Compositions containing nicotinamide and vitamin b6 and methods of using such compositions for rehabilitation

Abstract

The present invention provides compositions containing nicotinamide and vitamin B6. The compositions can be oral nutritional compositions, such as nutritional supplements, oral nutritional supplements. The compositions can be administered to an individual in need thereof for treating muscle injury in an individual having muscle injury, and / or promoting muscle repair, improving skeletal muscle regeneration, maintaining or increasing skeletal muscle function and / or skeletal muscle mass.

Description

Technical Field

[0001] This disclosure relates generally to compositions containing nicotinamide and vitamin B6, and also to methods for preparing and using such compositions. The composition may be an oral nutritional composition, such as a nutritional supplement or oral nutritional admixture. The composition may be administered to individuals in need to promote recovery from muscle injury, and / or promote muscle repair, improve skeletal muscle regeneration, maintain or increase skeletal muscle function and / or skeletal muscle mass. For example, the invention can be used to promote recovery after muscle injury caused by trauma or surgery. Background Technology

[0002] Skeletal muscle regeneration is an important mechanism for repairing and maintaining muscle mass and function throughout life. Skeletal muscle regeneration mainly requires the participation of myogenic progenitor cells (called muscle stem cells or satellite cells).

[0003] Non-proliferative quiescent satellite cells adjacent to resting skeletal muscle can be identified by their distinct locations between the sarcolemma and basal layer, high nucleus-to-cytoplasm volume ratio, few organelles (e.g., ribosomes, endoplasmic reticulum, mitochondria, Golgi apparatus), small nuclear size, and abundant heterochromatin relative to the muscle nucleus. Activated satellite cells, on the other hand, exhibit an increased number of caveolae, cytoplasmic organelles, and reduced levels of heterochromatin.

[0004] These muscle satellite cells are part of the adult stem cell nest and are involved in normal muscle growth, as well as regeneration after injury or disease. Therefore, they are potential targets for enhancing muscle regeneration in both healthy and diseased conditions. Skeletal muscle regeneration follows a series of steps that recapitulate developmental stages. Muscle progenitor cells must leave a quiescent state, become active, proliferate, and participate in myogenic differentiation.

[0005] Satellite cells express genetic markers at different stages of myogenesis and proliferation. Pax7 and Pax3 are considered satellite cell markers. For example, activated satellite cells expressing low levels of Pax7 are more prone to differentiation, while high levels of Pax7 are associated with cells less prone to differentiation and exhibit more undifferentiated stem cell characteristics. Activation and induction of myogenesis are generally regulated by myogenic regulatory factors such as MyoD, Myf5, myopoietin, and MRF4. Negative regulation by myostatin and TGF-β inhibits satellite cell differentiation (Almeida et al., 2016).

[0006] Previous experimental therapies that included myoblast transplantation have not been entirely successful because myoblasts, which are more directed and differentiated, have lower regenerative potential compared to muscle stem cells.

[0007] Therefore, there remains a great need to find compositions and methods that directly regulate muscle stem cells to maintain muscle health and improve muscle regeneration. Such compounds, compositions, and treatments can help subjects recover after muscle injury or surgery by maintaining or increasing muscle function (e.g., strength, endurance, contractile ability) and / or muscle mass. Summary of the Invention

[0008] As described in the experimental examples disclosed later herein, the inventors surprisingly identified nicotinamide as an enhancer of both the expansion and commitment of muscle stem cells, and vitamin B6 as an enhancer of their commitment. The inventors also surprisingly discovered that the effects of nicotinamide and vitamin B6 were enhanced when cells were treated with a combination of these two compounds, rather than when tested individually. Figure 3 The synergistic effect shown and described can be explained by the fact that nicotinamide and vitamin B6 have different effects on muscle stem cells; nicotinamide primarily increases the expansion step (Pax7 cells), while vitamin B6 specifically targets the stereotactic step (MyoD cells). This effect has been shown to be specific to B6 compared to other B vitamins (e.g., B9). Compositions containing this combination are advantageous in maintaining stem cell function. In particular, combinations of nicotinamide and vitamin B6 (e.g., pyridoxine), especially at specific concentrations and / or ratios, have unexpectedly shown a statistically significant synergistic association between nicotinamide and vitamin B6 and increased skeletal muscle regeneration through promoting muscle stem cell function, thus demonstrating the effect of these nutrients on maintaining or increasing muscle mass and / or skeletal muscle function in individuals in need, particularly for promoting muscle repair in individuals in need after muscle injury or surgery.

[0009] In one aspect of this disclosure, the composition comprises a combination of nicotinamide and vitamin B6 (e.g., pyridoxine), preferably in an amount that has a therapeutic effect on at least one of the physiological benefits disclosed herein.

[0010] In one embodiment, the composition contains vitamin B6 in amounts of 1.0 mg to 600 mg of vitamin B6 per day, for example, 1.0 mg to 200.0 mg of vitamin B6 per day, for example, 1.0 mg to 25.0 mg of vitamin B6 per day, for example, 1.0 mg to 15.0 mg of vitamin B6 per day, for example, 1.0 mg to 10 mg of vitamin B6 per day, for example, 1.0 mg to 7.0 mg of vitamin B6 per day.

[0011] In one embodiment, the composition comprises nicotinamide in amounts ranging from about 1 mg / day to about 3000 mg / day, for example from about 10 mg / day to about 2000 mg / day, for example from about 500 mg / day to about 1000 mg / day.

