Nutrient-based formulations usable in treating muscle atrophy

Abstract

A composition that comprises one or more nutrients capable of inhibiting myostatin signaling and a use thereof in treating muscle atrophy are provided. A method of identifying nutrients that are capable of inhibiting myostatin signaling and / or of treating muscle atrophy is also provided.

Description

[0001] NUTRIENT-BASED FORMULATIONS USABLE IN TREATING MUSCLE ATROPHY

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 649,479, filed on May 20, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to nutrient-based formulations, and more particularly, but not exclusively, to nutrient-based formulations which are usable in the treatment or prevention of muscle atrophy.

[0006] There are approximately 600 skeletal muscles in the human body, accounting for about 45 % of the total human body weight. Skeletal muscles play an important role in the body, including in thermoregulation, systemic metabolism, movement, posture, and visceral protection.

[0007] Muscle atrophy or wasting is a common condition characterized by the loss or decrease in muscle mass and / or strength, mitochondrial dysfunction, changes in muscle fiber type, and / or impaired physical function.

[0008] Muscle atrophy has a major impact on patients, by reducing the body's ability to respond to stress and chronic diseases, and promoting bone fracture and insulin resistance, thereby increasing morbidity and mortality.

[0009] Muscle atrophy can impair overall body movements, posing a potential risk to life as well as reduced quality of life for patients. The rapid loss of muscle mass and strength can lead to loss of muscle function, disability, frailty, reduced quality of life, and increased morbidity and mortality, thereby impacting patient prognosis and posing a huge socioeconomic burden.

[0010] Muscle atrophy can be caused by the imbalance between rates of protein synthesis and degradation occurring in various conditions such as aging, obesity, cachexia, malnutrition, immobility, and central nervous system damage, or can be associated with certain diseases, for example motor neuron diseases such as amyotrophic lateral sclerosis (ALS), diabetes, cancer, congestive heart failure, chronic obstructive pulmonary disease, AIDS, liver disease, renal failure and cardiac failure.

[0011] The loss of muscle mass results largely from the accelerated degradation of the contractile myofibrillar proteins, primarily by the ubiquitin-proteasome pathway. Their destruction accounts for the reduction in muscle strength and increased disability that reduce quality of life and contribute to mortality. Age-related muscle loss (sarcopenia) is a hallmark of aging, leading to weight loss, frailty, disability and reduced life expectancy, and preservation of muscle mass enhances health, quality of life, and longevity.

[0012] Loss of muscle mass and body weight in aging and cancer patients, known as cancer- associated cachexia, contribute to weakness, disability, frailty, and increased morbidity and mortality. Clinical studies indicate that cachexia is a major cause of death in cancer patients, and that preservation of muscle mass prolongs survival.

[0013] Currently, there are no effective FDA-approved therapies for muscle atrophy, and the only validated treatment is exercise, which reduces various types of atrophy and forms the mainstay of clinical management. However, exercise is not a practical option for bed-ridden, frail, sarcopenic or older individuals, or those with acute illnesses.

[0014] A few treatments for muscle atrophy have been explored, which are primarily focused on anti-inflammatory and anti-oxidation actions, promoting protein synthesis, inhibiting protein degradation, and promoting muscle regeneration [Huang et al. Antioxidants (Basel). 2022 Dec 26; 12( 1):44] . Salvia miltiorrhiza, a Traditional Chinese Medicine containing magnesius lithospermate B (MLB), has been used to prevent obesity-related skeletal muscle atrophy by inhibiting MAFbx and MuRFl -mediated muscle degeneration [Cheng et al., Nutrients. 2021; 14: 104]. The widely used drug for type-2 diabetes mellitus, Metformin, has also been found to alleviate skeletal muscle atrophy in grxl KO mice, reduce intramuscular lipid sediments, and increase glucose utilization through the AMPK / Sirtl pathway [Yang et al., Biochem. Biophys. Res. Commun. 2020;533:1226-1232],

[0015] Nutritional solutions (for example, Ensure, Fortimel, Boost), which contain a balanced diet of lipids, proteins and carbohydrates, have not been designed to specifically combat muscle atrophy, but to increase body weight. Other nutritional products (for example, HMB, FymBeam, Bodybio) that are consumed mainly by athletes, are drinkable protein enriched liquids aimed at promoting normal muscle growth. However, these products do not reverse the debilitating atrophy because they were not designed to specifically target catabolic mechanisms of an atrophying muscle, providing only a sub-optimal solution that is insufficient to block or treat atrophy.

[0016] Myostatin, a member of the TGF[3 superfamily of growth factors and a crucial regulator of muscle mass, has been explored as an attractive target for treating muscle atrophy [Smith et al., Curr Opin Support Palliat Care. 2013 Dec;7(4):352-60]. The signaling pathway for myostatin is shown in Background Art FIG. 1. Myostatin binds to type IIB activin receptor (ActRIIB) on muscle cells, which stimulates its dimerization, thus activating a signaling cascade involving the recruitment and activation of type I activin receptor transmembrane kinase ALK4 or ALK5; the phosphorylation of SMAD2 and SMAD3 and the recruitment of SMAD4, to form a SMAD complex; and the translocation of the SMAD complex into the nucleus, resulting in gene transcription changes of downstream genes, which results in muscle wasting. Concurrently, myostatin binding to ActRIIB reduces AKT activity, which results in reduced FOXO phosphorylation. Dephosphorylated FOXO enters the nucleus, thereby stimulating the transcription of atrophy-eliciting E3 ligases MuRFl and Atrogin-1, which results in muscle protein ubiquitination and degradation by the proteasome.

[0017] Inhibiting myostatin therefore offers a therapeutic strategy against muscle wasting by disrupting this pathway.

[0018] A number of myostatin inhibitors have been explored for muscle atrophy. sActRIIB, a potent inhibitor of myostatin and Activin A signaling, was found to potently inhibit both myostatin- and activin-mediated SMAD2 / 3 signal transduction. The administration of a single dose of sActRIIB to adult C57BI / 6 mice was found to lead to a dose-dependent increase in body weight and lean body mass [Zhou et al., Cell. 2010 Aug 20; 142(4):531-43] . BYM338, a fully human, receptor-neutralizing monoclonal antibody that blocks myostatin, demonstrated strong promyogenic effects leading to skeletal muscle hypertrophy [Lach-Trifilieff et al., Mol Cell Biol. 2014 Feb;34(4):606-18]. AMG 745, an antimyostatin peptibody, has been tested in mouse models, showing increased body weight gain, increased or improved maintenance of skeletal muscle mass, and increased strength compared with control mice [Padhi et al., J Clin Endocrinol Metab. 2014 Oct;99(10):E1967-75].

[0019] Review of myostatin inhibitors for the treatment of muscle wasting can be found, for example, in Smith et al., Curr Opin Support Palliat Care. 2013 Dec;7(4):352-60.

[0020] PCT International Patent Application Publication No. WO 2011 / 032109 discloses methods of predicting the severity of spinal muscular atrophy or disease progression in a patient or population of patients, which involve measuring the level of a marker and / or the ratio of at least two markers in a biological sample. Amongst the tested markers are glycine, 2-ketoglutaric acid (alpha-ketoglutaric acid), sodium butyrate and myoinositol, and mannitol.

[0021] PCT International Patent Application Publication No. WO 2022 / 082082 discloses lung surfactant therapeutic compositions comprising heat stable protein-phospholipid concentrates for the treatment of respiratory diseases in premature infants, children, and adults, including asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). These compositions may include excipients such as glycine, alpha-ketoglutaric acid, butyric acid, myoinositol, and mannitol. U.S. Patent Application Publication No. 2023 / 0346699 discloses a ketone body composition comprising beta-hydroxybutyrate, related compounds, and / or one or more compounds, such as butyrate, for administration to a non-human animal to raise ketones and improve animal health, for treating conditions such as obesity, muscle atrophy, body composition and gut health. The ketone body compositions may additionally include alpha-ketoglutaric acid and mannitol.

[0022] Buggeskov et al., J Clin Med. 2018 Nov 21;7(11):462, disclose a randomized controlled trial to study the effects of pulmonary artery perfusion with hypothermic histidine-tryptophan- ketoglutarate (HTK) solution or oxygenated blood, versus standard cardiopulmonary bypass (CPB), in protecting patients from lung ischemia-reperfusion injury, particularly those with COPD. The disclosed HTK solution contains 15 mM Na+, 9 mM K+, 4 mM Mg++, 0.015 mM Ca++, 198 mM histidine, 2 mM tryptophan, 1 mM 2-ketoglutarate, and 30 mM mannitol.

[0023] U.S. Patent No. 11,065,214 discloses compositions and methods for increasing lifespan, for preventing or treating a disease including an aging-related disorder, for reducing a symptom of aging, and / or boosting an immune system in a mammal, comprising at least a therapeutically effective amount of disulfiram and one or more additional ingredients, including glycine, ketoglutaric acid, i-inositol and D-mannitol.

[0024] Glycogen synthase kinase-3 (GSK-3) is recognized as an important target for drug discovery and its inhibition has been considered a promising therapeutic approach for treating several pathologies including neurodegenerative diseases and malignancies. In humans, GSK-3 is expressed as two isozymes, GSK-3a and GSK-3P (SEQ ID NO: 1) which are encoded by two genes and share high homology in their catalytic domains. The mechanisms by which GSK-3 is thought to contribute to pathogenesis are diverse. These include phosphorylation of the microtubule- associated protein tau, destabilization of the Wnt signaling component P-catenin, regulation of multiple transcription factors such as NF-KB, activation of pro-inflammatory factors, and impairment of clearance pathways.

[0025] Some of the present inventors have previously uncovered that an initial key event that triggers the atrophy process involves the phosphorylation of the desmin cytoskeleton by glycogen synthase kinase-3 beta (GSK-3 P), rendering this enzyme a target of interest in the treatment of muscle atrophy [Aweida et al., J Cell Biol. 2018 Oct 1 ;217( 10):3698-3714] . It has been uncovered that phosphorylation of desmin filaments, which are critical for muscle architecture and function, by GSK-3P, and their subsequent degradation promotes overall muscle protein breakdown and atrophy. It has been reported that GSK-3P inhibition in mice prevented desmin phosphorylation and depolymerization and blocked atrophy induced by fasting or denervation. Additionally, spaceflight, an activity known to cause muscle atrophy, was found to reduce GSK-3P content across all missions. It was further observed that inhibiting GSK-3P increases muscle mass, preserves muscle strength, and promotes the oxidative fiber type with Earth-based hindlimb unloading [Baranowski et al., iScience. 2023 Jun 8;26(7): 107047].

[0026] Bone marrow-derived miR-140 was found to inhibit endotoxic-induced glycolysis and atrophy of skeletal muscle by negatively regulating the WNT signaling pathway and simultaneously reducing the expression of Wnt family member 11, P-catenin, and GSK-3P [Liu et al., Am. J. Physiol. Cell Physiol. 2019;317:C189-C199],

[0027] GSK-3 inhibitors have been reviewed, for example, in Eldar-Finkelman et al., Front Mol Neurosci. 2011 ; 4: 32; and Arciniegas Ruiz et al., Front Mol Neurosci, 2022 Jan 21;14:792364, and include inorganic substances, and organic small molecules and peptides.

[0028] Among the first synthetic small molecule GSK-3 inhibitors reported were pyrimidine or pyridine- based compounds, developed by Chiron (see, for example, PCT International Patent Application Publication No. WO 99 / 65897 and U.S. Patent Application Publication No. 2002 / 0156087). Compounds known as CHIR98014 (CT98014), CHIR98023 (CT98023), CHIR99021 (CT99021) were shown as highly potent and selective inhibitors of GSK-3. Compounds of the CHIR family were reported, amongst other acti vities, to enhance the levels of the survival motor neuron protein (SMN) in spinal muscular atrophy (Makhortova et al. (2011) Nat. Chem. Biol. 7, 544-552. See also, PCT International Patent Application Publication No. WO 2010 / 048273. U.S. Patent Application Publication No. 2009 / 0306045 describes the use of these GSK-3P inhibitors as an effective therapy for several autoimmune diseases.

[0029] PCT International Patent Application Publication No. WO 2004 / 052404 describes substrate-competitive GSK-3P inhibitors, designed based on the recognition motif of the enzyme, and accordingly featuring a short peptide sequence that terminates by a fatty acid residue. An exemplary such inhibitor is also known as L8O3-mts having the following amino acid sequence: Myr-Gly-Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-GIn-{Ser(p)}-Pro-NH2. See also Plotkin et al., (2003) J. Pharmacol. Exp. Ther. 305, 974-980; and Kaidanovich-Beilin et al., J. Pharmacol. Exp. Ther. 316, 17-24.

[0030] Additional Background Art includes Kramer et al., Int J Alzheimer Dis.

[0031] 2012;2012:381029. SUMMARY OF THE INVENTION

[0032] According to an aspect of some embodiments of the present invention there is provided a composition for use in treating or preventing muscle atrophy in a subject in need thereof, the composition comprising at least one nutrient that exhibits inhibition of myostatin signaling.

[0033] According to some embodiments of any of the embodiments described herein, the at least one nutrient is characterized by at least one of: affecting at least 10, or at least 20, or at least 50, %, reduction in phosphorylated SMAD2,3 and / or in a ratio of phosphorylated SMAD2,3 to non-phosphorylated SMAD2,3 in atrophying muscle cells; and affecting a decrease of at least 10, or at least 20, or at least 50, % in expression of a muscle atrophy biomarker in atrophying muscle cells.

[0034] According to some embodiments of any of the embodiments described herein, a level of phosphorylated SMAD2,3 or a ratio as indicated in the atrophying muscle cells is determined by Western blotting.

[0035] According to some embodiments of any of the embodiments described herein, the muscle atrophy biomarker is associated with myostatin signaling pathway.

[0036] According to some embodiments of any of the embodiments described herein, the muscle atrophy biomarker is selected from Atrogin and / or Muscle RING-finger protein- 1 (MuRFl) mRNA.

[0037] According to some embodiments of any of the embodiments described herein, the composition comprises two or more of the nutrient as described herein.

[0038] According to some embodiments of any of the embodiments described herein, at least two of the nutrient act in synergy with one another.

[0039] According to some embodiments of any of the embodiments described herein, the at least one nutrient is selected from glycine, mannitol, butyric acid, pyruvic acid, myoinositol, uridine, adenosine, 3 -hydroxybutyric acid, alpha-ketoglutarate, and pharmaceutically acceptable salts thereof.

[0040] According to some embodiments of any of the embodiments described herein, the at least one nutrient is selected from adenosine, myoinositol, glycine, alpha-ketoglutarate, sodium butyrate and mannitol, and pharmaceutically acceptable salts thereof.

[0041] According to some embodiments of any of the embodiments described herein, an amount of the at least one nutrient is in a range of from 30 to 100, or from 40 to 100, or from 50 to 100, or from 60 to 100, or from 50 to 90, or from 60 to 90, % by weight of the total weight of the composition. According to some embodiments of any of the embodiments described herein, an amount of the at least one nutrient is in a range of from 0.01 to 50, or from 0.01 to 30, or from 0.01 to 20, or from 0.01 to 10, or from 1 to 50, or from 1 to 30, or from 1 to 20, or from 1 to 10 % by weight of the total weight of the composition.

[0042] According to some embodiments of any of the embodiments described herein, the composition is such that the subject receives each nutrient in an amount of from about 1 to about 1500, or from about 100 to about 1200, milligrams per day, when administered to a human subject (weighing about 70 kg), or an equivalent dose, when administered to a non-human subject.

[0043] According to some embodiments of any of the embodiments described herein, the at least one nutrient comprises glycine.

[0044] According to some embodiments of any of the embodiments described herein, a total daily amount of glycine in the composition is in a range of from about 0.2 to about 1.5, or from about 0.75 to about 1.5, grams per day, when the composition is administered to a human subject (weighing about 70 kg), or an equivalent dose, when administered to a non-human subject.

[0045] According to some embodiments of any of the embodiments described herein, the at least one nutrient comprises butyric acid and / or a salt thereof (e.g., sodium butyrate).

[0046] According to some embodiments of any of the embodiments described herein, a total daily amount of sodium butyrate in the composition is in a range of from about 0.3 to about 1.2, or from about 0.75 to about 1.1, grams per day, when the composition is administered to a human subject (weighing about 70 kg), or an equivalent dose, when administered to a non-human subject.

[0047] According to some embodiments of any of the embodiments described herein, the at least one nutrient comprises alpha-ketoglutarate.

[0048] According to some embodiments of any of the embodiments described herein, a total daily amount of alpha-ketoglutarate in the composition is in a range of from about 0.3 to about 1.5, or from about 0.75 to about 1.1, grams per day, when the composition is administered to a human subject (weighing about 70 kg), or an equivalent dose, when administered to a non-human subject.

[0049] According to some embodiments of any of the embodiments described herein, the at least one nutrient comprises adenosine.

[0050] According to some embodiments of any of the embodiments described herein, a total daily amount of adenosine in the composition is in a range of from about 0.75 to about 1.8, or from about 1.1 to about 1.5, grams per day, when the composition is administered to a human subject (weighing about 70 kg), or an equivalent dose, when administered to a non-human subject.

[0051] According to some embodiments of any of the embodiments described herein, the composition further comprises at least one of: a source of protein, a source of fatty acids, a source of one or more carbohydrates, sterol, vitamin, mineral, phenolic compound, carotenoid compound and odoriferous (scent) compound.

[0052] According to some embodiments of any of the embodiments described herein, the composition further comprises a physiologically acceptable carrier.

[0053] According to some embodiments of any of the embodiments described herein, the composition is formulated for administration by oral administration or oral ingestion.

[0054] According to some embodiments of any of the embodiments described herein, the composition is in a form selected from a tablet, a capsule (e.g., gelatin capsule), a powder, a granule, a bead, a pellet, a lozenge, a solution, a syrup, an edible product, and a chewable solid (e.g., chewing gum).

[0055] According to some embodiments of any of the embodiments described herein, treating the muscle atrophy comprises administering the composition to the subject by oral ingestion at least once, or at least twice, per day.

[0056] According to some embodiments of any of the embodiments described herein, treating the muscle atrophy comprises administering the composition to the subject by oral ingestion, such that the at least one nutrient is provided in an amount of from about 10 mg / kg to about 1,000 mg / kg per day, or from about 100 mg / kg to about 400 mg / kg per day.

[0057] According to some embodiments of any of the embodiments described herein, treating the muscle atrophy comprises administering the composition to the subject by oral administration or ingestion in a total amount of from 1 to 5, or from 1 to 3, grams of the composition, per day.

[0058] According to some embodiments of any of the embodiments described herein, the composition is formulated for topical administration.

[0059] According to some embodiments of any of the embodiments described herein, the composition is formulated in a form of a patch (e.g., a dermal patch as described herein).

[0060] According to some embodiments of any of the embodiments described herein, the composition is formulated as a medical device or a medical food.

[0061] According to some embodiments of any of the embodiments described herein, the composition further comprises a therapeutically active agent usable in treating or preventing muscle atrophy.

[0062] According to some embodiments of any of the embodiments described herein, the composition further comprises a GSK-3 inhibitor.

[0063] According to some embodiments of any of the embodiments described herein, treating or preventing the muscle atrophy further comprises administering to the subject a therapeutically active agent usable in treating or preventing muscle atrophy. According to some embodiments of any of the embodiments described herein, treating or preventing the muscle atrophy further comprises co-administering to the subject a GSK-3 inhibitor.

[0064] According to some embodiments of any of the embodiments described herein, the muscle atrophy is associated with at least one of ageing, cachexia, sedentary lifestyle, sarcopenia, malnutrition, disuse atrophy, neurogenic atrophy, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth disease, peripheral neuropathy, corticosteroid therapy, Emery-Dreifuss muscular dystrophy, Distal muscular dystrophy, Oculopharyngeal muscular dystrophy, Congenital muscular dystrophy, neuromuscular diseases, extended immobilization, trauma, alcoholism, cancer treatment, hyperthyroidism, heart failure, liver diseases, kidney diseases, diabetes, osteoarthritis, Cushing's syndrome, nutritional atrophy, severe burns, malabsorption syndromes, anorexia nervosa and ischemic atrophy, anorexia nervosa, rheumatoid arthritis and surgery.

[0065] According to some embodiments of any of the embodiments described herein, the muscle atrophy is associated with cachexia.

[0066] According to some embodiments of any of the embodiments described herein, cachexia is associated with cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), chronic kidney disease and Acquired Immune Deficiency Syndrome (AIDS).

[0067] According to an aspect of some embodiments of the present invention there is provided a method of identifying a nutrient capable of treating or preventing muscle atrophy in a subject in need thereof, the method comprising: determining if a biocompatible nutrient substance reduces a level of or inhibits phosphorylation of SMAD2,3 in atrophying muscle cells; and determining if a biocompatible nutrient substance reduces the level of or inhibits expression of a muscle atrophy biomarker in atrophying muscle cells, wherein a biocompatible nutrient substance that reduces the level of or inhibits phosphorylation of SMAD2,3 in atrophying muscle cells and reduces the level of or inhibits expression of the muscle atrophy biomarker in atrophying muscle cells, is identified as capable of treating or preventing muscle atrophy.

[0068] According to some embodiments of any of the embodiments described herein, determining if a biocompatible nutrient substance inhibits phosphorylation of SMAD2,3 in the atrophying muscle cells comprises contacting the nutrient substance with the atrophying muscle cells, and determining a level of phosphorylated SMAD2,3 (pSMAD2,3) in the atrophying muscle cells and / or determining an intensity ratio of phosphorylated SMAD2,3 to non-phosphorylated SMAD2,3 in the atrophying muscle cells.

[0069] According to some embodiments of any of the embodiments described herein, determining the level of phosphorylated SMAD2,3 and / or the intensity ratio is performed by immunoblotting (e.g., Western immunoblotting).

[0070] According to some embodiments of any of the embodiments described herein, a nutrient substance is determined as inhibiting or reducing a level of phosphorylation of SMAD2,3 in the atrophying muscle cells if the nutrient substance affects at least 10, or at least 20, or at least 50, %, reduction in the phosphorylated SMAD2,3 intensity and / or in the intensity ratio of phosphorylated SMAD2,3 to non-phosphorylated SMAD2,3, in the atrophying muscle cells.

[0071] According to some embodiments of any of the embodiments described herein, determining if a biocompatible nutrient substance inhibits expression of the muscle atrophy biomarker in atrophying muscle cells comprises contacting the nutrient substance with the atrophying muscle cells and determining a level of the expression of the muscle atrophy biomarker in the atrophying muscle cells.

[0072] According to some embodiments of any of the embodiments described herein, a nutrient substance is determined as inhibiting or reducing the level of the expression if it affects a decrease of at least 10, or at least 20, or at least 50, % in expression of the muscle atrophy biomarker in the atrophying muscle cells.

[0073] According to some embodiments of any of the embodiments described herein, determining a level of the expression is performed by determining biomarkers expression in atrophying muscle cells in the presence of the nutrient substance, wherein determining biomarkers expression in atrophying muscle cells in the presence of the nutrient is affected by a technique selected from quantitative PCR (e.g., RT-qPCR), Western blotting, ELISA, RNA sequencing, and immunohistochemistry.

[0074] According to some embodiments of any of the embodiments described herein, the muscle atrophy biomarker is selected from Atrogin and / or Muscle RING-finger protein- 1 (MuRFl) mRNA.

[0075] According to some embodiments of any of the embodiments described herein, the method comprises: determining if a biocompatible nutrient substance reduces the level of or inhibits phosphorylation of SMAD2,3 in atrophying muscle cells, to thereby identify nutrient substances capable of reducing the level of or inhibiting phosphorylation of SMAD2,3 in atrophying muscle cells; and determining if a nutrient substance identified as capable of reducing the level of or inhibiting phosphorylation of SMAD2,3 in atrophying muscle cells reduces the level of or inhibits expression of the muscle atrophy biomarker in atrophying muscle cells.

[0076] According to some embodiments of any of the embodiments described herein, the method further comprises determining an effect of the nutrient substance identified as reducing the level of or inhibiting phosphorylation of SMAD2,3 in atrophying muscle cells and as reducing the level of or inhibiting expression of a muscle atrophy biomarker in atrophying muscle cells, on muscle atrophy in an atrophying test animal (a model animal for muscle atrophy such as described herein).

[0077] According to some embodiments of any of the embodiments described herein, determining the effect of the nutrient substance on muscle atrophy in the test animal comprises administering the nutrient substance to the atrophying test animal and determining an effect of the administering on a weight of a muscle or muscle tissue of the animal and / or on a weight ratio of the muscle or muscle tissue and the body of the animal.

[0078] According to some embodiments of any of the embodiments described herein, a nutrient substance determined as increasing the weight or weight ratio by at least 5 %, is identified as capable of treating muscle atrophy.

[0079] According to an aspect of some embodiments of the present invention there is provided a nutrient usable in treating or preventing muscle atrophy in a subject in need thereof, identified by the method as described herein in any of the respective embodiments and in any combination thereof.

[0080] According to additional aspects of some embodiments of the present invention there are provided compositions and products which are usable in treating muscle atrophy, as described herein in any of the respective embodiments.

[0081] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0082] BRIEF DESCRIPTION OF THE SEVERAL VIEW OF THE DRAWINGS

[0083] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0084] In the drawings:

[0085] FIG. 1 (Background art) presents a diagram showing the myostatin signaling pathway that leads to muscle atrophy and currently available therapies directed thereto.

[0086] FIG. 2 is a schematic representation generally illustrating a screening method of identifying nutrients that inhibit muscle atrophy, according to some embodiments of the present invention, comprising (1) screening libraries and / or databases of biocompatible nutrient substances to thereby identify nutrient substances that are biocompatible, and feature inhibition of SMAD2,3 phosphorylation in C2C12 myotubes, as determined by Western blotting; (2) determining the Atrogin-1 and myostatin gene expression inhibition of each of the nutrient substances identified in step (1), by measuring the mRNA levels of Atrogin-1 and myostatin in the C2C12 myotubes treated with dexamethasone, using Reverse transcription PCR (RT-PCR) to thereby identify substances with significant inhibitory activity; (3) determining the effect of each of the nutrient substances identified in step (2) on muscle atrophy in vivo in a mouse model, to thereby identify nutrient substance lead candidates for treating or preventing muscle atrophy. The lead candidates are then used to produce a nutrient-based formulation for treating, preventing, or reducing muscle atrophy.

[0087] FIG. 3 presents an image of an immunoblotting image showing the effect of a vehicle (control), myostatin (1 microgram / mL), and myostatin (1 microgram / mL) and A83-O1 (a myostatin inhibitor; 1 micromolar), on pSMAD2,3 and SMAD2,3 levels in C2C12 myotubes (differentiated at 2 % horse serum (HS)).

[0088] FIGs. 4A-B present bar graphs showing the comparative fold increase in Atrogin-1 (FIG. 4A) and the comparative fold increase in MuRF-1 (FIG. 4B) mRNA levels in C2C12 myotubes (differentiated at 2 % horse serum (HS)) treated with dexamethasone (1 micromolar), n = 3 wells per condition. **, P < 0.01 vs. control by unpaired Student’s t-test.

[0089] FIG. 5 presents an image of representative Western blots from a screening of 400 screened nutrients, showing the effects of exemplary nutrients numbered 37 through 48 on the levels of pSMAD2,3, SMAD and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in C2C12 myotubes.

[0090] FIG. 6 is a bar graph showing the comparative fold increase in Atrogin-1 mRNA levels in C2C12 myotubes (differentiated at 2 % horse serum (HS)) treated with dexamethasone (1 micromolar) in the presence of varying glycine concentrations. Graph depicts mean ± SEM. n = 3 wells per condition. *, P < 0.05; **, P < 0.01 vs. dexamethasone alone; #, P < 0.05 vs. nutrient- untreated cells by unpaired Student’s t-test.

[0091] FIGs. 7A-C present an image of Western blots showing pSMAD2,3 and SMAD2,3 levels in C2C12 myotubes (differentiated at 2 % horse serum (HS)) cell extracts treated with dexamethasone (1 micromolar) in the presence or absence of glycine (5 micromolar), butyric acid (5, 10 and 20 micromolar), and pyruvic acid (2.5 and 5 micromolar) (FIG. 7A); a bar graph showing the pSMAD2,3 / SMAD2,3 densitometric measurement ratio as determined according to the Western blots shown in FIG. 7A (ratio is presented as mean ± SEM. n =3 wells per condition) (FIG. 7B); and a bar graph showing the comparative fold increase in Atrogin-1 mRNA levels in C2C12 myotubes, and in C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence and absence of 5 micromolar butyric acid (FIG. 7C). Graphs depict mean ± SEM. n = 4 wells per condition. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. dexamethasone alone; #, P

[0092] < 0.05 ##, P < 0.01; ###, P < 0.001 vs. nutrient-untreated control by unpaired Student’s t-test.

[0093] FIGs. 8A-B present a Western blot image showing pSMAD2,3 and SMAD2,3 levels in cultured C2C12 myotubes (differentiated at 10 % horse serum (HS)), and of the C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence or absence of myoinositol (2.5 and 5 and 10 micromolar) and uridine (2.5, 5 and 10 micromolar) (FIG. 8A); and a bar graph showing the pSMAD2,3 / SMAD2,3 densitometric measurement ratio as determined according to the Western blots shown in FIG. 8A (ratio is presented as mean ± SEM. n =3 wells per condition) (FIG. 8B). *, P < 0.05 vs. dexamethasone alone; #, P < 0.05; ##, P < 0.01 vs. nutrient-untreated control by unpaired Student’s t-test.

[0094] FIGs. 9A-B are bar graphs showing the comparative fold increase in myostatin gene (FIG. 9A) and Atrogin-1 (FIG. 9B) mRNA levels in C2C12 myotubes (differentiated at 10 % HS), and in C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence or absence of butyric acid (10 micromolar), pyruvic acid (2.5 micromolar) or uridine (2.5 micromolar). The graph depicts the mean ratio to nutrient-untreated cells ± SEM. n = 4 wells per condition. ****, P

[0095] < 0.0001 vs. dexamethasone; #, P < 0.05; ##, P < 0.01; ####, P < 0.0001 vs. nutrient-untreated control by unpaired Student’s t-test.

[0096] FIGs. 10A-D are bar graphs showing the comparative fold increase in Atrogin-1 mRNA levels in C2C12 myotubes (differentiated at 2 % horse serum (HS)), and in C2C12 myotubes (differentiated at 2 % HS) treated with dexamethasone (1 micromolar) in the presence or absence of Adenosine (2.5, 5, 10 and 15 micromolar) (FIG. 10A) or in the presence or absence of 3- hydroxybutyric acid (1 and 2.5 micromolar) (FIG. 10B); and the comparative fold increase in myostatin mRNA levels in C2C12 myotubes (differentiated at 2 % HS), and in C2C12 myotubes (differentiated at 2 % HS) treated with dexamethasone (1 micromolar) in the presence or absence of Adenosine (2.5, 5, 10 and 15 micromolar) (FIG. 10C) or in the presence or absence of 3- hydroxybuytic acid (1 and 2.5 micromolar) (FIG. 10D). Graphs depict mean ratio to nutrient- untreated cells ± SEM. n = 3-4 wells per condition. *, P < 0.05; **, P < 0.01; ***, P = 0.0001; ****, p < 0.0001 vs. dexamethasone; #, P < 0.05; ##, P < 0.01; ###, P < 0.001; ####, P < 0.0001 vs. control by unpaired Student’s t-test.

