WO2022225779A1

WO2022225779A1 - Methods and compositions for promoting wound healing

Info

Publication number: WO2022225779A1
Application number: PCT/US2022/024745
Authority: WO
Inventors: José María LÓPEZ PEDROSA; Ricardo Rueda Cabrera; Rachael Buck; Jorge GARCÍA MARTÍNEZ
Original assignee: Abbott Laboratories
Priority date: 2021-04-19
Filing date: 2022-04-14
Publication date: 2022-10-27

Abstract

A method for promoting wound healing in a subject comprises administering beta-hydroxy- beta-methylbutyrate (HMB), arginine, glutamine, and at least one human milk oligosaccharide (HMO) selected from the group consisting of an acidic HMO and a neutral HMO to the subject in need thereof. A nutritional composition comprises HMB, arginine, glutamine, and an HMO selected from the group consisting of an acidic HMO and a neutral HMO.

Description

METHODS AND COMPOSITIONS FOR PROMOTING WOUND HEALING

FIELD OF THE INVENTION

[0001] The present invention relates to methods for promoting wound healing in a subject. The present invention also relates to nutritional compositions which employ beta-hydroxy-beta- methylbutyrate (HMB), arginine, glutamine, and at least one human milk oligosaccharide (HMO) selected from the group consisting of an acidic HMO and a neutral HMO.

BACKGROUND OF THE INVENTION

[0002] Wound healing is generally divided into four phases: the hemostasis phase, the inflammatory phase, the proliferation phase, and the maturation phase. Hemostasis is defined as the mechanism that leads to cessation of bleeding from a blood vessel and involves multiple interlinked steps. During hemostasis, blood vessels constrict in order to limit blood flow, which is then followed by platelets sealing and plugging the break in the wall of the blood vessel. Blood coagulation, which is the final step of hemostasis, then occurs. The blood clot or thrombus serves to reinforce the plug created by the platelets.

[0003] The inflammatory phase, which is considered to be the defensive phase of wound healing in that it serves to both control bleeding and prevent infection, begins almost immediately after injury and typically lasts for a number of days thereafter. During the inflammatory phase, damaged cells, pathogens, and bacteria are removed from the wound site in order to prepare for the growth of new tissue. This phase of the healing process is normally associated with swelling, red skin, heat, and pain at the wound site.

[0004] The proliferative phase of wound healing encompasses rebuilding the wound with new tissue. The proliferative phase is divided into three stages: filling the wound, contracting the wound margins, and covering the wound with epithelium, otherwise known as epithelialization. [0005] Finally, during the maturation phase of wound healing, the wound closes. The tissue remodels and matures and the cells that had been used to repair the wound are removed by apoptosis. The maturation phase can last anywhere from several days to a year or more.

[0006] Unfortunately, there are a number of factors that can lead to impaired wound healing. Although this list is not exhaustive, some of these factors include necrosis, bleeding disorders, immobility, diet, medical conditions, age, medications, dryness, lifestyle choices, infection, and/or varicose veins. Necrosis, for example, impedes wound healing because dead skin and foreign materials interfere with the healing process. Infections also delay wound healing because, rather than working to heal the wound, the body works to fight the infection. In patients with bleeding disorders, defects at particular stages of hemostasis, such as platelet disorders, coagulation defects, and increased clot degradation, also affect the body’s ability to properly heal a wound. [0007] Age, diet, various medical conditions, and immobility also affect the wound healing process. For example, in patients with diabetes, vascular disease, or other conditions that are correlated with poor circulation and/or a compromised immune system, it becomes difficult for the body to deliver the proper nutrients to wounds. In elderly patients, wounds normally take longer to heal. Lifestyle choices, such as poor diet, prevent individuals from ingesting nutrients that are necessary for effective wound healing. It would therefore be advantageous to provide patients suffering from a lack of proper nutrition with appropriate nutrients to facilitate wound healing. [0008] While there are various treatment options available for patients suffering from impaired wound healing, for example cleaning the wound to remove debris, removing dead skin, treating signs of infection, and/or prescribing antibiotics, many of these treatment options involve invasive surgery, which carries a number of possible risks and complications. Further, increased antibiotic use is undesirable.