[0012] In one embodiment, vitamin B6 is administered at a dose of 10.0 mg to 20.0 mg of vitamin B6 per day, and / or nicotinamide is administered at a dose of about 500 mg to about 1000 mg of nicotinamide per day.

[0013] However, in any given case, the amount of compound administered will depend on factors such as the solubility of the active ingredient, the formulation used, the subject's condition (such as weight), and / or the route of administration. For example, the daily dose of vitamin B6 or nicotinamide disclosed above is not limiting and may vary in some embodiments.

[0014] In one embodiment, the composition is in the form of a solid powder, powder bar, capsule, or solution. The composition may be a nutritional composition, such as an oral nutritional composition.

[0015] In another aspect of this disclosure, a method for preparing a composition is provided. This method may include combining vitamin B6 (e.g., pyridoxine) and nicotinamide, and preferably the resulting combination is in an amount that has a therapeutic effect on at least one of the physiological benefits disclosed herein.

[0016] In another aspect of this disclosure, the nutritional supplement comprises a therapeutically effective amount of any of the compositions disclosed herein. In one embodiment, the nutritional supplement is an oral nutritional supplement (ONS). The nutritional supplement may be in the form of a solid powder, powder bar, capsule, or solution. In one embodiment, the nutritional supplement comprises vitamin B6 that effectively increases the amount of functional vitamin B6 in the supplement at a daily dose of 1.0 mg to 600 mg of vitamin B6, for example, 1.0 mg to 200 mg of vitamin B6, for example, 1.0 mg to 25.0 mg of vitamin B6. The nutritional supplement comprises nicotinamide at a total daily dose of about 1 mg / day to about 3000 mg / day, preferably about 10 mg / day to about 2000 mg / day, more preferably 500 mg / day to about 1000 mg / day.

[0017] In another aspect of this disclosure, the nutritional supplement contains nicotinamide in a total daily dose of about 1 mg / day to about 3000 mg / day, preferably about 10 mg / day to about 2000 mg / day, and more preferably 500 mg / day to about 1000 mg / day.

[0018] In another aspect of this disclosure, the kit comprises a therapeutically effective amount of any of the compositions disclosed herein. In one embodiment, the kit is configured for oral administration of the composition. For example, the kit may comprise at least two capsules, wherein the first capsule comprises vitamin B6 (preferably functional vitamin B6) and the second capsule comprises nicotinamide. In one embodiment, the kit comprises vitamin B6 in the first capsule at a daily dose of 1.0 mg to 600 mg of vitamin B6, for example, 1.0 mg to 200 mg of vitamin B6, for example, 1.0 mg to 25.0 mg of vitamin B6. In one embodiment, the kit comprises nicotinamide or a derivative in the second capsule at a total daily dose of about 1 mg / day to about 3000 mg / day, preferably about 10 mg / day to about 2000 mg / day, more preferably 500 mg / day to about 1000 mg / day.

[0019] In another aspect of this disclosure, methods are provided for treating muscle injury and / or promoting muscle repair, improving skeletal muscle regeneration, maintaining or increasing skeletal muscle function and / or skeletal muscle mass to promote recovery after muscle injury. The method comprises administering to an individual in need a therapeutically effective amount of a combination of vitamin B6 and nicotinamide and / or its derivatives. In one embodiment, the administration is oral. In another embodiment, the administration is intravenous.

[0020] The present invention also relates to a method for promoting recovery in an individual following muscle injury, the method comprising administering an effective amount of the composition of the present invention to an individual in need of such treatment.

[0021] In one implementation, muscle injury is associated with muscle trauma or surgery.

[0022] In one implementation, the subject is a human subject. In another implementation, the subject is a companion animal, preferably a dog. Attached Figure Description

[0023] Figures 1 to 5 —Myogenic expansion and shaping of muscle stem cells

[0024] Human skeletal muscle myoblasts were purchased from Lonza (https: / / bioscience.lonza.com). These cells were isolated from upper arm or leg muscle tissue from normal donors and used after a second passage. Several donors were tested to ensure cell viability and purity before selecting the final donors: a 20-year-old Caucasian female (hereinafter referred to as Donor 1), a 36-year-old Caucasian female (hereinafter referred to as Donor 2), and an 18-year-old Caucasian male (hereinafter referred to as Donor 3). Human primary myoblasts were seeded at a density of 1,000 cells per well in skeletal muscle growth medium (SKM-M, AMSbio) in 384-well plates. For treatment, the compound was added directly to the myoblast culture 16 hours after initial seeding.

[0025] All cultures were then grown for 96 hours. Cells were stained with antibodies against Pax7 and MyoD to determine Pax7 and MyoD expression, and counterstained with Hoechst 33342 to visualize the nuclei. Pax7+ cells were defined as cells expressing Pax7, independent of MyoD expression. MyoD+ cells were defined as cells expressing MyoD but not Pax7. Images were acquired using the ImageXpress (Molecular Devices) platform. Quantitative analysis was performed using a custom module for multi-wavelength cell scoring based on MetaXpress software. Differences from the control were indicated by one-way ANOVA, with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Data are expressed as mean ± SEM.