[0097] FIGs. 11 A-B present a scatter plot showing the mean body weight (FIG. 11 A) and the mean ratio of TA muscle weight (FIG. 11B) of mice administered with water soluble dexamethasone (10 mg / kg) over a period of 10 days in comparison to non-treated mice (control), n = 19 mice per group. ****, P < 0.0001 vs. dexamethasone by unpaired Student’s t-test.

[0098] FIGs. 12A-D present graphs showing the mean TA / BW ratios in mice treated with water- soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day) or glycine (4.1 grams / kg / day), in comparison to a nutrient-untreated control (n = 6-11 mice per group. *, P < 0.05; ****, P < 0.0001 vs. dexamethasone treated control; ##, P < 0.005, ###, P < 0.0005, ####, P < 0.0001 vs. nutrient- untreated control by one-way ANOVA) (FIG. 12A); in mice treated with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence of presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day), glycine (4.1 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day) or D-mannitol (12.96 grams / kg / day), in comparison with a nutrient-untreated control (Body weights were normalized to control mice (n =

[0099] 4-14 mice per group. *, P < 0.05, **, P > 0.01, ****, p < 0.0001 vs. dexamethasone treated control, #, P < 0.05; ##, P < 0.01, ####, P < 0.0001 vs. nutrient-untreated control by one-way ANOVA) (FIG. 12B); in mice treated with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of myoinositol (8.3 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), or D-mannitol (12.96 grams / kg / day), and a combination of D-mannitol (12.96 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day) and myoinositol (8.3 grams / kg / day), in comparison to a nutrient-untreated control (Body weights were normalized to control mice (n =

[0100] 5-19 mice per group. ****, p < 0.0001 vs. dexamethasone treated control, ####, P < 0.0001 vs. nutrient-untreated control by one-way ANOVA) (FIG. 12C); and in mice orally administered with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of glycine (4.1 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), adenosine (1.17 grams / kg / day) and sodium butyrate (3.3 grams / kg / day), and various combinations thereof as indicated, in comparison to a nutrient-untreated control. Adenosine dose within the combination is 0.23 gram / kg / day. Body weights were normalized to control mice (n = 6-8 mice per group); *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001 vs. dexamethasone treated control; ##, P < 0.005; ###, P < 0.0005; ####, P < 0.0001 vs. nutrient-untreated control by one-way ANOVA. The mean values for each dataset are also presented. Post-hoc tests: Brown-Forsythe test and Bartlett’s test (FIG. 12D).

[0101] FIG. 13 is a bar graph showing the mean heart weight (mg) of the mice treated with water soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day) or myoinositol (8.3 grams / kg / day).

[0102] FIGs. 14A-B present comparative plots showing the mean body weight (grams) of mice administered with water soluble dexamethasone (10 mg / kg), over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day), or glycine (4.1 grams / kg / day) in comparison with non-treated mice (control) (FIG. 14A); and the mean body weight (grams) of mice administered with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day) or D-mannitol (12.96 grams / kg / day), in comparison to nutrient-untreated mice (control) (FIG. 14B).

[0103] FIG. 15 is a bar graph showing the comparative fold increase in myostatin mRNA levels in mouse Tibialis Anterior (TA) muscle, and in Tibialis Anterior muscle from mice orally treated with water soluble dexamethasone (10 mg / kg body weight) over a period of 10 days, in the absence or presence of myoinositol (8.3 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day) and D- mannitol (10.96 grams / kg / day). Mean fold change ± SEM is presented, n = 5 mice per group. *, P < 0.05, **, P < 0.01 vs. dexamethasone-treated (muscle atrophying) control, #, P < 0.05 vs. nutrient-untreated control by one-way ANOVA.

[0104] FIGs. 16A-G present a representative photograph of a mouse subjected to the grip strength test (FIG. 16A); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day) or glycine (4.1 grams / kg / day), where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 (control) in each group (n = 5 mice per group. #, P < 0.05 vs. time 0 by unpaired Student’s t-test) (FIG. 16B); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day) or D-mannitol (12.96 grams / kg / day), where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 (control) in each group (n = 5-10 mice per group. #, P < 0.05 vs. time 0 by unpaired Student’s t-test) (FIG. 16C); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of alpha-ketoglutarate (3.3 grams / kg / day), D-mannitol (12.96 grams / kg / day), glycine (4.1 grams / kg / day), or a combination of D-mannitol (12.96 grams / kg / day), myoinositol (8.3 grams / kg / day) and alpha- ketoglutarate (3.3 grams / kg / day), where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 control in each group (FIG. 16D); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of glycine (4.1 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day), adenosine (1.17 grams / kg / day), or combinations thereof as indicated, where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 in each group (n = 6-8 mice per group). #, P < 0.05 vs. vehicle control by one-way ANOVA (FIG. 16E); a bar graph showing the mean heart weights in mice treated with dexamethasone over a period of 10 days, in the presence of glycine (4.1 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day), or adenosine (1.17 grams / kg / day), or combinations thereof as indicated, in comparison to a nutrient-untreated control (n = 6-8 mice per group). **, P < 0.005; ****, P < 0.0001 vs. dexamethasone-treated (muscle atrophy-induced) control; #, P < 0.05; ####, P < 0.0001 vs. nutrient-untreated control by one-way ANOVA (FIG. 16F)); and a bar graph showing the mean loss of body weights in mice treated with dexamethasone over a period of 10 days, in the absence or presence of glycine (4.1 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day), adenosine (1.17 grams / kg / day), or combinations thereof as indicated, in comparison to a dexamethasone-treated (muscle atrophy-induced) control (n = 6-8 mice per group). *, P < 0.05; **, P < 0.0001 vs. dexamethasone treated control by one-way ANOVA (FIG. 16G).

[0105] FIGs. 17A-C present bar graphs showing the mean Tibialis Anterior (TA) muscle weight to body weight (BW) ratios (FIG. 17A), the mean cardiac muscle weights (FIG. 17B) and grip test (FIG. 17C) following oral administration of a low-dose (“low cone”) combination of glycine and sodium butyrate to dexamethasone-induced muscle and cardiac atrophying mice. Mice were treated with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of glycine (0.21 grams / kg / day) and sodium butyrate (0.17 grams / kg / day). In FIGs. 17A- B, body weights were normalized to control (nutrient-untreated) mice; mean values ± standard deviation (SD) are indicated (****, p < 0.0001 vs. dexamethasone treated control; ###, P < 0.001, ####, P < 0.0001 vs. vehicle control by one-way ANOVA). In FIG. 7C, “P=0.01” marks the force at day 10 vs. day 2 of intervention; and “ns” denotes no significant difference in force between day 10 and day 2 of intervention. Muscle strength is depicted as gram force (gf) normalized to body weight, where gram force (gf) is the force exerted by one gram of mass under standard gravity (9.80665 m / s2).

[0106] FIGs. 18A-B present a photograph (FIG. 18 A) of two different batches of an exemplary formulation comprising glycine, agarose and alginate, prepared according to an exemplary process as described in the Examples section, and a scatter plot (FIG. 18B) showing the time-dependent dissolution percentage of 500 ppm glycine from the two batches of the exemplary formulation.

[0107] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0108] The present invention, in some embodiments thereof, relates to nutrient-based formulations, and more particularly, but not exclusively, to nutrient-based formulations which are usable in the treatment (reduction, elimination or prevention) of muscle atrophy.

[0109] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0110] As discussed hereinabove and shown in Background Art FIG. 1, while myostatin is a regulator of muscle mass, current methodologies for interfering with the myostatin signaling pathway are non- selective and are associated with severe side effects. The present inventors have conceived a new approach for designing a therapeutic strategy against muscle wasting, which is based on disrupting the myostatin signaling pathway.

[0111] In a search for a nutritional solution for handling muscle atrophy, the present inventors have designed a screening method for identifying nutrients which specifically target the debilitating effects of atrophy pathways in muscle. The three-step screening method, shown in FIG. 2, comprises (1) screening of a library of about 400 nutrients for their effects on the myostatin signaling based on the level of phosphorylated SMAD2,3; (2) evaluating the selected hit nutrients to assess their effects on Atrogin-1 and myostatin gene expression; and (3) conducting in vivo assays in mice to evaluate the effect of the selected hit nutrients in reducing muscle atrophy.

[0112] Based on this screening method, the present inventors have designed a nutrient-based formulation for treating, reducing or preventing muscle atrophy, which specifically targets the debilitating effects of atrophy pathways in muscle, and thereby increases muscle mass and strength and consequently survival of the affected individuals. The nutrient-based supplement was developed to specifically inhibit myostatin signaling and also the expression of MuRFl and Atrogin-1. Embodiments of the present invention relate to the use of nutrients that are capable inhibiting myostatin signaling, for attenuating muscle atrophy in a subject in need thereof, and to compositions and products comprising such nutrients.

[0113] Embodiments of the present invention further relate to a method of identifying a nutrient or a combination of nutrients that inhibits myostatin signaling and / or is capable of treating or preventing muscle atrophy (MA), which is also referred to herein as a “screening” method.

[0114] According to an aspect of some embodiments of the present invention, there is provided a method of identifying a nutrient that acts as a myostatin inhibitor and / or is capable of treating or preventing muscle atrophy in a subject in need thereof.

[0115] According to some of these embodiments, the method is effected by screening a library and / or a database of biocompatible nutrient substances to thereby identify biocompatible nutrient substances that inhibit phosphorylation of SMAD2,3 in atrophying (e.g., dexamethasone-treated) muscle cells (also referred to herein as step 1); and screening the identified biocompatible nutrient substances that inhibit phosphorylation of SMAD2,3 in the muscle cells, to thereby identify biocompatible nutrient substances that inhibit an expression of a muscle atrophy biomarker (also referred to herein as step 2), thereby identifying the nutrient.

[0116] As used herein and in the art, the term “nutrient”, which is also referred to herein interchangeably as “nutrient substance”, describes a biologically active compound that provides a metabolic, structural, and / or regulatory function in cells or tissues, and which supports cellular maintenance, growth, energy production, or physiological homeostasis. In the context of the present embodiments, this term includes compounds that may serve as energy substrates, precursors for biosynthetic pathways, signaling molecules, or cofactors, and may optionally be naturally occurring in humans and / or other organisms.

[0117] Non-limiting examples of nutrients include glycine, mannitol, butyric acid, pyruvic acid, myoinositol, uridine, adenosine, 3-hydroxybutyric acid (also referred to herein as 3 -hydrobutyric acid), alpha-ketoglutarate, Met-Trp, D-malic acid, sedoheptulosan, D-fructose, D-galactose, Leu- Asp, D-raffinose, D-lactitol, D,L-beta-hydroxy-butyric acid, gamma-hydroxy-butyric acid, Met- Pro, Meso-tartaric acid, N-acetyl- neuraminic acid, chondroitin-6 sulfate, L-rhamnose, mesoerythritol, pectin, Met-Thr, D-melezitose, succinamic acid, D-melibiose, propylene glycol, 2,3- butanediol, stachyose, beta-methyl-D-galactoside, palatinose, thymidine, methyl pyruvate, D,L- alpha-glycerol-phosphate, D-glucuronic acid, xylitol, inosine, methyl D-lactate, Leu-His, D- arabinose, mannan, D-fucose, succinic acid, alpha-methyl-D-galactoside, alpha-hydroxy-butyric acid, L-arabinose, hexanoic acid, alpha-methyl-D-mannoside, ethanolamine, N-acetyl-beta-D- mannosamine, D-fructose-6-phosphate, L-fucose, glycerol, beta-methyl-D-xylopyranoside, Met- Lys, adonitol, alpha-keto-butyric acid, gamma-amino-N-butyric acid, sucrose, acetic acid, Met- Met, Lys-Phe, 3-O-methyl-D-glucose, D-sorbitol, Met-Leu, D,L-lactic acid, lactulose, propionic acid, L-sorbose, maltitol, L-malic acid^ L-glucose, alpha-D-lactose, alpha-methyl-D-glucoside, Leu-Gly, D-turanose^ acetoacetic acid, Lys-Ser, Leu-Glu, alpha-cyclodextrin, Met-Tyr, L- histidine, 3-hydroxy-2-butanone, 11c- 11caD-cellobiose, D-salicin, Met-Val, tricarballylic acid, His- Trp, and any salts thereof (e.g., pharmaceutically acceptable salts, e.g., sodium salt).

[0118] The phrase “biocompatible nutrient substance” as used herein and in the art describes a physiologically acceptable nutrient that is non-toxic, non-immunogenic, and does not cause any adverse local or systemic effect when administered to a subject. In some embodiments, the biocompatible nutrient substance is a naturally-occurring nutrient substance.

[0119] According to some embodiments of any of the embodiments of this aspect of the present invention, identifying biocompatible nutrient substances that inhibit phosphorylation of SMAD2,3 in muscle cells (step 1) is effected by contacting a tested nutrient with atrophying muscle cells, and determining the level of phosphorylated SMAD2,3 (pSMAD2,3) in the muscle cells, for example by immunoblotting (e.g., Western Blotting).

[0120] According to some embodiments of any of the embodiments described herein, atrophying muscle cells are obtained by treating muscle cells with, for example, by culturing muscle cells in the presence of, an agent known to induce atrophy. An exemplary acceptable such an agent is dexamethasone, and according to some embodiments the method is effected by contacting the tested nutrient with dexamethasone-treated muscle cells as atrophying muscle cells.

[0121] Other exemplary atrophy-inducing agents include, without limitation, glucocorticoids (e.g., prednisolone or cortisol), cancer chemotherapeutic agents (e.g., doxorubicin), tumor necrosis factor-alpha (TNF-a), lipopolysaccharide (LPS), pro-inflammatory cytokines (e.g., interleukin-6 (IL-6)), myostatin, angiotensin II, and conditions such as starvation (e.g., serum starvation when used in vitro) or mechanical unloading (e.g., bed rest, hindlimb unloading, immobilization).

[0122] According to some embodiments, muscle cells are contacted (e.g., cultured) with the atrophy-inducing agent (e.g., dexamethasone) and with the tested nutrient, to thereby determine a level of phosphorylated SMAD2,3 in atrophying muscle cells. The contacting can be effected, for example, for 1 to 14 days, or from 1 to 5 days, or from 1 to 2 days, or from 1 to 24 hours.

[0123] Non-limiting methods of measuring a level of phosphorylated SMAD2,3 intensity and / or a ratio of phosphorylated SMAD2,3 to (non-phosphorylated) SMAD2,3 in cells include immunoblotting (e.g., by Western blotting). Immunoblotting techniques enable quantitative or semi-quantitative comparison of phosphorylation status under both conditions, thereby determining the level of the reduction in phosphorylation. According to some embodiments, determining the level of phosphorylated SMAD2,3 (pSMAD2,3) in the muscle cells is effected, following the contacting, by immunoblotting (e.g., Western Blotting).

[0124] In some embodiments, determining a level of pSMAD2,3 is effected by determining a ratio of phosphorylated SMAD2,3 (pSMAD2,3) to unmodified (non-phosphorylated) SMAD2,3 in the tested atrophying (e.g., dexamethasone-treated) cells, following the contacting, in the presence and absence of a tested nutrient. A pSMAD2,3 to SMAD2,3 ratio lower by at least 10 %, or at least 20 %, or at least 30 % or at least 40 %, preferably at least 50 %, for example, at least 60 %, or at least 70 %, or at least 80 %, compared to nutrient-untreated atrophying muscle cells is indicative of myostatin signaling inhibition.

[0125] In some embodiments, quantitatively determining a level of pSMAD2,3 is effected by densitometric measurements (such as described in the Examples section that follows) of an immunoblot (e.g., Western Blot), for example, by determining a ratio of phosphorylated SMAD2,3 (pSMAD2,3) to unmodified (non-phosphorylated) SMAD2,3 in the tested atrophying muscle cells, in the presence and absence of the tested nutrients. A pSMAD2,3 to SMAD2,3 ratio lower by at least 10 %, or at least 20 %, or at least 30 % or at least 40 %, preferably at least 50 %, for example, at least 60 %, or at least 70 %, or at least 80 %, compared to nutrient-untreated atrophying muscle cells is indicative of myostatin signaling inhibition.

[0126] In some embodiments, determining the level of phosphorylated SMAD2,3 (pSMAD2,3) in the atrophying muscle cells is affected in vitro (e.g., in cultured myotubes, for example, by adding a tested nutrient to the atrophying, e.g. dexamethasone-treated, cultured myotubes).

[0127] According to some embodiments of any of the embodiments of this aspect of the present invention, the method proceeds to identifying biocompatible nutrient substances that inhibit expression of a muscle atrophy biomarker in atrophying (e.g., dexamethasone-treated) muscle cells (step 2). Nutrient substances identified in step 1 as described herein as capable of inhibiting phosphorylation of SMAD2,3, as described herein are screened to identify those nutrients that inhibit expression of a muscle atrophy biomarker in atrophying cells.

[0128] According to these embodiments, identifying nutrient substances that inhibit expression of a muscle atrophy biomarker is effected by contacting a tested nutrient with atrophying (e.g., dexamethasone-treated) muscle cells, and determining an expression level of the biomarker, typically by PCR (e.g., RT-PCR) in the cells.

[0129] In some embodiments, a level of (e.g., decrease in) expression of a muscle atrophy biomarker(s) is measured by determining biomarkers expression in atrophying (e.g., dexamethasone-treated) muscle cells in the presence of the nutrient (i.e., versus atrophying (e.g., dexamethasone-treated) muscle cells, in the absence of a nutrient). Non-limiting methods for measuring the level of muscle atrophy biomarker expression in the muscle cells include quantitative PCR (e.g., RT-qPCR), Western blotting, ELISA, RNA sequencing, and immunohistochemistry.

[0130] The phrase “muscle atrophy biomarker” describes a measurable molecular indicator that reflects the activation of catabolic pathways leading to muscle protein degradation and / or loss of muscle mass or function. Non-limiting examples of muscle atrophy biomarkers include Atrogin (also known as FBXO32), MuRFl (Muscle RING-finger protein- 1), and other transcriptional or post-translational targets associated with myostatin signaling, such as SMAD2 / 3 phosphorylation levels, and other transcription factors associated with atrophy such as FoxOl / 3a activity, PAX4 or NRF1, and any combination thereof.

[0131] The biomarker may include one, or two, or more, molecular indicators.

[0132] In some embodiments, the muscle atrophy biomarker is associated with myostatin signaling pathway, for example, as presented in FIG. 1.

[0133] In some embodiments, the biomarker is Atrogin- 1 and a level of Atrogin- 1 mRNA is measured. In some embodiments, the biomarker is a myostatin gene and a level of myostatin gene mRNA is measured. In some embodiments, the biomarker is MuRFl and a level of MuRFl gene mRNA is measured.

[0134] In some embodiments, the measured mRNA levels of the biomarker (e.g., Atrogin-1, MuRFl and / or myostatin gene) are compared between atrophying, e.g. dexamethasone-treated muscle cells that have been contacted with the nutrient(s) (nutrient-treated) and atrophying, e.g. dexamethasone-treated, muscle cells that have not been contacted with any nutrient (nutrient- untreated). This comparison allows for determining the effect of the nutrient(s) on the expression of one or more biomarkers associated with muscle atrophy. A reduction in the expression level in the nutrient-treated cells, relative to the nutrient-untreated control, is indicative of the nutrient’s potential to inhibit atrophic pathways and / or mitigate (e.g., dexamethasone-induced) muscle wasting.

[0135] Nutrients that feature a decrease in expression of one or more muscle atrophy biomarkers by at least 10 %, or by at least 20 %, or by at least 30 %, or by at least 40 %, or by at least 50 %, when contacted with atrophying, e.g. dexamethasone-treated, muscle cells, as compared to an expression level of a respective biomarker in muscle cells treated with the atrophy-inducing agent (e.g., dexamethasone) alone, are identified as capable of treating or preventing muscle atrophy in a subject in need thereof. In some embodiments, determining the expression level of a muscle atrophy biomarker in the atrophying muscle cells is effected in vitro (e.g., in cultured myotubes, for example, by adding a tested nutrient to the atrophying, e.g. dexamethasone-treated, cultured myotubes).

[0136] The nutrients identified after step 2 are characterized as capable of interfering with myostatin signaling by being capable of inhibiting phosphorylation of SMAD2,3, and by being capable of inhibiting expression of a muscle atrophy biomarker.

[0137] It is to be noted that while the method is described herein as effected by performing step 1 and then step 2, embodiments of this aspect of the present invention also encompass a screening method as described herein in which step 2 precedes step 1, such that nutrients are first tested for their capability of reducing / inhibiting expression of a muscle atrophy biomarker, and nutrients identified as capable of inhibiting expression of a muscle atrophy biomarker are then tested for their capability of inhibiting phosphorylation of SMAD2,3, as described herein in any of the respective embodiments.

[0138] The method as described herein results in nutrients identified as usable in treating muscle atrophy.

[0139] According to some embodiments of any of the embodiments described herein, the method further comprises determining an effect of an identified nutrient or a combination of two or more nutrients (for example, a combination of nutrients that provides a synergistic or additive effect as described herein) on muscle atrophy in a test animal (e.g., mice) (also referred to herein as step 3).

[0140] The test animal can be an animal afflicted by muscle atrophy or an animal that was treated with an atrophy-inducing agent in an amount that causes muscle atrophy, to thereby provide a model animal afflicted by muscle atrophy. In some embodiments, the tested model animal is treated with an atrophy-inducing agent, as described herein, for example, with dexamethasone.

[0141] According to some of these embodiments, the animal is a dexamethasone-treated (muscle atrophy-induced) animal, although animals treated by other atrophy-inducing agents (as described herein) and / or other models of muscle atrophy-afflicted animals are also contemplated.

[0142] Exemplary test animals include, without limitation, mice, rats, guinea pigs, hamsters, rabbits, ferrets, dogs, cats, and other animal (e.g., mammalian) models commonly used in pharmacological or toxicological studies, including genetically modified or disease-model animals.

[0143] According to some of these embodiments, a tested nutrient, identified as described herein (e.g., in steps 1 and 2 as described herein), is administered to a (muscle atrophy-induced) dexamethasone-treated test animal (e.g., mice), and its effect on muscle atrophy is determined. According to some of these embodiments, the tested nutrient, identified as described herein (e.g., in steps 1 and 2 as described herein), and the muscle atrophy-inducing agent (e.g., dexamethasone) are co-administered to the test animal (e.g., mice), and following the coadministration (e.g., following 1 to 14 days), an effect on muscle atrophy is determined and optionally compared to animals administered with the atrophy-inducing agent only and / or with intact, non-treated animals.

[0144] An effect on muscle atrophy can be determined by determining an effect on muscle (e.g., Tibias Anterior) weight or volume or on a ratio of a muscle weight or volume to body weight or volume in a test (model) animal, compared to, for example non-treated animals and animals administered with the atrophy-inducing agent only.

[0145] According to some of these embodiments, a tested nutrient, identified as described herein (e.g., in steps 1 and 2 as described herein), is administered to a muscle atrophy-induced (e.g., dexamethasone-treated) test animal (e.g., mice), and its effect on muscle weight or on a ratio of a muscle weight to body weight in a test (model) animal is determined.

[0146] Muscle weight can be determined, for example, by isolating a selected muscle following treatment (e.g., TA), preferably following determining the body weight of the animal.

[0147] Direct measurement of isolated muscle weight may be used to quantify gross anatomical changes in muscle mass; provides a clear endpoint to assess the protective or restorative effect of nutrients against atrophy in specific muscles.

[0148] Exemplary muscles to be assessed include, for example, the Tibias Anterior, the gastrocnemius, quadriceps, soleus, extensor digitorum longus, as well as other muscle tissues as described hereinunder. In some embodiments, the measured muscle is the Tibialis Anterior (TA).

[0149] Nutrients exhibiting a significant increase in a muscle weight or weight ratio of at least 5 %, or at least 10 %, or at least 15 %, or at least 20 %, compared to a test animal treated with an atrophy-inducing agent (e.g., dexamethasone) alone are identified as usable in treating muscle atrophy.

[0150] According to some of these embodiments, a tested nutrient, identified as described herein (e.g., in steps 1 and 2 as described herein), is administered to a muscle atrophy-induced (e.g., dexamethasone-treated) test animal (e.g., mice), and its effect on muscle volume or on a ratio of a muscle volume to body volume in a test (model) animal is determined.

[0151] Muscle volume can be determined, for example, by isolating a selected muscle following treatment, preferably following determining the body weight of the animal, or, alternatively, using imaging methods such as Computed Tomography (CT) and / or magnetic resonance imaging (MRI). Exemplary muscles to be assessed include, for example, the Tibias Anterior, the gastrocnemius, quadriceps, soleus, or extensor digitorum longus, as well as other muscle tissues as described hereinunder. In some embodiments, the measured muscle is the Tibialis Anterior (TA).

[0152] Nutrients exhibiting a significant increase in a muscle volume or volume ratio of at least 5 %, or at least 10 %, or at least 15 %, or at least 20 %, compared to a test animal treated with an atrophy-inducing agent (e.g., dexamethasone) alone are identified as usable in treating muscle atrophy.

[0153] An effect on muscle atrophy can be determined, alternatively or in addition, using one or more acceptable models.

[0154] Non-limiting examples for tests which may be used to determining an effect on muscle include grip strength testing (e.g., hanging test), histological analysis of muscle fiber size, magnetic resonance imaging (MRI), and quantitative PCR for muscle atrophy-related gene expression.

[0155] In some embodiments, the effect on muscle atrophy is determined using one or more acceptable models, for example, grip strength by a hanging test.

[0156] A nutrient exhibiting an increase in hanging time of at least 5 %, or at least 10 %, or at least 15 %, or at least 20 %, compared to a test animal treated with dexamethasone alone, are identified as usable in treating muscle atrophy.

[0157] The phrase “grip strength” as used herein describes the functional capacity of a test animal to maintain a hold on a bar, grid, or similar structure, serving as a surrogate for neuromuscular performance and overall muscle strength. Grip strength is commonly tested using a hanging test.

[0158] The phrase “hanging test” as used herein describes an in vivo method in which a test animal is suspended by its forelimbs on a horizontal bar or grid, and the time until release is recorded as a measure of muscular endurance and strength. Non-limiting examples for additional tests which may be used to determine grip strength include wire-hang tests, digital grip strength meters, forelimb grip force assays, and inclined plane tests.

[0159] Histological analysis of muscle fiber size may be used to evaluate structural changes at the cellular level; provides insight into fiber atrophy or hypertrophy and allows assessment of the integrity and morphology of muscle tissue in response to nutrient treatment.

[0160] MRI may be used to assess muscle volume and structure non-invasively; allows longitudinal monitoring of muscle preservation or degeneration in live animals and may be particularly useful for evaluating the sustained effects of extended-release nutrient formulations. Quantitative PCR for muscle atrophy-related gene expression may be used to measure expression levels of molecular markers associated with muscle wasting, such as MuRFl and Atrogin-1; enables early detection of atrophic pathways and helps elucidate the mechanism of action of the tested nutrients.

[0161] In some embodiments of any of the embodiments described herein, an effect of a nutrient on the heart weight or volume (e.g., upon isolating the heart muscle) is also determined, to assure that this muscle tissue is not affected by the nutrient treatment.

[0162] In some embodiments of any of the embodiments described herein, an effect of a nutrient on muscle atrophy in a test animal as described herein (e.g., muscle atrophying (e.g., dexamethasone-treated) animal) is determined by one or more, preferably by two or more, or three or more, or four or more, or all, of the following: determining an effect on a muscle (e.g., Tibias Anterior) weight or on a ratio of a muscle weight to body weight in a treated atrophying (e.g., dexamethasone-treated) animal; determining an effect on a muscle (e.g., Tibias Anterior) volume or on a ratio of a muscle volume to body volume in a treated atrophying (e.g., dexamethasone-treated) animal; determining a lack of an effect on the heart tissue (e.g., a lack of an effect of a weight of an isolated heart; determining an expression of one or more muscle atrophy biomarker(s)) (e.g., biomarkers associated with myostatin signaling pathway, for example, Atrogin-1 and / or MuRFl mRNA) in a treated atrophying (e.g., dexamethasone-treated) animal; determining a level of SMAD2 / 3 phosphorylation or a ratio of phosphorylated SMAD2 / 3 to (non-phosphorylated) SMAD2 / 3 a treated atrophying (e.g., dexamethasone-treated) animal; and determining an effect on an overall muscle strength (e.g., grip strength), in a nutrient-treated atrophying animal, preferably compared to a nutrient-untreated atrophying animal.

[0163] Nutrients exhibiting at least one, or two, or three, or four, or five, or all, of: an increased muscle weight; an increased ratio of muscle weight to body weight; an increased or at least maintained isolated muscle weight (e.g., preserved cardiac muscle mass); a decrease in the expression of one or more muscle atrophy biomarker(s) (e.g., Atrogin-1, MuRFl); a reduction in the level of SMAD2 / 3 phosphorylation or a decreased pSMAD2 / 3-to-total SMAD2 / 3 ratio; and an increase in muscle strength (e.g., improved grip strength), compared to a test animal which is muscle atrophying and is nutrient-untreated (a test animal treated with dexamethasone alone) are identified as usable in treating muscle atrophy. In some embodiments of any of the embodiments described herein, an effect of a combination of (two or more) nutrients on muscle atrophy in a test animal as described herein (e.g., muscle atrophying (e.g., dexamethasone-treated) animal) is tested and determined, in accordance with any of the above-described methods / parameters.

[0164] In some embodiments of any of the embodiments described herein, an effect of a combination of (two or more) nutrients on muscle atrophy in a test animal as described herein (e.g., muscle atrophying (e.g., dexamethasone-treated) animal) is determined by one or more, preferably by two or more, or three or more, or four or more, or all, of the following: determining an effect on a muscle (e.g., Tibias Anterior) weight or on a ratio of a muscle weight to body weight in a treated atrophying (e.g., dexamethasone-treated) animal; determining an effect on a muscle (e.g., Tibias Anterior) volume or on a ratio of a muscle volume to body volume in a treated atrophying (e.g., dexamethasone-treated) animal; determining a lack of an effect on the heart tissue (e.g., a lack of an effect of a weight of an isolated heart; determining an expression of one or more muscle atrophy biomarker(s)) (e.g., biomarkers associated with myostatin signaling pathway, for example, Atrogin-1 and / or MuRFl mRNA) in a treated atrophying (e.g., dexamethasone-treated) animal; determining a level of SMAD2 / 3 phosphorylation or a ratio of phosphorylated SMAD2 / 3 to (non-phosphorylated) SMAD2 / 3 a treated atrophying (e.g., dexamethasone-treated) animal; and determining an effect on an overall muscle strength (e.g., grip strength), in a nutrient combination-treated atrophying animal, preferably compared to a nutrient- untreated atrophying animal.

[0165] Combinations of two or more nutrients that exhibit at least one, or two, or three, or four, or five, or all, of: an increased muscle weight; an increased ratio of muscle weight to body weight; an increased isolated muscle weight; a decrease in expression of one or more muscle atrophy biomarker(s); a reduction in SMAD2 / 3 phosphorylation or in pSMAD2 / 3-to-total SMAD2 / 3 ratio; and an improvement in muscle strength, as compared to dexamethasone-treated animals not administered with the nutrient combination, are identified as combinations usable in treating or preventing muscle atrophy. In some embodiments, the combination exhibits an additive effect or a synergistic effect, as those are described herein.