[0009] In view of the above limitations, it is desirable to provide methods and compositions that would promote wound healing in a simple and non-invasive manner. This is particularly true when it comes to promoting wound healing following surgical incisions, radiotherapy-induced injuries, diabetic foot ulcers, burns, and/or other acute or chronic wounds.

[0010] Abbott Laboratories’ Juven® product, and nutritional compositions as described in US 10,064,835 B2, promote wound healing, and more particularly promote the healing of diabetic ulcers in individuals suffering from diabetes and having at least one of a serum albumin level of less than or equal to 4.0 g/dL and an Ankle-Brachial Index of less than 1.0. However, further improvements in wound healing in general are desired. Accordingly, methods for promoting wound healing are desirable, including, but not limited to, methods for healing wounds such as surgical incisions, radiotherapy-induced injuries, diabetic foot ulcers, burns, and/or other acute or chronic wounds. A nutritional intervention that can help promote wound healing is also desirable.

SUMMARY OF THE INVENTION

[0011] Accordingly, it is an object of the invention to provide a convenient means for promoting wound healing in a subject in need thereof.

[0012] In one embodiment, the invention is directed to a method for promoting wound healing in a subject, comprising administering beta-hydroxy-beta-methylbutyrate (HMB), arginine, glutamine, and at least one human milk oligosaccharide (HMO) selected from the group consisting of an acidic HMO and a neutral HMO to the subject in need thereof.

[0013] In additional embodiments, the present invention is directed to a nutritional composition, comprising HMB, arginine, glutamine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO.

[0014] The methods for promoting wound healing, as well as the nutritional compositions according to the present invention, are advantageous in that they, for example, increase wound closure and/or reduce the amount of time needed to complete would healing, or otherwise promote wound healing, in a patient-convenient manner. These and additional objects and advantages of the invention will be more fully apparent in view of the following detailed description. BREIF DESCRIPTION OF THE DRAWINGS

[0015] The Detailed Description will be more fully understood in view of the drawings, in which: [0016] Fig. 1 shows the results of a fibroblast proliferation assay as described in the Example; [0017] Fig. 2 shows digital micrographs of a monolayer of human dermal fibroblasts CCD1064Sk (ATCC® CRL2076™) derived from normal male skin tissue, as described in the Example; and

[0018] Fig. 3 shows a graphical representation of the extent of wound closure presented in the digital micrographs as described in the Example.

DETAILED DESCRIPTION

[0019] Specific embodiments of the invention are described herein. The invention can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to illustrate more specific features of certain aspects of the invention to those skilled in the art.

[0020] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the disclosure as a whole. All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably. Furthermore, as used in the description and the appended claims, the singular forms “a,” “an,” and “the” are inclusive of their plural forms, unless the context clearly indicates otherwise.

[0021] To the extent that the term “includes” or “including” is used in the description or the claims, it is intended to be inclusive of additional elements or steps, in a manner similar to the term “comprising” as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term “or” is employed (e.g., A or B), it is intended to mean “A or B or both.” When the “only A or B but not both” is intended, then the term “only A or B but not both” is employed. Thus, use of the term “or” herein is the inclusive, and not the exclusive use. When the term “and” as well as “or” are used together, as in “A and/or B” this indicates A or B as well as A and B.

[0022] The methods and compositions described in the present disclosure can comprise, consist of, or consist essentially of any of the elements and steps as described herein.

[0023] All ranges and parameters, including but not limited to percentages, parts, and ratios disclosed herein are understood to encompass any and all sub-ranges subsumed therein, and every number between the endpoints. For example, a stated range of “1 to 10” should be considered to include any and all sub-ranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less (e.g., 1 to 6.1, or 2.3 to 9.4), and to each integer (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) contained within the range.

[0024] Any combination of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0025] All percentages are percentages by weight unless otherwise indicated.

[0026] The term ΉMB” as used herein, unless otherwise specified, refers to beta- hydroxy- beta- methylbutyrate (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.

[0027] The term “calcium HMB” as used herein, unless otherwise specified, 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. [0028] The term “human milk oligosaccharide” or ΉMO”, as used herein, unless otherwise specified, refers generally to any one or more of a number of complex carbohydrates found in human breast milk that can be acidic or neutral in nature, and to precursors thereof.