[0026] Figure 1 In vitro dose response of nicotinamide. Data were pooled from human primary myoblasts obtained from donors 1 and 2. For each condition, the total cell count was determined to assess compound toxicity, and the number of Pax7+ or MyoD+ cells was normalized to the total cell count to assess the proportion of this population and expressed as a fold change compared to the control condition (DMSO 1%). Figure 1 A represents the proportion of Pax7+ cells, and Figure 1 B represents the proportion of MyoD+ cells.

[0027] Figure 2 In vitro dose response of pyridoxine. Data were pooled from human primary myoblasts obtained from donors 1 and 2. For each condition, the total cell count was determined to assess compound toxicity, and the number of Pax7+ or MyoD+ cells was normalized to the total cell count to assess the proportion of this population and expressed as a fold change compared to the control condition (DMSO 1%). Figure 2 A represents the proportion of Pax7+ cells, and Figure 2 B represents the proportion of MyoD+ cells.

[0028] Figure 3 Synergistic effects of nicotinamide (NAM) and pyridoxine (B6). The effects of nicotinamide and pyridoxine alone or in combination on MyoD+ cells were evaluated in human primary myoblasts derived from donor 3. For each condition, the number of MyoD+ cells was normalized to the number of MyoD+ cells in the control condition (DMSO 1%). Figure 3 A represents the number of MyoD+ cells normalized to the control condition. Figure 3B represents the increase in MyoD+ cell number compared to the control condition (DMSO 1%). ΔB6 or ΔNAM refers to the change after treatment with B6 or NAM, respectively, compared to the control condition. ΔB6+ΔNAM refers to the theoretical sum of the effects of B6 and NAM measured separately. Δ(B6+NAM) refers to the experimental effect of combined treatment with B6 and NAM. A statistically significant synergistic effect between nicotinamide and pyridoxine has been observed by applying a linear regression model (interaction term, p=0.05).

[0029] Figure 4 Nicotinamide (NAM) in combination with vitamin B9. The effects of nicotinamide and vitamin B9, alone or in combination, on MyoD+ cells were evaluated on human primary myoblasts derived from donor 3. For each condition, the number of MyoD+ cells was normalized to the number of MyoD+ cells in the control condition (DMSO 1%). Figure 4 A represents the number of MyoD+ cells normalized to the control condition. Figure 4 B indicates the increase in the number of MyoD+ cells compared to the control condition (DMSO 1%). ΔB9 or ΔNAM refers to the change after treatment with B9 or NAM, respectively, compared to the control condition. ΔB9+ΔNAM refers to the theoretical sum of the effects of B9 and NAM measured separately. Δ(B9+NAM) refers to the experimental effect of combined treatment with B9 and NAM.

[0030] Figure 5 This indicates the number of Pax7+ cells at different ratios of pyridoxine to nicotinamide (B6 / NAM ratio). Differences from the control were indicated by one-way ANOVA, with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Data are expressed as mean ± SEM.

[0031] Figures 6 to 8 In vivo effects of the combination of nicotinamide (NAM) and pyridoxine (B6) on muscle stem cell function

[0032] To recreate the physiological process of muscle regeneration in adult skeletal muscle in response to injury or disease, we intramuscularly injected cardiotoxin into the hind limb muscles of mice. One week prior to inducing muscle injury, mice were administered the compounds of interest (nicotinamide and pyridoxine, 200 mg / kg body weight and 4 mg / kg body weight, respectively) orally, relative to a water control group. Mice were treated once daily until the end of the experiment. To assess the efficiency of muscle regeneration, 5 days after injury (… Figure 6 and Figure 7 ) and 12 days ( Figure 8Previously injured muscle was harvested and frozen sections were prepared. Several myogenic markers were then measured. Frozen sections were stained with specific antibodies against Pax7, myopoietin, laminin (to delineate muscle fibers), and embryonic myosin heavy chain (to define the injured / regenerated area), and counterstained with Hoechst 33342 to visualize cell nuclei.

[0033] Figure 6 This indicates that by separately targeting Pax7+ cells ( Figure 6 A) and myocyte cytopoietin + cells ( Figure 6 B) The number of cells was counted to assess the early and later stages of myogenic differentiation of muscle stem cells. Data were expressed as the number of cells per unit area of ​​injured muscle and as a fold change compared to control conditions. Differences from the control were indicated by one-way ANOVA, with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Data are expressed as mean ± SEM.

[0034] Figure 7 This indicates that by separately targeting Pax7+ cells ( Figure 7 A) and myocyte cytopoietin + cells ( Figure 7 B) The number of cells was counted to assess the early and later stages of myogenic differentiation of muscle stem cells. This was performed in 24-month-old mice defined as an aged population, with “adult” mice as controls. Data are expressed as the number of cells per unit area of ​​injured muscle and are presented as fold changes compared to control conditions. Differences from the control were indicated by one-way ANOVA, with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Data are expressed as mean ± SEM.

[0035] Figure 8 This study represents the late stage of muscle fiber maturation in 24-month-old mice, defined as an aged population, with "adult" mice serving as controls. Assessment was performed by quantifying the size of each newly formed muscle fiber, measured based on the expression of embryonic myosin heavy chain and laminin, which allowed for the identification and mapping of these nascent muscle fibers. Results are presented as average muscle fiber cross-sectional area (µm²).

[0036] Differences from the control were indicated by one-way ANOVA, with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Data are expressed as mean ± SEM. Detailed Implementation

[0037] definition

[0038] The following provides some definitions. However, definitions may be located in the “Implementation” section below, and the title “Definitions” above does not imply that such disclosures in the “Implementation” section are not definitions.