[0166] According to some embodiments of any of the embodiments described in this aspect of the present invention, combinations of nutrients are also determined for an additive or synergistic effect on muscle atrophy, accordance with any of the above methods / models / parameters. As used herein throughout, the term “synergy” or “synergistic effect” describes, in the context of the present embodiments, an effect produced by the combination of two or more nutrients that is greater than the sum of the individual effects of each nutrient (when administered separately).

[0167] As used herein, the phrase “additive effect” describes, in the context of the present embodiments, the combined effect of the nutrients, which is about equal to the arithmetic sum of the individual effects of each nutrient (when administered separately).

[0168] Additive and synergistic effects can be determined using methods well known in the art, for example, by the Loewe additivity model (e.g., by means of isobolograms). According to this model, if two agents act via similar mechanisms, the expected additive effect should fall on a curve defined by interpolating the individual dose-effect data. If a combination produces an equal or higher effect at a lower combined dose, synergy is indicated. Accordingly, for synergy, effects fall below the additivity line on an isobologram due to an unexpectedly increased effect by the combination; and for additivity, effects fall on the expected additivity line on an isobolograms.

[0169] In some embodiments, an additive effect on muscle atrophy is determined using acceptable models, for example, grip strength by hanging test. A combination of two or more nutrients (for example, a combination of nutrients that provides an additive effect as described herein) exhibiting an increase in hanging time that is about equal to the arithmetic sum of the increases produced by each nutrient when administered individually, and / or exhibits a combined effect that falls at about the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram), compared to a test animal treated with dexamethasone alone, is identified as having an additive effect in treating muscle atrophy.

[0170] In some embodiments, a synergistic effect on muscle atrophy is determined using acceptable models, for example, grip strength by hanging test. A combination of two or more nutrients (for example, a combination of nutrients that provides a synergistic effect as described herein) exhibiting an increase in hanging time that is greater than the arithmetic sum of the increases produced by each nutrient when administered individually, and / or exhibits a combined effect that falls below the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram), is identified as having a synergistic effect in treating muscle atrophy.

[0171] In some embodiments, an additive effect on muscle atrophy is determined by determining a change in a muscle weight or a ratio of muscle to body weight, as described herein. A combination of two or more nutrients (for example, a combination of nutrients that provides an additive effect as described herein) exhibiting an increase in muscle weight or ratio that is about equal to the arithmetic sum of the increases produced by each nutrient when administered individually, and / or exhibits a combined effect that falls at about the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram), compared to a test animal treated with dexamethasone alone, is identified as having an additive effect in treating muscle atrophy.

[0172] In some embodiments, a synergistic effect on muscle atrophy is determined by determining a change in a muscle weight or a ratio of muscle to body weight, as described herein. A combination of two or more nutrients (for example, a combination of nutrients that provides a synergistic effect as described herein) exhibiting an increase in muscle weight or ratio that is greater than the arithmetic sum of the increases produced by each nutrient when administered individually, and / or exhibits a combined effect that falls below the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram), is identified as having a synergistic effect in treating muscle atrophy.

[0173] In some embodiments of any of the embodiments described herein, the method further comprises determining a suitable dose of a nutrient (e.g., an identified nutrient) or for each nutrient in a combination of two or more nutrients (e.g., a combination of nutrients that provides an additive or synergistic effect). The dose may be optimized based on the degree of improvement in one or more parameters described herein (e.g., isolated muscle weight to body weight ratio, for example, TA / BW ratio, grip strength, and / or gene expression levels), and may be selected to achieve an effect compared to a dexamethasone-treated (muscle atrophy-induced) controls as described herein. Dose titrations may be performed, in which various doses of nutrients are tested to evaluate the relationship between nutrient concentration and therapeutic effect. For example, the dose may be tested at levels corresponding to a fraction (e.g., 10 %, 20 %, 50 %) or a multiple (e.g., 2x, 5x) of the physiological level of the nutrient. For a combination of nutrients, the dose of each nutrient may be independently varied to identify the lowest effective dose and / or an optimal ratio between the two or more nutrients in the combination that yields an additive or synergistic effect.

[0174] According to an aspect of some embodiments of the present invention, there is provided a nutrient, or a combination of two or more nutrients (for example a combination of nutrients that provides a synergistic or additive effect as described herein), identified as capable of treating muscle atrophy by a screening method as described herein.

[0175] According to an aspect of some embodiments of the present invention, there is provided a nutrient, or a combination of two or more nutrients (for example, a combination of nutrients as described herein, e.g., that provides a synergistic or additive effect as described herein) (e.g., 2, 3, 4, or even more, nutrients), that is usable in treating muscle atrophy in a subject in need thereof. According to some embodiments of this aspect of the present invention, the nutrient, or each nutrient in the combination of nutrients, is characterized as a myostatin signaling inhibitor.

[0176] According to some embodiments of any of the embodiments described herein, the nutrient is identified or characterized as capable of treating or preventing muscle atrophy in a subject in need thereof.

[0177] In some of these embodiments, the nutrient is such that features one or more of the following: (i) capable of causing a reduction in phosphorylated SMAD2 / 3 intensity and / or in a ratio of phosphorylated SMAD2 / 3 to (non-phosphorylated) SMAD2 / 3 in atrophying (e.g., dexamethasone-treated) muscle cells, wherein the reduction can be determined as described herein under embodiments of the screening method; and / or (ii) capable of causing a decrease in expression of one or more muscle atrophy biomarker(s) in atrophying muscle cells, wherein the decrease in expression is determined as described herein under embodiments of the screening method; and / or (iii) capable of increasing a muscle weight and / or in a muscle weight to body weight ratio, wherein the increase in weight or weight ratio is determined as described herein under embodiments of the screening method; and / or (iv) capable of causing an increase in overall muscle strength (e.g., grip strength), as compared to an untreated (nutrient-untreated) subject (or a nutrient-untreated atrophying subject (e.g., a test, model animal), wherein the increase in muscle strength is determined as described herein under embodiments of the screening method; and / or (iii) capable of increasing a muscle volume and / or in a muscle volume to body volume ratio, wherein the increase in volume or volume ratio is determined as described herein under embodiments of the screening method.

[0178] In some embodiments of any of the embodiments described herein, the reduction in phosphorylated SMAD2 / 3 intensity and / or in the ratio of phosphorylated SMAD2 / 3 to (nonphosphorylated) SMAD2 / 3 in atrophying muscle cells is of at least 5 %, or at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, or at least 50 %, or at least 60 %, or at least 70 %, or at least 80 %, in comparison to nutrient-untreated atrophying cells. In some of these embodiments, the reduction is determined as described herein under the screening method embodiments, for example, by Western blotting.

[0179] In some embodiments of any of the embodiments described herein, the decrease in expression of one or more muscle atrophy biomarkers is of at least 5 %, or at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, or at least 50 %, or at least 60 %, or at least 70 %, or at least 80 %, in comparison to nutrient-untreated atrophying cells. In some of these embodiments, the decrease in expression is determined as described herein under the screening method embodiments, in vitro, or otherwise in vivo, as described herein. In some embodiments of any of the embodiments described herein, the increase in a muscle weight and / or an increase in a muscle weight to body weight ratio is of at least 5 %, or at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, or at least 50 %, or at least 60 %, or at least 70 %, or at least 80 %, in comparison to nutrient-untreated atrophying cells. In some of these embodiments, the increase in weight or weight ratio is determined as described herein under the screening method embodiments.

[0180] In some embodiments of any of the embodiments described herein, the increase in overall muscle strength is of at least 5 %, or at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, or at least 50 %, or at least 60 %, or at least 70 %, or at least 80 %, in comparison to nutrient- untreated atrophying cells. In some of these embodiments, the increase on muscle strength is determined as described herein under the screening method embodiments, for example, by a suitable model such as the hanging test.

[0181] According to some embodiments, the nutrient or combination of nutrients as described herein is characterized by modulating the myostatin signaling pathway and / or inhibiting downstream transcriptional activation associated with muscle catabolism, for example, is characterized by one or more of: causing a at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, preferably at least 50 % (e.g., at least 70 % or at least 80 %) reduction in phosphorylated SMAD2,3 intensity and / or in a ratio of phosphorylated SMAD2,3 to SMAD2,3 in dexamethasone-treated muscle cells, determined as described herein (for example, by immunoblotting (e.g., Western blotting)), in comparison to untreated control (dexamethasone-treated muscle cells not contacted with the nutrient); and at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, preferably at least 50 % (e.g., at least 70 % or at least 80 %) decrease in expression of one or more muscle atrophy biomarker(s) (e.g., biomarkers associated with myostatin signaling pathway, for example, Atrogin- 1 and / or MuRFl mRNA) compared to an untreated control (dexamethasone-treated muscle cells not exposed to the nutrient).

[0182] In some embodiments of any of the embodiments described herein, the nutrient, or each nutrient in the combination of nutrients, or the combination of two or more nutrients, is characterized as a myo statin signaling inhibitor.

[0183] In some embodiments, the reduction in phosphorylated SMAD2,3 intensity and / or in a ratio of phosphorylated SMAD2,3 to (non-phosphorylated) SMAD2,3 in atrophying (e.g., dexamethasone-treated) muscle cells as described herein is (nutrient) dose-dependent, and is optionally maintained across at least two tested concentrations. In some embodiments, the nutrient or combination of nutrients is as described herein in any of the respective embodiments. Additional suitable nutrients can be selected or identified in accordance with any of the features associated with inhibiting myostatin signaling and / or treating muscle atrophy, as described herein.

[0184] Embodiments of the present invention further relate to a composition comprising one or more nutrients that inhibit myostatin signaling and / or are capable of treating or preventing muscle atrophy (MA), and to the use and formulation of such compositions.

[0185] According to an aspect of some embodiments of the present invention, there is provided a composition for use in treating or preventing muscle atrophy in a subject in need thereof, the composition comprising at least one nutrient (e.g., 1, 2, 3, 4, or even more, nutrients) as described herein in any of the respective embodiments and any combination thereof, for example, a biocompatible nutrient identified or characterized as a myostatin signaling inhibitor as described herein in any of the respective embodiments and any combination thereof.

[0186] According to some embodiments of any of the embodiments described herein, the composition comprises two or more (e.g., 2, 3, 4, or even more) nutrients (herein also referred to as a combination of nutrients, a nutrient combination, or simply as a combination).

[0187] According to some embodiments of any of the embodiments described herein, the composition comprises two or more (e.g., 2, 3, 4, or even more) nutrients that act in synergy with one another or which provide an additive effect, in the context of treating muscle atrophy and / or inhibiting myostatin signaling as described herein.

[0188] According to some embodiments of any of the embodiments described herein, the composition comprises one or more nutrient(s), for example, one or more biocompatible nutrients as described herein (e.g., identified as capable of reducing SMAD2,3 phosphorylation and / or inhibiting expression of one or more biomarkers as described herein.

[0189] Exemplary nutrients include, but are not limited to, glycine, mannitol, butyric acid, pyruvic acid, myoinositol, uridine, adenosine, 3 -hydroxy butyric acid, alpha-ketoglutarate, Met-Trp, D- malic acid, sedoheptulosan, D-fructose, D-galactose, Leu-Asp, D-raffinose, D-lactitol, D,L-beta- hydroxy -butyric acid, gamma-hydroxy-butyric acid, Met- Pro, Meso-tartaric acid, N-acetyl- neuraminic acid, chondroitin-6 sulfate, L-rhamnose, meso-erythritol, pectin, Met-Thr, D- melezitose, succinamic acid, D-melibiose, propylene glycol, 2,3-butanediol, stachyose, beta- methyl-D-galactoside, palatinose, thymidine, methyl pyruvate, D,L-alpha-glycerol-phosphate, D- glucuronic acid, xylitol, inosine, methyl D-lactate, Leu-His, D-arabinose, mannan, D-fucose, succinic acid, alpha-methyl-D-galactoside, alpha-hydroxy- butyric acid, L-arabinose, hexanoic acid, alpha-methyl-D-mannoside, ethanolamine, N-acetyl-beta-D- mannosamine, D-fructose-6- phosphate, L-fucose, glycerol, beta-methyl-D-xylopyranoside, Met-Lys, adonitol, alpha-keto- butyric acid, gamma-amino-N-butyric acid, sucrose, acetic acid, Met-Met, Lys-Phe, 3-O-methyl- D-glucose, D-sorbitol, Met-Leu, D,L-lactic acid, lactulose, propionic acid, L-sorbose, maltitol, L- malic acid^ L-glucose, alpha-D-lactose, alpha-methyl-D-glucoside, Leu-Gly, D-turanose^ acetoacetic acid, Lys-Ser, Leu-Glu, alpha-cyclodextrin, Met-Tyr, L-histidine, 3-hydroxy-2- butanone, Ile-Ile^ D-cellobiose, D-salicin, Met-Val, tricarballylic acid and His-Trp.

[0190] According to some embodiments of any of the embodiments described herein, the one or more nutrient(s) is / are selected from glycine, mannitol, butyric acid, pyruvic acid, myoinositol, uridine, adenosine, 3-hydroxybutyric acid, alpha-ketoglutarate, and pharmaceutically acceptable salts thereof.

[0191] According to some embodiments of any of the embodiments described herein, the one or more nutrient(s) comprise one or more of glycine, butyric acid, myoinositol, uridine, adenosine, 3-hydroxybutyric acid, and pharmaceutically acceptable salts thereof.

[0192] According to some embodiments of the present invention, the composition comprises glycine as a nutrient.

[0193] According to some embodiments, the composition comprises alpha-ketoglutaric acid and / or alpha-ketoglutarate as the nutrient.

[0194] According to some embodiments, the composition comprises butyric acid or a salt thereof (e.g., a pharmaceutically acceptable salt, e.g., sodium butyrate), as a nutrient.

[0195] According to some embodiments, the composition comprises adenosine, as a nutrient.

[0196] According to some embodiments, the composition comprises myoinositol, as a nutrient.

[0197] According to some embodiments, the composition comprises mannitol (e.g., D-mannitol), as a nutrient.

[0198] According to some embodiments, the composition comprises uridine, as a nutrient.

[0199] According to some embodiments, the composition comprises 3-hydroxybutyric acid or a salt thereof, as a nutrient.

[0200] According to some embodiments of any of the embodiments described herein, the composition comprises a combination of two or more nutrients, wherein the combination is identified as capable of treating or preventing muscle atrophy in a subject in need thereof. In some embodiments, the combination acts in synergy, as described herein (e.g., determined by means of an isobologram).

[0201] According to some embodiments, the composition comprises one, two, three, four or more of glycine, pyruvic acid, butyric acid, myoinositol, uridine, adenosine, 3-hydroxybutyric acid, alpha-ketoglutarate, D-mannitol, Met-Trp, D-malic acid, sedoheptulosan, D-fructose, D-galactose, Leu-Asp, D-raffinose, D-lactitol, D,L-beta-hydroxy-butyric acid, gamma-hydroxy-butyric acid, Met-Pro, Meso-tartaric acid, N-acetyl- neuraminic acid, chondroitin-6 sulfate, L-rhamnose, mesoerythritol, pectin, Met-Thr, D-melezitose, succinamic acid, D-melibiose, propylene glycol, 2,3- butanediol, stachyose, beta-methyl-D-galactoside, palatinose, thymidine, methyl pyruvate, D,L- alpha-glycerol-phosphate, D-glucuronic acid, xylitol, inosine, methyl D-lactate, Leu-His, D- arabinose, mannan, D-fucose, succinic acid, alpha-methyl-D-galactoside, alpha-hydroxy-butyric acid, L-arabinose, hexanoic acid, alpha-methyl-D-mannoside, ethanolamine, N-acetyl-beta-D- mannosamine, D-fructose-6-phosphate, L-fucose, glycerol, beta-methyl-D-xylopyranoside, Met- Lys, adonitol, alpha-keto-butyric acid, gamma-amino-N-butyric acid, sucrose, acetic acid, Met- Met, Lys-Phe, 3-O-methyl-D-glucose, D-sorbitol, Met-Leu, D,L-lactic acid, lactulose, propionic acid, L-sorbose, maltitol, L-malic acid^ L-glucose, alpha-D-lactose, alpha-methyl-D-glucoside, Leu-Gly, D-turanose^ acetoacetic acid, Lys-Ser, Leu-Glu, alpha-cyclodextrin, Met-Tyr, L- histidine, 3-hydroxy-2-butanone, Ilc-IlcaD-cellobiose, D-salicin, Met-Val, tricarballylic acid and His-Trp, as a nutrient or a combination of nutrients.

[0202] In some of these embodiments, the composition is such that a subject and / or a test animal and / or atrophying muscle cells treated with the combination of nutrients exhibit one or more, or preferably two or more, or three or more, or all, of: (i) a reduction in phosphorylated SMAD2 / 3 intensity and / or in a ratio of phosphorylated SMAD2 / 3 to (non-phosphorylated) SMAD2 / 3 in atrophying muscle cells; and / or (ii) a decrease in expression of one or more muscle atrophy biomarker(s) in atrophying muscle cells; and / or (iii) an increase in a muscle weight and / or an increase in a muscle weight to body weight ratio; and / or (iv) an increase in overall muscle strength (e.g., grip strength), as compared to an untreated (nutrient-untreated) subject (or a nutrient- untreated atrophying animal), all are as described herein in any of the respective embodiments, and all can be determined as described herein in any of the respective embodiments.

[0203] It is to be noted that a composition which comprises a combination of two or more nutrients as described herein can be such that each nutrient features one or more of the above characteristics / parameters, and the combination provides an additive or synergistic effect, as defined herein, or can be such that one, two or each nutrient alone does not exhibit the activity but the combination of these nutrients does.

[0204] According to some embodiments of any of the embodiments described herein, the combination comprises glycine and butyric acid or a salt thereof.

[0205] According to some embodiments of any of the embodiments described herein, the combination comprises glycine and butyric acid or a salt thereof, wherein the effect of the combination is equal to or greater than the sum of the effects of each nutrient when administered alone, and / or exhibits a combined effect that falls on or below the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram).

[0206] According to some embodiments of any of the embodiments described herein, the combination comprises glycine, alpha-ketoglutarate, and butyric acid or a salt thereof.

[0207] According to some embodiments of any of the embodiments described herein, the combination comprises glycine, alpha-ketoglutarate, and butyric acid or a salt thereof, wherein the effect of the combination is equal to or greater than the sum of the effects of each nutrient when administered alone, and / or exhibits a combined effect that falls on or below the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram).

[0208] According to some embodiments of any of the embodiments described herein, the combination comprises alpha-ketoglutarate and butyric acid or a salt thereof.

[0209] According to some embodiments of any of the embodiments described herein, the combination comprises alpha-ketoglutarate and butyric acid or a salt thereof, wherein the effect of the combination is equal to or greater than the sum of the effects of each nutrient when administered alone, and / or exhibits a combined effect that falls on or below the expected additivity line according to the Loewe additivity model (e.g., by means of an isobologram).

[0210] According to some embodiments of any of the embodiments described herein, the composition comprises one or more, two or more, three or more, or four or more of glycine, butyric acid, myoinositol, uridine, adenosine, 3-hydroxybutyric acid, and pharmaceutically acceptable salts thereof.

[0211] According to some embodiments of any of the embodiments described herein, the composition comprises glycine and butyric acid or any salt thereof; glycine, alpha-ketoglutarate and 3-hydroxybutyric acid or any salt thereof; or adenosine and butyric acid or any salt thereof.

[0212] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof and myoinositol.

[0213] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof and uridine.

[0214] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof and adenosine.

[0215] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof and myoinositol.

[0216] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof and D-mannitol. According to some embodiments of any of the embodiments described herein, the composition comprises glycine, alpha-ketoglutarate thereof and uridine.

[0217] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, alpha-ketoglutarate and adenosine.

[0218] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, alpha-ketoglutarate and myoinositol.

[0219] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, alpha-ketoglutarate and D-mannitol.

[0220] According to some embodiments of any of the embodiments described herein, the composition comprises butyric acid or a salt thereof, alpha-ketoglutarate thereof and uridine.

[0221] According to some embodiments of any of the embodiments described herein, the composition comprises butyric acid or a salt thereof, alpha-ketoglutarate and adenosine.

[0222] According to some embodiments of any of the embodiments described herein, the composition comprises 3butyric acid or a salt thereof, alpha-ketoglutarate and myoinositol.

[0223] According to some embodiments of any of the embodiments described herein, the composition comprises butyric acid or a salt thereof, alpha-ketoglutarate and D-mannitol.

[0224] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof, alpha-ketoglutarate thereof and uridine.

[0225] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof, alpha-ketoglutarate and adenosine.

[0226] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof, alpha-ketoglutarate and myoinositol.

[0227] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof, alpha-ketoglutarate and D-mannitol.

[0228] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, butyric acid or a salt thereof, alpha-ketoglutarate thereof and uridine.

[0229] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, uridine and adenosine.

[0230] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, uridine and myoinositol.

[0231] According to some embodiments of any of the embodiments described herein, the composition comprises glycine, uridine and D-mannitol. Any other combinations are contemplated. The amounts of each nutrient is determined and / or is in accordance with any of the embodiments as described herein. According to some embodiments of any of the embodiments described herein, the composition does not comprise at least one of heat stable protein-phospholipids, disulfiram and / or tert-Butylhydroquinone.

[0232] According to some embodiments of any of the embodiments described herein, a total amount of the one or more nutrients as described herein ranges from 0.01 % to 10 % by weight, of the total weight of the composition, including any intermediate values and subranges therebetween.

[0233] Depending on the form of the composition, higher and lower concentrations are also contemplated.

[0234] According to some embodiments of any of the embodiments described herein, a total amount of the one or more nutrients as described herein ranges from 1 % to 100 %, or from 1 to 80, or from 1 to 70, or from 1 to 60, or from 1 to 50, or from 1 to 40, or from 1 to 30, or from 1 to 20, or from 1 to 10, % by weight, of the total weight of the composition, including any intermediate values and subranges therebetween.

[0235] According to some embodiments of any of the embodiments described herein, a total amount of the one or more nutrients as described herein ranges from 10 % to 100 %, or from 10 to 80, or from 10 to 70, or from 10 to 60, or from 10 to 50, or from 10 to 40, or from 10 to 30, or from 10 to 20, % by weight, of the total weight of the composition, including any intermediate values and subranges therebetween.

[0236] According to some embodiments of any of the embodiments described herein, a total amount of the one or more nutrients as described herein ranges from 20 % to 100 %, or from 20 to 80, or from 20 to 70, or from 20 to 60, or from 20 to 50, or from 20 to 40, or from 20 to 30, % by weight, of the total weight of the composition, including any intermediate values and subranges therebetween.

[0237] According to some embodiments of any of the embodiments described herein, a total amount of the at least one nutrient is in a range of from 30 to 100, or from 40 to 100, or from 50 to 100, or from 60 to 100, or from 30 to 90, or from 40 to 90, or from 50 to 90, or from 60 to 90, or from 30 to 80, or from 40 to 80, or from 50 to 80, or from 60 to 80, or from 30 to 70, or from 40 to 70, or from 50 to 70, or from 60 to 70, % by weight of the total weight of the composition, including any intermediate values and subranges therebetween.

[0238] According to some of these embodiments, the composition is in a form of a solution, for example, an aqueous solution that comprises an aqueous carrier, and a concentration of each nutrient is no more than 50 %, or no more than 20 % of the maximal soluble concentration of the nutrient in the carrier. In some of these embodiments, the composition is in a liquid form, as described in further detail hereinunder.

[0239] According to some embodiments of any of the embodiments described herein, the composition is formulated and / or administered to a human subject (weighing about 70 kg) such that the subject receives each nutrient in an amount of from about 1 to about 1500, or from about 1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 300, or from about 1 to about 200, or from about 1 to about 100, or from about 10 to about 1500, or from about 10 to about 1000, or from about 10 to about 750, or from about 10 to about 500, or from about 10 to about 300, or from about 10 to about 200, or from about 10 to about 100, or from about 50 to about 1500, or from about 50 to about 1000, or from about 50 to about 750, or from about 50 to about 500, or from about 50 to about 250, or from about 50 to about 150, or from about 100 to about 1500, or from about 100 to about 1000, or from about 100 to about 750, or from about 100 to about 150, or from about 500 to about 1500, or from about 500 to about 1200, or from about 500 to about 1000, or from about 500 to about 750, or from about 800 to about 1200, or from about 850 to about 1100, or from about 900 to about 1000, or from about 900 to about 950, or from about 1100 to about 1200, or from about 1150 to about 1200, milligrams per day, including any intermediate values and subranges therebetween.

[0240] In some embodiments, the composition is a unit dosage form (e.g., capsule), such that a total daily dose is administered as a single capsule or is divided into two, or three, or four, or more, unit dosage forms (e.g., capsules), each unit dosage form contains a portion of the total daily dose. The unit dosage forms may contain equal or different amounts of the active nutrient(s), and may be administered concurrently or at different times throughout the day. In some embodiments, the total daily dose is administered as two or more, or three or more unit dosage forms. In some such embodiments, each unit dosage form (e.g., capsule) comprising a total amount of nutrients of at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, or at least 0.6, or at least 0.7, grams, including any intermediate values and subranges therebetween. In some embodiments, each capsule has a total weight of from about 0.5 to about 2, or from about 0.5 to about 1.75, or from about 0.5 to about 1.5, or from about 0.5 to about 1.2, or from about 0.7 to about 2, or from about 0.7 to about 1.75, or from about 0.7 to about 1.5, or from about 0.7 to about 1.2, or from about 0.8 to about 2, or from about 0.8 to about 1.75, or from about 0.8 to about 1.5, or from about 0.8 to about 1.2, or from about 0.9 to about 2, or from about 0.9 to about 1.75, or from about 0.9 to about 1.5, or from about 0.9 to about 1.2, grams, including any intermediate values and subranges therebetween. In some embodiments, each capsule has a total weight of about 1 gram. In some embodiments of any of the embodiments relating to composition which comprises two or more nutrients, a weight ratio between each pair of nutrients in the composition is in a range of from about 1000:1 to about 1:1000, or from about 1000:1 to 1:100, or from about 100:1 to 1:1000, or from about 100:1 to 1:100, or from about 100:1 to 1:75, or from about 100:1 to 1:50, or from about 100:1 to 1:25, or from about 100:1 to 1:10, or from about 100:1 to 1:5, or from about 100:1 to 1:3, or from about 100:1 to 1:2, or from about 100:1 to 1:1, or from about 75:1 to 1:100, or from about 75:1 to 1:75, or from about 75:1 to 1:50, or from about 75:1 to 1:25, or from about 75:1 to 1:10, or from about 75:1 to 1:5, or from about 75:1 to 1:3, or from about 75:1 to 1:2, or from about 75:1 to 1:1, or from about 50:1 to 1:100, or from about 50:1 to 1:75, or from about 50:1 to 1:50, or from about 50:1 to 1:25, or from about 50:1 to 1:10, or from about 50:1 to 1:5, or from about 50:1 to 1:3, or from about 50:1 to 1:2, or from about 50:1 to 1:1, or from about 25:1 to 1:100, or from about 25:1 to 1:75, or from about 25:1 to 1:50, or from about 25:1 to 1:25, or from about 25:1 to 1:10, or from about 25:1 to 1:5, or from about 25:1 to 1:3, or from about 25:1 to 1:2, or from about 25:1 to 1: 1, or from about 10:1 to 1:10, or from about 10:1 to 1:5, or from about 10:1 to 1:3, or from about 10:1 to 1:2, or from about 10:1 to 1:1, or from about 7.5:1 to 1:7.5, or from about 7.5:1 to 1:5, or from about 7.5:1 to 1:3, or from about 7.5:1 to 1:2, or from about 7.5:1 to 1:1.75, or from about 7.5:1 to 1:1.5, or from about 7.5:1 to 1:1.4, or from about 7.5:1 to 1:1.3, or from about 7.5:1 to 1:1.2, or from about 7.5:1 to 1:1.1, or from about 7.5:1 to 1:1, or from about 5:1 to 1:5, or from about 5:1 to 1:3, or from about 5:1 to 1:2, or from about 5:1 to 1:1.75, or from about 5:1 to 1:1.5, or from about 5:1 to 1:1.4, or from about 5:1 to 1:1.3, or from about 5:1 to 1:1.2, or from about 5:1 to 1:1.1, or from about 5:1 to 1:1, or from about 3:1 to 1:3, or from about 3:1 to 1:2, or from about 3:1 to 1:1.75, or from about 3:1 to 1:1.5, or from about 3:1 to 1:1.4, or from about 3:1 to 1:1.3, or from about 3:1 to 1:1.2, or from about 3:1 to 1:1.1, or from about 3:1 to 1:1, or from about 2:1 to 1:2, or from about 2:1 to 1:1.75, or from about 2:1 to 1:1.5, or from about 2:1 to 1:1.4, or from about 2:1 to 1:1.3, or from about 2:1 to 1:1.2, or from about 2:1 to 1:1.1, or from about 2:1 to 1:1, or from about 1.75:1 to 1:1.75, or from about 1.75:1 to 1:1.5, or from about 1.75:1 to 1:1.4, or from about 1.75:1 to 1:1.3, or from about 1.75:1 to 1:1.2, or from about 1.75:1 to 1:1.1, or from about 1.75:1 to 1:1, or from about 1.5:1 to 1:1.5, or from about 1.5:1 to 1:1.4, or from about 1.5:1 to 1:1.3, or from about 1.5:1 to 1:1.2, or from about 1.5:1 to 1:1.1, or from about 1.5:1 to 1:1, or from about 1.4:1 to 1:1.4, or from about 1.4:1 to 1:1.3, or from about 1.4:1 to 1:1.2, or from about 1.4:1 to 1:1.1, or from about 1.4:1 to 1:1, or from about 1.3:1 to 1:1.3, or from about 1.3:1 to 1:1.2, or from about 1.3:1 to 1:1.1, or from about 1.3:1 to 1:1, or from about 1.2:1 to 1:1.2, or from about 1.2:1 to 1:1.1, or from about 1.2:1 to 1:1, or from about 1.1:1 to 1:1.1, or from about 1.1:1 to 1:1, or from about 1:1, including any intermediate values and subranges therebetween. In some embodiments, the composition comprises glycine as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of glycine is in a range of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.3 to about 1.2, or from about 0.3 to about 1.0, or from about 0.2 to about 1.5, or from about 0.2 to about 1.25, or from about 0.2 to about 1.22, or from about 0.5 to about 1.5, or from about 0.5 to about 1.25, or from about 0.5 to about 1.22, or from about 0.75 to about 1.5, or from about 0.75 to about 1.25, or from about 0.75 to about 1.22, or from about 1.0 to about 1.5, or from about 1.0 to about 1.25, or from about 1.0 to about 1.22, or from about 1.1 to about 1.5, or from about 1.1 to about 1.25, or from about 1.15 to about 1.25, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0241] In some embodiments, the composition comprises glycine and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of glycine is administered is divided into two or more, or three or more unit dosage forms (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises glycine in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms). In some embodiments, the composition is formulated such that a total daily amount of glycine is in a range of from about 5 to about 23.5, or from about 5 to about 24, or from about 5 to about 25, or from about 5 to about 27, or from about 5 to about 30, or from about 5 to about 35, or from about 5 to about 50, or from about 10 to about 23.5, or from about 10 to about 24, or from about 10 to about 25, or from about 10 to about 27, or from about 10 to about 30, or from about 10 to about 35, or from about 10 to about 50, or from about 12 to about 23.5, or from about 12 to about 24, or from about 12 to about 25, or from about 12 to about 27, or from about 12 to about 30, or from about 12 to about 35, or from about 12 to about 50, or from about 15 to about 23.5, or from about 15 to about 24, or from about 15 to about 25, or from about 15 to about 27, or from about 15 to about 30, or from about 15 to about 35, or from about 15 to about 50, or from about 18 to about 23.5, or from about 18 to about 24, or from about 18 to about 25, or from about 18 to about 27, or from about 18 to about 30, or from about 18 to about 35, or from about 18 to about 50, or from about 20 to about 23.5, or from about 20 to about 24, or from about 20 to about 25, or from about 20 to about 27, or from about 20 to about 30, or from about 20 to about 35, or from about 20 to about 50, or from about 22 to about 23.5, or from about 22 to about 24, or from about 22 to about 25, or from about 22 to about 27, or from about 22 to about 30, or from about 22 to about 35, or from about 22 to about 50, or from about 23 to about 23.5, or from about 23 to about 24, or from about 23 to about 25, or from about 23 to about 27, or from about 23 to about 30, or from about 23 to about 35, or from about 23 to about 50, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0242] In some embodiments, the composition comprises butyric acid and / or a salt thereof (e.g., sodium butyrate) as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of sodium butyrate is in a range of from about 0.3 to about 1.2, or from about 0.3 to about 1.0, or from about 0.3 to about 1.1, or from about 0.4 to about 1.2, or from about 0.4 to about 1.0, or from about 0.4 to about 1.1, or from about 0.5 to about 1.2, or from about 0.5 to about 1.0, or from about 0.5 to about 1.1, or from about 0.6 to about 1.2, or from about 0.6 to about 1.0, or from about 0.6 to about 1.1, or from about 0.7 to about 1.2, or from about 0.7 to about 1.0, or from about 0.7 to about 1.1, or from about 0.8 to about 1.2, or from about 0.8 to about 1.0, or from about 0.8 to about 1.1, or from about 0.85 to about 1.2, or from about 0.85 to about 1.0, or from about 0.85 to about 1.1, or from about 0.9 to about 1.2, or from about 0.9 to about 1.0, or from about 0.9 to about 1.1, or from about 0.95 to about 1.0, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises sodium butyrate and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of sodium butyrate is administered is divided into two or more, or three or more unit dosage forms (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises sodium butyrate in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms).