[0029] The term “acidic HMO” as used herein, unless otherwise specified, refers to HMOs that contain sialic acid, i.e., sialylated HMOs.

[0030] The term “neutral HMO” as used herein, unless otherwise specified, refers to HMOs that lack sialic acid.

[0031] The term “nutritional powder” as used herein, unless otherwise specified, 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. [0032] The term “nutritional liquid” as used herein, unless otherwise specified, 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.

[0033] The terms “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 to form a nutritional liquid, and are suitable for oral consumption by a human.

[0034] The term “promoting wound healing” as used herein, unless otherwise specified, refers to obtaining an improved wound healing metric as compared with wound healing in the absence of the present methods and/or compositions. In certain embodiments, improved wound healing comprises increased wound closure and/or a reduction in the amount of time needed to complete wound healing. Those of ordinary skill in the art are familiar with other means of assessing improved wound healing and such means may be used to evidence improved wound healing according to the invention, for example through the use of scratch assay and/or a cell-proliferation assay, such as an epithelial proliferation assay. Therefore, in a more specific embodiment, promotion of wound healing can be evidenced by increased fibroblast proliferation and/or migration. [0035] In one embodiment, a method for promoting wound healing in a subject is provided. The method comprises administering HMB, arginine, glutamine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO to the subject. In additional embodiments of the invention, nutritional compositions are provided. According to one embodiment, the nutritional composition comprises HMB, arginine, glutamine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO.

[0036] Examples of HMOs that may be included in the compositions of the invention include N- acetylglucosamine (GlcNAc); L-fucose (L-Fuc); D-fucose (D-Fuc); fucosyl oligosaccharides (i.e. , Lacto-N-fucopentaose I; Lacto-N-fucopentaose II; Lacto-N-fucopentaose III; Lacto-N- difucohexaose I; and Lactodifucotetraose); non-fucosylated, non-sialylated oligosaccharides (i.e., Lacto-N-neotetraose); sialyl fucosyl oligosaccharides (i.e., 3'-Sialyl-3-fucosyllactose; Disialomonofucosyllacto-N-neohexaose; Monofucosylmonosialyllacto-N-octaose (sialyl Lea); Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II; Monofucosyldisialyllacto-N- tetraose); and sialyl oligosaccharides (i.e., 3'-Sialyllactose; 3-Sialyllactosamine; 6'-Sialyllactose; 6'-Sialyllactosamine; Sialyllacto-N-neotetraose c; Monosialyllacto-N-hexaose; Disialyllacto-N- hexaose I; Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II; Disialyllacto-N- neohexaose; Disialyllacto-N-tetraose; Disialyllacto-N-hexaose II; Sialyllacto-N-tetraose a; Disialyllacto-N-hexaose I; and Sialyllacto-N-tetraose b). Also useful are variants in which the glucose Glc at the reducing end is replaced by N-acetylglucosamine (e.g., 2'-fucosyl-N- acetylglucosamine (2'-FLNac) is such a variant to 2'-fucosyllactose). Other suitable examples of HMOs that may be included in the compositions according to the general inventive concepts include lacto-N-fucopentaose V, lacto-N-hexaose, para-lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-neohexaose, monofucosyllacto-N-hexaose II, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylated lacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto-N-neohexaose, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, lacto-N-neoocataose, para-lacto-N-octanose, iso-lacto-N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose, monofucosyllacto-N-ocataose, difucosyllacto-N-octaose I, difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II, difucosyllacto-N-neoocataose I, lacto-N-decaose, trifucosyllacto-N-neooctaose, trifucosyllacto-N-octaose, trifucosyl-iso-lacto-N- octaose, lacto-N-difuco-hexaose II, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto- N-tetraose c, sialyl-fucosyl-lacto-N-tetraose I, sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto- N-tetraose, and combinations thereof.