[0039] All percentages expressed herein are by weight of the total weight of the composition, unless otherwise stated. When pH is mentioned herein, the value corresponds to the pH measured using standard equipment at 25°C.

[0040] As used herein, “about,” “approximately,” and “basically” should be understood to refer to a number within a certain numerical range, such as -10% to +10% of the mentioned number, preferably -5% to +5% of the mentioned number, more preferably -1% to +1% of the mentioned number, and most preferably -0.1% to +0.1% of the mentioned number.

[0041] All numerical ranges herein should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be understood to support claims that involve any number or subset of numbers within that range. For example, disclosures of 1 to 10 should be understood to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0042] As used in this disclosure and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, references to “a component” or “the component” include two or more components.

[0043] The terms "including / containing" will be interpreted as inclusive rather than exclusive. Similarly, the terms "include," "including," "containing," and "having" should be considered inclusive unless the context explicitly prohibits this interpretation. Furthermore, in this respect, these terms specify the presence of the stated feature but do not exclude the presence of additional or other features.

[0044] However, the compositions and methods disclosed herein may not contain any elements not expressly disclosed herein. Therefore, the disclosure of embodiments using the term "comprising" refers to: (i) embodiments having the identified component or step as well as additional components or steps; (ii) embodiments "consisting substantially of the identified component or step"; and (iii) embodiments "consisting of the identified component or step". Any embodiment disclosed herein may be combined with any other embodiment disclosed herein.

[0045] For example, the term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "X and Y".

[0046] In the context of this document, the terms “example” and “such as” (especially when followed by a list of terms) are exemplary and illustrative only and should not be considered exclusive or comprehensive.

[0047] "Subject" or "individual" refers to mammals, preferably humans. As used herein, "effective amount" is the amount in an individual that prevents defects, treats diseases or medical conditions, or more generally, the amount that reduces symptoms, manages disease progression, or provides nutritional, physiological, or medical benefits to the individual.

[0048] The term "treatment" includes both preventative or deterrent treatment (preventing and / or delaying the development of a target pathological condition or disorder) and curative, therapeutic, or disease-modifying treatment, including therapeutic measures that cure, delay, alleviate symptoms of a diagnosed pathological condition or disorder and / or halt its progression; and treatment of patients at risk of or suspected of having the disease, as well as treatment of patients who are ill or have been diagnosed with a disease or medical condition. The term "treatment" does not necessarily mean that a subject is treated until fully recovered. The term "treatment" also refers to health maintenance and / or promotion in individuals who do not have the disease but may be susceptible to developing unhealthy conditions. The term "treatment" is also intended to include intensifying or otherwise enhancing one or more major preventative or therapeutic measures. As a non-limiting example, treatment may be administered by a patient, caregiver, physician, nurse, or other healthcare professional.

[0049] A “kit” means that the multiple components of a kit are physically associated with, or physically associated with, one or more containers and are regarded as a unit for manufacture, packaging, sale or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, outer packaging, shrink wrap, attachments (e.g., binding components, adhesive components, etc.), packaging of any type, any design or material, or combinations thereof.

[0050] The term "oral nutritional supplements (ONS)" refers to sterile liquids, semi-solids, or powders that provide macronutrients and micronutrients. They are widely used in acute and community health settings for individuals whose nutritional needs cannot be met by oral diet alone.

[0051] As used herein, “vitamin B6” may include one or more of the following: pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate (P5P), pyridoxal (PL), pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), 4-pyridinic acid, and pyrithiol. In a preferred embodiment, at least a portion of any vitamin B6 is PN. At least a portion of vitamin B6 may be PLP. Absorbed pyridoxamine is converted to PMP by pyridoxal kinase, and the PMP is further converted to PLP by pyridoxamine-phosphotransaminase or pyridoxine 5'-phosphooxidase, which also catalyzes the conversion of PNP to PLP. [2] Pyridoxine 5'-phosphooxidase depends on flavin mononucleotide (FMN) as a cofactor for the production of riboflavin (vitamin B2).

[0052] Implementation Plan

[0053] One aspect of this disclosure is a composition comprising nicotinamide and vitamin B6. Compositions comprising nicotinamide and vitamin B6 are beneficial for treating muscle injuries and / or promoting muscle repair, and / or improving skeletal muscle regeneration, and / or maintaining or increasing skeletal muscle function and / or skeletal muscle mass. For example, compositions comprising nicotinamide and vitamin B6 can be used to promote muscle repair and / or regeneration in individuals with muscle injuries following muscle trauma or surgery.

[0054] Composition

[0055] Niacinamide

[0056] Nicotinamide, also known as nicotinamide or nicotinic acid amide, is the water-soluble active form of vitamin B3.

[0057] Nicotinamide can be administered in doses of approximately 0.001 mg / day to approximately 3000 mg / day, for example, approximately 1 mg / day to approximately 3000 mg / day, for example, approximately 10 mg / day to approximately 2000 mg / day, for example, approximately 500 mg / day to approximately 1000 mg / day. Of course, the daily dose can be administered in divided doses throughout the day. However, in any given case, the amount of compound administered will depend on factors such as the solubility of the active ingredient, the formulation used, the subject's condition (such as weight), and / or the route of administration. For example, the daily dose of nicotinamide disclosed above is non-limiting and can vary in some embodiments.