[0243] In some embodiments of any of the embodiments described herein, when the amount of a nutrient (e.g., butyric acid) and / or a salt thereof (e.g., sodium salt, e.g., sodium butyrate) is / are described, a respective amount of the nutrient and / or the salt thereof can be used (i.e., using a similar mol amount of the indicated weight).

[0244] In some embodiments, the composition comprises alpha-ketoglutarate as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of alpha- ketoglutarate is in a range of from about 0.3 to about 1.5, or from about 0.3 to about 1.3, or from about 0.3 to about 1.2, or from about 0.3 to about 1.1, or from about 0.3 to about 1.0, or from about 0.3 to about 0.97, or from about 0.5 to about 1.5, or from about 0.5 to about 1.3, or from about 0.5 to about 1.2, or from about 0.5 to about 1.1, or from about 0.5 to about 1.0, or from about 0.5 to about 0.97, or from about 0.6 to about 1.5, or from about 0.6 to about 1.3, or from about 0.6 to about 1.2, or from about 0.6 to about 1.1, or from about 0.6 to about 1.0, or from about 0.6 to about 0.97, or from about 0.7 to about 1.5, or from about 0.7 to about 1.3, or from about 0.7 to about 1.2, or from about 0.7 to about 1.1, or from about 0.7 to about 1.0, or from about 0.7 to about 0.97, or from about 0.8 to about 1.5, or from about 0.8 to about 1.3, or from about 0.8 to about 1.2, or from about 0.8 to about 1.1, or from about 0.8 to about 1.0, or from about 0.8 to about 0.97, or from about 0.9 to about 1.5, or from about 0.9 to about 1.3, or from about 0.9 to about 1.2, or from about 0.9 to about 1.1, or from about 0.9 to about 1.0, or from about 0.9 to about 0.97, or from about 0.93 to about 1.5, or from about 0.93 to about 1.3, or from about 0.93 to about 1.2, or from about 0.93 to about 1.1, or from about 0.93 to about 1.0, or from about 0.93 to about 0.97, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises alphaketoglutarate and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of alpha-ketoglutarate is administered is divided into two, or three, or more, unit dosage form (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises alpha-ketoglutarate in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms).

[0245] In some embodiments, the composition is formulated such that a total daily amount of the alpha-ketoglutarate is in a range of from about 10 to about 100, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 35, or from about 10 to about 30, or from about 10 to about 29, or from about 15 to about 100, or from about 15 to about 50, or from about 15 to about 40, or from about 15 to about 35, or from about 15 to about 30, or from about 15 to about 29, or from about 20 to about 100, or from about 20 to about 50, or from about 20 to about 40, or from about 20 to about 35, or from about 20 to about 30, or from about 20 to about 29, or from about 25 to about 100, or from about 25 to about 50, or from about 25 to about 40, or from about 25 to about 35, or from about 25 to about 30, or from about 25 to about 29, or from about 27 to about 100, or from about 27 to about 50, or from about 27 to about 40, or from about 27 to about 35, or from about 27 to about 30, or from about 27 to about 29, or from about 28 to about 100, or from about 28 to about 50, or from about 28 to about 40, or from about 28 to about 35, or from about 28 to about 30, or from about 28 to about 29, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition is formulated such that a total daily amount of the alpha-ketoglutarate is in a range of from about 5 to about 50, or from about 5 to about 25, or from about 5 to about 20, or from about 5 to about 17, or from about 5 to about 16.5, or from about 5 to about 16, or from about 7 to about 50, or from about 7 to about 25, or from about 7 to about 20, or from about 7 to about 17, or from about 7 to about 16.5, or from about 7 to about 16, or from about 10 to about 50, or from about 10 to about 25, or from about 10 to about 20, or from about 10 to about 17, or from about 10 to about 16.5, or from about 10 to about 16, or from about 13 to about 50, or from about 13 to about 25, or from about 13 to about 20, or from about 13 to about 17, or from about 13 to about 16.5, or from about 13 to about 16, or from about 15 to about 50, or from about 15 to about 25, or from about 15 to about 20, or from about 15 to about 17, or from about 15 to about 16.5, or from about 15 to about 16, or from about 16 to about 50, or from about 16 to about 25, or from about 16 to about 20, or from about 16 to about 17, or from about 16 to about 16.5, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0246] In some embodiments, the composition comprises adenosine as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of adenosine is in a range of from about 0.1 to about 2, or from about 0.1 to about 2.5, or from about 0.1 to about 5, or from about 0.1 to about 10, or from about 0.1 to about 25, or from about 0.1 to about 50, or from about 0.75 to about 1.8, or from about 1.0 to about 1.8, or from about 1.0 to about 1.6, or from about 1.0 to about 1.5, or from about 1.0 to about 1.35, or from about 1.1 to about 1.8, or from about 1.1 to about 1.6, or from about 1.1 to about 1.5, or from about 1.1 to about 1.35, or from about 1.2 to about 1.8, or from about 1.2 to about 1.6, or from about 1.2 to about 1.5, or from about 1.2 to about 1.35, or from about 1.25 to about 1.8, or from about 1.25 to about 1.6, or from about 1.25 to about 1.5, or from about 1.25 to about 1.35, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises adenosine and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of adenosine is administered is divided into two, or three, or more, unit dosage form (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises adenosine in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms). In some embodiments, the composition is formulated such that a total daily amount of adenosine is in a range of from about 2 to about 10, or from about 3 to about 10, or from about 5 to about 10, or from about 5.5 to about 10, or from about 6 to about 10, or from about 6.5 to about 10, or from about 7 to about 10, or from about 7.5 to about 10, or from about 5 to about 8, or from about 5.5 to about 8, or from about 6 to about 8, or from about 6.5 to about 8, or from about 7 to about 8, or from about 6 to about 7.5, or from about 6.5 to about 7.5, or from about 6 to about 7, or from about 6.5 to about 7, or from about 6.3 to about 6.9, or from about 6.4 to about 6.8, or from about 6.5 to about 6.7, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0247] In some embodiments, the composition comprises mannitol (e.g., D-mannitol) as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of mannitol is in a range of from about 0.1 to about 1, or from about 0.1 to about 1.2, or from about 0.1 to about 1.5, or from about 0.1 to about 5, or from about 0.1 to about 10, or from about 0.1 to about 25, or from about 0.1 to about 50, or from about 0.1 to about 100, or from about 0.2 to about 1, or from about 0.2 to about 1.2, or from about 0.2 to about 1.5, or from about 0.2 to about 5, or from about 0.2 to about 10, or from about 0.2 to about 25, or from about 0.2 to about 50, or from about 0.2 to about 100, or from about 0.5 to about 5, or from about 0.5 to about 10, or from about 0.5 to about 25, or from about 0.5 to about 50, or from about 0.5 to about 100, or from about 0.7 to about 5, or from about 0.7 to about 10, or from about 0.7 to about 25, or from about 0.7 to about 50, or from about 0.7 to about 100, or from about 0.8 to about 5, or from about 0.8 to about 10, or from about 0.8 to about 25, or from about 0.8 to about 50, or from about 0.8 to about 100, or from about 1 to about 5, or from about 1 to about 10, or from about 1 to about 25, or from about 1 to about 50, or from about 1 to about 100, or from about 1.5 to about 5, or from about 1.5 to about 10, or from about 1.5 to about 25, or from about 1.5 to about 50, or from about 1.5 to about 100, or from about 5 to about 10, or from about 5 to about 25, or from about 5 to about 50, or from about 5 to about 100, or from about 10 to about 25, or from about 10 to about 50, or from about 10 to about 100, or from about 25 to about 50, or from about 25 to about 100, or from about 50 to about 100, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises mannitol and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of mannitol is administered is divided into two, or three, or more, unit dosage form (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises mannitol in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms).

[0248] In some embodiments, the composition comprises myoinositol as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of myoinositol is in a range of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.1 to about 2, or from about 0.1 to about 1.5, or from about 0.1 to about 1, or from about 0.1 to about 1.2, or from about 0.1 to about 1.5, or from about 0.1 to about 5, or from about 0.1 to about 10, or from about 0.1 to about 25, or from about 0.1 to about 50, or from about 0.1 to about 100, or from about 0.2 to about 1, or from about 0.2 to about 1.2, or from about 0.2 to about 1.5, or from about 0.2 to about 5, or from about 0.2 to about 10, or from about 0.2 to about 25, or from about 0.2 to about 50, or from about 0.2 to about 100, or from about 0.5 to about 5, or from about 0.5 to about 10, or from about 0.5 to about 25, or from about 0.5 to about 50, or from about 0.5 to about 100, or from about 0.7 to about 5, or from about 0.7 to about 10, or from about 0.7 to about 25, or from about 0.7 to about 50, or from about 0.7 to about 100, or from about 0.8 to about 5, or from about 0.8 to about 10, or from about 0.8 to about 25, or from about 0.8 to about 50, or from about 0.8 to about 100, or from about 1 to about 5, or from about 1 to about 10, or from about 1 to about 25, or from about 1 to about 50, or from about 1 to about 100, or from about 1.5 to about 5, or from about 1.5 to about 10, or from about 1.5 to about 25, or from about 1.5 to about 50, or from about 1.5 to about 100, or from about 5 to about 10, or from about 5 to about 25, or from about 5 to about 50, or from about 5 to about 100, or from about 10 to about 25, or from about 10 to about 50, or from about 10 to about 100, or from about 25 to about 50, or from about 25 to about 100, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises myoinositol and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of myoinositol is administered is divided into two, or three, or more, unit dosage form (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises myoinositol in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms).

[0249] In some embodiments, the composition comprises uridine as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of uridine is in a range of from about 0.1 to about 2.5, or from about 0.1 to about 3, or from about 0.1 to about 5, or from about 0.1 to about 10, or from about 0.1 to about 20, or from about 0.1 to about 50, or from about 0.1 to about 100, or from about 0.3 to about 2.5, or from about 0.3 to about 3, or from about 0.3 to about 5, or from about 0.3 to about 10, or from about 0.3 to about 20, or from about 0.3 to about 50, or from about 0.3 to about 100, or from about 0.5 to about 2.5, or from about 0.5 to about 3, or from about 0.5 to about 5, or from about 0.5 to about 10, or from about 0.5 to about 20, or from about 0.5 to about 50, or from about 0.5 to about 100, or from about 1 to about 2.5, or from about 1 to about 3, or from about 1 to about 5, or from about 1 to about 10, or from about 1 to about 20, or from about 1 to about 50, or from about 1 to about 100, or from about 1.5 to about 2.5, or from about 1.5 to about 3, or from about 1.5 to about 5, or from about 1.5 to about 10, or from about 1.5 to about 20, or from about 1.5 to about 50, or from about 1.5 to about 100, or from about 2 to about 2.5, or from about 2 to about 3, or from about 2 to about 5, or from about 2 to about 10, or from about 2 to about 20, or from about 2 to about 50, or from about 2 to about 100, or from about 2.1 to about 2.5, or from about 2.1 to about 3, or from about 2.1 to about 5, or from about 2.1 to about 10, or from about 2.1 to about 20, or from about 2.1 to about 50, or from about 2.1 to about 100, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises uridine and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of uridine is administered is divided into two, or three, or more, unit dosage form (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises uridine in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms).

[0250] In some such embodiments, the composition is in an aqueous solution, and a concentration of uridine in the composition is at least 5.1, or at least 5.5, or at least 6, or at least 7, or at least 9, or at least 10, e.g., is in a range of from 5.1 to 1000, or from 5.5 to 1000, or from 5.5 to 100, or from 5.5 to 50, or from 5.5 to 25, or from 5.5 to 10, or from 6 to 1000, or from 6 to 100, or from 6 to 50, or from 6 to 25, or from 6 to 10, or from 7 to 1000, or from 7 to 100, or from 7 to 50, or from 7 to 25, or from 7 to 10, or from 8 to 1000, or from 8 to 100, or from 8 to 50, or from 8 to 25, or from 8 to 10, or from 9 to 1000, or from 9 to 100, or from 9 to 50, or from 9 to 25, or from 9 to 10, or from 9.9 to 1000, or from 9.9 to 100, or from 9.9 to 50, or from 9.9 to 25, or from 9.9 to 10, or from 10 to 1000, or from 10 to 100, or from 10 to 50, or from 10 to 25, mM.

[0251] In some embodiments, the composition comprises 3-hydroxybutyric acid and / or a salt thereof as a nutrient. In some embodiments, the composition is formulated such that a total daily amount of 3-hydroxybutyric acid is in a range of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.1 to about 2, or from about 0.1 to about 1.5, or from about 0.1 to about 1, or from about 0.1 to about 1.2, or from about 0.1 to about 1.5, or from about 0.1 to about 5, or from about 0.1 to about 10, or from about 0.1 to about 25, or from about 0.1 to about 50, or from about 0.1 to about 100, or from about 0.2 to about 1, or from about 0.2 to about 1.2, or from about 0.2 to about 1.5, or from about 0.2 to about 5, or from about 0.2 to about 10, or from about 0.2 to about 25, or from about 0.2 to about 50, or from about 0.2 to about 100, or from about 0.5 to about 5, or from about 0.5 to about 10, or from about 0.5 to about 25, or from about 0.5 to about 50, or from about 0.5 to about 100, or from about 0.7 to about 5, or from about 0.7 to about 10, or from about 0.7 to about 25, or from about 0.7 to about 50, or from about 0.7 to about 100, or from about 0.8 to about 5, or from about 0.8 to about 10, or from about 0.8 to about 25, or from about 0.8 to about 50, or from about 0.8 to about 100, or from about 1 to about 5, or from about 1 to about 10, or from about 1 to about 25, or from about 1 to about 50, or from about 1 to about 100, or from about 1.5 to about 5, or from about 1.5 to about 10, or from about 1.5 to about 25, or from about 1.5 to about 50, or from about 1.5 to about 100, or from about 5 to about 10, or from about 5 to about 25, or from about 5 to about 50, or from about 5 to about 100, or from about 10 to about 25, or from about 10 to about 50, or from about 10 to about 100, or from about 25 to about 50, or from about 25 to about 100, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween. In some embodiments, the composition comprises 3-hydroxybutyric acid and is in a unit dosage form (e.g., capsule). In some embodiments, the unit dosage form is such that the total daily dose of 3-hydroxybutyric acid is administered is divided into two, or three, or more, unit dosage form (e.g., capsules). In some such embodiments, each of the unit dosage forms comprises 3-hydroxybutyric acid in an equal amount, respectively to the daily amounts as described herein (that is, the total daily amount is equally divided between the two or more or three or more unit dosage forms).

[0252] In some such embodiments, the composition is in an aqueous solution, and a concentration of 3-hydroxybutyric acid in the composition is up to 4.9, or up to 4.5, or up to 4, or up to 3, or up to 2.5, e.g., is in a range of from 0.1 to 4.9, or from 0.25 to 4.9, or from 0.5 to 4.9, or from 0.75 to 4.9, or from 1 to 4.9, or from 0.1 to 4.5, or from 0.25 to 4.5, or from 0.5 to 4.5, or from 0.75 to 4.5, or from 1 to 4.5, or from 0.1 to 4, or from 0.25 to 4, or from 0.5 to 4, or from 0.75 to 4, or from 1 to 4, or from 0.1 to 3, or from 0.25 to 3, or from 0.5 to 3, or from 0.75 to 3, or from 1 to 3, or from 0.1 to 2.75, or from 0.25 to 2.75, or from 0.5 to 2.75, or from 0.75 to 2.75, or from 1 to 2.75, or from 1 to 2.5, mM.

[0253] Herein throughout, “g” denotes “gram”, unless otherwise indicated.

[0254] In some embodiments, the composition comprises a combination of at least glycine and sodium butyrate, as nutrients. According to some embodiments of any of the embodiments described herein, the composition is formulated and / or administered to a human subject (e.g., weighing about 70 kg) such that the subject receives a total daily amount of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 0.1 to about 100, or from about 1 to about 100, or from about 0.1 to about 75, or from about 1 to about 75, or from about 0.1 to about 50, or from about 1 to about 50, or from about 0.1 to about 30, or from about 1 to about 30, or from about 0.1 to about 20, or from about 1 to about 20, or from about 0.1 to about 15, or from about 1 to about 15, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.2 to about 10, or from about 0.2 to about 5, or from about 0.5 to about 10, or from about 0.5 to about 5, or from about 1 to about 3, or from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 1 to about 2.5, or from about 1 to about 2.2, or from about 1.5 to about 3, or from about 1.5 to about 2.5, or from about 1.5 to about 2.2, or from about 2.0 to about 2.2, grams, of the one or more nutrients as described herein, including any intermediate values and subranges therebetween. In some of these embodiments, the composition is formulated such that a total daily amount of glycine is in a range of from about 0.1 to about 100, or from about 0.1 to about 70, or from about 0.1 to about 50, or from about 0.1 to about 30, or from about 0.1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.1 to about 2, or from about 0.1 to about 1.5, or from about 0.2 to about 1.5, or from about 0.2 to about 1.25, or from about 0.5 to about 1.5, or from about 0.5 to about 1.25, or from about 0.75 to about 1.5, or from about 0.75 to about 1.25, or from about 1.0 to about 1.5, or from about 1.0 to about 1.25, or from about 1.1 to about 1.5, or from about 1.1 to about 1.25, or from about 1.15 to about 1.25, grams per day, and a total daily amount of sodium butyrate is in a range of in an amount of from about 0.1 to about 1000, or from about 0.1 to about 100, or from about 0.1 to about 70, or from about 0.1 to about 50, or from about 0.1 to about 30, or from about 0.1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.1 to about 2, or from about 0.1 to about 1.5, or from about 0.3 to about 1.2, or from about 0.3 to about 1.0, or from about 0.3 to about 1.1, or from about 0.4 to about 1.2, or from about 0.4 to about 1.0, or from about 0.4 to about 1.1, or from about 0.5 to about 1.2, or from about 0.5 to about 1.0, or from about 0.5 to about 1.1 , or from about 0.6 to about 1.2, or from about 0.6 to about 1.0, or from about 0.6 to about 1.1, or from about 0.7 to about 1.2, or from about 0.7 to about 1.0, or from about 0.7 to about 1.1, or from about 0.8 to about 1.2, or from about 0.8 to about 1.0, or from about 0.8 to about 1.1, or from about 0.85 to about 1.2, or from about 0.85 to about 1.0, or from about 0.85 to about 1.1, or from about 0.9 to about 1.2, or from about 0.9 to about 1.0, or from about 0.9 to about 1.1, or from about 0.95 to about 1.0, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0255] In some of these embodiments, a weight ratio of the glycine and the sodium butyrate in the composition ranges from about 1000:1 to 1:1000, or from 100:1 to 1:100, or from 50:1 to 1:50, or from 20:1 to 1:20, or from 10:1 to 1:10, or from 5:1 to 1:5, or from 3:1 to 1:3, or from about 20:1 to 1:2, or from about 20:1 to 1:1.75, or from about 20:1 to 1:1.5, or from about 20:1 to 1:1.4, or from about 20:1 to 1:1.3, or from about 20:1 to 1:1.2, or from about 20:1 to 1:1.1, or from about 20:1 to 1:1, or from about 10:1 to 1:2, or from about 10:1 to 1:1.75, or from about 10:1 to 1:1.5, or from about 10: 1 to 1 : 1.4, or from about 10: 1 to 1 : 1.3, or from about 10:1 to 1:1.2, or from about 10:1 to 1:1.1, or from about 10:1 to 1:1, or from about 5:1 to 1:2, or from about 5:1 to 1:1.75, or from about 5:1 to 1:1.5, or from about 5:1 to 1:1.4, or from about 5:1 to 1:1.3, or from about 5:1 to 1:1.2, or from about 5:1 to 1:1.1, or from about 5:1 to 1:1, or from about 3:1 to 1:2, or from about 3:1 to 1:1.75, or from about 3:1 to 1:1.5, or from about 3:1 to 1:1.4, or from about 3:1 to 1:1.3, or from about 3:1 to 1:1.2, or from about 3:1 to 1:1.1, or from about 3:1 to 1:1 2:1 to 1:2, or from about 1.75:1 to 1:2, or from about 1.5:1 to 1:2, or from about 1.4:1 to 1:2, or from about 1.3:1 to 1 :2, or from about 1.2: 1 to 1 :2, or from about 1.17:1 to 1:2, or from about 2:1 to 1 : 1.75, or from about 1.75:1 to 1:1.75, or from about 1.5:1 to 1:1.75, or from about 1.4:1 to 1:1.75, or from about 1.3:1 to 1:1.75, or from about 1.2:1 to 1:1.75, or from about 1.17:1 to 1:1.75, or from about 2:1 to 1:1.5, or from about 1.75:1 to 1:1.5, or from about 1.5:1 to 1:1.5, or from about 1.4:1 to 1:1.5, or from about 1.3:1 to 1:1.5, or from about 1.2:1 to 1:1.5, or from about 1.17:1 to 1:1.5, or from about 2:1 to 1:1.4, or from about 1.75:1 to 1:1.4, or from about 1.5:1 to 1:1.4, or from about 1.4:1 to 1:1.4, or from about 1.3:1 to 1:1.4, or from about 1.2:1 to 1:1.4, or from about 1.17:1 to 1:1.4, or from about 2:1 to 1:1.3, or from about 1.75:1 to 1:1.3, or from about 1.5:1 to 1:1.3, or from about 1.4:1 to 1:1.3, or from about 1.3:1 to 1:1.3, or from about 1.2:1 to 1:1.3, or from about 1.17:1 to 1:1.3, or from about 2:1 to 1:1.2, or from about 1.75:1 to 1:1.2, or from about 1.5:1 to 1:1.2, or from about 1.4:1 to 1:1.2, or from about 1.3:1 to 1:1.2, or from about 1.2:1 to 1:1.2, or from about 1.17:1 to 1:1.2, or from about 2:1 to 1:1.1, or from about 1.75:1 to 1:1.1, or from about 1.5:1 to 1:1.1, or from about 1.4:1 to 1:1.1, or from about 1.3:1 to 1:1.1, or from about 1.2:1 to 1:1.1, or from about 1.17:1 to 1:1.1, or from about 2:1 to 1:1, respectively, including any intermediate values and subranges therebetween.

[0256] In some embodiments, the composition comprises a combination of at least glycine and alpha-ketoglutarate as nutrients. In some embodiments, the composition is formulated such that a total daily amount of glycine is in a range of from about 0.1 to about 1000, or from about 0.1 to about 100, or from about 0.1 to about 70, or from about 0.1 to about 50, or from about 0.1 to about 30, or from about 0.1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.2 to about 1.5, or from about 0.2 to about 1.25, or from about 0.2 to about 1.2, or from about 0.5 to about 1.5, or from about 0.5 to about 1.25, or from about 0.5 to about 1.2, or from about 0.75 to about 1.5, or from about 0.75 to about 1.25, or from about 0.75 to about 1.2, or from about 1.0 to about 1.5, or from about 1.0 to about 1.25, or from about 1.0 to about 1.2, or from about 1.1 to about 1.5, or from about 1.1 to about 1.25, or from about 1.1 to about 1.2, or from about 1.15 to about 1.25, grams per day, and a total daily amount of alpha-ketoglutarate is in a range of in an amount of from about 0.1 to about 1000, or from about 0.1 to about 100, or from about 0.1 to about 70, or from about 0.1 to about 50, or from about 0.1 to about 30, or from about 0.1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.3 to about 1.5, or from about 0.3 to about 1.3, or from about 0.3 to about 1.2, or from about 0.3 to about 1.1, or from about 0.3 to about 1.0, or from about 0.3 to about 0.97, or from about 0.5 to about 1.5, or from about 0.5 to about 1.3, or from about 0.5 to about 1.2, or from about 0.5 to about 1.1, or from about 0.5 to about 1.0, or from about 0.5 to about 0.97, or from about 0.6 to about 1.5, or from about 0.6 to about 1.3, or from about 0.6 to about 1.2, or from about 0.6 to about 1.1, or from about 0.6 to about 1.0, or from about 0.6 to about 0.97, or from about 0.7 to about 1.5, or from about 0.7 to about 1.3, or from about 0.7 to about 1.2, or from about 0.7 to about 1.1, or from about 0.7 to about 1.0, or from about 0.7 to about 0.97, or from about 0.8 to about 1.5, or from about 0.8 to about 1.3, or from about 0.8 to about 1.2, or from about 0.8 to about 1.1, or from about 0.8 to about 1.0, or from about 0.8 to about 0.97, or from about 0.9 to about 1.5, or from about 0.9 to about 1.3, or from about 0.9 to about 1.2, or from about 0.9 to about 1.1, or from about 0.9 to about 1.0, or from about 0.9 to about 0.97, or from about 0.93 to about 1.5, or from about 0.93 to about 1.3, or from about 0.93 to about 1.2, or from about 0.93 to about 1.1, or from about 0.93 to about 1.0, or from about 0.93 to about 0.97, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0257] In some of these embodiments, a weight ratio of the glycine and the alpha-ketoglutarate in the composition ranges from 1000:1 to 1:1000, or from 100:1 to 1:100, or from 50:1 to 1:50, or from 20:1 to 1:20, or from 10:1 to 1:10, or from 5:1 to 1:5, or from 3:1 to 1:3, or from about 20:1 to 1:2, or from about 20:1 to 1:1.75, or from about 20:1 to 1:1.5, or from about 20:1 to 1:1.4, or from about 20:1 to 1:1.3, or from about 20:1 to 1:1.2, or from about 20:1 to 1:1.1, or from about 20:1 to 1:1, or from about 10:1 to 1:2, or from about 10:1 to 1:1.75, or from about 10:1 to 1:1.5, or from about 10: 1 to 1 : 1.4, or from about 10: 1 to 1 : 1.3, or from about 10: 1 to 1 : 1.2, or from about 10:1 to 1:1.1, or from about 10:1 to 1:1, or from about 5:1 to 1:2, or from about 5:1 to 1:1.75, or from about 5:1 to 1:1.5, or from about 5:1 to 1:1.4, or from about 5:1 to 1:1.3, or from about 5:1 to 1:1.2, or from about 5:1 to 1:1.1, or from about 5:1 to 1:1, or from about 3:1 to 1:2, or from about 3:1 to 1:1.75, or from about 3:1 to 1:1.5, or from about 3:1 to 1:1.4, or from about 3:1 to 1:1.3, or from about 3:1 to 1:1.2, or from about 3:1 to 1:1.1, or from about 3:1 to 1:1, or from about 2:1 to 1:2, or from about 1.75:1 to 1:2, or from about 1.5:1 to 1:2, or from about 1.4:1 to 1:2, or from about 1.3:1 to 1:2, or from about 1.2:1 to 1:2, or from about 1.17:1 to 1:2, or from about 2:1 to 1:1.75, or from about 1.75:1 to 1:1.75, or from about 1.5:1 to 1:1.75, or from about 1.4:1 to 1:1.75, or from about 1.3:1 to 1:1.75, or from about 1.2:1 to 1:1.75, or from about 1.17:1 to 1:1.75, or from about 2:1 to 1:1.5, or from about 1.75:1 to 1:1.5, or from about 1.5:1 to 1:1.5, or from about 1.4:1 to 1:1.5, or from about 1.3:1 to 1:1.5, or from about 1.2:1 to 1:1.5, or from about 1.17:1 to 1:1.5, or from about 2: 1 to 1:1.4, or from about 1.75:1 to 1:1.4, or from about 1.5:1 to 1:1.4, or from about 1.4:1 to 1:1.4, or from about 1.3:1 to 1:1.4, or from about 1.2:1 to 1:1.4, or from about 1.17:1 to 1:1.4, or from about 2:1 to 1:1.3, or from about 1.75:1 to 1:1.3, or from about 1.5:1 to 1:1.3, or from about 1.4:1 to 1:1.3, or from about 1.3:1 to 1:1.3, or from about 1.2:1 to 1:1.3, or from about 1.17:1 to 1:1.3, or from about 2:1 to 1:1.2, or from about 1.75:1 to 1:1.2, or from about 1.5:1 to 1:1.2, or from about 1.4:1 to 1:1.2, or from about 1.3:1 to 1:1.2, or from about 1.2:1 to 1:1.2, or from about 1.17:1 to 1:1.2, or from about 2:1 to 1:1.1, or from about 1.75:1 to 1:1.1, or from about 1.5:1 to 1:1.1, or from about 1.4:1 to 1:1.1, or from about 1.3:1 to 1:1.1, or from about 1.2: 1 to 1 : 1.1 , or from about 1.17:1 to 1:1.1, or from about 2:1 to 1:1, respectively, including any intermediate values and subranges therebetween.

