to confluent monolayer in DMEM. Cell monolayers were disrupted by scraping them with a sterile plastic pipette tip in the center of the well.
  • Treated cells were kept at 37°C for 24 hours and photographs were captured with an inverted microscope equipped with a digital camera.
  • the size of the wound was measured using Image J software (version 1 .40 National Institutes of Health, Bethesda, MD, USA) and closure was expressed as a percentage of the initial wound size.
  • fibroblasts incubated with either an exosome-enriched product comprising intact bovine milk-derived exosomes (20 pg/mL) alone or HMB, arginine, and glutamine in the absence of the exosome-enriched product increased wound closure at 24 hours by 24.3% and 6.7%, respectively.
  • FIG. 3 Representative micrographs of the wound healing assay are illustrated in FIG. 3. Dotted lines are shown for ease of viewing, with the corresponding numerical data presented in FIG. 2. As illustrated in FIG. 3, the combination of HMB, arginine, and glutamine (labeled Abound®) and the exosome enriched product comprising intact bovine milk-derived exosomes improved wound closure at 24 hours, as compared to administration of the individual components. Again, these results indicate that the combination of the exosome-enriched product comprising intact bovine milk-derived exosomes and HMB, arginine, and glutamine provide a synergistic improvement on wound healing over the administration of each of the individual components.
  • an exosome-enriched product comprising intact bovine milk-derived exosomes in combination with HMB, arginine, and glutamine increases fibroblast proliferation and migration.
  • the increase in fibroblast proliferation and migration leads to improved wound healing, which has a significant application in the prevention or treatment of chronic or hard-to-heal wounds, such as pressure ulcers, diabetic foot ulcers, burns, surgical wounds, or venous leg ulcers.
  • Improved wound healing also has a significant application in treating patients suffering from conditions such as diabetes, neuropathy, and/or vascular disease.

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Abstract

Une composition nutritionnelle comprend du HMB, de l'arginine, de la glutamine et un produit enrichi en exosomes comprenant des exosomes dérivés du lait de vache intacts. Un procédé d'amélioration de la cicatrisation de plaies chez un sujet comprend l'administration de bêta-hydroxy-bêta-méthylbutyrate (HMB), d'arginine, de glutamine et d'un produit enrichi en exosomes comprenant des exosomes dérivés du lait de vache intacts au sujet en ayant besoin. Un procédé d'amélioration de la cicatrisation de plaies chez un sujet comprend l'administration d'une composition nutritionnelle comprenant du HMB, de l'arginine, de la glutamine et un produit enrichi en exosomes comprenant des exosomes dérivés du lait de vache intacts.
PCT/US2022/038620 2021-08-26 2022-07-28 Méthodes et compositions permettant d'améliorer la cicatrisation de plaies WO2023027859A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130209528A1 (en) * 2010-10-18 2013-08-15 Agency For Science, Technology And Research Use of exosomes to promote or enhance hair growth
US20150133548A1 (en) * 2012-01-11 2015-05-14 Abbott Laboratories Combination of beta-hydroxy-beta-methylbutyrate, arginine and glutamine for use in treating diabetic ulcers
WO2021107706A1 (fr) * 2019-11-28 2021-06-03 한국과학기술연구원 Nouvelle utilisation d'exosomes du lait

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130209528A1 (en) * 2010-10-18 2013-08-15 Agency For Science, Technology And Research Use of exosomes to promote or enhance hair growth
US20150133548A1 (en) * 2012-01-11 2015-05-14 Abbott Laboratories Combination of beta-hydroxy-beta-methylbutyrate, arginine and glutamine for use in treating diabetic ulcers
US10064835B2 (en) 2012-01-11 2018-09-04 Abbott Laboratories Combination of beta-hydroxy-beta-methylbutyrate, arginine and glutamine for use in treating diabetic ulcers
WO2021107706A1 (fr) * 2019-11-28 2021-06-03 한국과학기술연구원 Nouvelle utilisation d'exosomes du lait

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