[0037] Specific non-limiting examples of acidic HMOs that may be included individually or in combination in the compositions of the present invention include 3'-sialyl-3-fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, 2'-sialyllactose (2’-SL), 2-sialyllactosamine (2-SL), 3'-sialyllactose (3’-SL), 3'-sialyllactosamine, 6'- sialyllactose (6’-SL), 6'-sialyllactosamine, sialyllacto-N-neotetraose c, monosialyllacto-N- hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N- neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, and sialyllacto-N-tetraose b, sialyl-fucosyl- lacto-N-tetraose I, sialyl-fucosyl-lacto-N-tetraose II. In specific embodiments of the methods and compositions of the invention, the at least one acidic HMO comprises 3’-SL, 6’-SL, or a combination thereof.

[0038] In other specific embodiments, the neutral HMO comprises a fucosylated HMO and/or an N-acetylated HMO. Specific non-limiting examples of fucosylated and N-acetylated HMOs that may be included individually or in combination in the compositions of the present invention include lacto-N-fucopentaose I, lacto-N-fucopentaose II, 2'-fucosyllactose (2’-FL), 3-fucosyllactose (3- FL), lacto-N-fucopentaose III, lacto-N-difucohexaose I, lactodifucotetraose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 2'-fucosyl-N-acetylglucosamine (2'-FLNac), lacto-N- fucopentaose V, lacto-N-hexaose, para-lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N- neohexaose, monofucosyllacto-N-hexaose II, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylated lacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto- N-neohexaose, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, lacto-N-neoocataose, para-lacto-N-octanose, iso-lacto-N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose, monofucosyllacto-N-ocataose, difucosyllacto-N-octaose I, difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II, difucosyllacto-N-neoocataose I, lacto-N-decaose, trifucosyllacto-N-neooctaose, trifucosyllacto-N-octaose, trifucosyl-iso-lacto-N-octaose, lacto-N- difucohexaose II. In additional specific embodiments, the at least one neutral HMO comprises 2’- FL, 3-FL, LNT, LNnT, or a combination of two or more thereof.

[0039] In specific embodiments of the methods and compositions of the invention, the acidic HMO comprises 3’-SL, 6’-SL, or a combination thereof, and/or the neutral HMO comprises 2’-FL, 3-FL, LNT, LNnT, or a combination of two or more thereof. In a more specific embodiment of the methods and compositions of the invention, the acidic HMO comprises 6’-SL and/or the neutral HMO comprises 2’-FL, optionally in combination with at least one N-acetylated HMO, or more specifically, LNT and/or LNnT. In a more specific embodiment, the acidic HMO comprises 3’-SL and 6’-SL, and the neutral HMO comprises 2’-FL, 3-FL, and LNT.

[0040] The methods and nutritional compositions of the present invention employ HMB, which 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 promote wound healing in certain individuals, the present inventors have surprisingly discovered that HMB, in combination with arginine, glutamine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO, further promote wound healing.

[0041] 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. In specific embodiments of the methods and compositions of the invention, 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, or the 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 the HMB is calcium HMB monohydrate.

[0042] In specific embodiments of the methods of the invention, the HMB, arginine, glutamine, the at least one acidic HMO, and the at the least one neutral HMO are administered to the subject orally. In a further specific embodiment, the HMB, arginine, glutamine, and the at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO are administered to the subject in a nutritional composition.

[0043] In specific embodiments of the methods and compositions of the invention, the nutritional compositions further comprise one or more vitamins. Non-limiting examples of suitable vitamins for use in the methods and nutritional compositions of the invention include vitamin B12, vitamin C, and/or vitamin E. 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. In a specific embodiment, the vitamin E is dl-alpha- tocopheryl acetate.

[0044] In additional specific embodiments of the methods and compositions of the invention, the nutritional compositions further comprise one or more minerals as desired. In a specific embodiment, 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 a combinations thereof. In a specific embodiment of the methods and compositions of the invention, the nutritional compositions further comprise zinc.

[0045] In additional specific embodiments of the methods of the invention, the methods further comprise administering collagen to the subject. In specific embodiments of the compositions of the invention, 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.

[0046] In additional specific embodiments of the methods and compositions of the invention, the nutritional compositions further comprise carbohydrate. Suitable carbohydrates for use in the present invention include, inter alia, sugar, aspartame, acesulfame potassium, and combinations of two or more thereof.

[0047] As indicated above, in specific embodiments of the methods of the invention, the HMB, arginine, glutamine, and the at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO are administered to the subject orally. In a more specific embodiment, the HMB , arginine, glutamine, and the at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO are provided in a nutritional composition, which can be administered to the subject orally.