[0258] In some embodiments, the composition comprises a combination of at least glycine, sodium butyrate and alpha-ketoglutarate as nutrients. According to some embodiments of any of the embodiments described herein, the composition is formulated and / or administered to a human subject (e.g., weighing about 70 kg) such that the subject receives a total daily amount of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 0.1 to about 100, or from about 1 to about 100, or from about 0.1 to about 75, or from about 1 to about 75, or from about 0.1 to about 50, or from about 1 to about 50, or from about 0.1 to about 30, or from about 1 to about 30, or from about 0.1 to about 20, or from about 1 to about 20, or from about 0.1 to about 15, or from about 1 to about 15, or from about 0.1 to about 100, or from about 1 to about 100, or from about 0.1 to about 75, or from about 1 to about 75, or from about 0.1 to about 50, or from about 1 to about 50, or from about 0.1 to about 30, or from about 1 to about 30, or from about 0.1 to about 20, or from about 1 to about 20, or from about 0.1 to about 15, or from about 1 to about 15, or from about 0.1 to about 5, or from about 0.2 to about 10, or from about 0.2 to about 5, or from about 0.5 to about 10, or from about 0.5 to about 5, or from about 1 to about 3, or from about 1 to about 2.5, or from about 1 to about 2.2, or from about 1.5 to about 3, or from about 1.5 to about 2.5, or from about 1.5 to about 2.2, or from about 2.0 to about 2.2, grams, of the one or more nutrients as described herein, including any intermediate values and subranges therebetween. In some of these embodiments, the composition is formulated such that a total daily amount of glycine is in a range of from about 0.1 to about 100, or from about 0.1 to about 70, or from about 0.1 to about 50, or from about 0.1 to about 30, or from about 0.1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.1 to about 2, or from about 0.1 to about 1.5, or from about 0.2 to about 1.5, or from about 0.2 to about 1.25, or from about 0.5 to about 1.5, or from about 0.5 to about 1.25, or from about 0.75 to about 1.5, or from about 0.75 to about 1.25, or from about 1.0 to about 1.5, or from about 1.0 to about 1.25, or from about 1.1 to about 1.5, or from about 1.1 to about 1.25, or from about 1.15 to about 1.25, grams per day, and a total daily amount of sodium butyrate is in a range of in an amount of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.1 to about 2, or from about 0.1 to about 1.5, or from about 0.3 to about 1.2, or from about 0.3 to about 1.0, or from about 0.3 to about 1.1, or from about 0.4 to about 1.2, or from about 0.4 to about 1.0, or from about 0.4 to about 1.1, or from about 0.5 to about 1.2, or from about 0.5 to about 1.0, or from about 0.5 to about 1.1, or from about 0.6 to about 1.2, or from about 0.6 to about 1.0, or from about 0.6 to about 1.1 , or from about 0.7 to about 1.2, or from about 0.7 to about 1.0, or from about 0.7 to about 1.1, or from about 0.8 to about 1.2, or from about 0.8 to about 1.0, or from about 0.8 to about 1.1, or from about 0.85 to about 1.2, or from about 0.85 to about 1.0, or from about 0.85 to about 1.1, or from about 0.9 to about 1.2, or from about 0.9 to about 1.0, or from about 0.9 to about 1.1, or from about 0.95 to about 1.0, grams per day, and a total daily amount of alpha-ketoglutarate is in a range of from about 10 to about 100, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 35, or from about 10 to about 30, or from about 10 to about 29, or from about 15 to about 100, or from about 15 to about 50, or from about 15 to about 40, or from about 15 to about 35, or from about 15 to about 30, or from about 15 to about 29, or from about 20 to about 100, or from about 20 to about 50, or from about 20 to about 40, or from about 20 to about 35, or from about 20 to about 30, or from about 20 to about 29, or from about 25 to about 100, or from about 25 to about 50, or from about 25 to about 40, or from about 25 to about 35, or from about 25 to about 30, or from about 25 to about 29, or from about 27 to about 100, or from about 27 to about 50, or from about 27 to about 40, or from about 27 to about 35, or from about 27 to about 30, or from about 27 to about 29, or from about 28 to about 100, or from about 28 to about 50, or from about 28 to about 40, or from about 28 to about 35, or from about 28 to about 30, or from about 28 to about 29, or from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.75 to about 1.8, or from about 1.0 to about 1.8, or from about 1.0 to about 1.6, or from about 1.0 to about 1.5, or from about 1.0 to about 1.35, or from about 1.1 to about 1.8, or from about 1.1 to about 1.6, or from about 1.1 to about 1.5, or from about 1.1 to about 1.35, or from about 1.2 to about 1.8, or from about 1.2 to about 1.6, or from about 1.2 to about 1.5, or from about 1.2 to about 1.35, or from about 1.25 to about 1.8, or from about 1.25 to about 1.6, or from about 1.25 to about 1.5, or from about 1.25 to about 1.35, grams per day, and a total daily amount of sodium butyrate is in a range of from about 0.1 to about 1000, or from about 1 to about 750, or from about 1 to about 500, or from about 1 to about 250, or from about 1 to about 100, or from about 1 to about 50, or from about 1 to about 20, or from about 0.1 to about 10, or from about 0.1 to about 5, or from about 0.1 to about 2.5, or from about 0.3 to about 1.2, or from about 0.3 to about 1.0, or from about 0.3 to about 1.1, or from about 0.4 to about 1.2, or from about 0.4 to about 1.0, or from about 0.4 to about 1.1, or from about 0.5 to about 1.2, or from about 0.5 to about 1.0, or from about 0.5 to about 1.1, or from about 0.6 to about 1.2, or from about 0.6 to about 1.0, or from about 0.6 to about 1.1, or from about 0.7 to about 1.2, or from about 0.7 to about 1.0, or from about 0.7 to about 1.1, or from about 0.8 to about 1.2, or from about 0.8 to about 1.0, or from about 0.8 to about 1.1, or from about 0.85 to about 1.2, or from about 0.85 to about 1.0, or from about 0.85 to about 1.1, or from about 0.9 to about 1.2, or from about 0.9 to about 1.0, or from about 0.9 to about 1.1 , or from about 0.95 to about 1.0, grams per day, when administered to a human subject (weighing about 70 kg), including any intermediate values and subranges therebetween.

[0259] According to some embodiments of any of the embodiments described herein, the composition as described herein in any of the respective embodiments is a nutritional composition or a pharmaceutical composition, in which the one or more nutrients, and optionally other active agents as described herein (e.g., therapeutically active agents), are mixed with one or more components (physiologically acceptable carriers (e.g., a solid or liquid (e.g., aqueous) carrier) and / or excipients).

[0260] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier”, which may be interchangeably used, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Pharmaceutical or nutritional compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0261] Pharmaceutical or nutritional compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0262] Suitable routes of administration may, for example, include oral, rectal, topical, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0263] Alternately, one may administer the nutritional or pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0264] The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue. In some embodiments, the tissue is a muscle tissue.

[0265] The phrase “muscle tissue”, as known in the art, describes a biological tissue composed primarily of contractile cells (muscle fibers) that generate force and facilitate movement, posture, and structural support. Muscle tissue includes skeletal muscle tissue, cardiac muscle tissue, and smooth muscle tissue as the major types, and muscle-like cells, which, although not traditionally classified as muscle tissue, exhibit contractile behavior and contribute to various physiological or pathological processes.

[0266] According to some embodiments of any of the embodiments described herein, the muscle tissue is a skeletal muscle tissue. Skeletal muscles are voluntary, striated muscles attached to bones and are responsible for movement, posture, and joint stability. In the head and neck, muscles include the frontalis, occipitalis, orbicularis oculi, orbicularis oris, zygomaticus major and minor, buccinator, platysma, masseter, temporalis, medial and lateral pterygoids, sternocleidomastoid, trapezius, splenius capitis, scalenes, and levator scapulae. In the thorax and abdomen, key muscles are the pectoralis major and minor, serratus anterior, intercostals (external, internal, and innermost), rectus abdominis, external and internal obliques, and transversus abdominis. Back muscles include the erector spinae group (iliocostalis, longissimus, spinalis), latissimus dorsi, rhomboids, multifidus, and quadratus lumborum. Upper limb muscles encompass the deltoid, rotator cuff muscles (supraspinatus, infraspinatus, teres minor, subscapularis), biceps brachii, triceps brachii, brachialis, coracobrachialis, and numerous forearm and hand muscles such as the flexors and extensors of the wrist and fingers, pronator teres, supinator, lumbricals, interossei, and thenar and hypothenar muscles. Lower limb muscles include the gluteus maximus, medius, and minimus, iliopsoas, tensor fasciae latae, sartorius, quadriceps femoris group (rectus femoris, vastus lateralis, vastus medialis, vastus intermedins), hamstrings (biceps femoris, semitendinosus, semimembranosus), adductors (longus, brevis, magnus), gracilis, pectineus, as well as leg and foot muscles such as tibialis anterior and posterior, gastrocnemius, soleus, fibularis longus and brevis, extensor and flexor digitorum longus, flexor hallucis longus, and intrinsic muscles of the foot like abductor hallucis, abductor digiti minimi, and interossei.

[0267] According to some embodiments, the skeletal muscle tissue is of the upper or lower limb, and / or the leg and foot. According to some embodiments, the skeletal muscle tissue is of the head and neck.

[0268] Non-limiting examples of muscle-like cells include myofibroblasts (involved in wound contraction and fibrosis), myoepithelial cells (found in secretory glands such as mammary and salivary glands), and pericytes (associated with capillaries and microvessels, contributing to vascular stability and tone).

[0269] For injection, the active ingredients of the nutritional or pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Polymeric materials (e.g., organic and / or inorganic) and other carriers can be added to facilitate dissolution and / or provide a sustained-release effect, as described herein.

[0270] For oral administration or ingestion, the nutritional or pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the nutritional or pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, solutions, edible products, dehydrated forms that can be hydrated before use, and the like, for oral administration or ingestion by a patient. Pharmacological or nutritional preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0271] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0272] Pharmaceutical or nutritional compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0273] Additional embodiments of compositions formulated for oral administration or ingestion are described hereinafter.

[0274] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0275] Alternatively, the active ingredient may be in powder or other dehydrated form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0276] Pharmaceutical or nutritional compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., a nutrient as described herein) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., muscle atrophy) or prolong the survival of the subject being treated.

[0277] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0278] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

[0279] Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0280] Exemplary amounts are as described hereinabove.

[0281] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is achieved or diminution of the disease state is achieved.

[0282] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0283] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0284] In some embodiments, the composition as described herein is packaged in a kit and is identified for use in treating or preventing muscle atrophy, in accordance with any of the embodiments described herein, and is optionally identified for use in combination with an additional therapeutically active agent as described herein in any of the respective embodiments and any combination thereof.

[0285] Herein, the terms “composition” and “formulation” are used interchangeably.

[0286] According to some embodiments of any of the embodiments described herein, the composition is formulated for administration by oral ingestion or oral administration.

[0287] The phrase “oral ingestion” as used herein describes, in the context of the present embodiments, the administration of the composition through the mouth, such that it is swallowed and reaches the gastrointestinal (GI) tract, where it is absorbed via the digestive system. Nonlimiting examples of administration by oral ingestion include administration of the composition in the form of a solid (e.g., tablet, capsule, powder), semi-solid (e.g., gel, paste), or liquid (e.g., solution, suspension), whether swallowed directly or mixed with food or drink.

[0288] According to some embodiments of any of the embodiments described herein, the composition is formulated for administration by oral administration. The phrase “oral administration” as used herein describes, in the context of the present embodiments, the placement of the composition in the oral cavity, including buccal or sublingual routes, where absorption may occur through the mucosal lining without necessarily involving swallowing or GI absorption. Non-limiting examples of oral administration include oral films, mouth sprays, buccal tablets, lozenges, and sublingual drops.

[0289] Non-limiting examples of forms in which the composition may be provided include a tablet, a capsule (e.g., a gelatin capsule), a powder, a granule, a bead, a pellet, a lozenge, and a chewable solid (e.g., chewing gum).

[0290] In some embodiments, the composition is in a form of a capsule. For example, the capsule may be a 1-gram capsule comprising from about 50 % to about 90 % (e.g., about 70 %) by weight, the nutrient composition as described herein in any of the respective embodiments and in any combination thereof.

[0291] According to some embodiments of any of the embodiments described herein, the composition is in a form of a solid or a semi-solid dosage form. In some embodiments, the composition is provided as a unit dosage form, such as a tablet, a capsule (e.g., gelatin capsule), a granule or a plurality of granules bound together, a bead or a plurality of beads bound together, a pellet, a lozenge, or a chewable solid or semi-solid (e.g., chewing gum).

[0292] In some embodiments, the composition is formulated as a multi-particulate or multi- particulate-filled capsule. In some embodiments, the solid or semi-solid unit dosage form is configured for oral administration and may include one or more pharmaceutically acceptable excipients, binders, or matrix-forming agents suitable for delivering the at least one nutrient in a stable and bioavailable form.

[0293] Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0294] The phrase “matrix-forming agents” describes agents that form a three-dimensional network or scaffold within the composition, into which active ingredients can be embedded or entrapped. Non-limiting examples of matrix-forming agents include hydrogel-forming agents, natural or synthetic polysaccharides (e.g., alginate, agarose, carrageenan), synthetic polymers (e.g., polyvinyl alcohol), and protein-based materials (e.g., gelatin).

[0295] The phrase “hydrogel-forming agents” describes agents that form a polymeric network upon hydration or crosslinking, which retains water and controls the release of incorporated active ingredients. Non-limiting examples of hydrogel-forming agents include alginate, agarose, gellan gum, carrageenan, and polyvinyl alcohol.

[0296] According to some embodiments of any of the embodiments described herein, the composition is a nutritional supplement, and can be in a form of a power, a solution, a tablet, a capsule, granules, sachet, and any other form suitable for oral ingestion or administration. According to some embodiments, the composition is in a form of a shake, or in a form of a mixable powder, or in a form of a bar, or in a form of a chewable formulation. According to some embodiments, the composition is in a form of an edible product as described in further detail hereinbelow.

[0297] In some embodiments, the composition is provided in a ready-to-use or reconstitutable format.

[0298] A “ready-to-use” format describes a composition that is pre-prepared and requires no further mixing or processing prior to administration (e.g., a liquid supplement, pre-filled capsule, edible bar).

[0299] A “reconstitutable” format describes a composition that is provided in a stable form (e.g., a dry powder, a dehydrated concentrate, dehydrated liposomes, dehydrated hydrogel) and is intended to be mixed with a liquid such as water or milk prior to ingestion, typically at the time of use.

[0300] In some embodiments, the composition is shelf-stable. The phrase “shelf- stable” as used herein describes a composition that maintains its physical, chemical, and microbiological integrity under ambient storage conditions for an extended period (e.g., several months to years) without requiring refrigeration. Shelf stability may be achieved, for example, through formulation with low-moisture content, appropriate pH control, stabilizing excipients, antioxidants, or the use of moisture-resistant and light-protective packaging.

[0301] In some embodiments, the composition is configured for individual or multi-dose packaging. The phrase “individual-dose package” describes a unitized presentation containing a single administration amount (e.g., a capsule, sachet, or single-use bottle), which may facilitate convenience, accurate dosing, and hygiene. The phrase “individual-dose package” is also referred to herein as a “unit dosage form”. The phrase “multi-dose package” describes a container holding multiple administrations of the composition (e.g., a bulk powder, a bottle of tablets, or a multi-use liquid bottle), optionally provided with a measuring device such as a scoop or dropper to assist in accurate dose delivery.

[0302] According to some embodiments of any of the embodiments described herein, the composition further comprises at least one of: a source of protein, a source of fatty acids, a source of one or more carbohydrates, a sterol, a vitamin, a mineral, a phenolic compound, a carotenoid compound, and an odiferous (scent) compound, for example, for masking undesired odors. If present, the additional component(s) are selected, e.g., to support nutritional balance, improve organoleptic properties, enhance absorption or bioavailability of the active nutrient(s), or to increase palatability and compliance. In some embodiments, the composition is free of animal- derived ingredients, artificial preservatives, and / or common allergens.

[0303] In some embodiments of any of the embodiments described herein, the formulation as described herein comprises an odor-masking agent. The phrase “odor-masking agent” describes an ingredient that suppresses, neutralizes, or masks undesirable odors of active or excipient components in the composition. Non-limiting examples of odor-masking agents include menthol, vanillin, essential oils (e.g., peppermint oil, eucalyptus oil, citrus oils), cyclodextrins, and aromatic compounds such as ethyl vanillin or linalool.

[0304] Herein the term “active ingredient” refers to the compounds or materials accountable for the biological effect (e.g., a nutrient as described herein).

[0305] The amount of the nutrient(s) in the composition may depend on the form of the composition. Liquid compositions that comprise a liquid carrier should preferably comprise an amount of the nutrient(s) that is soluble or dispersible. Solid compositions may include higher amounts of the nutrient(s), including beyond the maximal soluble concentration that would apply in liquid formulations. Without being bound by any particular theory, in liquid formulations, it is assumed that maintaining the nutrient concentration below its maximal solubility in the aqueous carrier enhances formulation stability, prevents crystallization or precipitation, and / or supports a more predictable or sustained release profile upon administration.

[0306] According to some embodiments of any of the embodiments described herein, the composition is in a liquid form and comprises an aqueous carrier. In some of these embodiments, a concentration of the at least one (e.g., 1, 2, 3, or more) nutrient(s) is up to 50 %, or up to 40 %, or up to 30 %, or up to 20 %, of the maximal soluble concentration of the nutrient in the carrier, including any intermediate values and subranges therebetween.

[0307] According to some embodiments of any of the embodiments described herein, a composition as described herein is formulated for releasing therefrom (e.g., by means of extended release as described herein) the one or more nutrients as described herein so as to maintain a physiological concentration that is at least equal to and preferably higher than the physiological concentration in a healthy subject.

[0308] In some embodiments, the formulation is configured to release the one or more nutrient / s so as to achieve a level equal to or higher than physiological level, optionally in an extended release (sustained release) mode.

[0309] In some such embodiments, the composition is formulated for a controlled release as described herein in any of the respective embodiments.

[0310] For extended release, the formulation of the nutritional or pharmaceutical composition may include the encapsulation or entrapment of the nutrients within a matrix or carrier system that controls their release over an extended period of time, using, microencapsulation, nanotechnology, matrix systems, coating technologies, ion exchange resins, liposomes, spray drying, nanolaminated systems, emulsions, Solid Lipid Particles (SLPs), Self-dispersing lipid formulations (SDLFs) and Biopolymer-based delivery systems.

[0311] In some embodiments, the composition or formulation is designed for slow (extended, sustained or delayed) release. In some such embodiments, the delayed release is achieved by incorporating in the composition one or more release-modifying agents. In exemplary embodiments, the composition or formulation comprises one or more nutrient (e.g., glycine) and one or more matrix-forming agents (e.g., alginate and agarose, as exemplified in Example 4), which forms a structure (e.g., gel-like bead) that slows diffusion of the one or more nutrient (e.g., glycine) upon gelation (e.g., crosslinking, for example in a calcium-containing aqueous solution such as CaCE).

[0312] The phrases “sustained release”, “extended release” and “slow release” are used herein throughout interchangeably, and describe, in the context of the present embodiments, a release profile wherein the one or more nutrients are released from the composition at a controlled rate over an extended period of time (e.g., hours), so as to maintain a therapeutically effective concentration of the nutrient(s) in the bloodstream, muscle tissue, or other relevant biological compartment for longer than would occur following immediate release administration. Sustained release formulations may reduce dosing frequency, mitigate peak-trough fluctuations in plasma or tissue concentration, improve compliance, and / or optimize the bioavailability and physiological effect of the nutrient(s).

[0313] The phrase “release-modifying agents” describes components that influence the rate and / or location at which an active ingredient is released from a formulation. Non-limiting examples of release-modifying agents include hydrogels, gelling polymers, and crosslinkable excipients (e.g., alginate, agarose, gelatin, chitosan, pectin, xanthan gum).

[0314] In some embodiments, the sustained release is configured to occur over a period of from about 2 to about 48 hours, or from about 4 to about 36 hours, or from about 6 to about 24 hours, or from about 8 to about 18 hours, or from about 10 to about 16 hours, or from about 12 to about 24 hours, or from about 12 to about 20 hours, or from about 12 to about 18 hours, or from about 12 to about 16 hours, or from about 14 to about 24 hours, or from about 16 to about 24 hours, or from about 18 to about 24 hours, including any intermediate values and subranges therebetween.

[0315] In some embodiments, the composition is configured such that the one or more nutrient(s) reach a detectable or therapeutically effective concentration in blood, plasma, or muscle tissue within 0.5 to 2 hours post-administration, and remain above a threshold level (e.g., at least about 50 %, of Cmax) for a duration of at least 8, or at least 12, or at least 16, or at least 24 hours.

[0316] Non-limiting examples of sustained release formulations include formulation as described in Goncalves et al., 2018. Trends in Food Science & Technology. 10.1016 / j.tifs.2018.06.011, and in Gleeson et al., Trends in Food Science & Technology, Volume 53, 2016, Pages 90-101, ISSN 0924-2244.

[0317] In some embodiments, the nutrients in the formulation are encapsulated or entrapped within a matrix or carrier system. Such matrix or carrier system may control their release over an extended period of time using one or more of the following: microencapsulation, nanotechnology, matrix systems, coating technologies, ion exchange resins, liposomes, spray drying, nanolaminated systems, emulsions, Solid Lipid Particles (SLPs), Self-dispersing lipid formulations (SDLFs) and Biopolymer-based delivery systems.

[0318] For microencapsulation, nutrients may be encapsulated within microscopic capsules or particles made of materials such as polymers, lipids, proteins, or carbohydrates. These capsules can be designed to release their contents gradually, either through diffusion or degradation of the capsule material.

[0319] Nutrients can be encapsulated at the nanoscale using nanoparticles or nanocapsules. These nanoparticles can be engineered to have specific properties for controlled release, such as surface modifications or responsive materials that release nutrients in response to changes in pH or temperature.

[0320] For matrix systems, nutrients may be dispersed within a solid matrix made of materials like polymers, waxes, or hydrogels. The release of nutrients from the matrix occurs as the matrix erodes or swells in the presence of water or other physiological fluids.

[0321] Nutrients can be coated with thin layers of materials that control their release. This can include coating with polymers or lipid-based materials that dissolve or degrade at specific rates, allowing for sustained release of the encapsulated nutrients.

[0322] Nutrients can be bound to ion exchange resins, which release them gradually as they exchange ions with their surroundings. This technique is particularly useful for water-soluble nutrients like minerals.

[0323] Liposomes are spherical vesicles composed of lipid bilayers. Nutrients can be encapsulated within these vesicles, and their release can be controlled by modifying the lipid composition or adding surface-modifying agents.

[0324] Spray Drying involves atomizing a nutrient solution into droplets, which are then dried to form micron-sized particles. These particles can be designed to release nutrients gradually by controlling their size, composition, and porosity.

[0325] Nanolaminated systems can involve assembling multilayers of different materials on top of pre-existent delivery systems (e.g. emulsions, liposomes, polymeric particles), forming nanolaminated systems. Layer-by layer (LbL) is the most used method for producing nanolaminated systems and consists in the adsorption of charged layers of polyelectrolytes on top of the core material.

[0326] Emulsions can be oil-in-water (O / W ; preferred) or water-in-oil (W / O) and liquid in liquid or liquid in solid. The droplet size and emulsion stability are dependent on the oil type and composition, the surfactant / oil ratio and co-solvents or co-solutes used. Lipids, low molecular weight surfactants, proteins and polysaccharides are the main encapsulating agents used. Solid Lipid Particles (SLPs) are composed by lipid droplets that are fully crystallized and have an organized crystalline structure with the bioactive components accommodated within the lipid matrix. The most used lipids are fatty acids, glycerides, paraffin, triacylglycerol and waxes. SLPs present several advantages such as controlled and targeted release, good stability, good biodegradability and biocompatibility, high loading capacity and low cost. They can be produced by hot or cold homogenization.

[0327] Self-dispersing lipid formulations (SDLFs) are isotropic oil solutions or isotropic mixtures of oils, surfactants, co-surfactants and co solvents. The principal advantage of this system is the improvement of nutraceuticals’ solubility in the GI tract which can enhance their adsorption. SDLF bioavailability depends on the digestion of lipids, lipophilicity of nutraceuticals, type of lipids and on the mean droplet diameter.

[0328] Biopolymer-based delivery systems (e.g., polysaccharides and proteins) can be used to create a wide range of delivery systems for nutraceuticals, either individually or combined. Polymeric particles can be produced with common food biopolymers through self-assembly and can be used to encapsulate both hydrophilic and hydrophobic nutraceuticals. Their main advantages are high loading efficiency, high stability, biocompatibility, biodegradability and controlled release.

[0329] According to some of any of the embodiments described herein, a formulation for controlled (e.g., sustained) release of the nutrient(s) is selected suitable for controlled release of water-soluble agents (that exhibit high dissolution rate in water), such as glycine.

[0330] Sustained release formulations of water-soluble agents, such as glycine, are designed to extend the duration of drug release and absorption, thereby maintaining therapeutically effective concentrations over prolonged periods and reducing the frequency of dosing. High aqueous solubility typically requires formulation strategies that prevent rapid dissolution and absorption.

[0331] An exemplary approach involves embedding the active agent within a polymeric matrix system, which may be either hydrophilic - such as hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose, polyvinyl alcohol, or xanthan gum, or hydrophobic - such as ethylcellulose, stearates, or wax-based materials. In such systems, drug release is governed by diffusion through a gel layer and / or matrix erosion.

[0332] Alternatively, the active agent can be incorporated into coated granules, pellets, or particles, including nanoparticles and / or microparticles, using polymers such as ethylcellulose, methacrylate copolymers (e.g., Eudragit RS / RL), polyurethane, or biodegradable polymers like PLGA to provide diffusion-controlled release via semi-permeable or insoluble coatings. Ion-exchange resins may also be used to bind the active agent (nutrient) electrostatically, with the drug released gradually through ionic exchange in the gastrointestinal tract.

[0333] For precise zero-order release kinetics, osmotic pump systems utilizing a semi-permeable membrane and an orifice can deliver water-soluble nutrients in a controlled manner, independent of pH and motility.

[0334] In situ gelling systems that transform from solution to gel under physiological conditions (e.g., temperature or calcium ion presence) offer another approach to sustained delivery. Additionally, lipid-based systems, including liposomes, solid lipid nanoparticles, and nanostructured lipid carriers, can encapsulate glycine to control its diffusion and protect it from rapid dissolution.

[0335] Alternatively or in addition, techniques such as cross-linking, complexation, or use of enteric coatings may be required to modulate release rates effectively.

[0336] Sustained release formulations can also be achieved using inorganic silica-based carriers, particularly those derived from sol-gel technology. In sol-gel systems, organosilicon precursors such as tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS) are hydrolyzed and condensed under mild conditions to form a porous silica network that can encapsulate hydrophilic molecules. The nutrient(s) may be physically entrapped within the silica matrix or covalently bonded to the silicate framework using functionalized silanes. The resulting xerogels or aerogels exhibit high surface area and tunable porosity, allowing precise control over the drug release profile by adjusting pore size, surface chemistry, and degree of crosslinking. These materials are chemically inert, biocompatible, and can offer pH-independent release kinetics, which is particularly beneficial for maintaining consistent drug exposure. Mesoporous silica particles (MSPs) such as MCM-41, SBA-15, or modified variants can also be loaded with the nutrient(s) and further functionalized with gatekeeping molecules or polymer coatings to achieve stimuli- responsive or prolonged release. These carriers often permit adsorptive loading of small, polar molecules and sustained diffusion through well-defined mesoporous channels. Additionally, solgel materials can be combined with biodegradable polymers (e.g., forming hybrid organic- inorganic composites).

[0337] Sustained release formulations can be obtained using the Stober technology, for synthesizing monodisperse, spherical silica particles through controlled hydrolysis and condensation of alkoxysilanes such as tetraethyl orthosilicate (TEOS) in an alcohol-water- ammonia medium. This sol-gel-derived method enables the formation of uniform, amorphous silica particles with tunable sizes and surface properties, typically ranging from nanometers to several microns. These particles are inherently biocompatible and chemically stable, making them highly suitable for pharmaceutical applications, particularly in sustained drug delivery. Stober- derived silica particles can be utilized to retard drug release through physical entrapment within their porous matrix. The porous structure allows for controlled diffusion, and the release profile can be further modified through post-synthesis surface functionalization or coating with hydrophobic or biodegradable polymers. Additionally, these silica carriers may be integrated into hybrid organic-inorganic delivery platforms or modified with stimuli-responsive gatekeeping molecules to create intelligent, controlled-release systems.

[0338] Sustained release formulations can be obtained using Ludox® particles, a family of colloidal silica nanoparticles produced by Grace Davison, consisting of amorphous silica dispersed in aqueous media, or similar technologies. These particles are monodisperse, stable, and available in various sizes, typically ranging from 5 nm to 30 nm, with a high surface area and negative surface charge under neutral to basic pH conditions. Due to their nanoscale size, chemical inertness, and tunable surface chemistry, Ludox particles have attracted considerable interest as carriers for drug delivery systems, particularly for the encapsulation or adsorption of hydrophilic, water-soluble agents.

[0339] In sustained release applications, Ludox particles can be employed to immobilize the nutrient(s) via electrostatic interactions, hydrogen bonding, or covalent attachment. Their porous silica structure and high surface reactivity allow for the formation of composite materials, where the drug is either incorporated into a silica matrix or adsorbed onto the surface and then coated with rate-controlling polymers (e.g., ethylcellulose, Eudragit®, PLGA). Additionally, Ludox dispersions can be used as precursors in sol-gel processing, enabling the formation of xerogels or hybrid materials that entrap the drug within a silica network.

[0340] In some embodiments, any of the above technologies, can be combined. For example, silica-based carriers can be formulated in a water-in-oil emulsion and / or particulate formulations can be encapsulated in any of the carriers, matrices and / or coatings as described herein.

[0341] In some embodiments, the formulation as described herein is designed so as to release the one or more nutrients such that a plasma level thereof is above a physiological level of this nutrient in the treated subject.

[0342] In some embodiments, the formulation as described herein is designed so as to release (e.g., by slow or extended release) the nutrients in the intestine (e.g., by means of enteric coating or matrix and / or by means of particles-based formulation), so as to achieve slower elimination from the blood stream.

[0343] In some embodiments, extended or slowed down absorption to the blood (e.g., from the intestines), and / or extended or slowed down elimination from the blood is achieved by complexation of the one or more nutrients with a counter-charged substance (e.g., a positively charged compound).

[0344] According to some of any of the embodiments described herein, the composition is formulated for topical administration.

[0345] For topical administration, an appropriate carrier may be selected and optionally other ingredients that can be included in the composition or formulation, as is detailed herein. Hence, the compositions can be, for example, in a form of a cream, an ointment, a paste, a gel, a lotion, and / or a soap.

[0346] Ointments are semisolid preparations, typically based on vegetable oil (e.g., shea butter and / or cocoa butter), petrolatum or petroleum derivatives. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and non-sensitizing.

[0347] Lotions are preparations that may to be applied to the skin without friction. Lotions are typically liquid or semiliquid preparations with a water or alcohol base, for example, an emulsion of the oil-in-water type. Lotions are typically preferred for treating large areas (e.g., as is frequently desirable for sunscreen compositions), due to the ease of applying a more fluid composition.

[0348] Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases typically contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the “lipophilic” phase, optionally comprises petrolatum and / or a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase optionally contains a humectant. The emulsifier in a cream formulation is optionally a nonionic, anionic, cationic or amphoteric surfactant.