[0058] Vitamin B6

[0059] Pyridoxine is the 4-methanol form of vitamin B6, an important water-soluble vitamin that is naturally found in many foods.

[0060] In one embodiment, vitamin B6 may include one or more of the following: pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate (P5P), pyridoxal (PL), pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), 4-pyridinic acid, and pyrithiol. In a preferred embodiment, at least a portion of any vitamin B6 is PN. At least a portion of vitamin B6 may be PLP. Absorbed pyridoxamine is converted to PMP by pyridoxal kinase, and the pyridoxal kinase is further converted to PLP by pyridoxamine-phosphotransaminase or pyridoxine 5'-phosphooxidase, which also catalyzes the conversion of PNP to PLP. Pyridoxine 5'-phosphooxidase depends on flavin mononucleotide (FMN), which is a cofactor produced from riboflavin (vitamin B2). In one embodiment, vitamin B6 is pyridoxine.

[0061] In one embodiment, vitamin B6 may be administered in the following amounts: a daily dose of vitamin B6 of about 1.0 mg to 600 mg / day, for example, about 1.0 mg to 200 mg / day, for example, about 1.0 mg to 25.0 mg / day, for example, about 10 mg to 20 mg / day.

[0062] In one embodiment, the combination is particularly effective, especially for the expansion and shaping of muscle cells, when vitamin B6:nicotinamide is present in the following ratio: about 1:100 to about 1:9, preferably about 1:80 to about 1:20, preferably about 1:75 to about 1:25, more preferably about 1:60 to about 1:30. In one embodiment, pyridoxine:nicotinamide is present in a ratio of about 1:45 to about 1:30.

[0063] In some embodiments, the composition containing the combination of nicotinamide and vitamin B6 is in the form of a nutritional composition.

[0064] In some embodiments, the composition containing the combination of nicotinamide and vitamin B6 is in the form of a nutritional product, nutritional supplement, milk-based beverage, low-volume liquid supplement, or meal replacement drink.

[0065] In some embodiments, the composition comprising the combination of nicotinamide and vitamin B6 is in the form of a drug.

[0066] The drug may be in the form of, for example, tablets, capsules, lozenges, or liquids. The drug is preferably provided as a sustained-release formulation, thereby allowing a constant supply of the active ingredient over a long period.

[0067] The composition may be selected from the following group: milk powder-based products; instant beverages; ready-to-drink formulations; nutritional powders; nutritional liquids; milk-based products, especially yogurt or ice cream; cereal products; beverages; water; coffee; hot milk coffee; malt beverages; chocolate-flavored beverages; cooking products; soups; tablets; and / or syrups.

[0068] The composition may also include protective aqueous colloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulation agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavor masking agents, weighting agents, gelling agents, gelling agents, antioxidants, and antimicrobial agents.

[0069] In addition, as recommended by government agencies (such as the USRDA), the composition may contain organic or inorganic carrier materials suitable for oral or enteral administration, as well as vitamins, trace minerals and other micronutrients.

[0070] The compositions of the present invention may contain a protein source, a carbohydrate source, and / or a lipid source.

[0071] Any suitable dietary protein can be used, such as animal proteins (e.g., milk proteins, meat proteins, and egg proteins); plant proteins (e.g., soy proteins, wheat proteins, rice proteins, and pea proteins); mixtures of free amino acids; or combinations thereof. Milk proteins (e.g., casein and whey) and soy proteins are particularly preferred.

[0072] If the composition contains a fat source, the fat source preferably provides 5% to 40% of the energy in the formulated food; for example, 20% to 30% of the energy. DHA may be added. A suitable fat distribution can be obtained using a blend of low-erucic acid rapeseed oil, corn oil, and high-oleic acid sunflower oil.

[0073] The carbohydrate source can preferably provide between 40% and 80% of the energy of the composition. Any suitable carbohydrate can be used, such as sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, and mixtures thereof.

[0074] Another aspect of this disclosure is a kit containing a therapeutically effective amount of any of the compositions disclosed herein. In one embodiment, the kit is configured for oral administration of the composition. For example, the kit may be in the form of two capsules, wherein the first capsule contains vitamin B6 and the second capsule contains nicotinamide.

[0075] Another aspect of this disclosure is a method for preparing a composition. This method may include combining a therapeutically effective amount of nicotinamide and vitamin B6, preferably in an amount that has a therapeutic effect on at least one of the physiological benefits disclosed herein.

[0076] Recovery after muscle injury from surgery and muscle trauma

[0077] Muscle injuries can be caused by abrasions, stretching, or tearing, resulting in acute or chronic soft tissue damage to muscles, tendons, or both. They can occur due to muscle fatigue, overuse, or misuse. They can occur after physical trauma such as falls, fractures, or overuse during physical activity. Muscle injuries can also occur after surgery such as arthroscopic joint replacement surgery.

[0078] It should be understood that the compositions and methods of the present invention can be beneficial in treating the above-mentioned conditions and in promoting recovery from muscle damage following surgery and / or muscle trauma, particularly in promoting muscle repair and / or muscle regeneration and / or maintaining or improving skeletal muscle mass and / or muscle function.

[0079] In one embodiment of the invention, the invention provides a method for promoting recovery from muscle injury in an individual suffering from muscle injury, and / or promoting muscle repair, improving skeletal muscle regeneration, maintaining or increasing skeletal muscle function and / or skeletal muscle mass. The method comprises administering an effective amount of the composition of the invention to a human or animal subject.