[0048] 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 at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO. 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. In specific embodiments of the methods and compositions of the invention, the nutritional composition is in the form of a powder. In other specific embodiments, the nutritional composition is in the form of a liquid.

[0049] When the nutritional composition is a powder, for example, in specific embodiments, a serving size is from about 20 g to about 60 g, about 25 g to about 55 g, or about 45 g to about 50 g, to be administered as a powder or to be reconstituted in from about 1 ml to about 500 ml of liquid.

[0050] When the nutritional composition is in the form of a liquid, for example, reconstituted from a powder or manufactured as a ready-to-drink product, in specific embodiments, 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. In specific embodiments, the serving is about 1 ml, or about 100 ml, or about 225 ml, or about 237 ml, or about 500 ml.

[0051] In accordance with the methods of the present invention, to promote wound healing, the HMB, glutamine, arginine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO, may be administered to the subject for a time period of at least 2 days, or at least 3 days, or at least 5 days, or at least 6 days, or 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. In specific embodiments of the methods of the invention, the HMB, glutamine, arginine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO, are administered to a subject once or multiple times daily or weekly. In specific embodiments, the HMB, glutamine, arginine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO, 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. In other specific embodiments, the HMB, glutamine, arginine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO, are administered once or twice daily for a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days or at least 6 days, or at least one week, at least two weeks, at least three weeks, or at least four weeks.

[0052] In specific embodiments of the methods of the invention, the desired amounts of HMB, glutamine, arginine, and at least one HMO selected from the group consisting of an acidic HMO and a neutral HMO, 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.

[0053] In specific embodiments of the methods of the invention, the subject is administered about 0.5 grams to about 55 grams, or about 0.5 grams to about 30 grams, or about 2.5 grams to about 30 grams, or about 2.5 grams to about 20 grams, or about 5 grams to about 15 grams per day of arginine, about 0.5 grams to about 55 grams, or about 0.5 grams to about 30 grams, or about 2.5 grams to about 30 grams, or about 2.5 grams to about 20 grams, or about 5 grams to about 15 grams per day of glutamine, about 0.001 to about 5500 mg, or about 0.01 to about 3000 mg, or about 0.01 to about 300 mg, or about 0.01 to about 75 mg per day of the acidic HMO, about 0.001 to about 5500 mg, or about 0.01 to about 3000 mg, or about 0.01 to about 300 mg, or about 0.01 to about 75 mg per day of the neutral HMO, and about 0.1 grams to about 10 grams, or about 0.1 grams to about 5 grams, or about 0.5 grams to about 5 grams, or about 0.5 grams to about 3 grams, or about 0.5 grams to about 1.5 grams per day of HMB. In further specific embodiments, the subject is administered about 5 grams to about 15 grams per day of arginine, about 5 grams to about 15 grams per day of glutamine, about 0.01 to about 75 mg per day of the acidic HMO, about 0.01 to about 75 mg per day of the neutral HMO, and about 0.5 grams to about 1.5 grams per day of HMB. [0054] In further specific embodiments, the subject is administered about 5 grams to about 15 grams per day of arginine, about 5 grams to about 15 grams per day of glutamine, and about 0.5 grams to about 1.5 grams per day of HMB.

[0055] In further specific embodiments of the invention, the nutritional composition comprises from about 0.04% to about 5%, from about 0.04% to about 2.5%, from about 0.2% to about 2.5%, or from about 0.2% to about 1.5% of HMB, based on the weight of the nutritional composition. In additional specific embodiments, the nutritional composition comprises from about 0.2 to about 1.5 wt % of HMB, based on the weight of the nutritional composition.

[0056] In another specific embodiment, the nutritional composition comprises from about 0.1 to about 50 wt %, about 0.1 to about 30 wt %, about 10 to about 50 wt %, or about 10 to about 30 wt % of arginine, based on the weight of the nutritional composition. In a more specific embodiment, the nutritional composition is a liquid and comprises from about 1 to about 5 wt % of arginine, based on the weight of the nutritional composition. In an additional specific embodiment, 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.