[0349] Herein, the term “emulsion” refers to a composition comprising liquids in two or more distinct phases (e.g., a hydrophilic phase and a lipophilic phase). Non-liquid substances (e.g., dispersed solids and / or gas bubbles) may optionally also be present.

[0350] As used herein and in the art, a “water-in-oil emulsion” is an emulsion characterized by an aqueous phase which is dispersed within a lipophilic phase.

[0351] As used herein and in the art, an “oil-in-water emulsion” is an emulsion characterized by a lipophilic phase which is dispersed within an aqueous phase.

[0352] Pastes are semisolid dosage forms which, depending on the nature of the base, may be a fatty paste or a paste made from a single-phase aqueous gel. The base in a fatty paste is generally petrolatum, hydrophilic petrolatum, and the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

[0353] Gel formulations are semisolid, suspension-type systems. Single-phase gels optionally contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous; but also, preferably, contains a non-aqueous solvent, and optionally an oil. Preferred organic macromolecules (e.g.. gelling agents) include crosslinked acrylic acid polymers such as the family of carbomer polymers, e.g., carboxypolyalkylenes, that may be obtained commercially under the trademark Carbopol®. Other types of preferred polymers in this context are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.

[0354] A composition formulated for topical administration may optionally be present in a patch, a swab, a pledget, and / or a pad.

[0355] Dermal patches and the like may comprise some or all of the following components: a composition to be applied (e.g. , as described herein); a liner for protecting the patch during storage, which is optionally removed prior to use; an adhesive for adhering different components together and / or adhering the patch to the skin; a backing which protects the patch from the outer environment; and / or a membrane which controls release of a drug to the skin. In some embodiments, a dermal patch is configured for controlled release of the nutrient(s), so as to act a depot. The dermal patch can be configured for a daily use, such that it is replaced every day, or for an hourly basis, such that it is contacted with the skin for a pre-determined time, a few times a day (for example, it is replaced by a new one every 3, 4, 5, 6, 7, 8 or more hours, or for a weekly basis.

[0356] The composition as described herein in any of the respective embodiments can be provided to a skin of the subject in a form of a patch, for example, a dermal patch configured to deliver the nutrient(s), e.g., a patch that acts as depot for slow release of the nutrient(s), as described herein. The patch may be applied to an area of intact skin and worn for a predetermined period (e.g., hours to days), during which the formulation diffuses through the skin layers and enters systemic or local circulation, depending on the formulation and delivery goals. The adhesive and backing layer optionally ensure prolonged adherence and controlled moisture conditions at the application site. The patch can be designed for variable administration schedules, wherein the subject replaces the patch with a new one at predetermined intervals, such as once daily, multiple times per day (e.g., every 4, 6, 8, or 12 hours), or less frequently (e.g., every 2, 3, 4, 5, 6, or 7 days), depending on the specific pharmacokinetic properties of the nutrient(s) and the specific use. Dermal patches, also referred to as transdermal therapeutic systems (TTS), are drug delivery devices designed to administer active pharmaceutical ingredients across the skin barrier in a sustained and controlled manner, enabling either local or systemic therapeutic effects.

[0357] Exemplary patch designs include, without limitation, matrix-type patches in which the drug is dispersed within a polymeric matrix (e.g., polyacrylates, ethylene-vinyl acetate, or hydrophilic cellulosic polymers), and release occurs primarily via diffusion through the polymer and into the skin; Reservoir-type systems that comprise a drug-loaded gel or solution held within a compartment and separated from the skin by a semi-permeable membrane, through which drug diffuses at a controlled rate. Alternatively, adhesive dispersion systems incorporate the drug directly into the adhesive layer, offering simplified construction suitable for low-dose applications. For more complex release profiles, multilayer or gradient systems may be used, incorporating multiple functional layers and components such as rate-controlling membranes, backing films, and release liners. To enhance dermal delivery, the formulation may incorporate permeation enhancers such as ethanol, oleic acid, or Azone®, as well as physical enhancement techniques including microneedle arrays or iontophoresis.

[0358] In any of these designs, the nutrient(s) may be encapsulated in carriers such as liposomes, nanoparticles, silica-based and other matrices, oil-in-water emulsion, and more, as described herein.

[0359] According to some embodiments of any of the embodiments described herein, a composition as described herein in any of the respective embodiments and in any combination thereof is for use in treating or preventing muscle atrophy in a subject need thereof.

[0360] Herein, treating or preventing muscle atrophy encompasses treatment or preventing muscle loss or reduced muscle mass and / or strength.

[0361] Subject to be treated according to the present embodiments include subjects identified as exhibiting or at risk of developing one or more symptoms and / or markers (e.g., biomarkers) of muscle atrophy, and / or diagnosed as afflicted by a disease or disorder that is known to cause muscle atrophy or that is treated by medications that are known to cause muscle atrophy.

[0362] In some embodiments of any of the embodiments described herein, the composition as described herein in any of the respective embodiments and in any combination thereof is for increasing muscle mass and / or strength or for preventing loss of muscle mass and / or strength (regardless of muscle atrophy).

[0363] According to some embodiments of any of the embodiments described herein, the subject is a healthy subject who is in need of, or is willing to increase muscle mass and / or strength or prevent loss of muscle mass and / or strength. Subject to be treated according to the present embodiments include subjects identified as exhibiting or as being at risk of developing one or more symptoms and / or markers (e.g., biomarkers) of muscle atrophy, and / or (diagnosed as) afflicted by a disease or disorder that is known to cause muscle atrophy or that is treated by medications that are known to cause muscle atrophy.

[0364] Non-limiting examples of symptoms and / or markers of muscle atrophy include reduced muscle mass, decreased muscle strength, diminished physical endurance, impaired mobility, elevated circulating myostatin levels, elevated expression of muscle atrophy biomarkers (e.g., Atrogin-1, MuRFl) (compared to healthy subjects), and increased SMAD2 / 3 phosphorylation (compared to healthy subjects).

[0365] Non-limiting examples of conditions, diseases or disorders that are known to cause muscle atrophy or that are treated by medications that are known to cause muscle atrophy include cachexia, spaceflight, sedentary lifestyle, sarcopenia, malnutrition, disuse atrophy, neurogenic atrophy, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth disease, peripheral neuropathy, corticosteroid therapy, Emery- Dreifuss muscular dystrophy, Distal muscular dystrophy, Oculopharyngeal muscular dystrophy, Congenital muscular dystrophy, neuromuscular diseases, extended immobilization, trauma, alcoholism, cancer treatment, hyperthyroidism, heart failure, liver diseases, kidney diseases, diabetes, osteoarthritis, Cushing's syndrome, nutritional atrophy, severe burns, malabsorption syndromes, anorexia nervosa and ischemic atrophy, rheumatoid arthritis, spaceflight and surgery.

[0366] According to some embodiments of any of the embodiments described herein, the muscle atrophy is associated with spaceflight, such that the subject participates or participated in a spaceflight and exhibits muscle atrophy or is prone to exhibit muscle atrophy. According to some of these embodiments, a method as described herein is for increasing muscle mass and / or strength or for preventing a reduction in muscle mass and / or strength in the subject.

[0367] According to some embodiments of any of the embodiments described herein, the muscle atrophy is associated with cachexia.

[0368] According to some embodiments of any of the embodiments described herein, the cachexia is associated with cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), chronic kidney disease and Acquired Immune Deficiency Syndrome (AIDS), bums, trauma, sarcopenia, denervation, disuse, and fasting. According to some of these embodiments, the subject is afflicted by cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), chronic kidney disease, Acquired Immune Deficiency Syndrome (AIDS), bums, trauma, sarcopenia, denervation, disuse, or is a fasting subject, and exhibits cachexia. According to some of these embodiments, the subject is afflicted with cancer, and is treated with anti-cancer therapy (e.g., chemotherapy, radiation) that can lead to cachexia or muscle loss.

[0369] The phrase “sedentary lifestyle”, as known in the art, describes a behavioral pattern characterized by low levels of physical activity and prolonged periods of inactivity, which may lead to disuse-related muscle atrophy. According to some of these embodiments, the subject exhibits such a behavioral pattern.

[0370] The term “malnutrition”, as known in the art, describes a condition resulting from inadequate intake or absorption of nutrients necessary for health and muscle maintenance. According to some of these embodiments, the subject suffers from malnutrition.

[0371] The phrase “extended immobilization”, as known in the art, describes a state in which a limb or the body is kept inactive for prolonged periods, often resulting in muscle atrophy. According to some of these embodiments, the subject suffers from extended immobilization.

[0372] The term “trauma”, as known in the art, describes physical injury that may result in disuse or direct damage to a muscle tissue. According to some of these embodiments, the subject suffers from such an injury.

[0373] The term “alcoholism”, as known in the art, describes chronic alcohol use disorder, which may lead to nutritional deficiencies and muscle wasting. According to some of these embodiments, the subject is a chronic alcohol user.

[0374] The phrase “cancer treatment”, as known in the art, describes therapeutic interventions for cancer (e.g., chemotherapy, radiation) that can lead to cachexia or muscle loss. According to some of these embodiments, the subject suffers from cancer or is treated with anti-cancer preventing therapy (e.g., to avoid recurrency).

[0375] The term “hyperthyroidism”, as known in the art, describes excessive thyroid hormone levels, which may lead to increased metabolism and muscle wasting. According to some of these embodiments, the subject suffers from hyperthyroidism.

[0376] The phrase “heart failure”, as known in the art, describes a condition where the heart cannot pump sufficient blood, often associated with cachexia and muscle loss. Non-limiting examples of heart failure conditions include systolic heart failure, diastolic heart failure, congestive heart failure, left-sided heart failure, and right-sided heart failure. According to some of these embodiments, the subject suffers or suffered from heart failure as described herein. According to some of these embodiments, the subject suffered from heart failure and is administered with medications to prevent heart failure recurrence. The phrase “liver diseases”, as known in the art, describes disorders affecting liver function, which may disrupt metabolism and contribute to muscle wasting. Non-limiting examples of liver diseases include cirrhosis, hepatitis (e.g., hepatitis B and C), non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease, and hepatocellular carcinoma. According to some of these embodiments, the subject suffers from a liver disease as described herein.

[0377] The phrase “kidney diseases”, as known in the art, describes chronic or acute impairments in kidney function, often leading to uremic cachexia and muscle loss. Non-limiting examples of kidney diseases include chronic kidney disease (CKD), acute kidney injury (AKI), end-stage renal disease (ESRD), glomerulonephritis, and diabetic nephropathy. According to some of these embodiments, the subject suffers from a kidney disease, as described herein.

[0378] The term “diabetes”, as known in the art, describes a metabolic disorder characterized by hyperglycemia, often associated with altered protein metabolism and muscle atrophy. Nonlimiting types of diabetes include type 1 diabetes (insulin-dependent diabetes mellitus), type 2 diabetes (non-insulin-dependent diabetes mellitus), gestational diabetes, latent autoimmune diabetes in adults (LADA), maturity-onset diabetes of the young (MODY), and secondary diabetes (resulting from conditions such as pancreatitis, endocrine disorders, or medication use (e.g., corticosteroids)). According to some of these embodiments, the subject is a diabetic subject.

[0379] The term “osteoarthritis”, as known in the art, describes degenerative joint disease that may lead to decreased mobility and disuse atrophy. According to some of these embodiments, the subject suffers from osteoarthritis or osteopenia.

[0380] The phrase “nutritional atrophy”, as known in the art, describes muscle wasting due to inadequate dietary intake of essential nutrients.

[0381] The phrase “severe burns”, as known in the art, describes traumatic injuries causing systemic hypermetabolism and muscle catabolism.

[0382] The phrase “malabsorption syndromes”, as known in the art, describes conditions impairing nutrient absorption in the gastrointestinal tract, leading to muscle loss.

[0383] The phrase “anorexia nervosa”, as known in the art, describes an eating disorder characterized by self-induced starvation, leading to severe muscle and fat loss.

[0384] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with a neurodegenerative disease or disorder, a genetic disease or disorder, and / or with motor neuron-related conditions.

[0385] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with a genetic disease or disorder. The phrase “genetic disease or disorder” in the context of the present embodiments describes a pathological condition that arises from a heritable or de novo mutation, deletion, duplication, or chromosomal rearrangement affecting one or more genes, and that contributes to the etiology or phenotype of the disorder. Non-limiting examples of genetic diseases or disorders include Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular atrophy (SMA), and Charcot-Marie- Tooth disease.

[0386] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with a neurodegenerative disease or disorder. By "neurodegenerative disorder" in the context of the present embodiments it is meant any disease or disorder caused by or associated with the deterioration of cells or tissues of the nervous system. Exemplary neurodegenerative disorders include polyglutamine expansion disorders (e.g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also referred to as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., type 1, type 2, type 3 (also referred to as Machado- Joseph disease), type 6, type 7, and type 17)), other trinucleotide repeat expansion disorders (e.g., fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease (also referred to as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt- Jakob disease, ischemia stroke, Krabbe disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, Pelizaeus- Merzbacher disease, Pick's disease, primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), SteeleRichardson- Olszewski disease, and Tabes dorsalis.

[0387] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with motor neurons. The phrase “motor neurons” describes neurons that originate in the spinal cord or brainstem and project to muscles to control voluntary movement. As known in the art, the survival of motor neurons may affect a number of neurodegenerative disorders. Non-limiting such neurodegenerative disorders include polyglutamine expansion disorders (e.g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also referred to as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., type 1, type 2, type 3 (also referred to as Machado-Joseph disease), type 6, type 7, and type 17)), other trinucleotide repeat expansion disorders (e.g., fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease (also referred to as Spielmeyer- Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, ischemia stroke, Krabbe disease, Lewy body dementia, multiple sclerosis, multiple system atrophy, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), SteeleRichardson-Olszewski disease, and Tabes dorsalis. Motor neuron diseases (MNDs) are a group of neurodegenerative disorders that selectively target motor neurons, which are the nerve cells responsible for controlling voluntary muscle activities such as speaking, walking, breathing, swallowing, and general body movement. While these motor neuron diseases typically present distinct variations in their origins and causative factors, they share a common outcome for patients: profound muscle weakness. The classification of these disorders primarily depends on the principal location of motor neuron degeneration. As used herein, the phrase "motor neuron degeneration" means a condition of deterioration of motor neurons, wherein the neurons die or change to a lower or less functionally-active form. Non-limiting examples of MNDs include amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), pseudobulbar palsy, progressive bulbar palsy, spinal muscular atrophy (SMA) and postpolio syndrome. Other systemic conditions such as cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), AIDS, liver disease, renal failure, and cardiac failure may affect neuromuscular function secondarily, although they are not primarily classified as motor neuron disorders.

[0388] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with downregulation of activity and / or expression of a Survival of Motor Neuron protein and / or is not treatable by upregulating a Survival of Motor Neuron protein. The phrase “Survival of Motor Neuron protein” (SMN protein), as used herein describes a protein essential for the maintenance and function of motor neurons, the deficiency of which is implicated in genetic motor neuron disorders such as spinal muscular atrophy (SMA).

[0389] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with a neurodegenerative or genetic disorder, or with motor neuron- related conditions. Non-limiting examples of excluded disorders include spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), post-polio syndrome, multiple system atrophy, and spinal cord injury.

[0390] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with a motor neuron disease or with motor neuron degeneration, that is, a condition of deterioration of motor neurons, wherein the neurons die or change to a lower or less functionally- active form.

[0391] According to some embodiments of any of the embodiments described herein, the muscle atrophy is not associated with Spinal Muscular Atrophy (SMA).

[0392] Spinal Muscular Atrophy (SMA) refers to a number of different disorders, all having in common a genetic cause and the manifestation of weakness due to loss of the motor neurons of the spinal cord and brainstem. The most common form of SMA is caused by mutation of the SMN gene. Exemplary conditions include Werdnig-Hoffmann disease, scoliosis (curvature of the spine) and other skeletal abnormalities.

[0393] According to some embodiments of any of the embodiments described herein, treating the muscle atrophy comprises administering the composition to the subject in need thereof by oral ingestion or oral administration in a total amount as described herein (e.g., of from 1 to 5, or from 1 to 4, or from 1 to 3, grams of the composition, per day).

[0394] In some embodiments of any of the embodiments described herein, the composition or formulation as these are described herein in any of the respective embodiments and in any combination thereof comprises and / or is co-administered to the subject with an additional (e.g., therapeutically active) agent.

[0395] Non-limiting examples of additional agents include agents usable in treating muscle atrophy, GSK-3 inhibitors, and agents that induce muscle atrophy.

[0396] The phrase “agents usable in treating muscle atrophy” encompasses therapeutically active agents such as those described herein in any of the respective embodiments (i.e., which differ from the nutrient / s as described herein in any of the respective embodiments) (e.g., GSK-3 inhibitors), and any other nutritional and / or pharmacological agent / s known to support muscle mass, function, and / or recovery. Non-limiting examples for therapeutically active agents usable in treating or preventing muscle atrophy include anti-catabolic agents (e.g., myostatin inhibitors such as follistatin or monoclonal antibodies against myostatin, activin receptor antagonists such as ACE- 031); anabolic agents (e.g., testosterone, nandrolone, selective androgen receptor modulators such as ostarine (MK-2866), growth hormone secretagogues (GHS)); anti-inflammatory agents (e.g., corticosteroids such as dexamethasone, non-steroidal anti-inflammatory drugs (NSAIDs), cytokine inhibitors such as IL-6 or TNF-a inhibitors); antioxidants and mitochondrial function modulators (e.g., coenzyme Q10, L-carnitine, N-acetyl cysteine, mitochondrial-targeted peptides); P2-adrenergic agonists (e.g., clenbuterol, formoterol); neuromuscular activity modulators (e.g., cholinesterase inhibitors or agents that enhance neuromuscular transmission in neurogenic atrophy); antidiabetic agents (e.g., metformin and thiazolidinediones such as pioglitazone); nutritional supplements (e.g., leucine, HMB (P-hydroxy-P-methylbutyrate), creatine, and omega- 3 fatty acids); and other small molecules or peptides (e.g., proteasome inhibitors such as MG- 132, and agents that modulate autophagy or lysosomal pathways such as rapamycin, trehalose, spermidine).

[0397] According to some embodiments of any of the embodiments described herein, the composition further comprises an (additional) therapeutically active agent. In some such embodiments, the (additional) therapeutically active agent is an agent usable in treating muscle atrophy and an agent that induces muscle atrophy.

[0398] According to some embodiments, the one or more (additional) therapeutically active agent is an agent or a therapy that causes muscle atrophy, for example, anti-cancer therapy, and the GSK- 3 inhibitor is co-administered or co-formulated with such an agent so as to attenuate the adverse side effect caused thereby.

[0399] Exemplary agents (medications) that induce or contribute to muscle atrophy or cachexia, either through direct catabolic effects on muscle tissue or via systemic metabolic disturbances, include, without limitation, glucocorticoids such as prednisone, dexamethasone, and hydrocortisone; chemotherapeutic agents, such as, for example, cisplatin, doxorubicin, paclitaxel, and methotrexate, and any other agent that causes cancer-associated cachexia; Immunosuppressants such as cyclosporine, sirolimus, and tacrolimus; antiretroviral drugs used in HIV therapy, such as zidovudine and stavudine; statins such as simvastatin and atorvastatin; biologic agents targeting tumor necrosis factor-alpha, such as infliximab and etanercept; Loop diuretics like furosemide; Antiepileptic drugs such as phenytoin and valproic acid; heart failure medications such as beta-blockers and ACE inhibitors; and androgen-suppressing therapies including flutamide and leuprolide.

[0400] According to some embodiments of any of the embodiments described herein, the composition further comprises an (additional) therapeutically active agent usable in treating or preventing muscle atrophy.

[0401] According to some embodiments of any of the embodiments described herein, treating or preventing the muscle atrophy further comprises administering to the subject a therapeutically active agent (an additional therapeutically active agent) usable in treating or preventing muscle atrophy (i.e., an agent that is not the nutrient composition as described herein in any of the respective embodiments and in any combination thereof).

[0402] According to some embodiments of any of the embodiments described herein, the (additional) therapeutically active agent, as described herein in any of the respective embodiments and in any combination thereof, is present in the composition or formulation, as described herein in any of the respective embodiments and in any combination thereof, in a therapeutically effective amount (i.e., an amount sufficient to achieve a desired therapeutic effect in treating or preventing muscle atrophy). Alternatively, the therapeutically active agent is co-administered or coformulated with a composition as described herein. In some embodiments, the composition and optionally the (additional) therapeutically active agent are administered or co-administered to the subject by oral ingestion.

[0403] According to some embodiments, the (additional) therapeutically active agent is a GSK-3 inhibitor.

[0404] Exemplary GSK-3 inhibitors include, but are not limited to, those of the CHIR family, for example, CHIR98014, CHIR98023, CHIR99021 , or as collectively represented by Formula. 1, II or III herein, peptide inhibitors such as L803-mts, indirubin-3’ -oxime, SB-216763, SB-415286, BIP-135, AZD1080, SAR502250, IMID1, IMID2, TWS119, AZD1080, SAR502250, JGK-263, AR-A014418, VP2.51, VP2.54, Kenpaullone, Alsterpaullone, Cazpaullone, Azakenpaullone, AZD2858, MMBO, TCS2002, PF-04802367, BRD0705, BRD3731, AF3581, TDZD-8, Tideglusib, VP0.7, VP3.35, SC100, L807mts, 5-imino-l,2,4-thiadiazoles, VP 1.14, and VP 1.16. Additional GSK-3 inhibitors are described, e.g., in PCT International Patent Application Publication Nos. WO 2001 / 049709, WO 2004 / 052404, WO 2012 / 101599, WO 2012 / 101601, WO 2014 / 207743, and WO 2022 / 044024.

[0405] In some embodiments of any of the embodiments described herein, treating or preventing the muscle atrophy further comprises co-administering to the subject, along with a nutrientcontaining composition as described herein, a GSK-3 inhibitor as described herein.

[0406] In some embodiments of any of the embodiments described herein, a composition as described herein further comprises a GSK-3 inhibitor as described herein.

[0407] According to some embodiments of any of the embodiments described herein, the GSK-3 inhibitor is a small molecule inhibitor, for example, a GSK-3 inhibitor of the CHIR family, such as disclosed in WO 99 / 65897.

[0408] According to some embodiments of any of the embodiments described herein, GSK-3 inhibitors usable in the method as described herein can be collectively represented by Formula I:

[0409]

[0410] Formula I wherein:

[0411] X is O, NR15, or CR15R16;

[0412] Ri, R2, R3 and R4 are independently selected from hydrogen, hydroxy, thiol, alkyl, cycloalkyl, alkoxy, amine, aryl, alkaryl, heteroaryl, and heteroalicyclic;

[0413] Rs is selected from hydrogen, halo, alkyl, cycloalkyl, alkoxy, thioalkoxy, amine, aryl, alkaryl, heteroaryl, heteroalicyclic, amide, thioamide and sulfonamide;

[0414] Re is selected from hydrogen, hydroxy, thiol, halo, carboxy, nitro, amine, amide, thioamide, cyano, alkyl, cycloalkyl, aryl, alkaryl, heteroaryl, heteroalicyclic, alkoxy, thioalkoxy, formyl, amide, sulfonyl, sulfonamide, and guanidinyl;

[0415] Rs and R9 are independently selected from hydrogen, nitro, amine, cyano, halo, thioamide, amide, oxime, guanidinyl, sulfonamide, carboxy, formyl, alkyl, cycloalkyl, aryl, alkaryl, heteroaryl and heteroalicyclic;

[0416] Rio, R11, R12, R13 and R14 are independently selected from hydrogen, nitro, amine, cyano, halo, thioamide, carboxy, hydroxy, thiol, amide, thioamide, alkyl, cycloalkyl, aryl, alkaryl, heteroaryl and heteroalicyclic; and

[0417] R15 and Ri6 are each independently selected from hydrogen, hydroxy, thiol, alkyl, cycloalkyl, alkoxy, amine, aryl, alkaryl, heteroaryl, and heteroalicyclic, or pharmaceutically acceptable salts thereof.

[0418] According to some embodiments of any of the embodiments described herein for Formula I, X is NR15.

[0419] According to some embodiments of any of the embodiments described herein, R15 is hydrogen.

[0420] According to some embodiments of any of these embodiments, X is NR15 and R15 is selected from hydrogen, alkyl, cycloalkyl, aryl, alkaryl, heteroaryl, and heteroalicyclic.

[0421] According to some embodiments of any of these embodiments, X is NR15 and R15 is hydrogen. According to some embodiments of any of the embodiments described herein, at least one of Rs and R9 is selected from nitro, amine, cyano, alkyl and alkoxy.

[0422] According to some of any of these embodiments, Rs and R9 are each independently selected from amine, cyano, nitro and alkyl.

[0423] According to some embodiments of any of the embodiments described herein, at least one or each of Rs and R9 is independently selected from nitro, amine, alkyl and alkoxy.

[0424] According to some embodiments of any of the embodiments described herein, at least one of Rs and R9 is nitro or amine.

[0425] According to some embodiments of any of the embodiments described herein, one of Rs and R9 is nitro and the other one of Rs and R9 is amine.

[0426] According to some embodiments of any of the embodiments described herein, one of Rs and R9 is nitro and the other one of Rs and R9 is amine.

[0427] According to some embodiments of any of the embodiments described herein, Rs is amine, preferably primary amine as described herein and R9 is nitro.

[0428] According to some embodiments of any of these embodiments, at least one of Rs and R9 is selected from nitro, amine, cyano, alkyl and alkoxy, X is NR15 and R15 is selected from hydrogen, alkyl, cycloalkyl, aryl, alkaryl, heteroaryl, and heteroalicyclic.

[0429] According to some embodiments of any of these embodiments, at least one of Rs and R9 is selected from nitro, amine, cyano, alkyl and alkoxy, X is NR15 and R15 is hydrogen.

[0430] According to some embodiments of any of these embodiments, at least one of Rs and R9 is selected from nitro, amine, alkyl and alkoxy, X is NR15 and R15 is hydrogen.

[0431] According to some embodiments of any of these embodiments, at least one of Rs and R9 is nitro, and the other one is amine, as described herein, X is NR15 and R15 is hydrogen.

[0432] According to some embodiments of any of the embodiments described herein, at least one, or at least two, or at least three, or each of Ri, R2, R3 and R4 is hydrogen.

[0433] According to some embodiments of any of the embodiments described herein, each of Ri, R2, R3 and R4 is hydrogen.

[0434] According to some embodiments of any of these embodiments, at least one of Rs and R9 is nitro, and the other one is amine, as described herein, and each of Ri, R2, R3 and R4 is hydrogen. According to some of these embodiments, X is NR15 and R15 is hydrogen.

[0435] According to some embodiments of any of the embodiments described herein, at least one of Rio, R11, R12, R13 and R14 is selected from halo, alkyl, hydroxy, alkoxy, amide and cyano.

[0436] According to some embodiments of any of the embodiments described herein, at least one or at least two of Rio, R11, R12, R13 and R14 is / are independently halo (e.g., chloro). According to some embodiments of any of the embodiments described herein, Rio, Rn and R13 are hydrogen and R12 and R14 are each independently selected from halo, alkyl, hydroxy, alkoxy, amide and cyano.

[0437] According to some of any of these embodiments, at least one of R12 and R14 is halo (e.g., chloro). According to some embodiments of any of the embodiments described herein, R12 and R14 are each independently halo (e.g., chloro). According to some embodiments of any of the embodiments described herein, R12 and R14 are each chloro.

[0438] According to some embodiments of any of the embodiments described herein, Rio, R11 and R13 are hydrogen and at least one or each of R12 and R14 is halo (e.g., chloro).

[0439] According to some embodiments of any of the embodiments described herein, Rio, R11 and R13 are hydrogen and R12 and R14 are each independently halo (e.g., chloro).

[0440] According to some embodiments of any of the embodiments described herein, Rs and R9 are each independently other than hydrogen and selected from nitro, alkyl, alkoxy, amine and cyano; X is NR15 and R15 is hydrogen; each of Ri, R2, R3 and R4 is hydrogen; Rio, R11 and R13 are hydrogen and R12 and R14 are each independently selected from halo, alkyl, hydroxy, alkoxy, amide and cyano. According to some of these embodiments, R12 and R14 are each independently halo, and in some embodiments, R12 and R14 are each chloro.

[0441] According to some embodiments of any of the embodiments described herein, at least one of R5 and Re is other than hydrogen.

[0442] According to some embodiments of any of the embodiments described herein, at least one or each of R5 and Re is independently an aryl or heteroaryl, each can be substituted or unsubstituted.

[0443] According to some embodiments of any of the embodiments described herein, R5 is selected from hydrogen, aryl and heteroaryl and is preferably hydrogen. According to some embodiments of any of the embodiments described herein, R5 is hydrogen and Re is aryl or heteroaryl as described herein.

[0444] According to some embodiments of any of the embodiments described herein, Re is a heteroaryl as defined herein. According to some of these embodiments, R5 is hydrogen.

[0445] According to some embodiments of any of the embodiments described herein, Re is selected from pyridyl, pyrimidinyl, pyrrolindinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thienyl, furanyl, quinolinyl, pyrrolylpyridyl, benzothiazolyl, benzopyridyl, benzotriazolyl, and benzimidazolyl, each being unsubstituted or substituted, as defined herein. According to some of any of these embodiments, Re selected from an imidazolyl, a triazolyl, and a pyridyl, each being optionally substituted.

[0446] According to some embodiments of any of the embodiments described herein, R5 is hydrogen Re is an imidazole.

[0447] According to some embodiments of any of the embodiments described herein, Re is an imidazole.

[0448] According to some embodiments of any of the embodiments described herein, Re is an unsubstituted imidazole.

[0449] According to some embodiments, Re is a substituted imidazolyl. In some such embodiments, Re is an imidazolyl substituted by one or more of alkyl, cycloalkyl, aryl. In some embodiments, Re is an imidazolyl substituted by one or more alkyls, preferably lower alkyls (e.g., methyl).

[0450] According to some of any of these embodiments, the GSK-3 inhibitor is represented by Formula III: wherein:

[0451] R12 and Ru are each independently selected from halo, alkyl, hydroxy, alkoxy, amide and cyano;

[0452] Ris and R19 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl; and

[0453] Rs and R9 are each independently selected from hydrogen, amine, cyano, nitro and alkyl.

[0454] According to some of any of these embodiments relating to Formula III, one or each of R12 and R14 is independently halo and in some embodiments, each of R12 and R14 is chloro.

[0455] According to some of any of these embodiments relating to Formula III, Rs and R9 are as described herein in any of the respective embodiments relating to Formula I. According to some of any of these embodiments relating to Formula III, at least one of Rs and R9 is other than hydrogen, and in some embodiments, at least one or each of Rs and R9 is nitro or amine, as described herein in any of the respective embodiments relating to Formula I.

[0456] According to some of any of these embodiments relating to Formula III, Rs is an amine, preferably a primary amine.

[0457] According to some of any of these embodiments relating to Formula III, R9 is a nitro.

[0458] According to some of any of these embodiments relating to Formula III, R9 is a nitro and Rs is an amine.

[0459] According to some of any of these embodiments relating to Formula III, at least one, or both, of R9 and Rs is not cyano.