[0080] In one embodiment of the invention, a method for treating muscle injury is provided, the method comprising administering an effective amount of the composition of the invention to a human or animal subject. In one embodiment, the muscle injury is associated with muscle trauma or surgery.

[0081] In one embodiment of the invention, a method for promoting recovery after muscle injury is provided, the method comprising administering an effective amount of the composition of the invention to a human or animal subject. In one embodiment, the muscle injury is associated with muscle trauma or surgery.

[0082] Non-limiting examples of orthopedic surgery that may be associated with muscle injuries include carpal tunnel release, knee chondroplasty, removal of supporting implants, anterior cruciate ligament reconstruction of the knee, rotator cuff tendon repair, knee replacement, hip replacement, femoral neck fracture repair, trochanteric fracture repair, radius fracture repair, ankle fracture repair, femoral shaft fracture repair, trochanteric fracture repair, lower lumbar disc surgery, and orthopedic reconstruction.

[0083] Non-limiting examples of trauma-related muscle injuries include muscle contusions, muscle tears, and tendon injuries / ruptures.

[0084] Non-limiting examples of administration include oral administration and intravenous administration. In a preferred embodiment, the administration is oral administration. In one embodiment, the method includes administering a therapeutically effective amount of a combination of vitamin B6 and nicotinamide to the individual in need.

[0085] In another embodiment of the invention, the compounds or compositions of the invention may be combined with dietary interventions of high calories, high protein, high carbohydrates, vitamin B12 and / or vitamin D supplementation, antioxidants, omega-3 fatty acids, butyrate ester producers and / or polyphenols in a method for the prevention or treatment of cachexia or pre-cachexia.

[0086] In the context of this invention, the term "butyrate generating agent" refers to a substance or ingredient that, when administered to a subject, can deliver and / or stimulate the production of butyrate (e.g., in the subject's intestine). Non-limiting examples of butyrate generating agents include sodium butyrate, potassium butyrate, and / or butyrate-containing triglycerides, such as those described, for example, in the same applicant's patent application WO2019 / 228851.

[0087] As used herein, the term “combination” or the terms “in combination,” “used in combination with,” or “combination formulation” may refer to the simultaneous, sequential, or separate application of two or more reagents.

[0088] As used in this article, the term "simultaneously" means that these reagents are applied simultaneously (i.e., at the same time).

[0089] As used in this article, the term “successively” means applying these reagents one after another.

[0090] As used herein, the term “separate” means applying the reagent independently of each other but at a time interval so that the reagent can produce a combined (preferably synergistic) effect. Thus, “separate” application may allow, for example, the application of one reagent after another within 1 minute, 5 minutes, or 10 minutes.

[0091] Without requiring extensive experimentation, technicians can easily determine the appropriate dosage of one of the reagents of this invention to be administered to a subject. Typically, a physician will determine the most suitable practical dosage for an individual patient, and this dosage will depend on a variety of factors, including the activity of the specific active agent used, the metabolic stability and duration of action of the active agent, age, weight, general health condition, sex, diet, administration pattern and time, excretion rate, drug combination, severity of the specific condition, and the individual's ongoing therapy. Of course, there may also be individual cases where a beneficial higher or lower dosage range exists, which is within the scope of this invention.

[0092] In one embodiment, the method includes administering to an individual in need a therapeutically effective amount of a combination of vitamin B6 and nicotinamide, wherein the amount of vitamin B6 is about 1.0-200 mg / day, preferably about 1.0-25.0 mg / day, and the amount of nicotinamide is about 0.001 mg / day to about 2000 mg / day, preferably about 0.001 mg / day to about 1000 mg / day.

[0093] In one implementation, the combination is administered to an individual for a period of at least one month; preferably at least two months, more preferably at least three, four, five, or six months; and most preferably at least one year. During this period, the combination may be administered to the individual at least one day per week; preferably at least two days per week, more preferably at least three, four, five, or six days per week; and most preferably seven days per week. The combination may be administered daily as a single dose or daily as multiple individual doses.

[0094] The above application examples do not require uninterrupted, continuous daily application. Instead, brief interruptions may be allowed during application, such as two to four days during the application period. The ideal duration of application of the composition can be determined by those skilled in the art.

[0095] Subjects

[0096] In some implementations, the subjects are humans or non-human animals.

[0097] Examples of non-human animals include vertebrates such as mammals, such as non-human primates (especially higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs, and cats. Non-human animals can be companion animals.

[0098] Preferably, the subjects are humans.

[0099] Example

[0100] The following non-limiting examples support the unexpected effectiveness of compositions containing nicotinamide and vitamin B6 in promoting or improving muscle repair, skeletal muscle regeneration, muscle function, and / or muscle mass.

[0101] Example 1 - Myogen Expansion and Characterization of Muscle Stem Cells

[0102] Materials and methods

[0103] Human primary myoblasts from different donors (donor 1, donor 2, and donor 3) were seeded at a density of 1,000 cells per well in skeletal muscle growth medium (SKM-M, AMSbio) in 384-well plates. For treatment, the compound was added directly to the myoblast culture 16 hours after initial seeding.