[0057] In another specific embodiment of the invention, the nutritional composition comprises from about 0.1 to about 50 wt %, about 0.1 to about 30 wt %, about 10 to about 50 wt %, or about 10 to about 30 wt % of glutamine, based on the weight of the nutritional composition. In a more specific embodiment, the nutritional composition is a liquid and comprises from about 1 to about 5 wt % of glutamine, based on the weight of the nutritional composition. In an additional specific embodiment, 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.

[0058] In other specific embodiments of the invention, the nutritional composition comprises from about 0.0001 to about 2 wt %, about 0.001 to about 2 wt %, about 0.1 to about 1 wt %, about 0.2 to about 1 wt %, or about 0.25 to about 0.85 wt % of the acidic HMO, based on the weight of the nutritional composition. In a more specific embodiment, the nutritional composition comprises from about 0.2 to about 1 wt % of the acidic HMO, based on the weight of the nutritional composition.

[0059] In further specific embodiments of the invention, the nutritional composition comprises from about 0.0001 to about 2 wt %, about 0.001 to about 2 wt %, about 0.1 to about 1 wt %, about 0.2 to about 1 wt %, or about 0.25 to about 0.85 wt % of the neutral HMO, based on the weight of the nutritional composition. In a more specific embodiment, the nutritional composition comprises from about 0.2 to about 1 wt % of the neutral HMO, based on the weight of the nutritional composition.

[0060] In further specific embodiments of the compositions of the invention, the nutritional composition comprises from 0.04% to about 5 wt%, or from about 0.2 to about 1.5 wt %, of HMB; from about 0.1 to about 50 wt %, or from about 1 to about 30 wt %, of arginine; from about 0.1 to about 50 wt %, or from about 1 to about 30 wt %, of glutamine; from about 0.0001 to about 2 wt %, or from about 0.001 to about 2 wt % of the acidic HMO; and from about 0.0001 to about 2 wt %, or from about 0.001 to about 2 wt % of the neutral HMO, all based on the weight of the nutritional composition.

[0061] In an additional embodiment of the compositions of the invention, the nutritional composition comprises from 0.04% to about 5 wt%, or from about 0.2 to about 1.5 wt %, of HMB; from about 0.1 to about 50 wt %, or from about 1 to about 30 wt %, of arginine; and from about 0.1 to about 50 wt %, or from about 1 to about 30 wt %, of glutamine, all based on the weight of the nutritional composition.

[0062] In other specific embodiments of the invention, in addition to the protein, carbohydrate and fat contents provided by the HMB, amino acids and HMOs, or other components mentioned above, the nutritional composition further comprise additional sources of protein, carbohydrate, and/or fat. A wide variety of sources and types of protein, carbohydrate, and/or fat can be used in embodiments of nutritional compositions described herein. In a specific embodiment, the nutritional composition includes additional sources of protein, carbohydrate and fat. [0063] In further specific embodiments, an additional source 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.

[0064] 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 about 1 wt% to about 20 wt%, about 2 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 10 wt%, about 10 wt% to about 25 wt%, or 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. [0065] In other specific embodiments, 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, glucomannan, xanthan gum, alginate, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucans, psyllium, inulin, and combinations of two or more thereof.

[0066] 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 about 5 wt% to about 70 wt%, about 5 wt% to about 65 wt%, about 5 wt% to about 50 wt%, about 5 wt% to about 40 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 10 wt% to about 65 wt%, about 20 wt% to about 65 wt%, about 30 wt% to about 65 wt%, about 40 wt% to about 65 wt%, about 40 wt% to about 70 wt%, or about 15 wt% to about 25 wt%, of the nutritional composition.

[0067] The terms “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.

[0068] In further specific embodiments, 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. In a specific embodiment, 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. [0069] The 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.

[0070] 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. Non-limiting examples of 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.

[0071] In specific embodiments, 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.

[0072] The nutritional compositions may be formed using any techniques known in the art. In one embodiment, a powder product can be formed by dry blending ingredients. In another embodiment, 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 HMOs 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. Typically, 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

[0073] This Example demonstrates various improvements provided by the methods of the invention. Specifically, this Example evaluated the stimulatory effect of HMB, arginine, glutamine, and HMOs on fibroblast proliferation through MTT assays and on fibroblast migration in scratch assays.