[0460] According to some of any of these embodiments relating to Formula III, Ris and R19 are each independently hydrogen, such that the imidazole is an unsubstituted imidazole. In some embodiments, one or each of Ris and R19 is other than hydrogen, and is selected from alkyl, cycloalkyl, aryl. In some embodiments, one or each of Ris and R19 is alkyl, preferably a lower alkyl such as methyl.

[0461] According to some of any of these embodiments relating to Formula III, Rs is an amine and one or both of R12 and R14 are independently chloro.

[0462] According to some of any of these embodiments relating to Formula III, R9 is a nitro and one or both of R12 and R14 are independently chloro.

[0463] According to some of any of these embodiments relating to Formula III, R9 is a nitro, Rs is an amine, and one or both of R12 and R14 are independently chloro.

[0464] According to exemplary embodiments, the compound is:

[0465] CHIR98014.

[0466] According to some embodiments of any of the embodiments described herein, the compounds as described herein are collectively represented by Formula II:

[0467] wherein:

[0468] R12 and R 14 are each independently each independently selected from halo, alkyl, hydroxy, alkoxy, amide and cyano;

[0469] R17 is selected from hydrogen, alkyl, cycloalkyl, aryl, and is preferably selected from hydrogen and alkyl (e.g., methyl); and

[0470] R8 and R9 are each independently selected from hydrogen, amine, cyano, nitro and alkyl.

[0471] According to some of any of the embodiments described herein for Formula II, one or each of R12 and R14 is independently halo, and in some embodiments, each of R12 and R14 is independent halo, e.g.., chloro.

[0472] According to some of any of the embodiments described herein for Formula II, Rs is hydrogen.

[0473] According to some of any of the embodiments described herein for Formula II, one or both of Rs and R9 is other than hydrogen.

[0474] According to some of any of the embodiments described herein for Formula II, Rs is hydrogen and R9 is other than hydrogen.

[0475] According to some of any of these embodiments described herein for Formula II, R9 is a cyano.

[0476] According to some of any of these embodiments described herein for Formula II, R9 is a cyano and R8 is hydrogen.

[0477] According to some of any of these embodiments described herein for Formula II, R17 is an alkyl, preferably a lower alkyl such as methyl.

[0478] According to some of any of these embodiments described herein for Formula II, R17 is an alkyl and R8 is hydrogen.

[0479] According to some of any of these embodiments described herein for Formula II, R17 is an alkyl and R9 is a cyano.

[0480] According to some of any of these embodiments described herein for Formula II, R17 is an alkyl, R9 is a cyano and R8 is hydrogen. According to some of any of these embodiments described herein for Formula II, R17 is an alkyl, R9 is a cyano and R8 is hydrogen, and one or both of R12 and R14 are independently chloro.

[0481] According to an exemplary embodiments, the compound is:

[0482] CHIR99021.

[0483] It is to be noted that compounds which are encompassed by Formula II or Formula III are also encompassed by Formula I. Compounds of Formula II and compounds of Formula III generally differ in the connectivity of their Re (imidazolyl) group.

[0484] According to some of any of the embodiments described herein, the GSK-3 inhibitor is collectively represented by Formula IV :

[0485] Formula IV wherein:

[0486] R21 is an aryl, alkaryl or is collectively referred to as an aryl-containing substituent,

[0487] R30, R31, R22, R23, R24, R25, and R26 are each independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, -COR27, -C(O)OR27, -C(O)NR2?R28, - C=NR27, -CN, -OR27, -OC(O)R27, -S(O)t -R27, -NR27R28, -NR27C(O)R28, -NO2, -N=CR27R28 and halo, t is selected from 0, 1, 2 and 3, R27 and R28 are each independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, alkoxy, aryloxy, and halo; or a pharmaceutically acceptable salt thereof. As used herein, the phrase “aryl-containing substituent” describes a substituent group that contains at least one aryl (as defined herein), optionally substituted and / or fused, including an aryl per se, or an alkaryl, as defined herein.

[0488] Non-limiting examples include wherein the wavy lines indicate the connection of the substituent to the core structure.

[0489] According to some of any of these embodiments relating to Formula IV, R21 is

[0490] According to some of any of these embodiments relating to Formula IV, at least one or each of R30 and R31 is hydrogen.

[0491] According to some of any of these embodiments relating to Formula IV, at least one, or at least two, or at least three, or at least four, or all, of R22, R23, R24, R25, and R26, is each hydrogen.

[0492] According to some of any of these embodiments relating to Formula IV, R21 is least one or each of R30 and R31 is hydrogen. some of any of these embodiments relating to Formula IV, R21 is , at least one, or at least two, or at least three, or at least four, or all, of R22,

[0493] R23, R24, R25, and R26, is each hydrogen.

[0494] According to some of any of these embodiments relating to Formula IV, at least one or each of R30 and R31 is hydrogen, and at least one, or at least two, or at least three, or at least four, or all, of R22, R23, R24, R25, and R26, is each hydrogen. According to an exemplary embodiments, the compound is Tideglusib (see, FIG. 2).

[0495] It is to be noted that in all Formulae I, II, III and IV, any position at which no substituent is explicitly depicted is understood to bear one or more hydrogen atoms as substituents, in accordance with the valency requirements of the atom at that position. According to some embodiments of any of the embodiments described herein, a method as described herein is for treating or preventing muscle atrophy in a subject need thereof.

[0496] The composition as described herein in any of the respective embodiments can be provided to the subject in a form of an edible product, as a nutritional composition or supplement or as a pharmaceutical composition.

[0497] According to an aspect of some embodiments of the present invention, there is provided an edible (food) product comprising the composition or formulation as those are described herein in any of the respective embodiments and in any combination thereof. In some embodiments, the edible (food) product is solid, semi-solid or liquid. Non-limiting examples of solid edible (food) products include bars, biscuits, cookies, chews, and frozen confections (e.g., popsicles, frozen yoghurts). Non-limiting examples of liquid or semi-liquid edible (food) products include shakes, beverages, yoghurts, smoothies, and puddings. Any other forms of edible product are also contemplated.

[0498] In some embodiments, the edible (food) product is provided in the form of a powder or granulated material intended to be mixed with a solution (e.g., an aqueous solution, e.g., water, milk, juice) prior to consumption. Non-limiting examples of such powder-form edible products include protein powders, nutritional supplement powders, instant drink mixes, and reconstitutable nutritional preparations. Any other forms of edible product are also contemplated.

[0499] In some embodiments, the edible (food) product comprises a daily dose of the composition in a single unit. In other embodiments, the edible (food) product is formulated such that two, three, or more units are to be consumed daily to provide the intended daily dose of the composition. In some embodiments, the edible (food) product is packaged as two, or three, or more, units packaged together in a manner to provide a complete daily dose. In some embodiments, the edible (food) product is provided with instructions for consumption of two, or three, or more, units per day (to achieve the treatment or prevention of muscle atrophy in the subject).

[0500] The composition as described herein in any of the respective embodiments can be provided to the subject in a form of a medical food. The phrase “medical food” as used herein describes a food formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation. Medical foods are distinct from conventional foods in that they are specifically formulated and processed for a patient who has limited or impaired capacity to ingest, digest, absorb, or metabolize ordinary foodstuffs or certain nutrients, or who has other special medically determined nutrient requirements that cannot be achieved by modification of the normal diet alone. Medical foods can be administered orally or through enteral feeding tubes, and may be provided in various forms including, but not limited to, liquids, semi-solids, powders for reconstitution, or solid forms that can be consumed as provided or mixed with conventional food.

[0501] Any of the products described herein can be used in combination with an additional therapy that treats, prevents or induces muscle atrophy, as described herein.

[0502] According to an aspect of some embodiments of the present invention there is provided a composition that comprises one or more nutrients as described herein in any of the respective embodiments and any combination thereof, and one or more additional therapeutically active agent as described herein in any of the respective embodiments.

[0503] According to an aspect of some embodiments of the present invention there is provided a composition that comprises one or more nutrients as described herein in any of the respective embodiments and any combination thereof, which is identified for use in combination with one or more additional therapeutically active agent as described herein in any of the respective embodiments.

[0504] According to an aspect of some embodiments of the present invention there is provided a kit that comprises a composition that comprises one or more nutrients as described herein in any of the respective embodiments and any combination thereof, and one or more additional therapeutically active agent as described herein in any of the respective embodiments. The nutrient composition and the additional agent can be packaged individually in the kit.

[0505] According to some embodiments of these aspects, the composition can be in any of the forms as described herein, including any of the products as described herein.

[0506] According to some embodiments of these aspects, the compositions and kits are for use in treating muscle atrophy as described herein.

[0507] As used herein throughout, the term “subject” includes mammals, preferably human beings at any age which suffer from, or are at risk of developing muscle atrophy, for example, who are afflicted by a condition associated with muscle atrophy or treatable by a medication or therapy that may induce muscle atrophy, as described herein.

[0508] For any of the embodiments described herein, the compound (e.g., a nutrient or a GSK-3 inhibitor) described herein may be in a form of a salt, for example, a pharmaceutically acceptable salt (e.g., a sodium salt), and / or in a form of a prodrug. As used herein, the phrase “pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter-ion, which is typically used to modify the solubility characteristics of the parent compound and / or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. A pharmaceutically acceptable salt of a compound as described herein can alternatively be formed during the synthesis of the compound, e.g., in the course of isolating the compound from a reaction mixture or re-crystallizing the compound.

[0509] In the context of some of the present embodiments, a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt and / or a base addition salt.

[0510] An acid addition salt comprises at least one basic (e.g., amine and / or guanidinyl) group of the compound which is in a positively charged form (e.g., wherein the basic group is protonated), in combination with at least one counter-ion, derived from the selected acid, that forms a pharmaceutically acceptable salt. The acid addition salts of the compounds described herein may therefore be complexes formed between one or more basic groups of the compound and one or more equivalents of an acid.

[0511] A base addition salt comprises at least one acidic (e.g., carboxylic acid) group of the compound which is in a negatively charged form (e.g., wherein the acidic group is deprotonated), in combination with at least one counter-ion, derived from the selected base, that forms a pharmaceutically acceptable salt. The base addition salts of the compounds described herein may therefore be complexes formed between one or more acidic groups of the compound and one or more equivalents of a base.

[0512] Depending on the stoichiometric proportions between the charged group(s) in the compound and the counter-ion in the salt, the acid additions salts and / or base addition salts can be either mono-addition salts or poly-addition salts.

[0513] The phrase “mono-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and charged form of the compound is 1:1, such that the addition salt includes one molar equivalent of the counter-ion per one molar equivalent of the compound.

[0514] The phrase “poly-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1:1 and is, for example, 2: 1, 3: 1, 4: 1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound.

[0515] An example, without limitation, of a pharmaceutically acceptable salt would be an ammonium cation or guanidinium cation and an acid addition salt thereof, and / or a carboxylate anion and a base addition salt thereof. The base addition salts may include a cation counter-ion such as sodium, potassium, ammonium, calcium, magnesium and the like, that forms a pharmaceutically acceptable salt. The acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which affords a naphthalenesulfonic acid addition salt, oxalic acid which affords an oxalic acid addition salt, phosphoric acid which affords a phosphoric acid addition salt, toluenesulfonic acid which affords a p-toluenesulfonic acid addition salt, succinic acid which affords a succinic acid addition salt, sulfuric acid which affords a sulfuric acid addition salt, tartaric acid which affords a tartaric acid addition salt and trifluoroacetic acid which affords a trifluoroacetic acid addition salt. Each of these acid addition salts can be either a mono-addition salt or a poly-addition salt, as these terms are defined herein.

[0516] As used herein, the term “prodrug” refers to a compound which is converted in the body to an active compound (e.g., the compound of the formula described hereinabove). A prodrug is typically designed to facilitate administration, e.g., by enhancing absorption. A prodrug may comprise, for example, the active compound modified with ester groups, for example, wherein any one or more of the hydroxyl groups of a compound is modified by an acyl group, optionally (Ci- 4)-acyl (e.g., acetyl) group to form an ester group, and / or any one or more of the carboxylic acid groups of the compound is modified by an alkoxy or aryloxy group, optionally (Ci-4)-alkoxy (e.g., methyl, ethyl) group to form an ester group.

[0517] Further, each of the compounds described herein, including the salts thereof, can be in a form of a solvate or a hydrate thereof.

[0518] The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta- , hexa-, and so on), which is formed by a solute (the heterocyclic compounds described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.

[0519] The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water.

[0520] The compounds described herein can be used as polymorphs and the present embodiments further encompass any isomorph of the compounds and any combination thereof. The compounds and structures described herein encompass any stereoisomer, including enantiomers and diastereomers, of the compounds described herein, unless a particular stereoisomer is specifically indicated.

[0521] As used herein, the term “enantiomer” refers to a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion / reflection (mirror image) of each other. Enantiomers are said to have “handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems. In the context of the present embodiments, a compound may exhibit one or more chiral centers, each of which exhibiting an (R) or an (S) configuration and any combination, and compounds according to some embodiments of the present invention, can have any their chiral centers exhibit an (R) or an (S) configuration.

[0522] The term “diastereomers”, as used herein, refers to stereoisomers that are not enantiomers to one another. Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more, but not all of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter they are epimers. Each stereo-center (chiral center) gives rise to two different configurations and thus to two different stereoisomers. In the context of the present invention, embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of stereo-configuration, namely any diastereomer.

[0523] Structural analogs of any of the nutrients described herein, which exhibit the same activity / activities on the myostatin signaling pathway are also contemplated.

[0524] Herein throughout, the term “hydrocarbon” collectively describes a chemical group composed mainly of carbon and hydrogen atoms. A hydrocarbon can be comprised of alkyl, alkene, alkyne, aryl, and / or cycloalkyl, as defined herein, each can be substituted or unsubstituted, and can be interrupted by one or more heteroatoms. The number of carbon atoms can range from 2 to 20, and is preferably lower, e.g., from 1 to 10, or from 1 to 6, or from 1 to 4. A hydrocarbon can be a linking group or an end group.

[0525] As used herein throughout, the term “alkyl” refers to any saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1 to 20”, is stated herein, it implies that the group, in this case the hydrocarbon, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0526] Herein, the term “alkenyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon double bond, including straight chain and branched chain groups. Preferably, the alkenyl group has 2 to 20 carbon atoms. More preferably, the alkenyl is a medium size alkenyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkenyl is a lower alkenyl having 2 to 4 carbon atoms. The alkenyl group may be substituted or non-substituted. Substituted alkenyl may have one or more substituents, whereby each substituent group can independently be, for example, alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, S -thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0527] Herein, the term “alkynyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon triple bond, including straight chain and branched chain groups. Preferably, the alkynyl group has 2 to 20 carbon atoms. More preferably, the alkynyl is a medium size alkynyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkynyl is a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group may be substituted or non-substituted. Substituted alkynyl may have one or more substituents, whereby each substituent group can independently be, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0528] A “cycloalkyl” group refers to a saturated on unsaturated all-carbon monocyclic or fused ring (z.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S -thiocarbamyl, C- amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. When a cycloalkyl group is unsaturated, it may comprise at least one carbon-carbon double bond and / or at least one carboncarbon triple bond.

[0529] An “aryl” group refers to an all-carbon monocyclic or fused-ring polycyclic (z.e., rings which share adjacent pairs of carbon atoms) having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0530] A “heteroaryl” group refers to a monocyclic or fused ring (z.e., rings which share an adjacent pair of atoms) having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, pyrazine, pyridazine, indole, benzofuran, benzothiophene, benzoxazole, benzimidazole, benzothiazole, quinoxaline, and carbazole and purine. The heteroaryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, S -thiocarbamyl, C-amido, N-amido, C-carboxy, O- carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0531] A “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or non-substituted. When substituted, the substituted group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine, pyrrolidine, phthalimide, 1,4-dioxane, azepine, thiazolidine, and the like.

[0532] Herein, the terms “amine” and “amino” each refer to either a -NR’R” group or a - N+R’R”R’ ’ ’ group, wherein R’ , R” and R’ ’ ’ are each hydrogen or a substituted or non-substituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (linked to amine nitrogen via a ring carbon thereof), aryl, or heteroaryl (linked to amine nitrogen via a ring carbon thereof), as defined herein. Optionally, R’, R” and R’” are hydrogen or alkyl comprising 1 to 4 carbon atoms. Optionally, R’ and R” (and R”’, if present) are hydrogen. When substituted, the carbon atom of an R’, R” or R”’ hydrocarbon moiety which is bound to the nitrogen atom of the amine is not substituted by oxo (unless explicitly indicated otherwise), such that R’, R” and R’” are not (for example) carbonyl, C-carboxy or amide, as these groups are defined herein.

[0533] An “azide” group refers to a -N=N+=N“ group.

[0534] An “alkoxy” group refers to any of an -O-alkyl, -O-alkenyl, -O-alkynyl, -O-cycloalkyl, and -O-heteroalicyclic group, as defined herein.

[0535] An “aryloxy” group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.

[0536] A “hydroxy” group refers to a -OH group.

[0537] A “thiohydroxy” or “thiol” group refers to a -SH group.

[0538] A “thioalkoxy” group refers to any of an -S-alkyl, -S-alkenyl, -S-alkynyl, -S -cycloalkyl, and -S-heteroalicyclic group, as defined herein.

[0539] A “thioaryloxy” group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein. A “carbonyl” or “acyl” group refers to a -C(=O)-R’ group, where R’ is defined as hereinabove.

[0540] A “thiocarbonyl” group refers to a -C(=S)-R’ group, where R’ is as defined herein.

[0541] A “C-carboxy” group refers to a -C(=O)-O-R’ group, where R’ is as defined herein.

[0542] An “O-carboxy” group refers to an R’C(=O)-O- group, where R’ is as defined herein.

[0543] A “carboxylic acid” group refers to a -C(=O)OH group.

[0544] An “oxo” group refers to a =0 group.

[0545] An “imine” group refers to a =N-R’ group, where R’ is as defined herein.

[0546] An “oxime” group refers to a =N-0H group.

[0547] A “hydrazone” group refers to a =N-NR’R” group, where each of R’ and R” is as defined herein.

[0548] A “methyleneamine” group refers to an -NR’-CH2-CH=CR”R”’ end group or a -NR’-CH2-CH=CR”- linking group, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0549] A “halo” group refers to fluorine, chlorine, bromine or iodine.

[0550] A “sulfinyl” group refers to an -S(=O)-R’ group, where R’ is as defined herein.

[0551] A “sulfonyl” group refers to an -S(=O)2-R’ group, where R’ is as defined herein.

[0552] A “sulfonate” group refers to an -S(=O)2-O-R’ group, where R’ is as defined herein.

[0553] A “sulfate” group refers to an -O-S(=O)2-O-R’ group, where R’ is as defined as herein.

[0554] A “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamido and N- sulfonamido groups, as defined herein.

[0555] An “S-sulfonamido” group refers to a -S(=O)2-NR’R” group, with each of R’ and R” as defined herein.

[0556] An “N-sulfonamido” group refers to an R’S(=O)2-NR”- group, where each of R’ and R” is as defined herein.

[0557] An “O-carbamyl” group refers to an -0C(=0)-NR’R” group, where each of R’ and R” is as defined herein.

[0558] An “N-carbamyl” group refers to an R’0C(=0)-NR”- group, where each of R’ and R” is as defined herein.

[0559] An “O-thiocarbamyl” group refers to an -OC(=S)-NR’R” group, where each of R’ and R” is as defined herein.

[0560] An “N-thiocarbamyl” group refers to an R’OC(=S)NR”- group, where each of R’ and R” is as defined herein. An “S-thiocarbamyl” group refers to an -SC(=O)-NR’R” group, where each of R’ and R” is as defined herein.

[0561] An “amide” or “amido” group encompasses C-amido and N-amido groups, as defined herein.

[0562] A “C-amido” group refers to a -C(=O)-NR’R” group, where each of R’ and R” is as defined herein.

[0563] An “N-amido” group refers to an R’C(=O)-NR”- group, where each of R’ and R” is as defined herein.

[0564] A “urea group” refers to an -N(R’)-C(=O)-NR”R’” group, where each of R’, R” and R” is as defined herein.

[0565] A “thiourea group” refers to a -N(R’)-C(=S)-NR”R”’ group, where each of R’, R” and R” is as defined herein.

[0566] A “nitro” group refers to an -NO2 group.

[0567] A “cyano” group refers to a -C=N group.

[0568] A “isocyanate” group refers to a -N=C=O group.

[0569] The term “phosphonyl” or “phosphonate” describes a -P(=O)(OR’)(OR”) group, with R’ and R’ ’ as defined hereinabove.

[0570] The term “phosphate” describes an -O-P(=O)(OR’)(OR”) group, with each of R’ and R” as defined hereinabove.

[0571] The term “phosphinyl” describes a -PR’R” group, with each of R’ and R” as defined hereinabove.

[0572] The term “hydrazine” describes a -NR’-NR”R”’ group, with R’, R”, and R’” as defined herein.

[0573] As used herein, the term “hydrazide” describes a -C(=O)-NR’-NR”R”’ group, where R’, R” and R’” are as defined herein.

[0574] As used herein, the term “thiohydrazide” describes a -C(=S)-NR’-NR”R”’ group, where R’, R” and R’” are as defined herein.

[0575] A "azo" or “diazo” group refers to an -N=NR’ end group or an -N=N- linking group, as these phrases are defined hereinabove, with R’ as defined hereinabove.

[0576] A “guanidinyl” group refers to an -RaNC(=NRd)-NRbRc group, where each of Ra, Rb, Rc and Rd can be as defined herein for R’ and R’ ’ .

[0577] A “guanyl” or “guanine” group refers to an RaRbNC(=NRd)- group, where Ra, Rb and Rd are as defined herein. A “silyl” group refers to a -SiR’R”R”’ end group or a -SiR’R”- linking group, as these phrases are defined hereinabove, whereby each of R’, R” and R'" are as defined herein.

[0578] A “siloxy” or “siloxane” or “alkoxysilane” group refers to a -Si(OR’)R”R”’ end group or a -Si(OR’)R”- linking group, as these phrases are defined hereinabove, whereby each of R’, R" and R”' are as defined herein.

[0579] A “silaza” group refers to a -Si(NR’R”)R”’ end group or a -Si(NR’R”)- linking group, as these phrases are defined hereinabove, whereby each of R’, R” and R’” is as defined herein.

[0580] A “silicate” or “triorthosilicate” group refers to a -O-Si(OR’)(OR”)(OR"') end group or a -O-Si(OR’)(OR”)- linking group, as these phrases are defined hereinabove, with R’, R" and R”' as defined herein.

[0581] As used herein the term “about” refers to ± 10 % or ± 5 %.

[0582] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0583] The term “consisting of’ means “including and limited to”.

[0584] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0585] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0586] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0587] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0588] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0589] The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and / or causing the reduction, remission, or regression (e.g., preventing or reducing progression) of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

[0590] As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

[0591] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0592] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0593] EXAMPLES

[0594] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0595] MATERIALS AND EXPERIMENTAL METHODS

[0596] Materials:

[0597] C2C12 myotube cells were obtained from American Type Culture Collection (ATCC).

[0598] Myostatin was obtained from Mercury Ltd. A83-O1 was obtained from Sigma-Aldrich.

[0599] Dexamethasone was obtained from Sigma- Aldrich.

[0600] Horse serum (10 %) was obtained from Biological Industries.

[0601] Myoinositol was obtained from Sigma- Aldrich.

[0602] Uridine was obtained from Sigma- Aldrich.

[0603] Sodium butyrate was obtained from Sigma- Aldrich.

[0604] Pyruvic acid was obtained from Sigma- Aldrich.

[0605] 3-hydroxybutyric acid was obtained from Sigma- Aldrich.

[0606] Adenosine was obtained from Sigma- Aldrich.

[0607] Glycine was obtained from Sigma- Aldrich.

[0608] Alpha-ketoglutarate was obtained from Sigma- Aldrich.

[0609] D-mannitol was obtained from Sigma- Aldrich.

[0610] Acetonitrile (ACN), acetone, and methanol (MeOH), HPLC grade, were obtained from J.T Baker.

[0611] Water HPLC grade were obtained from Sigma- Aldrich.

[0612] Ammonium formate salt (HCOONH4) and formic acid (FA) HCOOH, LC-MS grade were obtained from Sigma- Aldrich.

[0613] Fmoc chloride (FMOC-C1), for HPLC derivatization, was obtained from Sigma- Aldrich.

[0614] Alginic acid sodium salt (alginate) was obtained from Acros Organics.

[0615] Standard Agarose 8100 (KI 8100-500) was obtained from Conda.

[0616] Sodium bicarbonate, 99.8 % was obtained from Chem-IMPEX International.

[0617] Sodium citrate dihydrate was obtained from J.T Baker.

[0618] EDTA-Na2 (Titriplex III) was obtained from Merck.

[0619] 0.2 pm membrane cellulose syringe filters were obtained from OlimPeak by Teknokroma.

[0620] Animals:

[0621] About 300 HsdJCR mice (25-29 grams), aged about 12 weeks, were obtained from Envigo RMS LLC.

[0622] All animal experiments were consistent with Israel Council on Animal Experiments guidelines and the Institutional Regulations of Animal Care and Use. Specialized personnel provided mice care in the institutional animal facility.

[0623] EXAMPLE 1

[0624] Design

[0625] As discussed hereinabove, myostatin is a regulator of muscle mass [Smith et al., Curr Opin Support Palliat Care. 2013 Dec;7(4):352-60]. Myostatin binds to the ActRIIB receptor on muscle cells, initiating a signaling cascade involving ALK4 / ALK5, SMAD2 / 3, and SMAD4, leading to muscle wasting gene activation (see, Background Art FIG. 1). Concurrently, myostatin reduces AKT activity, increasing FOXO's entry into the nucleus, which upregulates E3 ligases MuRFl and Atrogin-1, promoting muscle protein breakdown (see, Background Art FIG. 1). Inhibiting myostatin offers a therapeutic strategy against muscle wasting by disrupting this pathway.

[0626] The present inventors have now developed a nutrition solution to handle atrophy, that comprises nutrients which specifically target the debilitating effects of atrophy pathways in muscle, and thereby increase muscle mass and strength and consequently survival of the affected individuals. The nutrient-based supplement was developed to specifically inhibit myostatin signaling and also the expression of MuRFl and Atrogin-1.

[0627] To identify nutrients which inhibit myostatin signaling, a multi-step (e.g., three-step) process was conducted, as schematically depicted in FIG. 2. An exemplary such process includes (1) screening of 400 nutrients for effects on SMAD2,3 phosphorylation on the myostatin signaling pathway; (2) evaluating the selected hit nutrients to assess their effects on myostatin signaling by determining the effect of various concentrations of each nutrient on SMAD2,3 phosphorylation, and / or on Atrogin-1, MuRF-1 and myostatin gene mRNA levels; and (3) conducting in vivo assays in mice to evaluate the effect of the selected hit nutrients in reducing muscle atrophy, to thereby obtain a nutrient-based formulation to inhibit muscle atrophy.

[0628] An in-vitro model was first developed to be utilized in screening the 400 nutrients for effects on myostatin signaling (Step 1), by evaluating the levels of SMAD2,3 and phosphorylated SMAD2,3 (pSMAD2,3) in C2C12 myotubes. Immunoblotting was used to evaluate the effects of a vehicle (control), myostatin (1 mg / mL) and myostatin (1 mg / mL) and the myostatin inhibitor, A83-O1 (1 pM), on pSMAD2,3 and SMAD2,3 protein levels in cultured C2C12 myotubes, to confirm that pSMAD2,3 was reduced following myostatin inhibition.

[0629] FIG. 3 presents an immunoblotting graph showing the effect of a vehicle (control), myostatin (1 microgram / mL), and myostatin (1 pM) and A83-O1 (1 pM), on pSMAD2,3 and SMAD2,3.

[0630] As can be seen in FIG. 3, elevated level of pSMAD2,3 was observed in the presence of myostatin (1 pM), showing a clear indication that the muscle atrophy pathway is activated by myostatin. In contrast, in the presence of myostatin (1 pM) and A83-O1 (1 pM) no pSMAD2,3 is observed, indicating that A83-O1 disrupts the phosphorylation of SMAD2,3.

[0631] These data confirmed that pSMAD2,3 and SMAD2,3 levels in C2C12 myotubes treated with dexamethasone in the presence of various nutrients can be used to evaluate their effectiveness in inhibiting myo statin signaling. An in-vitro dexamethasone-induced muscle atrophy model was developed to further evaluate the effect of the nutrients that were selected based on the Step (1) model on mRNA levels of Atrogin-1 and MuRF-1 in C2C12 myotubes.

[0632] FIGs. 4A-B present a bar graph showing the comparative fold increase in Atrogin-1 (FIG. 4A) and MuRF-1 (FIG. 4B) mRNA levels in C2C12 myotubes treated with dexamethasone (1 pM).

[0633] As can be seen in FIGs. 4A-B, C2C12 myotubes treated with dexamethasone (atrophying cells) exhibited a significant increase in Atrogin-1 and MurF-1 mRNA levels in comparison to the nutrient-untreated control.

[0634] These data confirmed that an effect on the Atrogin-1 and MurF-1 mRNA levels in C2C12 myotubes treated with dexamethasone can be used to further evaluate the effect of various nutrients in inhibiting muscle atrophy.

[0635] EXAMPLE 2

[0636] In vitro screening (Steps (1) and (2))

[0637] The in vitro model described in Example 1, for Step (1), was used for screening about 400 nutrients, to evaluate their effect on the level of pSMAD2,3.

[0638] FIG. 5 presents a representative Western blot image from this screening, showing the effects of exemplary nutrients, each at a concentration of 5-20 mM on the levels of pSMAD2,3, SMAD and on the levels of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; control).

[0639] As can be seen in FIG. 5, nutrients 43 (D-Fructose) and 45 (D-Raffinose) exhibited the most significant reduction in pSMAD2,3 levels, indicating that these nutrients exhibited substantial inhibition of the myostatin signaling.

[0640] Nutrients that reduced the ratio of pSMAD2,3 / SMAD2,3 intensity in C2C12 myotubes compared with nutrient-untreated control cells, by 80 % or more (were selected as “hits” for step (2) to evaluate their effects on myostatin signaling and Atrogin-1 and myostatin mRNA levels.

[0641] Exemplary nutrients that “passed” step 1 of the screening include glycine, pyruvic acid, butyric acid, myoinositol, uridine, adenosine, 3-hydroxybutyric acid, alpha-ketoglutarate, D- mannitol, Met-Trp, D-malic acid, sedoheptulosan, D-fructose, D-galactose, Leu-Asp, D-raffinose, D-lactitol, D,L-beta-hydroxy-butyric acid, gamma-hydroxy-butyric acid, Met-Pro, Meso-tartaric acid, N-acetyl- neuraminic acid, chondroitin-6 sulfate, L-rhamnose, meso-erythritol, pectin, Met- Thr, D-melezitose, succinamic acid, D-melibiose, propylene glycol, 2,3-butanediol, stachyose, beta-methyl-D-galactoside, palatinose, thymidine, methyl pyruvate, D,L-alpha-glycerol- phosphate, D-glucuronic acid, xylitol, inosine, methyl D-lactate, Leu-His, D-arabinose, mannan, D-fucose, succinic acid, alpha-methyl-D-galactoside, alpha-hydroxy -butyric acid, L-arabinose, hexanoic acid, alpha-methyl-D-mannoside, ethanolamine, N-acetyl-beta-D-mannosamine, D- fructose-6-phosphate, L-fucose, glycerol, beta-methyl-D-xylopyranoside, Met-Lys, adonitol, alpha-keto-butyric acid, gamma-amino-N-butyric acid, sucrose, acetic acid, Met-Met, Lys-Phe, 3- O-methyl-D-glucose, D-sorbitol, Met-Leu, D,L-lactic acid, lactulose, propionic acid, L-sorbose, maltitol, L-malic acid5L-glucose, alpha-D-lactose, alpha-methyl-D-glucoside, Leu-Gly, D- turanose^ acetoacetic acid, Lys-Ser, Leu-Glu, alpha-cyclodextrin, Met-Tyr, L-histidine, 3- hydroxy-2-butanone, Ile-Ile^ D-cellobiose, D-salicin, Met-Val, tricarballylic acid and His-Trp.