[0104] All cultures were then grown for 96 hours. Cells were stained with antibodies against Pax7 and MyoD to determine Pax7 and MyoD expression, and counterstained with Hoechst 33342 to visualize the nuclei. Pax7+ cells were defined as cells expressing Pax7, regardless of MyoD expression. MyoD+ cells were defined as cells expressing MyoD but not Pax7. Images were acquired using the ImageXpress (Molecular Devices) platform. Quantification was performed using a custom module analysis of multi-wavelength cell scoring based on MetaXpress software. Additionally, several ratios (vitamin B6 / NAM ratios) of pyridoxine to nicotinamide, ranging from 1:2 to 1:80, were tested. Figure 5 This represents the number of Pax7+ cells at these specific ratios in the same model.

[0105] Differences from the control were indicated by one-way ANOVA, with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Data are expressed as mean ± SEM.

[0106] result

[0107] The results are presented in Figures 1 to 5 middle.

[0108] Data collected from human primary myoblasts from donors 1 and 2 (see [link to data]). Figure 1 For each condition, the total cell count was determined to assess compound toxicity, and the number of Pax7+ or MyoD+ cells was normalized to the total cell count to assess the proportion of the population and expressed as a fold change compared to the control condition (DMSO 1%). Figure 1 A represents the proportion of Pax7+ cells, and Figure 1 B represents the proportion of MyoD+ cells. These data indicate that nicotinamide promotes muscle stem cell function by increasing the ratio of both expanded cells (Pax7+) and differentiated cells (MyoD+) in a dose-dependent manner.

[0109] Similarly, for pyridoxine, data were pooled from human primary myoblasts from donors 1 and 2. For each condition, the total cell count was determined to assess compound toxicity, and the number of Pax7+ or MyoD+ cells was normalized to the total cell count to assess the proportion of this population and expressed as a fold change compared to the control condition (DMSO 1%). Figure 2 A represents the proportion of Pax7+ cells, and Figure 2B represents the proportion of MyoD+ cells. These data indicate that pyridoxine promotes muscle stem cell function by increasing the proportion of differentiated (MyoD+) cells in a dose-dependent manner.

[0110] Figure 3 The effects of nicotinamide and pyridoxine, alone or in combination, on MyoD+ cells (from donor 3) are indicated. For each condition, the number of MyoD+ cells was normalized to the number of MyoD+ cells in the control condition (DMSO 1%). Figure 3 A represents the number of MyoD+ cells normalized to the control condition. Figure 3 B represents the increase in the number of MyoD+ cells compared to the control condition (DMSO 1%). These data indicate that the combined effect of nicotinamide and pyridoxine is greater than the sum of the effects of nicotinamide and pyridoxine alone, demonstrating a synergistic effect. In fact, by applying a linear regression model (interaction term, p=0.05), we were able to observe a statistically significant synergistic effect between nicotinamide and pyridoxine.

[0111] As a comparison, the combination of nicotinamide (NAM) and vitamin B9 was measured similarly to the above (see...). Figure 4 Unlike pyridoxine (vitamin B6), vitamin B9 (another member of the B vitamin complex) exhibits neither additive nor synergistic effects when added in combination with nicotinamide. Additionally, Figure 5 This indicates that the ratio of pyridoxine to nicotinamide (the vitamin B6 / NAM ratio) has a relevant effect on promoting muscle stem cell function.

[0112] Example 2: In vivo effects of the combination of nicotinamide (NAM) and pyridoxine (B6) on muscle stem cell function. Materials and methods

[0113] To recreate the physiological process of muscle regeneration in adult skeletal muscle in response to injury or disease, we intramuscularly injected cardiotoxin into the hind limb muscles of mice. One week prior to inducing muscle injury, mice were administered the compounds of interest (nicotinamide and pyridoxine, 200 mg / kg body weight and 4 mg / kg body weight, respectively) orally, relative to a water control group. Mice were treated once daily until the end of the experiment. To assess the efficiency of muscle regeneration, 5 days after injury (… Figure 6 and Figure 7 ) and 12 days ( Figure 8 Previously injured muscle was harvested and frozen sections were prepared. Several myogenic markers were then measured. Frozen sections were stained using specific antibodies against Pax7, myopoietin, laminin (to delineate muscle fibers), and embryonic myosin heavy chain (to define the injured / regenerated area), and counterstained with Hoechst 33342 to visualize cell nuclei. Pax7+ cells (…) were further stained separately. Figure 6 A and Figure 7 A) and myocyte cytopoietin + cells ( Figure 6 B and Figure 7 The number of cells (B) was counted to assess the early and later stages of myogenic differentiation of muscle stem cells. Data were expressed as the number of cells per unit area of ​​injured muscle and as a fold change compared to control conditions. Late stages of muscle fiber maturation ( Figure 8 The assessment was performed by quantifying the size of each newly formed muscle fiber, which was measured based on the expression of embryonic myosin heavy chain and laminin, allowing for the identification and mapping of these new muscle fibers. The results are shown as the cross-sectional area of ​​the muscle fiber (µm2). Figure 6 The experiment shown was conducted using 3-month-old mice defined as an adult population. Figure 7 and Figure 8 The experiment shown was conducted using 24-month-old mice, which were defined as an older population, and "adult" mice as a control.

[0114] result

[0115] These data indicate that, in preclinical in vivo models of muscle repair / regeneration, the combination of nicotinamide and pyridoxine promotes muscle stem cell function by increasing both the number of expanded cells (Pax7+) and differentiated cells (MyoD+). Figure 6 Similar experiments were also conducted in older animals. Figure 7 Furthermore, it was shown that, under aging conditions, the combination of nicotinamide and pyridoxine also promoted muscle stem cell function by increasing both the number of proliferating cells (Pax7+) and differentiated cells (MyoD+), thereby restoring these bioreadings to adult animal levels. Additionally, Figure 8 This indicates that the combination of nicotinamide and pyridoxine can promote the muscle repair process by increasing the size of newly formed muscle fibers.