[0074] The first study employed the MTT cell proliferation assay. Human dermal fibroblasts CCD1064Sk (ATCC® CRL2076™) derived from normal male skin tissue were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% (v/v) FBS, 2 mmol/l glutamine and 0.1 mg/ml penicillin. The cells were grown and maintained in an incubator at 37°C with 95% humidity and 5% of CO2. The cells were seeded in 48-well plates at a density of 15x10³ cells/well. After 24 h of incubation, cells were treated with an HMO mixture comprising 3’-SL, 6’-SL, 2’-FL, 3-FL, and LNT (“5HMOs” or “5-HMOs”) in amounts of 0.1, 0.5 and 5 g/L, or with 5 mM of a mixture of HMB, arginine and glutamine comprising 5 pM HMB, 19.1 pM arginine, and 22.7 pM glutamine (“Abound”), or with a combination of 5-HMOs and Abound for 48 h. After the treatment, MTT reagent was diluted (1:100) in DMEM without FBS and 200 pL were added per well. Cells were incubated at 37°C for 45 min. The supernatant was replaced with 200 pL acidic isopropanol to dissolve the formazan crystals. The absorbance was measured at 595 nm with a microplate reader Multiskan Spectrum (Thermo Labsystems). Absorbances from both treated and control cells were calculated and expressed as percentage of cell proliferation. The results are shown in Fig. 1.

[0075] Fig. 1 shows the percentage of cell proliferation for a Control - bar A at time 0; and after 48 hours: Control - bar B; 5 pM Abound - bar C; 0.1 g/L 5-HMOs - bar D; 0.5 g/L 5-HMOs - bar E; 5 g/L 5-HMOs - bar F; 0.1 g/L 5-HMOs and 5 pM Abound - bar G; 0.5 g/L 5-HMOs and 5 pM Abound - bar H; and 5 g/L 5-HMOs and 5 pM Abound - bar I. A synergistic and dose-dependent effect was found when fibroblasts were incubated with a combination of Abound plus increasing amounts of 5-HMOs. At the highest 5-HMOs dose assayed (5 g/L 5-HMOs + Abound, bar I), fibroblast proliferation was increased by 26% as compared with the untreated control. In Fig. 1, * represents p<0.05 for Control vs Treatment; # represents p<0.05 for Abound vs Abound + 5g/L 5-HMOs; and a represents p<0.05 for 5g/L 5-HMOs vs Abound+ 5g/L 5-HMOs. [0076] The second study employed a scratch assay. Human dermal fibroblasts CCD1064Sk (ATCC® CRL2076™) derived from normal male skin tissue were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% (v/v) FBS, 2 mmol/l glutamine and 0.1 mg/ml penicillin. Cells were grown and maintained in an incubator at 37°C with 95% humidity and 5% of CO2.

[0077] The CCD1064Sk cells were seeded in 12-well plates at a density of 15x10^s 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. Cells were washed twice with PBS to remove cellular debris and treated with 5 mM Abound (5 pM HMB, 19.1 pM Arg, 22.7 pM Gin), 5-HMOs (5 g/L) and a combination of 5 pM Abound and 5 g/L 5-HMOs. Treated cells were kept at 37°C for 24 h and photographs were captured with an inverted microscope equipped with a digital camera. Fig. 2 presents the photographs. The wounded area 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, as shown in Fig. 3, where bar A represents the Control, bar B represents 5 pM Abound, bar C represents 5 g/L 5-HMOs, and bar D represents 5 pM Abound and 5 g/L 5-HMOs. In Fig. 3, * represents p<0.05 for Control vs Treatment, and a represents p<0.05 for 5g/L 5- HMOs vs Abound and 5g/L 5-HMOs

[0078] Compared with the untreated controls, a synergistic effect was found when fibroblasts were incubated with a blend of Abound plus 5-HMOs (bar D in Fig. 3). At the dose assayed (5 g/L 5- HMOs + Abound), fibroblast migration capacity was enhanced by 28%.