[0642] Of the above-listed exemplary nutrients that “passed” step 1 of the screening, glycine, pyruvic acid, butyric acid, myoinositol, uridine, adenosine, 3 -hydroxybutyric acid, alphaketoglutarate and D-mannitol, were further tested in the preliminary studies described herein.

[0643] The in-vitro dexamethasone-induced muscle atrophy model, as described in Example 1 hereinabove, for Step (2) was used to evaluate the effect of various concentrations of glycine on Atrogin-1 mRNA levels.

[0644] FIG. 6 is a bar graph showing the comparative fold increase in Atrogin-1 mRNA levels in C2C12 myotubes treated with dexamethasone (1 pM) in the presence of varying glycine concentrations.

[0645] As can be seen in FIG. 6, C2C12 myotubes treated with dexamethasone led to a marked increase in Atrogin-1 gene expression mRNA levels in comparison to the nutrient-untreated control, whereby the presence of glycine led to Atrogin-1 gene expression mRNA levels which are lower than in the dexamethasone-treated (atrophy-induced) myotubes, and similar to those of the nutrient-untreated control.

[0646] The in-vitro dexamethasone-induced muscle atrophy model (Step (2)), as described hereinabove, was used to evaluate the effect of glycine (5 mM), butyric acid (5, 10 and 20 mM) and pyruvic acid (2.5 and 5 mM) on pSMAD2,3 and SMAD2,3 protein levels and Atrogin-1 gene expression mRNA levels and some of the obtained data is shown in FIGs. 7A-C. As can see seen in FIGs. 7A-B, in the presence of these nutrients, at almost all tested concentrations, reduced pSMAD2,3 levels were observed. As can be seen in FIG. 7C, butyric acid, at 5 mM concentration, substantially reduced expression of Atrogin-1 in the tested C2C12 myotubes treated with dexamethasone (1 micro molar).

[0647] FIGs. 8A-B present additional exemplary data obtained in Step (2) for varying concentrations of myoinositol and uridine.

[0648] FIGs. 8A-B present a Western blot image of pSMAD2,3 and SMAD2,3 levels in cultured myotubes (differentiated at 10 % horse serum (HS)) and of the C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence or absence of myoinositol (2.5 and 5 and 10 micromolar) and uridine (2.5, 5 and 10 micromolar) (FIG. 8A); and a bar graph showing the pSMAD2,3 / SMAD2,3 densitometric measurement ratio as determined according to the Western blots shown in FIG. 8A (ratio is presented as mean ± SEM. n =3 wells per condition) (FIG. 8B).

[0649] As can be seen in FIGs. 8 A and 8B, myotubes treated with dexamethasone in the presence of certain concentrations of myoinositol or uridine showed a lower level of pSMAD2,3 in comparison to myotubes treated with only dexamethasone.

[0650] The in-vitro dexamethasone-induced muscle atrophy model, as described hereinabove (Step (2)), was further used to evaluate the effect of butyric acid, pyruvic acid and uridine on myostatin and Atrogin-1 mRNA levels.

[0651] The gene expression mRNA levels of Myostatin and Atrogin-1 in C2C12 myotubes (differentiated at 10 % Horse Serum (HS)) were analyzed by Reverse transcription polymerase chain reaction (RTPCT).

[0652] FIGs. 9A-B are bar graphs showing the comparative fold increase in myostatin (FIG. 9A) and Atrogin-1 (FIG. 9B) gene expression mRNA levels in C2C12 myotubes (differentiated at 10 % HS), and in C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence or absence of butyric acid (10 micromolar), pyruvic acid (2.5 micromolar) and uridine (2.5 micromolar).

[0653] As can be seen in FIG. 9A, C2C12 myotubes treated with dexamethasone in the presence of butyric acid displayed a lower myostatin gene expression mRNA levels in comparison to myotubes treated with only dexamethasone, whereas myotubes treated with dexamethasone in the presence of pyruvic acid and uridine displayed no difference in myostatin gene expression mRNA levels.

[0654] As can be seen in FIG. 9B, C2C12 myotubes treated with dexamethasone in the presence of butyric acid displayed a lower Atrogin-1 gene expression mRNA levels in comparison to myotubes treated with only dexamethasone, whereas myotubes treated with dexamethasone in the presence of pyruvic acid and uridine displayed no difference in Atrogin-1 gene expression mRNA levels.

[0655] The in-vitro dexamethasone-induced muscle atrophy model, as described hereinabove (Step (2)), was used to evaluate the effect of adenosine and 3-hydroxybutyric acid on Atrogin-1 and myostatin gene expression mRNA levels.

[0656] FIGs. 10A-D present bar graphs showing the comparative fold increase in Atrogin-1 gene expression mRNA levels in C2C12 myotubes, and in C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence or absence of Adenosine (2.5, 5, 10 and 15 micromolar) (FIG. 10A) and in the presence or absence of 3-hydroxybutyric acid (1 and 2.5 micromolar) (FIG. 10B); and the comparative fold increase in myostatin gene expression mRNA levels in C2C12 myotubes, and in C2C12 myotubes treated with dexamethasone (1 micromolar) in the presence or absence of Adenosine (2.5, 5, 10 and 15 micromolar) (FIG. 10C) and in the presence or absence of 3 -hydroxy buy tic acid (1 and 2.5 micromolar) (FIG. 10D).

[0657] As can be seen in FIG. 10A, C2C12 myotubes treated with dexamethasone in the presence of adenosine displayed progressively lower Atrogin-1 gene expression mRNA levels with increasing concentration. C2C12 myotubes treated with dexamethasone in the presence of adenosine at concentrations of 2.5 and 5 micromolar displayed higher Atrogin-1 gene expression mRNA levels in comparison to the dexamethasone-treated (atrophy-induced) myotubes, whereas myotubes treated with dexamethasone in the presence of adenosine at a concentration of 10 micromolar displayed a lower Atrogin-1 gene expression mRNA levels in comparison to the dexamethasone-treated (atrophy-induced) myotubes, and those treated with adenosine at a concentration of 15 micromolar displayed a lower Atrogin-1 gene expression mRNA levels than the nutrient-untreated control.

[0658] As can be seen in FIG. 10B, C2C12 myotubes treated with dexamethasone in the presence of 3-hydroxybutyric acid at concentrations of 1 and 2.5 micromolar displayed lower Atrogin-1 gene expression mRNA levels in comparison to the dexamethasone-treated (atrophy-induced) myotubes, but higher in comparison to the nutrient-untreated control.

[0659] As can be seen in FIG. 10C, C2C12 myotubes treated with dexamethasone in the presence of adenosine displayed progressively lower myostatin gene expression mRNA levels with increasing concentration. C2C12 myotubes treated with dexamethasone in the presence of adenosine at concentrations of 2.5 and 5 and 10 micromolar displayed higher Atrogin-1 gene expression mRNA levels in comparison to the dexamethasone-treated (atrophy-induced) myotubes, whereas myotubes treated with dexamethasone in the presence of adenosine at a concentration of 15 micromolar displayed lower myostatin gene expression mRNA levels in comparison to the dexamethasone-treated (atrophy-induced) myotubes.

[0660] As can be seen in FIG. 10D, C2C12 myotubes treated with dexamethasone in the presence of 3-hydroxybutyric acid at concentrations of 1 and 2.5 micromolar displayed lower Atrogin-1 gene expression mRNA levels in comparison to the dexamethasone-treated (atrophy-induced) myotubes and in comparison to the nutrient-untreated control.

[0661] The data presented hereinabove, and additional data (not shown) obtained in in vitro screening Steps (1) and (2) identified the following nutrients as potential candidates for treating muscle atrophy: adenosine, myoinositol, glycine, alpha-ketoglutarate, sodium butyrate and mannitol. EXAMPLE 3

[0662] In vivo validation (Step (3))

[0663] The therapeutic effect of the nutrients identified in the in vitro screening Steps (1) and (2) were then further evaluated in vivo, in mice treated with dexamethasone (1 pM).

[0664] For each nutrient, a concentration lower by 5-folds (20 %) of the concentration that achieves saturation in water or an aqueous solution (0.9 % Saline) was used.

[0665] First, in-vivo dexamethasone-induced muscle atrophy model was developed to evaluate the effect of the identified nutrients on body weight and muscle weight (FIGs. 10A-B). Mice were orally administered with water-soluble dexamethasone (10 mg / kg) daily for 10 consecutive days via oral gavage. Throughout the treatment period, nutrients were co-administered with dexamethasone, dissolved or suspended in the drinking solution. Mice body weight was monitored periodically.

[0666] At the end of the treatment period, mice were euthanized using an approved method of terminal anesthesia. The Tibialis Anterior (TA) muscles from both hind limbs were dissected, cleaned of connective tissue, and immediately weighed to assess changes in skeletal muscle mass due to treatment with the nutrient(s). To determine heart weight, following euthanasia, the thoracic cavity was opened and the heart was excised, rinsed briefly in physiological saline to remove excess blood, and gently blotted dry. The whole heart was then weighed.

[0667] FIGs. 11 A-B present a scatter plot showing the mean body weight (FIG. 11 A) and the mean ratio of isolated tibialis anterior (TA) muscle weight (FIG. 11B) of mice administered with water soluble dexamethasone (10 mg / kg), over a period of 10 days in comparison to non-treated mice (control).

[0668] As can be seen in FIG. 11 A, the mean body weight (grams) of mice treated with dexamethasone was lower than in the nutrient-untreated mice.

[0669] As can be seen in FIG. 1 IB, the mean TA / BW ratio in mice treated with dexamethasone was lower than in the nutrient-untreated mice.

[0670] These data confirm that the mean body weight (grams) and TA / BW ratio in mice treated with dexamethasone in the presence of various nutrients can be used to evaluate the effect the nutrient(s) in inhibiting muscle atrophy.

[0671] This in vivo model was therefore used to validate the effect of the nutrients identified in Steps (1) and (2) on muscle atrophy. The following presents representative data obtained in these studies.

[0672] FIGs. 12A-D present graphs showing the mean TA / BW ratios in mice treated with water- soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day) and glycine (4.1 grams / kg / day), in comparison to a nutrient-untreated control (FIG. 12A); in mice treated with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence of presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day), glycine (4.1 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day) or D-mannitol (12.96 grams / kg / day), in comparison to a nutrient-untreated control (FIG. 12B); in mice treated with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of myoinositol (8.3 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day), or D-mannitol (12.96 grams / kg / day), or a combination of D-mannitol (12.96 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day) and myoinositol (8.3 grams / kg / day), in comparison to a nutrient-untreated control (FIG. 12C); and in mice treated with dexamethasone in the absence or presence of glycine (4.1 grams / kg / day), alpha- ketoglutarate (3.3 grams / kg / day), adenosine (1.17 grams / kg / day), or sodium butyrate (3.3 grams / kg / day) and various combinations thereof, as indicated; Adenosine dose within the combination is 0.23 gram / kg / day (FIG. 12D; number of tested mice for each tested nutrient or combination of nutrients indicated in parentheses).

[0673] As can be seen, the TA / BW ratio in mice administered with most of the tested nutrients was higher compared to mice treated with dexamethasone only, indicating an attenuated loss of muscle mass following nutrient treatment.

[0674] In particular, the combination shown in FIG. 12D, which included glycine, sodium butyrate, and alpha-ketoglutarate, was associated with reduced variability in response and a more consistent attenuation of muscle atrophy across the treated group.

[0675] In FIG. 12D, the mean values for administering either glycine or sodium butyrate to atrophying muscle cells are 1.4+0.01 and 1.4+0.02, respectively, while the co-administration of glycine and sodium butyrate to atrophying muscle cells has a mean value of 1.5+0.03. This higher TA / BW obtained by the combination of nutrients indicates a synergistic effect between the nutrients, for example, according to the Loewe additivity model.

[0676] FIG. 13 is a bar graph showing the mean heart weight (mg) of the mice treated with water soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day) or myoinositol (8.3 grams / kg / day). This indicates that nutrient treatment does not affect cardiac muscle weight.

[0677] FIGs. 14A-B present comparative plots showing the mean body weight (grams) of mice administered with water soluble dexamethasone (10 mg / kg), over a period of 10 days, in the presence or absence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day), and glycine (4.1 grams / kg / day) in comparison to non-treated mice (control) (FIG. 14A); and showing the mean body weight (grams) of mice administered with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, in the absence or presence of alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day) or D-mannitol (12.96 grams / kg / day), in comparison to nutrient-untreated mice (control) (FIG. 14B).

[0678] As can be seen, a reduction of 10 % in body weights of mice was observed in the presence of dexamethasone, with and without nutrients. Since the TA / BW ratio was shown to be higher in mice treated with dexamethasone and nutrients compared to dexamethasone alone, these data may suggest that mice treated with nutrients experience a greater reduction in fat compared to those treated with dexamethasone alone. Consequently, the nutrient treatment may confer additional benefits by preserving muscle weight and enhancing the muscle-to-fat ratio.

[0679] FIG. 15 presents a bar graph showing the comparative fold-increase in myostatin gene expression mRNA levels in mouse Tibialis Anterior (TA) muscle, and in Tibialis Anterior muscle from mice orally treated with water soluble dexamethasone (10 mg / kg body weight) over a period of 10 days, in the absence or presence of myoinositol (8.3 grams / kg / day), alpha-ketoglutarate (3.3 grams / kg / day) or D-mannitol (10.96 grams / kg / day).

[0680] As can be seen in FIG. 15, mice treated with dexamethasone in the presence of myoinositol, alpha-ketoglutarate or D-mannitol, displayed lower myostatin gene expression in comparison to the dexamethasone-treated (atrophy-induced) myotubes and in comparison to the nutrient- untreated control.

[0681] Studies were also conducted to evaluate the effect of selected nutrients in Grip strength test, tested using a grip strength meter. When pulled by the tail, rodents instinctively grasp a bar until the pulling force overcomes their grip strength. After the animal loses its grip, the device records and displays the peak force achieved by the limbs. The following presents representative data obtained in these studies.

[0682] FIGs. 16A-G present a representative photograph of a mouse subjected to the grip strength test (FIG. 16A); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of adenosine (1.17 grams / kg / day), myoinositol (8.3 grams / kg / day) and glycine (4.1 grams / kg / day), where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 (control) in each group (n = 5 mice per group. #, P < 0.05 vs. time 0 by unpaired Student’s t-test) (FIG. 16B); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of alpha-ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day) and D-mannitol (12.96 grams / kg / day), where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 (control) in each group (n = 5-10 mice per group. #, P < 0.05 vs. time 0 by unpaired Student’s t-test) (FIG. 16C); a bar graph showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone over a period of 10 days, in the absence or presence of alpha-ketoglutarate (3.3 grams / kg / day), D-mannitol (12.96 grams / kg / day), glycine (4.1 grams / kg / day), and D- mannitol (12.96 grams / kg / day), myoinositol (8.3 grams / kg / day) and alpha-ketoglutarate (3.3 grams / kg / day), where muscle strength is expressed as hanging time x BW (seconds x grams) in day 11 in comparison to time 0 control in each group (FIG. 16D).

[0683] As can be seen, treatment with all tested nutrients resulted in improved performance compared to dexamethasone alone.

[0684] FIGs. 16E-G present bar graphs showing the grip strength at day 11 as % of time 0 in mice treated with dexamethasone in the absence or presence of a glycine (4.1 grams / kg / day), alpha- ketoglutarate (3.3 grams / kg / day), sodium butyrate (3.3 grams / kg / day), adenosine (1.17 grams / kg / day), and combinations thereof as indicated (FIG. 16E); a bar graph showing the mean heart weights in the same treatment groups (FIG. 16F); and a bar graph showing the mean loss of body weights (FIG. 16G).

[0685] As can be seen, the tested nutrients and their combinations maintained muscle strength in mice treated with dexamethasone (FIG. 16E), protected cardiac muscle from dexamethasone- induced atrophy (FIG. 16F), and attenuated the loss of body weight during dexamethasone-induced atrophy (FIG. 16G).

[0686] The effect of the tested nutrients, in lower doses, which may facilitate formulating the nutrients for oral administration, was then tested. Doses were reduced by more than 100-fold, and tested on dexamethasone-induced muscle atrophy.

[0687] Thus, a combination of glycine and sodium butyrate was tested at lower concentrations of 0.21 grams / kg / day and 0.17 grams / kg / day, respectively, administered orally to dexamethasone- treated mice, as described herein.

[0688] FIGs. 17A-B present bar graphs showing the mean Tibialis Anterior (TA) muscle weight to body weight (BW) ratios (FIG. 17A) and the mean cardiac muscle weights (FIG. 17B) in mice orally administered with water-soluble dexamethasone (10 mg / kg) over a period of 10 days, with or without the low-dose of a nutrient combination (glycine (0.21 grams / kg / day) and sodium butyrate (0.17 grams / kg / day), which are equivalent to 1.17 grams / day glycine and 0.93 grams / day sodium butyrate).

[0689] Grip strength measurements were performed using a grip strength meter. To this, mice were orally administered with water soluble dexamethasone (10 mg / kg) in the presence or absence of nutrients combination for 10 days. A combination of glycine (0.21 gram / kg / day) and Na- butyrate (0.17 gram / kg / day) was used, as in FIGs. 17A-B, and the results are presented in FIG.

[0690] 17C.

[0691] As can be seen, this low-dose formulation significantly attenuated both skeletal and cardiac muscle atrophy in dexamethasone-treated (atrophy-induced) mice, compared to nutrient-untreated and vehicle controls. These findings support the synergistic action of glycine and sodium butyrate at low-dose levels and provide a strong rationale for advancing such an exemplary combination towards formulating it for human use, for example, in capsule formulations totaling about 2 grams / day in a 70 kg adult (e.g., divided into 3 capsules, each containing 0.7 gram of active nutrients in a 1-gram capsule). Additional studies are performed with a formulation comprising glycine (0.208 gram / kg / day) and alpha-keto glutamate (0.167 gram / kg / day), using the same protocol.

[0692] EXAMPLE 4

[0693] Formulation Chromatography. Shimadzu HPLC system with a UV DAD detector was used, using the parameters as follows:

[0694] HPLC derivatization (FMOC-Cl derivatization): The purpose of this method is to identify and quantify glycine as a free substance, and when formulated in a slow-release formulation, using HPLC with a UV detector. Glycine (NH2-CH2-COOH) has no absorbance in the UV-VIS range, and it is therefore difficult to detect using the above-mentioned HPLC DAD detector (an exemplary UV detector). However, glycine is detectable and quantifiable using HPLC after derivatization with a light-absorbing structure such as FMOC-Cl.

[0695] FMOC-Cl solution was prepared as follows: 100 mg FMOC-Cl was weighed into 50 ml acetone and mixed at room temperature using a magnetic stirrer for 5 minutes with the lid closed.

[0696] Carbonate buffer solution (pH 8.3-8.4) was prepared as follows: 5 grams of sodium bicarbonate was added to 95 ml H2O and stirred with a magnetic stirrer at medium speed. After about 45 minutes, when the sodium bicarbonate dissolved, the pH was measured. If the pH was below 8.3, additional sodium bicarbonate was added. Diluent solution was prepared as follows: methanol and water were mixed at a 20:80 ratio.

[0697] Derivatization process: If the sample had not been filtered, a disposable syringe was used to draw about 1 ml, which was then filtered through a syringe filter, discarding the first few drops. The filtered sample was then diluted with diluent to reach an estimated concentration between 12.5-625 ppm (the dilution could also be performed directly in the derivatization tube). Then, into a 2 ml plastic Eppendorf tube, 200 pl carbonate buffer, 1 ml FMOC-Cl solution, and 800 pl sample were added. The total volume was 2 ml. If dilution was performed in the derivatization tube, the diluent and filtered sample were added to the carbonate buffer before adding the FMOC-Cl. The...

Claims

WHAT IS CLAIMED IS:

1. A composition for use in treating or preventing muscle atrophy in a subject in need thereof, the composition comprising at least one nutrient that exhibits inhibition of myostatin signaling.

2. The composition for use of claim 1 , wherein the at least one nutrient is characterized by at least one of: affecting at least 10, or at least 20, or at least 50, %, reduction in phosphorylated SMAD2,3 and / or in a ratio of phosphorylated SMAD2,3 to non-phosphorylated SMAD2,3 in atrophying muscle cells; and affecting a decrease of at least 10, or at least 20, or at least 50, % in expression of a muscle atrophy biomarker in atrophying muscle cells.

3. The composition for use of claim 2, wherein a level of phosphorylated SMAD2,3 in the atrophying muscle cells is determined by Western blotting.

4. The composition for use of claim 2 or 3, wherein said muscle atrophy biomarker is associated with myostatin signaling pathway.

5. The composition for use of any one of claims 2 to 4, wherein said muscle atrophy biomarker is selected from Atrogin and / or Muscle RING-finger protein- 1 (MuRFl) mRNA.

6. The composition for use of any one of claims 1 to 5, comprising two or more of said nutrient.

7. The composition for use of claim 6, wherein at least two of said nutrient act in synergy with one another.

8. The composition for use of any one of claims 1 to 7, wherein said at least one nutrient is selected from glycine, mannitol, butyric acid, pyruvic acid, myoinositol, uridine, adenosine, 3 -hydroxybutyric acid, alpha-ketoglutarate, and pharmaceutically acceptable salts thereof.

9. The composition for use of any one of claims 1 to 6, wherein said at least one nutrient is selected from adenosine, myoinositol, glycine, alpha-ketoglutarate, sodium butyrate and mannitol, and pharmaceutically acceptable salts thereof.

10. The composition for use of any one of claims 1 to 9, wherein an amount of the at least one nutrient is in a range of from 30 to 100, or from 40 to 100, or from 50 to 100, or from 60 to 100, or from 50 to 90, or from 60 to 90, % by weight of the total weight of the composition.

11. The composition for use of any one of claims 1 to 10, wherein the composition is such that said subject receives each nutrient in an amount of from about 1 to about 1500, or from about 100 to about 1200, milligrams per day, when administered to a human subject (weighing about 70 kg).

12. The composition for use of claim 11, wherein the at least one nutrient comprises glycine.

13. The composition for use of claim 12, wherein a total daily amount of glycine in the composition is in a range of from about 0.2 to about 1.5, or from about 0.75 to about 1.5, grams per day, when the composition is administered to a human subject (weighing about 70 kg).

14. The composition for use of any one of claims 11 to 13, wherein the at least one nutrient comprises butyric acid and / or a salt thereof.

15. The composition for use of claim 14, wherein a total daily amount of sodium butyrate in the composition is in a range of from about 0.3 to about 1.2, or from about 0.75 to about 1.1, grams per day, when the composition is administered to a human subject (weighing about 70 kg).

16. The composition for use of any one of claims 11 to 15, wherein the at least one nutrient comprises alpha-ketoglutarate.

17. The composition for use of claim 16, wherein a total daily amount of alpha- ketoglutarate in the composition is in a range of from about 0.3 to about 1.5, or from about 0.75 toabout 1.1, grams per day, when the composition is administered to a human subject (weighing about 70 kg).

18. The composition for use of any one of claims 11 to 17, wherein the at least one nutrient comprises adenosine.

19. The composition for use of claim 18, wherein a total daily amount of adenosine in the composition is in a range of from about 0.75 to about 1.8, or from about 1.1 to about 1.5, grams per day, when the composition is administered to a human subject (weighing about 70 kg).

20. The composition for use of any one of claims 1 to 19, further comprising at least one of: a source of protein, a source of fatty acids, a source of one or more carbohydrates, sterol, vitamin, mineral, phenolic compound, carotenoid compound and odoriferous (scent) compound.

21. The composition for use of any one of claims 1 to 20, further comprising a physiologically acceptable carrier.

22. The composition for use of any one of claims 1 to 21, being formulated for administration by oral administration or oral ingestion.

23. The composition for use of claim 22, wherein the composition is in a form selected from a tablet, a capsule (e.g., gelatin capsule), a powder, a granule, a bead, a pellet, a lozenge, a solution, a syrup, an edible product, and a chewable solid (e.g., chewing gum).

24. The composition for use of claim 22 or 23, wherein treating the muscle atrophy comprises administering the composition to the subject by oral ingestion at least once, or at least twice, per day.

25. The composition for use of any one of claims 1 to 24, wherein treating the muscle atrophy comprises administering the composition to the subject by oral ingestion, such that said at least one nutrient is provided in an amount of from about 10 mg / kg to about 1,000 mg / kg per day, or from about 100 mg / kg to about 400 mg / kg per day.

26. The composition for use of any one of claims 1 to 25, wherein treating the muscle atrophy comprises administering the composition to the subject by oral administration or ingestion in a total amount of from 1 to 5, or from 1 to 3, grams of the composition, per day.

27. The composition for use of any one of claims 1 to 21, being formulated for topical administration.

28. The composition for use of claim 27, being formulated in a form of a patch.

29. The composition for use of any one of claims 1 to 28, being formulated as a medical device or a medical food.

30. The composition for use of any one of claims 1 to 29, further comprising a therapeutically active agent usable in treating or preventing muscle atrophy.

31. The composition for use of any one of claims 1 to 30, further comprising a GSK-3 inhibitor.

32. The composition for use of any one of claims 1 to 31 , wherein treating or preventing the muscle atrophy further comprises administering to the subject a therapeutically active agent usable in treating or preventing muscle atrophy.

33. The composition for use of any one of claims 1 to 32, wherein treating or preventing the muscle atrophy further comprises administering to the subject a GSK-3 inhibitor.

34. The composition for use of any one of claims 1 to 33, wherein the muscle atrophy is associated with at least one of ageing, cachexia, sedentary lifestyle, sarcopenia, malnutrition, disuse atrophy, neurogenic atrophy, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth disease, peripheral neuropathy, corticosteroid therapy, Emery-Dreifuss muscular dystrophy, Distal muscular dystrophy, Oculopharyngeal muscular dystrophy, Congenital muscular dystrophy, neuromuscular diseases, extended immobilization, trauma, alcoholism, cancer treatment, hyperthyroidism, heart failure, liver diseases, kidney diseases, diabetes, osteoarthritis, Cushing's syndrome, nutritional atrophy,severe bums, malabsorption syndromes, anorexia nervosa and ischemic atrophy, anorexia nervosa, rheumatoid arthritis and surgery.

35. The composition for use of any one of claims 1 to 34, wherein the muscle atrophy is associated with cachexia.

36. The composition for use of claim 34 or 35, wherein the cachexia is associated with cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), chronic kidney disease and Acquired Immune Deficiency Syndrome (AIDS).

37. A method of identifying a nutrient capable of treating or preventing muscle atrophy in a subject in need thereof, the method comprising: determining if a biocompatible nutrient substance reduces a level of or inhibits phosphorylation of SMAD2,3 in atrophying muscle cells; and determining if a biocompatible nutrient substance reduces a level of or inhibits expression of a muscle atrophy biomarker in atrophying muscle cells, wherein a biocompatible nutrient substance that reduces a level of or inhibits phosphorylation of SMAD2,3 in atrophying muscle cells and reduces a level of or inhibits expression of said muscle atrophy biomarker in atrophying muscle cells, is identified as capable of treating or preventing muscle atrophy.

38. The method of claim 37, wherein determining if a biocompatible nutrient substance reduces a level of or inhibits phosphorylation of SMAD2,3 in the atrophying muscle cells comprises contacting the nutrient substance with the atrophying muscle cells determining a level of phosphorylated SMAD2,3 (pSMAD2,3) in the atrophying muscle cells and / or determining an intensity ratio of phosphorylated SMAD2,3 to non-phosphorylated SMAD2,3 in the atrophying muscle cells.

39. The method of claim 38, wherein determining said level of phosphorylated SMAD2,3 and / or said intensity ratio is performed by immunoblotting.

40. The method of claim 38 or 39, wherein nutrient substance is determined as reducing a level of or inhibiting phosphorylation of SMAD2,3 in the atrophying muscle cells if the nutrient substance affects at least 10, or at least 20, or at least 50, %, reduction in said level ofphosphorylated SMAD2,3 and / or in said intensity ratio of phosphorylated SMAD2,3 to nonphosphorylated SMAD2,3, in said atrophying muscle cells.

41. The method of any one of claims 37 to 40, wherein determining if a biocompatible nutrient substance reduces a level of or inhibits expression of said muscle atrophy biomarker in atrophying muscle cells comprises contacting the nutrient substance with the atrophying muscle cells and determining a level of the expression of said muscle atrophy biomarker in the atrophying muscle cells.

42. The method of claim 41, wherein a nutrient substance is determined as reducing a level of or inhibiting said expression if it affects a decrease of at least 10, or at least 20, or at least 50, % in a level of expression of the muscle atrophy biomarker in the atrophying muscle cells.

43. The method of claim 41 or 42, wherein determining a level of the expression is performed by determining biomarkers expression in atrophying muscle cells in the presence of the nutrient, wherein determining biomarkers expression in atrophying muscle cells in the presence of the nutrient is effected by a technique selected from quantitative PCR (e.g., RT-qPCR), Western blotting, ELISA, RNA sequencing, and immunohistochemistry.

44. The method of any one of claims 37 to 43, wherein the muscle atrophy biomarker is selected from Atrogin and / or Muscle RING-finger protein- 1 (MuRFl) mRNA.

45. The method of any one of claims 37 to 44, comprising: determining if a biocompatible nutrient substance reduces a level of or inhibits phosphorylation of SMAD2,3 in atrophying muscle cells, to thereby identify nutrient substances capable of reducing a level of or inhibiting phosphorylation of SMAD2,3 in atrophying muscle cells; and determining if a nutrient substance identified as capable of reducing a level of or inhibiting phosphorylation of SMAD2,3 in atrophying muscle cells reduces a level of or inhibits a level of expression of said muscle atrophy biomarker in atrophying muscle cells.

46. The method of any one of claims 37 to 45, further comprising determining an effect of the nutrient substance identified as reducing a level of or inhibiting phosphorylation ofSMAD2,3 in atrophying muscle cells and reducing a level of or inhibiting expression of a muscle atrophy biomarker in atrophying muscle cells, on muscle atrophy in an atrophying test animal.

47. The method of claim 37, wherein determining said effect of the nutrient substance on muscle atrophy in the test animal comprises administering the nutrient substance to the test animal and determining an effect of said administering on a weight of a muscle of the animal and / or on a weight ratio of said muscle and the body of the animal.

48. The method of claim 47, wherein a nutrient substance determined as increasing said weight or weight ratio by at least 5 %, is identified as capable of treating muscle atrophy.

49. A nutrient usable in treating or preventing muscle atrophy in a subject in need thereof, identified by the method of any one of claims 37 to 48.

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