[0116] Various changes and modifications to the presently preferred embodiments disclosed herein will be apparent to those skilled in the art. These changes and modifications can be made without departing from the spirit and scope of the subject matter of the invention and without diminishing its intended advantages. Therefore, such changes and modifications are intended to be covered by the appended claims.

Claims

1. A composition, characterized in that, The composition comprises a therapeutically effective amount of a combination of vitamin B6 and nicotinamide, wherein the vitamin B6:nicotinamide is present in a ratio of about 1:80 to about 1:20, and the composition is intended to treat muscle damage in an individual suffering from muscle injury, and / or promote muscle repair, improve skeletal muscle regeneration, maintain or increase skeletal muscle function and / or skeletal muscle mass.

2. The composition according to claim 1, wherein the vitamin B6: nicotinamide is present in a ratio of about 1:75 to about 1:

25.

3. The composition according to claim 1, wherein the vitamin B6: nicotinamide is present in a ratio of about 1:60 to about 1:

30.

4. The composition according to claim 1, wherein the vitamin B6: nicotinamide is present in a ratio of about 1:45 to about 1:

30.

5. The composition according to claim 1, wherein the composition is an oral nutritional composition.

6. The composition of claim 1, wherein the composition is a nutritional supplement.

7. The composition of claim 6, wherein the composition is an oral nutritional supplement.

8. The composition according to claim 1, wherein the composition is in the form of a solid powder, capsule, or liquid.

9. The composition of claim 8, wherein the composition is in the form of a powder rod.

10. The composition according to any one of claims 1 to 9, wherein the composition further comprises a protein source, a carbohydrate source and / or a lipid source.

11. Use of the composition according to any one of claims 1 to 10 in the preparation of a product for treating muscle injury in an individual suffering from muscle injury, and / or promoting muscle repair, improving skeletal muscle regeneration, maintaining or increasing skeletal muscle function and / or skeletal muscle mass.

12. Use of the combination of vitamin B6 and nicotinamide in the preparation of a composition for treating muscle injury in an individual suffering from muscle damage, and / or promoting muscle repair, improving skeletal muscle regeneration, maintaining or increasing skeletal muscle function and / or skeletal muscle mass, wherein said vitamin B6:nicotinamide is present in a ratio of about 1:80 to about 1:

20.

13. The use according to claim 11 or 12, wherein the vitamin B6 is administered in an amount of about 1.0 mg to 600 mg of vitamin B6 per day.

14. The use according to claim 13, wherein the vitamin B6 is administered in an amount of about 1.0 mg to 200 mg of vitamin B6 per day.

15. The use according to claim 13, wherein the vitamin B6 is administered in the following amounts: from about 1.0 mg to 25.0 mg of vitamin B6 per day.

16. The use according to claim 11 or 12, wherein the nicotinamide is administered in an amount of about 1 mg / day to about 3000 mg / day.

17. The use according to claim 16, wherein the nicotinamide is administered in an amount of about 10 mg / day to about 2000 mg / day.

18. The use according to claim 16, wherein the nicotinamide is administered in an amount of about 100 mg / day to about 1000 mg / day.

19. The use according to claim 16, wherein the nicotinamide is administered in an amount of about 500 mg / day to about 1000 mg / day.

20. The use according to claim 11 or 12, wherein the vitamin B6 is administered in an amount of 10 mg to 20.0 mg vitamin B6 / day, and / or the nicotinamide is administered in an amount of about 500 mg to about 1000 mg nicotinamide / day.

21. The use according to claim 11 or 12, wherein the product or composition is used to promote recovery from muscle injury in an individual in need of it.

22. The use according to claim 21, wherein the muscle injury is related to muscle trauma and / or surgery.

23. The use according to claim 12, wherein the vitamin B6: nicotinamide is present in a ratio of about 1:75 to about 1:

25.

24. The use according to claim 12, wherein the vitamin B6: nicotinamide is present in a ratio of about 1:60 to about 1:

30.

25. The use according to claim 12, wherein the vitamin B6: nicotinamide is present in a ratio of about 1:45 to about 1:

30.

26. The use according to claim 12, wherein the composition is an oral nutritional composition.

27. The use according to claim 12, wherein the composition is a nutritional supplement.

28. The use according to claim 27, wherein the composition is an oral nutritional supplement.

29. The use according to claim 12, wherein the composition is in the form of a solid powder, capsules or liquid.

30. The use according to claim 29, wherein the composition is in the form of a powder rod.

31. The composition according to claim 12 further comprises a protein source, a carbohydrate source, and / or a lipid source.

32. The kit, characterized in that, The kit contains therapeutically effective amounts of nicotinamide and vitamin B6, wherein the vitamin B6:nicotinamide is present in a ratio of about 1:80 to about 1:20, and the kit is intended to treat muscle damage in individuals suffering from muscle injury, and / or promote muscle repair, improve skeletal muscle regeneration, maintain or increase skeletal muscle function and / or skeletal muscle mass.

33. The kit of claim 32, wherein the muscle injury is related to muscle trauma and / or surgery.

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