[0079] While the present application has been illustrated by the description of embodiments thereof, and while the embodiments have been described in considerable detail, such descriptions are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the invention, in its broader aspects, is not limited to the specific details, the representative methods or compositions, or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

Claims

WHAT IS CLAIMED IS:

1. A method for promoting wound healing in a subject, the method comprising administering beta-hydroxy-beta-methylbutyrate (HMB), arginine, glutamine, and at least one human milk oligosaccharide (HMO) selected from the group consisting of an acidic HMO and a neutral HMO.

2. The method of claim 1, wherein the acidic HMO comprises 3’-sialyllactose (3’-SL), 6’- sialyllactose (6’-SL), or a combination thereof, and/or the neutral HMO comprises 2’- fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT) and lacto-N- neotetraose (LNnT), or a combination of two or more thereof.

3. The method of claim 2, wherein the acidic HMO comprises 6’-SL, and/or the neutral HMO comprises 2’-FL.

4. The method of claim 3, wherein the neutral HMO further comprises at least one N-acetylated HMO, or more specifically, lacto-N-tetraose (LNT) and/or lacto-N-neotetraose (LNnT).

5. The method of any one of claims 1-4, wherein the subject is administered about 0.5 grams to about 55 grams per day of arginine, about 0.5 grams to about 55 grams per day of glutamine, about 0.001 to about 5500 mg per day of the acidic HMO, about 0.001 to about 5500 mg per day of the neutral HMO, and about 0.1 gram to about 10 grams per day of

HMB.

6. The method of claim 5, wherein the subject is administered about 5 grams to about 15 grams per day of arginine, about 5 grams to about 15 grams per day of glutamine, about

0.01 to about 75 mg per day of the acidic HMO, about 0.01 to about 75 g per day of the neutral HMO, and about 0.5 grams to about 1.5 grams per day of HMB.

7. The method of any one of claims 1-6, wherein the HMB, arginine, glutamine, the acidic HMO, and/or the neutral HMO are administered to the subject orally.

8. The method of any one of claims 1-7, wherein the HMB, arginine, glutamine, the acidic HMO, and/or the neutral HMO are administered to the subject in a nutritional composition.

9. The method of any one of claims 1-8, wherein the 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, or the 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 the HMB is calcium HMB monohydrate.

10. The method of any one of claim 8 or claim 9, wherein the nutritional composition further comprises one or more vitamins.

11. The method of any one of claims 1-10, wherein the method further comprises administering collagen to the subject.

12. A nutritional composition comprising beta-hydroxy-beta-methylbutyrate (HMB), arginine, glutamine, and at least one human milk oligosaccharide (HMO) selected from the group consisting of an acidic HMO and a neutral HMO.

13. The nutritional composition of claim 12, wherein the acidic HMO comprises 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), or a combination thereof, and/or the neutral HMO comprises 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT) lacto-N-neotetraose (LNnT), or a combination of two or more thereof.

14. The nutritional composition of claim 12 or 13, wherein the acidic HMO comprises 6’-SL and/or the neutral HMO comprises 2’-FL.

15. The nutritional composition of claim 14, wherein the neutral HMO further comprises at least one N-acetylated HMO, or more specifically, lacto-N-tetraose (LNT) and/or lacto-N- neotetraose (LNnT).

16. The nutritional composition of any one of claims 12-15, wherein the nutritional composition comprises, based on the weight of the nutritional composition:

(a) from about 0.4 to about 5 wt %, or from about 0.2 to about 1.5 wt %, of HMB; and/or

(b) from about 0.1 to about 50 wt %, or from about 10 to about 30 wt %, of arginine; and/or

(c) from about 0.1 to about 50 wt %, or from about 10 to about 30 wt %, of glutamine; and/or

(d) from about 0.0001 to about 2 wt %, or from about 0.001 to about 2 wt %, of the acidic HMO; and/or

(e) from about 0.0001 to about 2 wt %, or from about 0.001 to about 2 wt %, of the neutral HMO.

17. The nutritional composition of any one of claims 12-16, wherein the 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, or the 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 the HMB is calcium HMB monohydrate.

18. The nutritional composition of any one of claims 12-17, wherein the nutritional composition further comprises carbohydrate.

19. The nutritional composition of any one of claims 12-18, wherein the nutritional composition further comprises collagen.

20. The nutritional composition of any one of claims 12-19, wherein the nutritional composition further comprises zinc.